Principles of Biochemistry - Lehninger 5th ed

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PRINCIPLESOF BIOCHEMISTRY FIFTH

EDITION

David L. Ne lson Professorof Bio chemistry Uniuersity of Wisconsin-Madison

Michael M. Cox Professor of Bi,ochemistry Uni,uersity of Wi,sconsin-Madi,son

l= ANDCOMPANY W.H.FREEMAN N e wY o r k

4

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On the cover: RNA polymeraseII from yeast, bound to DNA and in the act of transcribing it into RNA. Imagecreatedby H. Adam Steinbergusing PDB ID 1I6Has modi.fledby Seth Darst.

Library of CongressControl Number: 2007941224 ISBN-13:978-0-7167-7108-1 ISBN-I0: 0-7167-7r08-X @2008by W. H. Freemanand Company All rights reserved Printed in the United States of America First printing W H. Freeman and Company 41 MadisonAvenue New York,NY 10010 Houndmills,BasingstokeRG216XS,England www.whfreeman.com

To Our Teachers PauLR. Burton Albert Fi,ntuolt Wi,LLi,am P. Jencks Eugene P. Kennedy Homer Knoss ArtLtur Kornberg L RobertLeltman EarL K, I{elson Dauid E, Sh,eppard Harold B. Wti,te

DaVid L. NelSOn,bornin Fairmont, Minnesora, received his BS in Chemistry and Biology from St. Olaf College in 1964 and earned his PhD in Biochemistry at Stanford Medical School under Arthur Kornberg. He was a postdoctoral felLowat the Harvard Medical School with Eugene P. Kennedy, who was one of Albert Lehninger's first graduate students. Nelson joined the faculty of the University of Wisconsin-Madison h 1971 and became a full professor of blochemrstry in 1982. He is the Director of the Center for Biology Education at the University of Wisconsin-Madison. Nelson's research has focused on the signal transductions that regulate ciliary motion and exocytosis in the protozoan Parameci,um. The enzymes of signal transductions, including a variety ofprotein kinases, are primary targets of study. His research group has used enzyme purification, immunological techniques, electron microscopy, genetics, molecuiar biology, and electrophysiologyto study these processes Dave Nelson has a distinguished record as a lecturer and research superuisor. For 36 years he has taught an intensive survey of brochemistry for advanced biochemistry undergraduates in the life sciences. He has also taught a survey of biochemistry for nursing students, and graduate courses on membrane structure and function and on molecular neurobiology. He has sponsored numerous PhD, MS, and undergraduate honors theses, and has received awards for his outstanding teaching, including the Dreyfus Teacher-Scholar Award, the Atwood Distinguished Professorship, and the Unterkofler Excellence in Teaching Award from the University of Wisconsin System.In 1991-1992he was a visiting professor of chemistry and biology at Spelman College. His second love is history and in his dotage he has begun to teach the history of biochemistry to r-mdergraduatesand to collect antique scientific instruments.

MiChagl M. COXwasbornin Wlmington, I)elaware. In his first biochemistry course,Lehninger's Biochem'istry was a major influence in refocusing his fascination with biology and inspiring him to pursue a career in biochemistry. After graduating from the University of Delaware inl974, Cox went to Brandeis University to do his doctoral work with MIIiam P Jencks, and then to Stanford in 1979 for postdoctoral study with I. Robert Lehman. He moved to the University of WisconsinMadison in 1983, and became a full professor of biochemistry in 1992. Cox's doctoral research was on general acid and base catalysis as a model for enz;,,rne-catalyzedreacvl

David [. Nelson andMichael M.Cox

tions. At Stanford,he beganwork on the enzymesinvolvedin geneticrecombination.The work focusedparticularly on the RecAprotein, designingpuriflcation and assaymethodsthat are still in use, and illuminating the processof DNAbranchmigration.Explorationof the en4,.rnesof genetic recombinationhas remained the central themeof his research. Mike Cox has coordinateda large and active researchteam at Wisconsin,investigatingthe enzymology, topology,and energeticsof genetic recombination.A primary focus has been the mechanism of RecA protein-mediatedDNA strand exchange,the role of ATP in the RecA system,and the regulationof recombinational DNA repair. Part of the researchprogram now focuseson organismsthat exhibit an especiallyrobust capacityfor DNA repair, such asDei,nococcusrad'i,odurans, and the applicationsof those repair systemsto biotechnology.For the past24 yearshe has taught (with DaveNelson)the suwey of biochemistryto undergraduatesand haslectured in graduatecourseson DNA structure and topology,protein-DNAinteractions,and the biochemistryof recombination.A more recent project has been the organizationof a new courseon professionalresponsibilityfor fi.rst-yeargraduatestudents.He has received awards for both his teaching and his research,including the Dreyfus Teacher-ScholarAward and the 1989EIi Lilly Award in BiologicalChemistry His hobbiesinclude gardening,wine collecting,and assisting in the designof laboratorybuildings.

I n this twenty-flrst century a typical scienceeducation I often leavesthe philosophicalunderpinningsof scienceunstated,or relies on oversimplifieddefinitions.As you contemplatea careerin science,it may be usefulto consideronce againthe terms science, scientist, and scientifie method. Science is both a way of thinking about the natural world and the sum of the information and theory that result from such thinking.The power and successof scienceflow directlyfrom its relianceon ideasthat can be tested: information on natural phenomenathat can be observed,measured,and reproducedand theoriesthat havepredictivevalue.The progressof sciencerestson a foundationalassumptionthat is often unstatedbut crucial to the enterprise:that the lawsgoverningforcesand phenomenaexisting in the universe are not subject to change.The NobellaureateJacquesMonodreferredto this underlyingassumptionasthe "postulateof objectivity." The natural world can therefore be understoodby applying a processof inquiry-the scientific method. Sciencecould not succeedin a universe that played tricks on us. Other than the postulateof objectivity,science makesno inviolate assumptionsabout the natural world.A usefiilscientiflcideais onethat (1) hasbeenor can be reproduciblysubstantiatedand (2) can be used to accuratelypredict new phenomena. Scientrflcideastake many forms.The terms that scientists use to describetheseforms havemeaningsquite differentfrom thoseappliedby nonscientists. Ahypothesesis an idea or assumptionthat providesa reasonable and testableexplanationfor one or more observations, but it may lack extensiveexperimentalsubstantiation.A sci,enti,fi,ctheorA is much more than a hunch. It is an idea that has been substantiatedto some extent and provides an explanationfor a body of experimentalobservations.A theory can be tested and built upon and is thus a basisfor further advanceand innovation.When a scientiflctheory has been repeatedlytested and validatedon manyfronts,it canbe acceptedas a fact. In one importantsense,what constitutesscienceor a scientiflc idea is defined by whether or not it is published in the scientiflc literature after peer review by other working scientists.About 16,000peer-reviewed scientific journals worldwide publish some 1.4 million articles eachyear, a continuing rich harvest of information that is the birthright of every human being. Scientists are indiredualswho rigorouslyapply the scientific method to understand the natural world. Merely having an advanceddegreein a scientiflc discipline doesnot makeone a scientist,nor doesthe lack of such a degreeprevent one from making important scientific contributions.A scientistmust be willing to challenge any idea when new findings demandit. The ideas that a scientistacceptsmust be basedon measurable,

reproducibleobservations, and the scientistmust report with completehonesty. theseobservations The scientific method is actually a collection of paths,all of wtuch may lead to scientificdiscovery.In the hypothesi,sand erperiment path,a scientistposesa hypothesis,then subjectsit to experimentaltest. Many of the processesthat biochemistswork with everyday were discoveredin this manner.The DNA structure elucidated by JamesWatsonand FrancisCrick led to the hypothesis that basepairjrg is the basisfor information transfer in po\mucleotide sS,nthesis. This hlpothesis helpedinspire the discoveryof DNA and RNA pol5.'rnerases. Watsonand Crick produced their DNA structure through a process of model bui,ldi,ng and calculat'ion. No actual experimentswere involved, although the model building and calculations used data collectedby other scientists.Many adventurousscientists haveappliedthe processoferp\oration and obseruat'ion as a path to discovery.Historicalvoyagesof discovery (Charles Darwin's 1831 voyage on H.M.S. Beagleamongthem) helpedto map the planet,catalog its living occupants,and changethe way we view the world. Modern scientists follow a similar path when they explore the ocean depths or launch probes to other planets.An analogof hypothesisand experiment is hypothesi,sand deduct'ion. Crick reasoned that there must be an adaptor molecule that facilitated translationof the information in messengerRNA into protein.This adaptorhypothesisled to the discoveryof transfer RNA by MahlonHoaglandand Paul Zamecnik. Not all paths to discoveryinvolve planrung.Serendipi,tg often plays a role. The discovery of penicilJin by Alexander Fleming in 1928, and of RNA catalysts by ThomasCechin the early 1980s,wereboth chancediscoveries,albeit by scientistswell preparedto exploit them. Irnpi,rati,on canalsoleadto important advances.The polymerasechainreaction(PCR),now a centralpart of biotechnology, was developedby Kary Mullis afler a flash of inspration dudng a road trip in northern Califomiain 1983. Thesemany paths to scientiflc discoverycan seem quite different, but they have some important things in common. They are focused on the natural world. They rely on reproducCbleobseruat'ion anilor erperiment. Nl of the ideas,insights, and experimentalfacts that arise from these endeavorscan be tested and reproducedby scientistsan5,wherein the world. All can be usedby other scientiststo build new hypothesesand make new discoveries.All lead to information that is properlyincludedin the realm of science.Understanding our universerequires hard work. At the sametime, no human endeavoris more exciting and potentially rewarding than trying, and occasionallysucceeding,to understandsomepart of the natural world. vtl

first edition of Pnnctples oJBi,ochenuistry, v'ritten Albert Lehningertwenty-flveyearsago,hasservedas the starting point and the model for our four subsequent editions.Overthat quarter-centurythe world of biochemistry haschangedenormously. yearsago,not a TWenty-flve singlegenomehadbeensequenced, not a singlemembrane proternhad beensolvedby crystallography, and not a sinjust beendishnockout mouse existed. RibozJrmes had $e covered, PCR technology introduced, and archaea recognizedasmembersof a kingdomseparatefrom bacteria Now,newgenomicsequences areannouncedweekly, new protern structures even more frequently, and researchershave engineeredthousandsof djfferent lcrockout mice, with enormouspromise for advancesin basic biochemistryphysiology,and medicine.This ffih edition containsthe photographsof 31 Nobellaureateswho have receivedtheirprizesfor Chemistryor for Physiologror Medicine sincethat first edition of Prhrciples of Binchemistry. One major challengeof each edition has been to reflect the torrent of new information without making the book overwhelmingfor students having their first encounterwith biochemistry.This hasrequiredmuch careful sifting aimed at emphasizingprinciples while still conveyingthe excitementof current researchand its promisefor the future. The cover of this new edition exempli-fles this excitementand promise:in the x-ray structure of RNA polymerase,we seeDNA, RNA, and protein in their informationalroles,in atomrcdimensions,caught in the central act of in-formationtransfer.

We are at the threshold of a new molecularphysiology in which processessuch as membrane excitation, secretion,hormoneaction,vision, gustation,olfaction, respiration,musclecontraction,and cell movementswill be explicablein molecularterms and will becomeaccessible to genetic dissectionand pharmacologicalmanipulation. Knowledgeof the molecular structures of the highly organizedmembrane complexes of oxidative phosphorylation and photophosphorylation,for example, will certainly bring deepenedinsight into those processes, so centralto life. (Thesedevelopments make us wish we were young again,just beginningour careers in biochemicalresearchand teaching. Our book is not the only thing that has acquired a touch of silver over the years!) In the past two decades,we have striven alwaysto maintain the qualitiesthat made the original Lehninger text a classic-clear wdting, careftrlexplanationsof difflcult concepts,and communicatingto studentsthe ways in which biochemistryis understoodand practicedtoday. Wehavewritten togetherfor twenty yearsand taught together for almosttwenty-flve.Our thousandsof students at the Universityof Wisconsin-Madison over thoseyears havebeen an endlesssourceof ideasabout how to present biochemistrymore clearly;they haveenlightenedand rnspired us. We hope that this twenty-flfth aruLiversary edition will erLlightenand inspire current studentsof biochemistryeverywhere,and perhapsleadsomeof them to Iovebiochemistryaswe do.

Major Recent Advances in Biochemistry Every chapter has been thoroughly revised and updated to include the most important advancesin biochemistryincluding: r

Conceptsof proteomes and proteomics, introducedearlierin the book (Chapter1)

r

New discussionof amyloid diseasesin the context of protein folding (Chapter 4)

r

New section on pharmaceuticals developedfrom an understandingof enzymemechanism,using penicillinand HIV proteaseinlLibitorsas examples (Chapter6)

r

New discussionof sugar analogs as drugs that target viral neuraminidase(Chapter 7)

r

New material on green fluorescent protein (Chapter9)

r

New sectionon lipidomics (Chapter10)

r

w descriptionsof volatile lipids used as signals vi

by plants, and of bird feather pigmentsderived from coloredlipids in plant foods (Chapter10) Expandedand updated sectionon lipid rafrts and caveolae to rncludenew material on membrane curvature and the proteins that influenceit, and introducng amphitropic proteins and anmrlar Iipids (Chapter11) New sectionon the emergingrole of ribulose 5-phosphate as a central regulator of $ycolysis (Chapter 15) andgluconeogenesis New Box 16-1, MoonlightingErzymes:Proteins with More Than One Job New sectionon the role of transcriptionfactors (PPARs) in regulationof lipid catabolism (Chapter17) Revisedand updated sectionon fatty acid synthase, including new structural information on FASI (Chapter21)

Preface tx

Updatedcoverageof the nitrogen cycle, including new Box 22-1, UnusualLife Stylesof the Obscure but Abundant, discussinganammox bacteria (Chapter22New Box 24-2, Epigenetics,Nucleosome Structure,and Histone Variantsdescribingthe role of histone modification and FIGURE 21-3 Thestructure typeI systems. offattyacidsynthase nucleosome deposition in the transmissionof New information on the roles of RNA epigeneticinformation in heredity in protein biosynthesis New information on the initiation of replication (Chapter 27) and the dymamicsat the replicationfork, New sectionon riboswitches introducing AAA+ ATPases and their functions (Chapter28) in replication and other aspectsof DNA metabolism(Chapter25) New Box 28-1, Of Fins,Whgs, Beaks,and Things, New sectionon the expandedunderstandingof the roles of RNA in cells (Chapter 26)

describingthe cormectionsbetweenevolution and development

Biochemical Methods An appreciation of biochemistry often requires an understanding of how biochemical information is obtained. Some of the new methodsor updatesdescribed in this editionare: r

Circulardicluoism (Chapter4)

r

Measurementof glycated hemoglobinas an indicator of averagebloodglucoseconcentration, over days,in personswith diabetes (Chapter7)

r

Useof MALDI-MSin determinationof oligosaccharide structure (Chapter7)

r

ForensicDNA analysis,a majorupdate coveringmodernSTRanalysis(Chapter 9)

r

More on microarrays(Chapter9)

r

Use of tags for protein analysisand purification (Chapter9)

r

r

PET combinedwith CT scansto pinpoint cancer (Chapter14)

Glutathione (GSH)

G€ne for tusion prctein

flcuflt9-12 The useof taggedproteinsin protein purification. The use of a CST tag is illustrated(a) Clu(CST)is a smallenzyme(depicted tathione-s-transferase (a Slutahereby the purpleicon)that bindsglutathione at materesidue to whicha Cys-Clydipeptideis attached the carboxylcarbonof theClu sidechain,hencetheabbreviationCSH) (b) The CST tag is fusedto the catr boxyl terminus of the target protein by Senetic engineeringThe taggedprotein is expressedin host cells,and is presentin the crudeextractwhen the cells are lysed The extract is subjectedto chromatography on a column containinga mediumwith immobilized SlutathioneThe CsT{aggedproteinbinds to the 8lutathione,retardinBits migrationthroughthe column, while the other proteinswash through rapidly The elutedfrom the column taggedproteinis subsequently or elevatedsaltconcentration with a solutioncontaining free glutathione

I

v

Express tu8ion Foteh h a cell

Add gotein

EIub

fusion plobin

IIGURE 9-12

Chromatinimmunoprecipitationand ChlP-chip experiments(Chapter24)

r

Developmentof bacterial strainswith altered ge netic codes,for site-specific insertion of novel amino acids into proteins (Chapter 27)

x

Preface

Medically Relevant Examples This icon is used throughoutthe book to denote materialof specialmedicalinterest.As teachers, our goal is for students to learn biochemistryand to understand its relevance to a healthier life and a healthier planet. We have included many new examples that relatebiochemistryto medicineand to health issuesin general.Someof the medicalapplicationsnew to this edition are: r r

r

r r

The role of polyunsaturatedfatty acidsand trans fatty acidsin cardiovascular disease(Chapter10) G protein-coupledreceptors(GCPRs)and the range of diseasesfor which drugs targeted to GPCRsare beingusedor developed(Chapter12) G proterns,the regulationof GTPaseactivity, and the medicalconsequences of defectiveG protein function(Chapter12),includingnew Box 12-2, G Proteins:Binary Switchesin Health and Disease Box 12-5,Developmentof ProteinKinaseInhibitors for CancerTleatment Box 14-1,HtghRateof Glycolysisin Tlmors Suggests Targetsfor Chemotherapyand FacrlitatesDiagnosis

r

Box 15-3, GeneticMutationsThat Leadto Rare Formsof Diabetes

r

Mutationsin citric acid cycle enzyrnesthat lead to cancer(Chapter16)

r

Perniciousanemiaand associatedproblemsin strict vegetarians(Chapter 18)

r

Updatedinformationon cyclooxygenase hhibitors (pain relieversVioxx, Celebrex,Bextra) (Chapter21)

r

HMG-CoAreductase(Chapter21) and Box 21-3, The Lipid Hypothesisand the Developmentof Statins

r

Box 24-1, CuringDiseaseby Inhibiting Topoisomerases, describingthe use of topoisomeraseinhibitors in the treatment of bacterial rnfectionsand cancer,including material on ciprofloxacin (the antibiotic effective for anthrax)

Special Theme: Understanding Metabolism through 0besity andDiabetes Obesity and its medical consequences-cardiovascular diseaseand diabetes-are fast becomingepidemic in the industrializedworld, and we include new material on the biochemicalconnectionsbetween obesity and health throughout this edition. Our focus on diabetes provides an integrating theme throughout the chapterson metabolismand its control, and this will, we hope, inspire some students to find solutions for this disease.Some of the sectionsand boxes that highlight the interplay of metabolism, obesity, and diabetesare:

@ Fatty acid oxidatioD Stawation response

Fat synthesie and storage

and storage Adipokineproduction

Fatty acid oxidati Themogenesis

r

Untreated DiabetesProducesLife-ThreatenineAci dosis(Chapter2)

r

Box 7-1 , Blood GlucoseMeasurementsin the Diagnosisand T?eatmentof Diabetes,introducing hemoglobinglycation and AGEsand their role in the pathologyof advanceddiabetes Box 11-2,DefectiveGlucoseand WaterT?ansport in TWoFormsof Diabetes

FIGURE 23-42 r

AdiposeTissueGeneratesGlycerol3-phosphate (Chapter21) by Glyceroneogenesis

r

GlucoseUptakeIs Deficientin T}pe 1 DiabetesMel Iitus (Chapter14)

r

DiabetesMellitus Arises from Defectsin Insulin Productionor Action (Chapter23)

r

KetoneBodiesAre Overproducedin Diabetesand during Starvation (Chapter 17)

r

r

SomeMutationsin MitochondrialGenomesCause Disease(Chapter19) DiabetesCan Resultfrom Defectsin the Mitochon dria ofPancreaticB Cells(Chapter19)

Section23.4,Obesityand the Regulationof Body Mass,discussesthe role of adiponectinand insulin sensitivity and tlpe 2 diabetes

r

Section23.5,Obesity,the MetabolicS;'ndrome,and T\pe 2 Diabetes,includesa discussionof managing type 2 diabeteswith exercise,diet, and medication

r

r

Advances in Teaching Biochemistry

1l-3 WORKED EXAMPII

f

Revisingtlus textbookis neverjust an updatingexercise.At Ieastasmuch time is spent reexamininghow the core topics of biochemrstryare presented.We haverevisedeachchapterwith an eyeto helpingstudentslearn and masterthe fundamentalsof biochemistry.Studentsencounteringbiochemistryfor the first trmeoften havedifflcultywith two key aspectsof the course:approachingquantitative problemsand drawingon what they learnedin orgarucchemistryto help them understandbiochemistry.Those samestudents must also learn a complex language,with conventionsthat are often unstated.Wehavemadesome major changesin the book to help studentscope with all these challenges: new problem-solvingtools, a focus on organic chemistry foundations, and highlightedkey conventions.

EnergeticsofPumping bYSYnPort

lglumseli, --= mtio that can be .': lglucosejour plasma membrme Na*-glucose symachieved by the porter of an epithelial cell, when [Na-]6 is 12 mM, -50 mV [Nat]."1 is 145 ro, the membrme potential is 'C (inside negative), and the temperature is 37 Cahulal,e lhe maimm

Soltrtion: Using Equation 11-4 (p 396), we can calcuNa+ late the energy inherent in an electrochemical gradient-that is, the cost of moving one Na- ion up gradientl this

AG. ' _ R?lnry+ + zr a,r, tNal,"

We then substitute standardvaluesfor-&, ?, and J, and the given valuesfor [Na-] (expressedas molar concentrations), +l for Z (because Na+ has a positive charge), md 0 050 V for a,y' Note that the membrane potential is -50 mV (inside negative),so the chmge in potential when m ion moves from inside to outside is 50 mV.

N e wPr o b l e m-5 o lTvionogl s

1 45 x 10-' 1.2xto 2 + 1(96,500 JV.mol)(0 050V)

AGt : (8 315J/mol K)(3to rcm

r New in-text Worked Examples help studentsimprove their quantitativeproblem-solvingskills, taking them through someof the most difficult equations.

= 112 kJ/mol This AGr is the potential energyper mole of Na- in the Na* $adient that is available to pmp glucose Given that two Na- ions passdom their electrochemicalgradient md into the cell for each glucose canied in by slmport, the energyavailableto pmp 1 mol of Llucose is2 x II2 kJ/mol = 22 4 kJ/mol We can now calculate the concentrationratio of Elucosethat cm be achieved by this pmp (from Equation l1-3, p 396):

r More than 100 new end-of-chapter problems give students further opportunity to practice what they have learned. r New Data Analysis Problems (one at the end of eachchapter), con tributed by BrianWhite of the Universityof Massachusetts-Boston, en couragestudentsto slmthesizewhat they have learnedand apply their knowledgeto the interpretationof datafrom the literature.

_ __. [glucose]r lG, = ft?ln:iLgrucoselour

Remnuing, then substitutingthe valuesof AGt,,&,and ?, gives 22.4kJ/mol . [g]ucosel* AG, = - o ot 'n n,r E:rs .llorot' t > 1, AGois largeandnegative;whenK"q 0; positive andunfavorable or more valuein the index)foraminoacidswithnonpolar h y d r o p h o b i c s i d e c hS ae i nesC h a p t e r l l . F r o m K y t&e D , Jo o l i t t l e , R . E ( 1 9 8 2 ) A s i m p l e m e t h o d f o r d i s p l a y i n g t h e h y d r o p a t h i c of a protein. character J. Mol.Biol.157, 105-132. occunence in morethan1,150proteins. Prediction FromDoolittle, R.E(1989)Redundancies in proteinsequences.ln fAverage of ProteinStructureand the Pilnciples (Fasman, of ProteinConfornation G.D.,ed ), pp 599-623,PlenumPress,NewYork. is generally classified as polardespite having a positive theabilityof thesulfhydryl hydropathy index. Thisreflects Scysteine groupt0 actas a weakacidandto forma weakhydrogen bondwithoxygen or nitrogen

(see Fig. 1-19). All molecules a chiral center are also optically active-that "r/ith is, they rotate planepolarizedlight (seeBox 1-2). KEYC0NVENTI0N: I\.voconventions are usedto identify the carbons in an amino acid-a Dracticethat can be

confusing. The additional carbons in an R group are commonly designatedB , 7,6, E, and so forth, proceeding out from the a carbon. For most other organic molecules, carbon atoms are simply numbered from one end, giving highest priority (C-1) to the carbon with the substituent containing the atom of highest atomic number.

Within this latter convention,the carboxyl carbon of an aminoacidwould be C-1and the a carbonwouldbe C-2. e6lpd 651321

cH2 -cH2 -cH2-CH2 -CH-COO-

tl

*NHa

*NHe Lysine

In some cases,such as amino acids with heterocyclic R groups(suchas histidine),the Greekletteringsystem is ambiguousand the numberingconventionis therefore used.For branchedamino acid side chains,equivalent carbons are given numbers after the Greek letters. Leucinethus has61 and62 carbons(seethe structurein Fig.3-b) r Specialnomenclaturehas been developedto specify the absolute configuration of the four substituents of asymmetric carbon atoms. The absolute configurationsof simple sugars and amino acids are specifiedby the D, L system (Fig. 3-4), basedon the absoluteconflgurationof the three-carbonsugarglyceraldehyde,a conventionproposed by Emil Fischer in 1891. (Fischer knew what groups surroundedthe as;nnmetriccarbon of glyceraldehydebut had to guess at their absoluteconflguration;his guesswas later confirmed by x-ray diffraction analysis.)For all chiral compounds, stereoisomershaving a configurationrelated to that of t -glyceraldehydeare designated l, and stereoisomersrelated to l-glyceraldehyde are designatedl. The functionalgroupsof l-alaninearematched with those of l-glyceraldehydeby aligning those that can be interconvertedby simple,one-stepchemicalreactions.Thus the carboxylgroup of l-alanine occupies the sameposition about the chiral carbonas does the aldehydegroup of r,-glyceraldehyde, becausean aldehyde is readily converted to a carboxyl group via a

lCHO

HOJC-H 3cH2oH

H--C'.OH cH2oH o-Glyceraldehyde

coo

coo

CH, r,-Alanine

TheAmino inProteins Acid Residues Arer Stereolsomers Nearly all biologicalcompoundswith a chiral center occur naturally in only one stereoisomericform, either n or l. The amino acid residuesin protein moleculesare exclusively l stereoisomers.D-Amino acid residues have been found in only a few, generally small peptides, including somepeptides of bacterial cell walls and certain peptideantibiotics. It is remarkable that virtually all amino acid residuesin proteins are l stereoisomers.When chiral compounds are formed by ordinary chemical reactions, the result is a racemic mixture of n and I isomers, which are difficult for a chemist to distinguish and separate.But to a living system,n and l isomers are as different as the right hand and the left. The formation of stable, repeating substructuresin proteins (Chapter 4) generallyrequires that their constituent amino acidsbe of one stereochemicalseries.Cellsare able to specificallysynthesizethe I isomersof amino acids becausethe active sites of enzymesare asymmetric, causingthe reactionsthey catalyzeto be stereospecific.

CHO

r,-Glyceraldehyde

n,fr-0-n

one-stepoxidation. Historically,the similar L and D designations were used for levorotatory (rotating planepolarizedlight to the left) and dextrorotatory (rotating light to the right). However,not all L-amino acids are levorotatory and the convention shown in Figure 3-4 was needed to avoid potential ambiguitiesabout absolute conflguration. By Fischer's convention, L and D refer only to the absolutecon-fi.guration of the four substituents around the chiral carbon,not to optical propertiesof the molecule. Another system of speci$ringcon-flgurationaround a chiral center is the RS system, which is used in the systematicnomenclatureof organic chemistry and describes more precisely the conflguration of molecules with more than one chiral center (seep. 17).

H-C-NHs CHt o-Alanine

FIGURE 3-4 Stericrelationshipof the stereoisomers of alanineto the absoluteconfigurationof l- and o-glyceraldehyde. In theseperspectiveformulas, the carbonsare linedup vertically, with the chiralatom in the center. Thecarbonsin thesemolecules arenumbered beginning with the terminalaldehydeor carboxylcarbon(red),I to 3 from top to bottom as shown.When presentedin this way, the R group of the aminoacid(in thiscasethe methylgroupof alanine)is alwaysbelow the o carbon.r-Aminoacidsarethosewith the a-aminogroupon the left,and o-aminoacidshavethe a-aminogroupon the right.

(an$eClassified Acids Amino byRGroup Ifuowledge of the chemical properties of the cornmon amino acids is central to an understandingof biochemistry. The topic can be simplifiedby groupng the amino acids into flve main classesbased on the properties of their R groups (Table3-1), in particular,their polarity, or tendencyto interact with water at biologicalpH (near pH 7.0). The polarity of the R groupsvarieswidely,from nonpolar and hydrophobic (water-insoluble)to highly polar and hydrophilic (water-soluble). The structures of the 20 corrrmonamino acids are shown in Figure 3-5, and someof their properties are listed in Table 3-1. Within each class there are gradations of polarity, size,and shapeof the R groups. Nonpolar, Aliphatic R Groups The R groups in this class of amino acids are nonpolar and hydrophobic.

Nonpolar, aliphatic

*l

coo

H3N-C-H 'l

H

R groups

coo

coo-

n,r(r-E-H J--CHs

H2C-CH2 Glycine

coo H3N-C-H

coo*l H3N-C-H

1t I/c{

coo

coo

H-C-CH3

H3N-C-H I CHO

CH, lCHr

CH,

t-

S

Positively charged R groups

I

Methionine

Isoleucine

+

coo Hrfr-c H

coo I -C-H

coo-

coo

H3N-C-H I CH,

gH"

t-

C-N H

l*

C:NHO

l-

Negatively

*l

charged R groups

coo-

H3N-C-H

CII* l-

coo-.

o

Glutamine

Histidine

Arginine

l

CHo

Asparagine

tq-\H ll ,cH

NH

Lysine

CHo

,\ HrN

CHo

t-

coo Hrfr c-H

O

H

NH,

Cysteine

t-

l" HzN

CH, {*NH,

SH

Threonine

*l

1-

H3N-C-H

H-C-OH

l-

CH.

coo-

H.N-C "l

tCH, tCH, t-

CHz

coo+' H3N-C-H

*l

I CHo

l-

ll CHS Serine

Tlyptophan

Tlrosine

Phenylalanine

CHo

rtt cH2oH

HSN-C-H I CHo

t-

Polar, uncharged R groups

coo-

coo

OH

+

HaN

*l H3N-C-H

C-H

*l

l-

CHe Leucine

H3N

coo-

Valine

l

cH3 cH3

*l

cH3 cH3

+l H3N-C-H

CHO

coo

H3N-C-H ll

CH

|'

Proline

Alanine

*L

+

,,['n -cH,

HrN-l

R groups

Arorratic

Aspartate

coo rl H3N-C-H -l

CH,

t-

CHO

tcooGlutamate

FIGURE 3-5 The 20 common amino acidsof proteins.The structural formulasshowthe stateof ionizationthat would oredominate at DH portionsarethosecommonto all the aminoacids; 7.0.Theunshaded the portionsshadedin pink arethe R groups. Althoughthe R groupof

histidineis shown uncharged,its pK" (seeTable3-.1) is such that a chargedat fractionof thesegroupsarepositively smallbut significant pH 7.0.The protonatedform of histidineis shownabovethe graphin Fie.3-12b.

The side chains of alanine, valine, leucine, and isoleucine tend to cluster together within proteins, stabilizingprotein structure by meansof hydrophobic interactions.Glycine has the simplest structure. Although it is most easily grouped with the nonpolar amino acids, its very small side chain makes no real contribution to hydrophobic interactions. Methionine, one of the two sulfur-containingaminoacids,has a nonpolar thioether group in its side chain. Proline has an aliphatic side chain with a distinctive cyclic structure. The secondaryamino (imino) group of pro-

line residuesis held in a rigid conformationthat reduces the structural flexibility of polypeptide regions containingproline. Aromatic R Groups Phenylalanine, tyrosine, and tryptophan, with their aromatic side chains, are relatively nonpolar (hydrophobic).All can participate in hydrophobic interactions.The hydroxyl group of tyrosine can form hydrogen bonds, and it is an important functional group in someenz).rnes.Tlrrosineand trlptophan are signiflcantlymore polar than phenylalanine,because

240 250 260 270 280 290 300 310 Wavelength(nm)

FIGURE 3-6 Absorptionof ultravioletlight by aromaticamino acids. Comparison of the lightabsorption spectra of thearomaticaminoacids tryptophanand tyrosineat pH 6.0. The amino acidsare presentin equimolaramounts(.1 0 3 la)underidentical conditions. Themeasured absorbanceof tryptophanis as much as four times that of tyrosine Notethatthe maximumlightabsorption for bothtryptophan andtyrosineoccursneara wavelength of 280 nm. Lightabsorption by thethird aromaticamino acid, phenylalanine(not shown),generallycontributeslittleto the spectroscopic properties of proteins.

of the tyrosine hydroxyl group and the nitrogen of the tr;,ptophanindolering. ftptophan and tyrosine,and to a much lesserextent phenylalanine,absorbultrauolet light (Fig. 3-6;

see alsoBox 3-1). This accountsfor the characteristic strong absorbanceof light by most proteins at a waveIengthof 280nm, a propertyexploitedby researchers in the characterization of oroteins.

A wide rangeof biomoleculesabsorblight at characterjust astryptophanabsorbslight at 280 istic wavelengths, nm (seeFiS.3-6). Measurementof light absorptionby a spectrophotometer is usedto detect and identify molecules and to measuretheir concentrationin solution. The fraction of the incident light absorbedby a solution at a given wavelengthis related to the thickness of the absorbinglayer (path length) and the concentrationof the absorbingspecies(Fig. 1). Thesetwo relationships are combinedinto the Lambert-Beerlaw'

species(in molesper liter), and I is the path length of the light-absorbingsample (in centimeters). The LambertBeer Iaw assurnesthat the incident light is parallel and monochromatic(of a singlewavelength)and that the solvent and solutemoleculesare randomlyoriented.The expressionlog (1611) is calledthe absorbance, designated,A. It is important to note that eachsuccessivemillimeter of path length of absorbingsolutionin a 1.0 cm cell absorbsnot a constantamountbut a constantfraction of the light that is incident upon it. However,with an absorbinglayer of fixed path length,the absorbance,A,,is di,rectly proportional to the concentration of the absorbi,ngsolute. The molar extinction coefflcientvarieswith the nature of the absorbingcompound,the solvent,and the wavelength,and also with pH if the light-absorbing speciesis in equilibriumwith an ionizationstatethat has properties. differentabsorbance

l o- et l :

"./

where 1e is the intensity of the incident light, 1is the intensity of the transmitted light, the ratio l/ls (the inverse of the ratio in the equation) is the transmittance, e is the molar extinction coefflcient (in units of liters per molecentimeter), c is the concentration of the absorbing Intensity of incident light

Lamp

Intensity of transmitted Iight

Monochromator

Detector Sample cuvette with c moles/Iiter of absorbing specles

FIGURE 1 The principalcomponents of a spectrophotometer. A lightsourceemitslightalonga broad spectrum,then the monochromator selectsand transmitslight of a particularwavelength.Themonochromatic lightpasses through the samplein a cuvetteof path length/ and is absorbed by thesamplein proportion to theconcentration of theabsorbing species. Thetransmitted light is measuredby a detector.

coo-

coo-

+l H.N-CH

*l H.N-CH

Cysteine"lr""l", -

2H*+2e

I

|

SH/S -=-

SH

2H*+2e-

|

cysteine

?t'

*

CH-NHs

tl coo-

cystine

| |

f"'

*

CH-NH3

coo-

FIGURt Disulfidebonds 3-7 Reversible formationof a disulfidebondby the oxidationof two molecules of cysteine. betweenCysresidues stabilize the structures of manyproteins.

Polar, Uncharged R Groups The R groupsof these amino acids are more soluble in water, or more hydrophilic, than those of the nonpolaramino acids,because they contain functional groups that form hydrogenbonds wrth water. This classof amino acids includes serine, threonine, cysteine, asparagine, and glutamine. The polarity of serine and threonine is contributed by their hydroxyl groups; that of cysteine by its sulfhydrylgroup, which is a weak acid and can make weak hydrogenbondswith oxygenor nitrogen; and that of asparagineand glutamine by their cmirla

drnrrnq

Asparagine and glutamine are the amides of two other amino acids also found in proteins, aspartate and glutamate, respectively, to which asparagine and glutamine are easily hydrolyzed by acid or base. Cysteine is readily oxidized to form a covalently linked dimeric amino acid called cystine, in which two cysteine molecules or residues are joined by a disulfide bond (Fig. 3-7). The disulfide-linked residues are strongly hydrophobic (nonpolar). Disulfide bonds play a special role in the structures of many proteins by forming covalent links between parts of a polypeptide molecule or between two different polypeptide chains. Positively Charged (Basic) R Groups The most hydrophilic R groups are those that are either positively or negatively charged. The amino acids in which the R groups have signiflcant positive charge at pH 7.0 are lysine, which has a second primary amino group at the a position on its aliphatic chain; arginine, which has a positively charged guanidinium group; and histidine, which has an aromatic imidazole group. As the only common amino acid having an ionizable side chain with pK" near neutrality, histidine may be positively charged (protonated form) or uncharged at pH 7.0. His residues facilitate many enz),Tne-catalyzedreactions by serving as proton donors/acceptors. Negatively Charged (Acidic) R Groups The two amino acids having R groups with a net negative charge

at pH 7.0 are aspartate and glutam&te, each of which has a secondcarboxylgroup.

Also Have lmportant Amino Acids Uncommon Functions In addition to the 20 common amino acids, proteins may contain residuescreatedby modificationof common residuesalreadyincorporatedinto a polypeptide (Fig. 3-8a). Among these uncommon amino acids are 4-hydroxyproline, a derivative of proline, and 5-hydroxylysine, derived from lysine. The former is found in plant cell wall proteins, and both are found in collagen,a fibrous protein of connective tissues. G-N-Methyllysine is a constituent of myosin, a contractile protein of muscle.Another important uncolnmon aminoacid is 1-carboxyglutamate, found in the bloodclotting protein prothrombin and in certain other proteins that bind Ca2+as part of their biologicalfunction. More complex is desmosine, a derivative of four Lys residues,which is found in the flbrous protein elastin. Selenoeysteine is a special case.This rare amino acid residue is introduced during protein synthesis rather than created through a postsynthetic modi-flcation. It containsseleniumrather than the sulfur of cysteine. Actually derived from serine, selenocysteineis a constituent of just a few known proteins. Some amino acid residues in a protein may be modified transiently to alter the protein's function. The addition of phosphoryl, methyl, acetyl, adenylyl, ADPribosyl, or other groups to particular amino acid residuescan increaseor decreasea protein's activity (FiC. 3-8b). Phosphorylationis a particularly corunon regulatorymoffication. Covalentmodi-ficationas a protein regulatory strategy is discussedin more detail in Chapter6. Some300 additional amino acids have been found in cells. They have a variety of functions but are not all constituents of proteins. Ornithine and citrulline (Fig. 3-8c) deservespecialnote becausethey are key intermediates(metabolites)in the biosynthesisof arginine (Chapter 22) and in the urea cycle (Chapter 18).

o li o-P-o-cH2-cH-coo-

H

I HO-c_cH, ll

ll

*NH,

O

Phosphoserine 4-Hydroxyproline

II l 9H' -o-P-o-cH-cH-cootl _O *NH, Phosphothreonine

5-Hydroxylysine

o cH3-NH-

CH2- CH2-CH2-

CH2-CH-

o $-o1\.'

COO-

r

*rts,

O

6-N-Methyllysine

*NH,

Phosphotyrosine

rrrN

coo ooc-cH

r'z-cH-coo-

\_/

+,) c-

cH2-cH

NH-CH2-CH2-CH2-CH-COO-

HNI ---l'

COO

-NHt

*NHt

CHt o-N-Methylarginine

7-Carboxyglutamate

HN - CH2 - CH2- CH2- CH2-CH-

r-l

H3N\

C_O

/COOCH

I

CHu

,ft,,,

H,fr

CH-(C H, ), --{,'\-

6-N-Acetyllysine

.fru,

(CH, )"- C'H

ilI

ooc

_.) \ -N-

\

'coo_ H3C - O

'?",'.

o tl -C-

I

NH,

i-

Desmosrne

NZC-C

(a)

FIGURE 3-8 Uncommonamino acids.(a) Someuncommonamino acidsfoundin proteins. All arederivedfromcommonaminoacids Extra functionalgroupsadded by modificationreactionsare shown in red.Desmosineis formedfrom four Lysresidues(thefour carbonbackbonesare shadedin yellow).Notethe useof eithernumbersor Creek lettersto identifythe carbonatomsin thesestructures(b) Reversible amino acid modifications involvedin regulationof proteinactivity. Phosphorylation is the mostcommontype of regulatorymodification. (c) Ornithineandcitrulline,whicharenotfoundin proteins, areintermediates in thebiosynthesis of arginineand in the ureacycre.

o -"-o1-\cHs-cH-coo-

ltl

CH-COO

NHt Selenocysteine

CIJ2- CH2-CH-COO

"NHt Glutamate methyl ester

CH u.tt/ \coo-

HSe-CH2

COO

*NH,

"t-*-'

\./l

O OH (b)

_NH,

OH Adenylyltyrosine

urfr-cur-cH2-cH2-cH-coo I -NHt Ornithine H2N-C-N-CH2-CH2-CH2-CH-COO-lllr (c)

*NH,

o H Citrulline

(anActasAcids Amino Acids andBases The amino and carboxyl groups of amino acids, along with the ionizable R groups of some amino acids, function as weak acids and bases. When an amino acid lacking an ionizable R group is dissolved in water at neutral

pH, it exists in solution as the dipolar ion, or zwitterion (German for "hybrid ion"), which can act as either an acid or a base (FiS. 3-9). Substanceshaving this dual (acid-base) nature are amphoteric and are often called

H

H

R-C-C:O

R-C-C:O

I

H,N OH Nonionic form

tcoo-

COOH form

PKz:9.60

I I

R-C-COO

*NHt

ttcooCH,

CH.

.-

H

H R-C-COO-

t-

NH, pK,

l"pK, CHo

H3N+ O Zwitterionic

rlH,

ttH"

I

+ H+

NH,

Zwitterion as acid

I R-C-COO I *NHt

pH

H

H

I R-C-COOH I *NHt

+ H+ i.-

PKr,:2.34

Zwitterion as base f IGURE l*9 Nonionicand zwitterionicforms of amino acids.The nonionicform doesnot occurin significant amountsin aqueoussolutions.Thezwitterionpredominates at neutralpH. A zwitterioncan act aseitheran acid (protondonor)or a base(protonacceptor)

empholytes (from "amphotericelectrolytes").A simple monoamino monocarboxylic a-amino acid, such as alanine,is a diprotic acid when fully protonated; it has two groups,the -COOH group and the -NHj group, that canyield protons: tIHHr{H t';t-;l R-C-COOH --z-+R-C-COO

ttl

*NH, Net charge: *1

*NH, 0

-z-+ R-C-COONH, -l

Aminn Acids l"iave Iharacteristie Titratisn Iurv*s Acid-basetitration involves the gradual addition or removal of protons (Chapter2). F igurt 3-ltl showsthe titration curve of the diprotic form of glycine. The two ionizablegroupsof glycine,the carboxylgroup and the amino group, are titrated with a strong base such as NaOH.The plot has two distinct stages,corresponding to deprotonationof two different groups on glycine. Each of the two stagesresemblesin shapethe titration curve of a monoprotic acid, such as acetic acid (see Fig. 2-16), and can be analyzedin the sameway. At very low pH, the predominantionic speciesof glycineis the fully protonatedform, *H3N-CH2-COOH. At the midpoint in the first stageof the titration, in which the -COOH group of glycine loses its proton, equimolar concentrationsof the proton-donor (+HBN-CH2COOH) and proton-acceptor(+H3N-CH2-COO-) speciesare present.At the midpoint of any titration, a point of inflectionis reachedwhere the pH is equal to the pK" of the protonated group being titrated (see Fig.2-I7). For glycine,the pH at the midpoint is 2.34,

0

00.5 OH

11.52 (equivalents)

FIGURE 3-10 Titrationof an aminoacid. Shownhereis the titration predominating at key curveof 0..1ruglycineat 25'C. Theionicspecies pointsin the titrationare shownabovethe graph.The shadedboxes, at aboutpKr : 2.34 andpK2: 9.60,indicatethe regionsof centered greatest bufferingpower Notethat 1 equivalentof OH = 0.1 la NaOH added.

thus its -COOH group has a pKu (labeled pKr in Fig. 3-10) of 2.34. (Recallfrom Chapter2 that pH and pKuaresimplyconvenientnotationsfor proton concentration and the equilibrium constant for ionization, respectively.The pK, is a measureof the tendencyof a group to give up a proton, with that tendencydecreasing tenfold as the pKu increasesby one unit.) As the titration proceeds,anotherimportant point is reached at pH 5.97.Here there is anotherpoint of inflection,at which removalof the flrst proton is essentiallycomplete and removalof the secondhas just begun.At this pH glycineis presentlargelyas the dipolarion (zwitterion) *HrN-CHr-COO . We shallreturn to the signiflcance of this inflection point in the titration curve (labeled pI in Fig. 3-10) shortly. The secondstageof the titration correspondsto the removal of a proton from the -NHJ group of glycine. The pH at the midpointof this stageis 9.60,equalto the pK, (labeledpK2inFig. 3-10) for the -NHi group.The titration is essentiallycompleteat a pH of about 12, at which point the predominant form of glycine is H2N-

cH2-coo-. From the titration curve of glycine we can derive several important pieces of information. First, it gives a quantitative measure of the pK, of each of the two ionizing groups: 2.34 for the -COOH group and 9.60 for the -NHJ group. Note that the carboxyl group of glycine is over 100 times more acidic (more easily ionized) than

10

PKt

Methyl-substituted carboxyl and amlno gToups

1

+

cH3-cooH

cH3-NH3

Il$thylamine i The nprmal pK, for {n aminogfoupis about!0.6. Carboxyl and amino gtoups in glycine

frn" t"

H-C-COOH

+

NH"

i/,

I

H

t"

H-C-COO-

t1 H1

1 2

{-Amino acid j(glycine) pK":9i60 i ElQctronegative ofygenatoms

,

t

t

in thei carboxyl grouf null electron{ 1 alT ay from the afrlino group, t lowering its pK". a

,

1 1

I

1

FIGURE 3-11 Effectof the chemicalenvironmenton pK.. The pKu valuesfor the ionizablegroupsin glycinearelowerthanthosefor simple, methyl-substituted aminoand carboxylgroups.Thesedownward

perturbations of pKuare due to intramolecular interactions. Similar effectscan be causedby chemicalgroupsthat happento be positioned nearby-for example,in the activesiteof an enzyme.

the carboxyl group of acetic acid, which, as we saw in Chapter2, has a pKu of 4.76-about averagefor a carboxyl group attached to an otherwise unsubstituted aliphatic hydrocarbon.The perturbed pK, of glycine is causedby repulsionbetweenthe departingproton andthe nearby positively chargedamino group on the a-carbon atom, as describedin Figure 3-11. The opposite chargeson the resulting zwitterion are stabilizing.Similarly, the pKuof the amino group in glycine is perturbed downward relative to the average pKu of an amino group. This effect is due partly to the electronegative oxygenatomsin the carboxylgroups,which tend to pull electronstoward them, increasingthe tendencyof the amino group to give up a proton. Hence,the a-amino group has a pKu that is lower than that of an aliphatic amine such as methylamine(Fig. 3-11). In short, the pKu of any functional group is greatly affected by its chemicalenvironment,a phenomenonsometimesexploited in the active sites of enz;.'rnes to promote exquisitely adaptedreactionmechanismsthat dependon the perturbed pKu valuesof proton donor/acceptorgroups of specificresidues. The secondpiece of informationprovided by the titration curve of glycine is that this amrno acidhas two regionsof buffering power. One of theseis the relatively flat portion of the curve, extendingfor approximately 1 pH unit on either side of the first pK, of 2.S{,indicating that glycine is a good buffer near this pH. The other bufferingzone is centeredaround pH 9.60. (Note that glycine is not a good buffer at the pH of intracellular fluid or blood, about 7.4.) Within the buffering rangesof

glycine, the Henderson-Hasselbalch equation (p. 60) can be used to calculate the proportions of protondonor and proton-acceptorspeciesof glycinerequired to make a buffer at a given pH.

(urves (harge Titration Predid theElefiric ofAmino Acids Another important piece of informationderivedfrom the titration curve of an amino acid is the relationshipbetween its net chargeand the pH of the solution.At pH 5.97,the point of ffiection betweenthe two stagesin its titration curve, glycine is present predominantly as its dipolarform, fully ionizedbut with no net electic charge (Fig. 3-10). The characteristic pH at which the net electric chargeis zero is calledthe isoeleetric point or isoelectric pH, designatedpI. For glycine, which has no ionizablegroup in its side chain, the isoelectricpoint is simply the arithmetic meanof the two pKuvalues: 11

pI :;tplfr + pK2\:

+ 9.60): 5.97 ,tZ.S+

As is evident in Figure 3-10, glycine has a net negative chargeat any pH aboveits pI and will thus move toward the positive electrode(the anode) when placed in an electric fleld. At any pH below its pI, glycine has a net positive chargeand will move toward the negativeelectrode (the cathode).The farther the pH of a glycinesolution is from its isoelectric point, the greater the net electric charge of the population of glycine molecules. At pH 1.0,for example,glycineexistsalmostentirelyas the form *H3N-CHr-COOH with a net positivecharge

,I H3N

cooH CH, 1,,

CH. 1.,

PAr

Plln

Y",:

cooH

coo

HBiI cH I CH,

H3N-CH

Y n ,: Net charge:

coo

coo

CH

cooH

I

CH, DA"

CH,:-

CH,

coo

coo

-1

+l

HrN-cH

_,

+2+10-1 10 Histidine

1 0 Glutamate

8

pH

PKz: 9.1 PKn 60

pH6

PKn =

A

2 1.0 (a)

2.0

0

3.0

OH- (equivalents)

(b)

2.0 1.0 OH (equivalents)

3.0

hereas pKp. FIGURE 3-12 Titration curvesfor (a)glutamate and (b) histidineThepKnof the R groupis designated

of 1.0.At pH 2.34,where there is an equalmixture of *HrN-cHr-CooH and +H3N-cHz-Coo , the average or net positive chargeis 0.5. The sign and the magnitudeof the net chargeof any aminoacid at any pH can be predicted in the sameway.

Acids Amino Differ inTheir Acid-Base Properties The sharedproperties of many amino acidspermit some simplifying generalizationsabout their acid-basebehaviors. First, all amino acidswith a singlea-amino group, a singlea-carboxylgroup, and an R group that doesnot ionize have titration curves resemblingthat of glycine (Fig. 3-10). These amino acids have very similar,although not identical,pK" values:pKu of the -COOH group in the rangeof 1.8to 2.4, andpKuof the -NH3+ groupin the rangeof 8.8to 1I .0 (Table3-l). The differencesin these pKu valuesreflect the effects of the R groups.Second,aminoacidswith an ionizableR group have more complex titration curves, with three stages correspondingto the three possibleionization steps; thus they havethree pKuvalues.The additionalstagefor the titration of the ionizableR group mergesto some extent with the other two. The titration curves for two amino acids of this type, glutamate and histidine, are shownin -F'igure3-12. The isoelectricpointsreflectthe nature of the ionizingR groupspresent.For example, glutamatehas a pI of 3.22,considerablylower than that of glycine This is due to the presenceof two carboxyl groups,which, at the averageof their pKuvalues (3.22), contributea net chargeof - I that balancesthe *1 contributed by the amrnogroup.Similarly,the pI of tustidine, with two groupsthat are positivelychargedwhen protonated,is 7.59(the averageof the pKuvaluesof the amino and imidazolegroups),much higher than that of glycine.

Finally, as pointed out earlier, under the general conditionof free and open exposureto the aqueousenvironment, only histidine has an R group (pK, : 6.0) providing signiflcant buffering power near the neutral pH usually found in the intracellular and extracellular fluids of most animalsand bacteria (Table 3-1).

S U M M A R3Y. 1 A m i n oA c i d s r

The 20 amino acidscommonlyfound as residuesin proteins contain an a-carboxyl group, an a-amino group, and a distinctive R group substituted on the a-carbon atom. The o-carbon atom of all amino acidsexcept glycine is asymmetric,and thus amino forms. acidscan exist in at leasttwo stereoisomeric Only the t, stereoisomers,with a conflguration relatedto the absoluteconflgurationof the reference moleculet -glyceraldehyde,are found in proteins.

r

Other,Iesscommonaminoacidsalsooccur,either as constituentsof proteins (through modiflcation of commonamino acid residuesafter protein s;,rrthesis)or as free metabolites.

r

Amino acids are classifledinto flve types on the basisof the polarity and charge (at pH 7) of their R groups. Amino acidsvary in their acid-baseproperties and have characteristictitration curves.Monoamino monocarboxylicamino acids (with nonionizable at R groups)are diprotic acids(+H3NCH(R)COOH) low pH and exist in severaldifferent ionic forms as the pH is increased.Amino acidswith ionizable R groups have additionalionic species,depending on the pH of the mediumand the pK, of the R group.

r

82

A m i nA o c i d sP,e p t i d ea sn,dP r o t e i n s

3.2 Peptides andProteins We now turn to pol}rmersof amino acids,the peptides and proteins. Biologicallyoccurringpolypeptidesrange in size from small to very large, consisting of two or three to thousandsof linked amino acid residues.Our focusis on the fundamentalchemicalpropertiesof these PolSrmers.

Peptides Are(hains ofAmino Acids Tlvo amino acid molecules can be covalentlyjoined through a substituted amide linkage,termed a peptide bond, to yield a dipeptide. Such a linkage is formed by removai of the elements of water (dehydration) from the a-carboxyl group of one amino acid and the a-amino group of another (Fig. 3-13). Peptidebond formation is an example of a condensationreaction, a cofiunon class of reactionsin living cells. Under standardbiochemicalconditions,the equilibrium for the reaction shown in Figure 3-13 favors the amino acids over the dipeptide. To make the reaction thermodynamically more favorable,the carboxyl group must be chemically modified or activated so that the hydroxyl group can be more readily eliminated. A chemical approach to this problem is outlined later in this chapter. The biological approachto peptide bond formation is a major topic of Chapter27. Three amino acids can be joined by two peptide bonds to form a tripeptide; simiiarly, four amino acids can be linked to form tetrapeptides,five to form pentapeptides, and so forth. When a few amino acids are joined in this fashion, the structure is called an oligopeptide. When many amino acids are joined, the product is called a polypeptide. Proteins may have thousandsof amino acid residues.Although the terms "protein" and "polypeptide" are sometimesused interchangeably,moleculesreferred to as polypeptidesgenerally have molecularweightsbelow 10,000,and those calledproteins have higher molecularweights.

*l

HFP

Rl

H3N-CH-C-OH

+

-CH-COO-

ll

o

",o ","-']f-*

R1

HR:

*lll

H3N-CH-Q*N-CH-COO-

6 FIGURE 3-13 Formationof a peptide bond by condensation.The a-aminogroupof oneaminoacid(withR2group)actsasa nucleophile to displacethe hydroxylgroupof anotheramino acid (with R1group), forminga peptidebond (shadedin yellow) Amino groupsaregoodnucleophiles,but the hydroxylgroup is a poor leavinggroupand is not readilydisplaced.At physiologicalpH, the reactionshownheredoes not occurto any appreciableextent.

ce3"cH3 CH +

CH,OH H ttl

I

H

HsN -c-c-N_c

rill

HOH

HO

Aminoterminal end

H Carboxylterminal end

FldURt 3*14 The pentapeptide serylglycyltyrosylalanylleucine, Ser-Cly-Tyr-Ala-Leu, or SCYAL.Peptidesare namedbeginningwith the amino-terminalresidue,which by conventionis placedat the left. The peptidebondsareshadedin yellow;the R groupsare in red.

[jigure 3-1 4 shows the structure of a pentapeptide. As alreadynoted, an amino acid unit in a peptide is often called a residue (the part left over after losing a hydrogen atom from its amino group and the hydroxyl moiety from its carboxylgroup). In a peptide, the amino acid residue at the end with a free a-amino group is the amino-terminal (orN-terminal) residue;the residueat the other end, which has a free carboxyl group, is the carboxyl-terminal (C-terminal) residue. KEY(0NV[NT|0N: When an aminoacid sequenceof a peptide,polypeptide,or proteinis displayed,the aminoterminal end is placed on the left, the carboxyl-terminal end on the right. The sequenceis read left to right, beSinningwith the amino-terminalend. r Althoughhydrolysisof a peptidebond is an exergonic reaction,it occursonly slowly becauseit has a high activation energy (seep. 25). As a result, the peptide bonds in proteinsare quite stable,with an averagehalf-life (1172) of about 7 yearsunder most intracellularconditions.

Peptid*s {anBeDistinguished byTh*ir ionication Behavi*r Peptidescontain only one free a-amino group and one free a-carboxyl group, at opposite ends of the chain ( l-is, -3- 1; ;. Thesegroupsionizeasthey do in free amino acids,althoughthe ionizationconstantsare different becausean oppositelychargedgroup is no longer linked to the o carbon.The o-amino and a-carboxylgroups of all nonterminalaminoacidsare covalentlyjoined in the peptide bonds, which do not ionize and thus do not contribute to the total acid-base behavior of peptides. However,the R groups of some amino acids can ionize (Table 3-1), and in a peptide these contribute to the overallacid-basepropertiesof the molecule(Fig. 3-15). Thusthe acid-basebehaviorofa peptidecanbe predicted from its free a-aminoand a-carboxylgroupsaswell asthe nature and nurnberof its ionizableR groups. Like free amino acids,peptides have characteristic titration curves and a characteristicisoelectric pH (pI) at which they do not move in an electric field. These propertiesare exploited in someof the techniquesused

tion to their functions. Naturally occuning peptides range in length from two to marry thousands of amino acid residues.Even the smallestpeptides can havebiologically important effects. Considerthe commerciallysynthesized dipeptidel-aspartyl-l-phenylalaninemethyl ester,the artiflcial sweetenerbetter lcrown asaspartameor NutraSweet.

NH" Ala

I

CH-CHa

o:c

I

NH Glu

CH-CH2-CH2-COO

I

o:c I

, respectively(not so for either examplehere).A period ends the pattern. Applying these rules to the sequencein (a), eitherA or G canbe foundat consensus position. Any amino acid can occupy the next the flrst positions, followed by an invariant G and an invarifour ant K. The last position is either S or T. Sequence logos provide a more informative and graphicrepresentationof an aminoacid (or nucleic acid) multiple sequencealignment.Each logo consistsof a for eachposition in the sequence.The stack of s).'rnbols overall height of the stack (in bits) indicatesthe degree of sequenceconservationat that position, while the in the stack indicatesthe relative height of each sSrmbol frequencyof that amino acid (or nucleotide). For amino acid sequences,the colors denote the characteristicsof the aminoacid:polar (G, S,T, Y,C, Q, N) green;basic(K, R, H) blue;acidic(D, E) red; andhydrophobic(A, V, L, I, of aminoacidsin this R W R M) black.The classi-fication schemeis somewhatdifferent from that in Table3-l and Figure 3-5. The amino acids with aromatic side chatns are subsumedinto the nonpolar (F, !V) and polar fl) classiflcations.Glycine,alwayshard to group, is assigned to the polar group. Note that when multiple amino acids are acceptableat a particular position,they rarely occur with equalprobability.Oneor a fewusuallypredominate. The logo representationmakesthe predominanceclear, and a conservedsequencein a protein is made obvious. However,the logo obscuressome amino acid residues that may be allowed at a position, such as the Cys that occasionallyoccurs at position 8 of the EF hand in (b).

and macromolecularstructures,has given rise to the new fleld of bioinformatics. One outcomeof this discipline is a growing suite of computer programs, many readily availableon the Internet, that canbe usedby any scientist, student, or knowledgeablelayperson.Each protein's function relies on its three-dimensionalstructure, which in turn is determinedlargely by its primary structure. Thus, the biochemicalinformation conveyed by a protein sequenceis limited only by our owrrunderstanding of structural and functional principles. The constantly evolving tools of bioinformatics make it possibleto identify functional segmentsin new proteins and help establishboth their sequenceand their structural relationshipsto proteins alreadyin the databases. On a different level of inquiry protein sequencesare beginningto tell us how the proteins evolvedand, ultimately,how life evolvedon this planet.

The field of molecular evolution is often traced to Emile Zuckerkandl and Linus Pauling, whose work in the mid-1960sadvancedthe use of nucleotideand protein sequencesto explore evolution.The premiseis deceptively straightforward. If two organismsare closely related, the sequencesof their genes and proteins shouldbe similar.The sequencesincreasirgly divergeas the evolutionarydistancebetween two organismsincreases.The promiseof this approachbeganto be realizedin the 1970s,when CarlWoeseusedribosomalRNA sequencesto define the Archaeaas a group of living organismsdistinct from the Bacteria and Eukarya (see Fig. 1-4). Protein sequencesoffer an opportunity to greatly refine the availableinformation. With the advent of genomeprojects investigatingorganismsfrom bacteria to humans,the number of availablesequencesis growing at an enormousrate. This information can be

tAGl-x(4lG-K-tSTl. 4 a

P9.

0 (a)

| 2 3 4 5 6 7 8 NC D.{W}- tDNSl -{ILVFYW}. TDENSTG]- TDNQGHRK]-{GP}ILIVMCI - TDENQSTAGCI-x(2)-tDEl - ILIVMFYWI.

i

J a

1

0 (b)

I234 N

LI t2 t3 C

FIGURE 1 Representations (a) p loop,an oftwo consensus sequences, (b) ATP-binding structure; EFhand,a Ca2*-binding structure.

[t

t-]

A m i nA o c i d sP,e p t i d ea sn,dp r o t e i n s

used to trace biological history. The challenge is in Iearning to read the genetic hieroglyphics. Evolution has not taken a simple linear path. Complexities abound in any attempt to mine the evolutionary information stored in protein sequences.For a given protein, the amino acid residues essential for the activity of the protein are conserved over evolutionary time. The residues that are Iess important to function may vary over time-that is, one amino acid may substitute for another-and these variable residues can provide the information to trace evolution. Amino acid substitutions are not always random, however. At some positions in the primary structure, the need to maintain protein function may mean that only particular amino acid substitutions can be tolerated. Some proteins have more variable amino acid residues than others. For these and other reasons,different proteins can evolve at different rates. Another complicating factor in tracing evolutionary history is the rare transfer of a gene or group of genes from one organism to another, a process called lateral gene transfer. The transferred genes may be quite similar to the genes they were derived from in the original organism, whereas most other genes in the same two organisms may be quite distantly related. An example of lateral gene transfer is the recent rapid spread of antibiotic-resistance genes in bacterial populations. The proteins derived from these transferred genes would not be good candidates for the study of bacterial evolution, because they share only a very limited evolutionary history with lheir "host" organisms. The study of molecular evolution generally focuses on families of closely related proteins. In most cases, the families chosen for analysis have essential functions in cellular metabolism that must have been present in the earliest viable cells, thus greatly reducing the chance that they were introduced relatively recently by lateral gene transfer. f'or example, a protein called EF-la (elongation factor la) is involved in the synthesis of proteins in all eukaryotes. A similar protein, EI'-T\r, with the same function, is found in bacteria. Similarities in sequence and function indicate that Ef-la and EF-TIr are members of a family of proteins that share a common ancestor. The members of protein families are called homologous proteins, or homologs. The concept of a homolog can be further refined. If two proteins in a family (that is, two homologs) are present in the same species, they are referred to as paralogs. Homologs from different species

are calledorthologs. The processof tracing evolution involves first identifying suitable families of homologousproteinsand then using them to reconstructevolutionary paths. Homologsare identified through the use of increasingly powerful computer programs that can directly comparetwo or more chosenprotein sequences, or can searchvast databasesto find the evolutionaryrelatives of one selectedprotein sequence.The electronicsearch processcan be thought of as slidingone sequencepast the other until a section with a good match is found. Within this sequencealignment, a positive score is assignedfor each position where the amino acid residues in the two sequencesare identical-the value of the scorevarying from one programto the next-to provide a measureof the quality of the alignment.The process has somecomplications.Sometimesthe proteinsbeing comparedmatch well at, say,two sequencesegments, and these segmentsare connectedby less related sequencesof different lengths. Thus the two matching segmentscannotbe alignedat the sametime. To handle this, the computerprogramintroduces"gaps"in one of the sequencesto bring the matching segmentsinto register(Fig. 3-30). Of course,if a sufflcientnumberof gapsare introduced,almostany two sequences couldbe brought into some sort of alignment.To avoid uninformative alignments,the programs include penalties for each gap introduced, thus lowering the overall alignment score.With electronictrial and error,the program selectsthe alignmentwith the optimal scorethat maximizes identical amino acid residueswhile minimizing the introductionof gaps. Identicalaminoacidsare often inadequateto identify related proteins or, more importantly,to determinehow closelyrelated the proteins are on an evolutionarytime scale.A more useful analysisincludesa considerationof the chemicalpropertiesof substitutedaminoacids.When amino acid substitutionsare found within a proteir family, many of the differencesmay be conservative-that is, an amino acid residueis replacedby a residuehaving similar chemicalproperties.For example,a Glu residue may substitutein one family memberfor the Asp residue found in another; both amino acids are negatively charged.Such a conservativesubstitution should logically garner a higher scorein a sequencealignmentthan doesa nonconservativesubstitution,suchasthe replacement of the Asp residuewith a hydrophobicPhe residue. For most efforts to find homologiesand exploreevoIutionaryrelationships,protein sequences(derivedeither

E , c o l i T G N R TI A V Y D L G G G T F D I S I I E I D E V D G E K T F E V L A T N G D T H L G G E D F D S R L H I YL B . S U b t i I i SD E D Q TI L L Y D L G G G T F D V SI L E L G D G T F E V R S T A G D N R L G G D D F D Q IV I D H L G.p

FIGURE 3-30 Aligningproteinsequences with the useof gaps.Shown hereis the sequence alignmentof a shortsectionof the Hsp70proteins (awidespread classof protein-folding chaperones) fromtwo well-studied

bacterialspecies, E.coli andBacillussubtilis.Introduction of a gap in the B subtilissequence allowsa betteralignmentof aminoacid residues on eithersideof thegap.ldentical aminoacidresidues areshaded.

a

r yt r u c t u r e 10 5 3 . 4T h eS t r u c t uor feP r o t e i nPsr:i m a S

l

Signature sequence I GHVD HGK S TMVG R I GHVD HGK ST LVG R

Gram-positive bacterium Gram-negative bacterium

Bacillus subtilis Escherichia coli

IGHVDSGKSTTTGH IGHVDSGKSTTTGH I G H V D H G KS T M V G R IGHVDHGKTTLTAA

I

KEA I EKF I EKF I TTV I TTV

FIGURE 3-31 A signaturesequencein the EF-lalEF-Tuproteinfamily. The signaturesequence(boxed)is a 12-residueinsertionnear the aminoterminusof the sequence. Residues thatalignin all species are shadedyellow Both archaeaand eukaryoteshave the signature,

of the insertionsare quite distinctfor the two althoughthe sequences reflects the significant in the signature sequence Thevariation groups. it first apat this site since has occurred divergence that evolutionary pearedin a commonancestorof both groups.

directly from protein sequencingor from the sequencing of the DNA encodingthe protein) are superior to nongenicnucleicacid sequences(thosethat do not encodea protein or functional RNA) For a nucleic acid, with its four differenttypesof residues,randomalignmentof nonhomologoussequenceswill generallyyield matchesfor at least2So/o of the positions.Introductionof a few gapscan often increasethe fraction of matchedresiduesto 40o/o or more, and the probability of chancealignrnentof unrelated sequencesbecomesquite high. The 20 different aminoacidresiduesin proteinsgreatlylower the probabilrty of uninformative chancealignmentsof this type. The programsused to generatea sequencealignment are complementedby methodsthat test the reliability of the alignments.A commoncomputerizedtest is to shufflethe amino acid sequenceof one of the proteins being comparedto produce a random sequence, then to instruct the program to align the shuffledsequencewith the other, unshuffledone. Scoresare assigned to the new alignment, and the shuffling and alignmentprocessis repeatedmanytimes.The original alignment,before shuffling, should have a score signiflcantly higher than any of those within the distribution of scoresgeneratedby the randomalignments;this increasesthe confldencethat the sequencealignmenthas identifleda pair of homologs.Note that the absenceof a signiflcantalignmentscoredoesnot necessarilymean that no evolutionaryrelationshipexists between two proteins.As we shallseein Chapter4, three-dimensional structural similarities sometimesreveal evolutionary relationships where sequence homology has been wiped awayby time. Use of a protein family to explore evolutionrequires the identiflcation of family memberswith similar molecular functions in the widest possiblerangeof organisms. Information from the family can then be used to trace the evolution of those organisms.By analyzingthe sequencedivergencein selectedprotein families,investigators can segregateorganismsinto classesbased on their evolutionaryrelationships.This information must be reconciled with more classicalexaminationsof the physiologyand biochemistryof the organisms. Certain segmentsof a protein sequencemay be found in the organismsof one taxonomicgroup but not in other groups;thesesegmentscan be used as signa-

ture sequences for the group in which they are found. An example of a signature sequenceis an insertion of 12 amino acids near the amino terminus of the EF1alEF-T\rproteins in all archaeaand eukaryotesbut not in bacteria(FiS. 3-3f ). This particularsignatureis one of many biochemicalclues that can help establishthe evolutionaryrelatednessof eukaryotesand archaea. Other signature sequencesallow the establishmentof evolutionary relationshipsamong groups of organisms at many different taxonomiclevels. By consideringthe entire sequenceof a protein, researcherscan now construct more elaborateevolutionary trees with many speciesin each taxonomic group.Figure 3-32 presentsone suchtree for bacteria, based on sequencedivergencein the protein GroEL (a protein presentin all bacteriathat assistsin the proper folding of proteins). The tree can be refined by basing it on the sequencesof multiple proteins and by supplementingthe sequenceinformation with data on the unique biochemicaland physiological propertiesof eachspecies.There are many methods for generating trees, each method with its own advantagesand shortcomings,and many ways to represent the resulting evolutionary relationships.In Figure 3-32, the free end points of lines are called "externalnodes";each representsan extant species, and each is so labeled. The points where two lines come together, the "internal nodes," represent extinct ancestorspecies.In most representations(including Fig. 3-32), the lengths of the lines connecting the nodes are proportional to the number of amino acid substitutionsseparatingone speciesfrom another. If we trace two extant speciesto a commoninternal node (representingthe common ancestorof the two species),the length of the branch connecting each external node to the internal node represents the number of amino acid substitutions separating one extant speciesfrom this ancestor.The sum of the lengthsof all the line segmentsthat connectan extant speciesto anotherextant speciesthrough a common ancestorreflects the number of substitutionsseparating the two extant species.To determine how much time was needed for the various speciesto diverge, the tree must be calibrated by comparingit with information from the fossil record and other sources.

10 6

A m i nA o c i d sP,e p t i d ea sn,dP a r t i c l e s

jily:i:':::1-""; chramydia I Lnlamvdta Dsrttacr I

Bacteroides 5/e

I Porphyromonas'gingiualis',

V

Botelia burgdoferi )

I Spirochaetes

,Leptospiraint"rrogon" )

I Helicobacter pylori

Legionella pneumop hila Themop hiLic bacterium P S -3

P seudomonas aer ug i nosa

Bacillus subtilis Staphylococcus aureus -CI o stri d.ium acetobuty I i cu m C lostridium p et'ft ingens

Yersinia enterocolitica SalmonelLa typht Escherichia coli

.F

p

lNeisseria gonorrhoeae

low G+C

(! L

Streptomyces coelicolor

a

- Mycobacterium leprae M y cobacter i u n t ubercuIosi s

al

h

B rady r hizobium j aponic um

Streptomyces albus [genel

Ag r oba cteri um t u mefaci en s Zymomonas mobilis

Cyanobacteria and chloroplasts

or Jo*.'l^'r" FIGURE 3-32 Evolutionarytree derived from amino acid sequence comparisons. A bacterialevolutionarytree,basedon the sequencediAs more sequence information

is made available in

databases,we can generateevolutionarytrees based on multiple proteins. And we can refine these trees as additional genomic information emergesfrom increasingly sophisticatedmethods of analysis.All of this work movesus toward the goal of creating a detailed tree of life that describesthe evolutionand relationshioof everv

vergenceobservedin the CroELfamilyof proteins. Also includedin this (chl.)of somenonbacterial tree(lowerright)arethechloroplasts species.

organismon Earth. The story is a work in progress,of course(Fig. 3-33). The questionsbeingaskedand answeredare ftndamental to how humansview themselves and the world aroundthem. The fleld of molecularevolution promisesto be amongthe most vibrant of the scientiflc frontiers in the twenty-fi-rstcentury.

LowG+C, gram-positive Aquificales

Eurryarchaeota

""*fl'tr"i81> frrgnG+u.,.-7

{y.-::y:":'^ ,

Cymobacteria

Prcteobacteria

0pisthokonta Fungi Glaucophl'tes Mycetozoans

Pelobionts Amoebozoa

^. , [[DlOmOnads

Chronalveolata

FIGURI3-33 A consensus tree of life.Thetreeshownhereis basedon analyses of manydifferentproteinsequences and additionalgenomic features. Branches shownas dashedlinesremainunderinvestigation.

Thetreepresents only a fractionof the availableinformation,aswell as only a fractionof the issuesremainingto be resolved.Eachextant groupshownis a complexevolutionary storyuntoitself.

['.1

F u r t h eRre a d i n g

SUMMAR 3 .Y4 T h eS t r u c t uor e fP r o t e i n s : P r i m a rSyt r u c t u r e r

r

r

r

Differencesin protein function result from differencesin aminoacid compositionand sequence. Somevariationsin sequenceare possiblefor a particularprotein,with little or no effect on furction. Amino acid sequencesare deducedby fragmenting polypeptidesinto smallerpeptideswith reagents known to cleavespeci-ficpeptide bonds;determining the amino acid sequenceof eachfragmentby the automatedEdmandegradationprocedure;then ordering the peptide fragmentsby finding sequence overlapsbetweenfragmentsgeneratedby different reagents.A protein sequencecan alsobe deduced from the nucleotidesequenceof its corresponding genein DNA. Shortproteinsand peptides(up to about 100 residues)can be chemicallysyerthesized. The peptideis built up, one aminoacidresidueat a time, whrle tethered to a solid support. Proteinsequences are a rich sourceof information about protein structure and function, as well as the evolutionof life on Earth. Sophisticated methods are being developedto trace evolutionby analyzing the resultant slow changesin amino acid sequences of homologousproteins.

homolog I04 paralog 104 ortholog I04 signaturesequence 105

Reading Further Amino Acids Dougherty, D.A. (2000) Unnaturalamino acidsas probesof protein structure and function. Cum Opi,n Chem.BioI 4,645-652 Greenstein, J.P. & lVinitz, M. (i961) Chemi'sttg of the Ami'no Acids, 3 Vols,John Wiley & Sons,New York. Kreil, G. (1997) n-Amino acidsin animalpeptides Annu Reu Bi.ochem.66,337-345. of Detailsthe occurrenceof theseunusualstereoisomers amino acids Meister, A. (1965)Bi,ochemi'strgoJthe Ami,no Aci'ds, 2nd edn, Vols 1 and 2, AcademicPress,Inc., New York. Encyclopedictreatment of the properties,occurrence,and metabolismof amino acids Peptides and Proteins Creighton, T.E. (1992)Protebrc: Structures and Molecular Propert'ies,2nd edn, W H. Freemanand Company,New York. Very usefui generalsource Working with Proteins Dunn, M,J. & Corbett, J.M. (1996) Tko-dimensionalpolyacrylamidegel electrophoresis.MefhodsEnzEmoI 271, 177-203. A detaileddescriptionof the technology. Kornberg, A. (1990) Why purify enrymes?MethodsEn'zEmol 182, 1-5. The critical role of classicalbiochemicalmethodsin a new age

KeyTerms Terms i,n boLdare defi,nedin tlte glossary. amino acids 72 R group 72 chiral center 72 enantiomers 72 absolute configuration 74 o, r, system 74 polarity 74 absorbance,-4 76 zwitterion 78 isoelectrie pII (isoelectric point, pI) 80 peptide 82 protein 82 peptide bond 82 oligopeptide 82 polyreptide 82 oligomeric protein 84 protomer 84 co4iugatedprotein 84 prosthetic group 84 crude extract 85 fraction 85 fractionation 85 dialysis 85

consensus sequence 102 103 bioinformatics Iateralgenetransfer I04 homologous proteins 104

column chromatography 85 ion-exchange chromatography 86 size-exclusion chromatography 87 afflnity chromatography 88 high-performance liquid chromatography (HPLC) 88 electrophoresis 88 sodiumdodecylsulfate (sDS) 8e isoelectricfocusing 90 primary structure 92 secondary structure 92 tertiary structure 92 quaternary structure 92 Edman degradation 95 proteases 95 proteome 100

Scopes, R,K. (i994) Protein Purifi,cati'on:PrinciQles and New York Practi,ce,3rd edn, Springer-Verlag, A good sourcefor more completedescriptionsof the principles and other methods. underlyingctLromatography Protein

Primary

Structure

and Evolution

Andersson, L., Blomberg, L., Flegel, M., Lepsa, L., Nilsson, B., of pept\des'Bi'opoly& Verlander, M. (2000) Large-scales],Trthesis mers 65,227-250. A discussionof approachesto manufacturingpeptidesas pharmaceuticals Dell, A. & Morris, H,B. (2001) Glycoproteinstructure determination by massspectrometry Sci'ence291,2351-2356. Glycoproteinscan be complex;massspectrometryis a preferred method for sortingthings out. Delsuc, F., Brinkmann, H., & Philippe H' (2005) Phylogenomics and the reconstructionof the tree of life. NaJ Reu Gertet.6, 361-375 Gogarten, J.P. & Townsend, J.P. (2005) Horizontalgenetransfer, genomelnnovationand evolution.Not Reu Mi'crobio| 3,679-687. Gygi, S.P.& Aebersold, B. (2000) Massspectrometryand proteomics Curr Opin Chem Bi,ol4,489-494 Usesof massspectrometryto identify and study cellularproteins. Koonin, E.V., Tbtusov, R.L., & Galperin, M.Y. (1998) Beyond completegenomes:from sequenceto structure and function. Curz: Opi.n.Struct BioI 8,355-363. A gooddiscussionabout the possibleusesof the increasing amount of inJormationon protein sequences. Li, W.-H. & Graur, D. (2000)Fundamentals oJMolecu\ar Euolution, 2nd edn, SinauerAssociates,Inc , Sunderland,MA.

!"1 A m i nAo c i d sP,e p t i d easn,dP r o t e i n s A very readabletext describingmethodsused to analyzeprotein and nucleic acid sequencesChapter5 providesone ofthe best availabledescriptionsof how evolutionarytrees are constructedfrom sequencedata Mann, M. & Tllilm, M. (1995) Electrospraymassspectrometryfor protein charactenzationTlends Bi,ochem Sci 20, 21,9-224. An approachablesummaryof this techniquefor beginners Mayo, K.H. (2000) Recentadvancesin the designand construction of synthetic peptides:for the love of basicsor just for the technology of it TtrendsB,iotechnoL18,212-217

(i) Glycine is completely titrated (second equivalence point). -H3N-CH2-COO. fi) The predominantspeciesis (k) The auerdge net chargeof glycine is - 1. (1) Glycineis presentpredominantlyas a 50:50mixture of * H3N-cH2-cooH and * H3N-cHz-coo- . (m) This is the isoelectricpoint. (n) This is the end of the titration (o) These are the worst pH regions for buffering power

Miranda, L.P. & Alewood, P.F. (2000) Chaliengesfor protein chemicals5'nthesrs in the 21st century: bridging genomicsand proteomics Bi,opolymers 55,21.7 226 This and Mayo,2000(above),describehow to makepeptides and splicethem together to addressa wide rangeof problemsin protein biochemistry Rokas, A., Williams, B.L., King, N., & Carroll, S.B. (2003) Genome-scale approachesto resolvingincongruencein molecular phylogeniesNature 425, 798-804 How sequencecomparisonsof multiple proteins can fleld accurate evolutionaryinformation Sanger,F. (1988)Sequences, sequences, sequencesAnnu Reu Biochem 57,I-28. A nice historicaiaccountof the developmentof sequencing methods

11.30 10

pH

9.60

5.97

il)

2.34 II)

Snel, B., Huynen, M.A., & Dutilh B.E. (200b)Genometreesand the natureofgenomeevolutionAnnu Reu Microbio| bg, 1gl-209 Zuckerkandl, E. & Pauling, L (1965) Moleculesas documentsof evolutionarytustory J Theor Bi,oL8,357-366 Many considerthis the foundingpaper in the fleld of molecular evolution.

0.5

1.0 1.5 2.0 OH- (equivalents)

3. How Much Alanine Is Present as the Completely Uncharged Species? At a pH equal to the isoelectric point of alanine, the net charge on alanine is zero. TWo structures can

Problems 1. Absolute Configuration of Citrulline The citrulline isolatedfrom watermelonshas the structure shownbelow.Is it a D- or L-aminoacid?Explain. CHz(CHz)zNH-C-NH2 H-O-NHg

O

coo 2. Relationship betrveen the Tltration Curve and the Acid-Base Properties of Glycine A 100 mL solution of 0.1 u giycineat pH L.72was titrated with 2 u NaOHsolution. The pH was monitored and the results were plotted as sho\4n in the following graph. The key points in the titration are designatedI to V. For eachofthe statements(a) to (o), identiJg the appropriate key point in the titration and justi,Jy yortr choice (a) Giycine is present predominantly as the species *H3N-cH2-cooH (b) The clueragenet chargeof glycine is + j. (c) Half of the aminogroups are ionized. (d) The pH is equal to the pK" ofthe carboxylgroup. (e) The pH is equal to the pK" of the protonated amino group (f) Glycinehas its maximum buffering capacity. (g) The aueragenet chargeofglycineis zero. (h) The carboxylgroup hasbeen completelytitrated (first equivalencepoint).

be drarnmthat have a net charge ofzero, but the predominant form of alanine at its pI is zwitterionic.

QH,

- 1 . / / t . / /o H3N-g-C\

'|b Zwitterionic

9H, o HrN-g-C\

,!bn Uncharged

(a) Why is alanine predominantly zwitterionic rather than completely uncharged at its pI? (b) What fraction of alanine is in the completely uncharged form at its pI? Justify your assumptions. 4. Ionization State of Ilistidine Each ionizable group of an amino acid can exist in one of two states, charged or neutral The electric charge on the functional group is determined by the relationship between its pK" and the pH of the solution. This reiationship is described by the Henderson-Hasselbalch equatlon. (a) Histidine has three ionizable functional groups Write the equilibrium equations for its three ionizations and assign the proper pK^for eachionization. Drawthe structure ofhistidine in each ionization state. What is the net charge on the histidine molecule in each ionization state? (b) Draw the structures of the predominant ionization state of histidine at pH 1, 4, 8, and 12. Note that the ionization state can be approximated by treating each ionizable group independently.

P r o b l e mlso s

(c) What is the net chargeof histidineat pH 1,4,8, and 12?For each pH, wili histidine migrate toward the anode (+) or cathode(-) when placedin an electric fleld?

8. The Size of Proteins What is the approximatemolecular weight of a protein with 682 amino acid residuesin a single polypeptidechain?

5. Separation of Amino Acids by Ion-Exchange Chromatography Mixtures of amino acidscan be anallzed by first separating the mixture into its components through ionexchangechromatography Amino acids placed on a cationexchange resin (see Fig.3-17a) containing sulfonate (-SOt) groups flow down the column at different rates becauseof two factors that influencetheir movement:(1) ionic attraction between the sulfonateresidueson the column and positively chargedfunctional groups on the amino acids,and (2) hydrophobicinteractionsbetween amino acid side chains and the strongly hydrophobicbackboneof the polystl'rene resin. For each pair of amino acids listed, determine which will be elutedfirst from the cation-exchange columnby a pH 7.0buffer. (a) Asp and Lys (b) Are and Met (c) Glu and Val (d) Gly and Leu (e) Ser and Ala

9. The Number of Tfyptophen Besidues in Bovine Serum Albumin A quantitative amino acid analysis reveals that bovine serum albumin (BSA) contains0.58%tryptophan (M,204) by weight. (a) Calcnlatethemini,mum molecularweightof BSA (i.e., assumethere is only one Ttp residueper protein molecule). (b) Gelfiltration of BSA givesa molecularweight estimate of 70,000.How many TYpresiduesare presentin a moleculeof serum albumin?

6. Naming the Stereoisomers of Isoleucine The structure of the amino acid isoleucineis

(a) What is the net chargeof the moleculeat pH 3, 8, and 11? (Use pK, values for side chains and terminal amino and carboxylgroupsas given in Table3-1.) (b) Estimatethe pI for this peptide.

H-C-CH"

I

CHO

t-

CH, (a) Howmany chiral centers does it have? (b) How many optical isomers? (c) Draw perspective formulas for all the optical isomers of isoleucine 7. Comparing the pI(" Values of Alanine and Polyalanine The titration curve of alanine shows the ionization of two functional groups with pK. values of 2.34 and 9.69, corresponding to the ionization of the carboxyl and the protonated amino groups, respectively. The titration of di-, tri-, and larger oiigopeptides of alanine also shows the ionization of only two functional groups, although the experimental pK. values are different The trend in pK. values is summarized in the table. Amino acid or peptide

pKr

DKz

Ala

2.34 3.12 3 39

9.69

3.42

7.94

Ala-Ala-A-la Ala-(Ala)"-Ala,

n > 4

8.30

8.03

(a) Draw the structure of A-la-Ala-Ala. Identify the functional groups associated with pKr andpK2. (b) \Mhv does the value of pKf increase with each additional Ala residue in the oligopeptide? (c) Why does the value of pK2 decrease with each additional Ala residue in the oligopeptide?

11. Net Electric Charge of Peptides A peptide has the sequence Glu-His-Tlp-S er-Gly-Leu-Arg-Pro-Gly

goo u"fr-c-H "l

Ala-Ala

10. Subunit Composition of a Protein A protein has a molecr-ilarmassof 400 kDa when measuredby gel flltration. When in the presenceof sodiumdodesubjectedto gel electrophoresis gives protein (SDS), three bands with molecular the cyl sulfate is carried When electrophoresis massesof 180,160,and 60 kDa. are ttree bands out in the presence of SDSand dithiothreitol, 160, 90, and 60 againformed,this time with molecularmassesof protein. kDa. Determine the subunit compositionof the

12. Isoelectric Point of Pepsin Pepsinis the namegivento a mix of severaldigestiveenzyrnessecreted(as larger precursor proteins) by glands that line the stomach.These glands also secretehydrochloricacid, which dissolvesthe particulate matter in food, allowing pepsin to enz;rmaticallycleaveindividual protein molecules.The resulting mixture of food, HCI, and has a pH near 1.5. and digestiveenzlmes is lcrown as ch].ryne pepsh proteins? What funcyou predict pI for the What would pI on pepsin?Which present groups ttLis to confer be must tional proteins such groups? contribute would amino acidsin the 13. The Isoelectric Point of Ilistones Histones are proteins found in eukaryotic cell nuclei, tightly bound to DNA, which has many phosphategroups. The pI of histonesis very high, about 10.8.What aminoacid residuesmust be presentin relatively large numbers in histones?In what way do these residuescontribute to the strong binding of histonesto DNA? 14. Solubility of Polypeptides One method for separating pollpeptides makesuse of their different solubilities.The solubility of large pollpeptides in water dependson the relative polarity of their R groups,particularly on the number of ionized groups:the more ionizedgroupsthere are, the more soluble the pollpeptide. Which of each pair of the polypeptides that follow is more solubleat the indicatedpH? (a) (Gly)zoor (Glu)2sat pH 7.0 (b) (Lys-Ala)3 or (Phe-Met)3at pH 7.0 (c) (Ala-Ser-Gly)s or (Asn-Ser-His)s at pH 6.0 (d) (Ala-Asp-Gly)s or (Asn-Ser-His)s at pH 3'0

-I

[

10

A m i nA o c i d sP,e p t i d ea sn,dP r o t e i n s

15. Purification of an Enzyme A biochemistdiscoversand puri-flesa new enzyrne,generatingthe puriflcation table below.

Procedure 1. Crudeextract 2. Precipitation (salt) 3. Precipitation (pH) 4. Ion-exchange chromatography 5. Affinity chromatography 6. Size-exclusion chromatography

Total protein (ng)

20,000 5,000 4,000 200

Activity (units)

4,000,000

3,ooo,ooo 1,000,000 800,000

50

750,000

Atr'

675,000

(a) From the information given in the table, calculate the speciflcactivity of the enzymeafter eachpurification procedure. (b) Which of the purification proceduresused for this enzlme is most effective (i.e.,givesthe greatestrelative increase in purity)? (c) Which of the puriication proceduresis least effective? (d) Is there any indication basedon the results shown in the table that the enzyrnea^fterstep 6 is now pure?What else cot-tldbe doneto estimatethe purity of the enz;rrnepreparation? 16. Dialysis. A puriied protein is in a Hepes(N-(2-hydroxyethyl)piperazine-N'-(2-ethanesu.lfonic acid)) buffer at pH Z with 500mu NaCl.A sample(1 mL) of the protein solutionis placed in a tube made of dialysismembraneand diagzed against 1 L of the sameHepesbu-fferwith 0 mu NaCLSmallmoleculesand ions (such as Na+, Cl-, and Hepes) can diffuse acrossthe dialysis membrane,but the protein cannot. (a) Oncethe dialysishas come to equilibrium,what is the concentrationof NaCIin the protein sample?Assumeno volume changesoccur in the sampleduring the dialysis. (b) If the original 1 mL samplewere dialyzedtwice, successively,against100mL of the sameHepesbuffer with 0 mll NaCl, what wou-ldbe the final NaCl concentration in the sample? 17. Peptide Purification At pH 7.0,in what order would the following three peptidesbe eluted from a column fllled with a cation-exchangepolymer?Their amino acid compositionsare: Protein A: Ala 10%,Glu 5%, Ser 5%0,Leu 10%0, Argl0%, pro b%o, His 5%, Ile 10%,Phe 5%, T\,r 5%o, Lys 10%,Gly 10%o, and Tlp 10%. ProteinB: Ala 5%,YalSo/0, Gly 10%,Asp 5%0, Leu 5%,Arg 50/o,Ile 5%,Phe5%o,Tlr5o/o,Lys5o/o,Trp 5%,Serbo/o,Tfubo/0, Glu 5%, Asn 5%, Pro 10%o , Met 5o/o , and Cys b% ProteinC: Ala 10%,Glu 10%,Gly 5o/o,Leu5o/o Asp 10%, ArgSo/o, Met 5%,CysSo/o,T\r5o/o,Phe5o/o,His Valb%,pro b%o, 5%o, Thr 5%, Ser 5%,Asn 5%0, and Gln 5%. 18. Sequence Determination of the Brain peptide Leueine Enkephalin A group of peptides that influence nerve transmissionin certain parts of the brah has been isolated from normal brain tissue. Thesepeptides are knolvn as opioids,becausethey bind to specificreceptorsthat alsobind opiate drugs, such as morphine and naloxone. Opioids thus

mimic some of the properties of opiates. Some researchers considerthesepeptidesto be the brain'sown painkillers.Using the information below,determine the amino acid sequenceof the opioid leucine enkephalin.Explain how your structure is consistentwith eachpiece of information. (a) Completehydrolysisby 6 u HCI at 110 "C followed by amino acid analysisindicated the presenceof Gly, Leu, Phe, and TF, in a 2:1:1:1molarratio. (b) Tteatment of the peptide with 1-fluoro-2,4dinitrobenzene followed by complete hydrolysis and chromatographyindicated the presence of the 2,4-dinitrophenyl derivativeoftyrosine. No free tlrosine could be found. (c) Completedigestionof the peptide with ch5rmotrypsin followedby chromatographyyielded free ty'r'osineand leuche, plus a tripeptide containingPhe and Gly in a I:2 ratio. 19. Strueture of a Peptide Antibiotic from Bacillus brevis Extracts from the bacteriumBoci,llus breui,scontain a peptide with antibiotic properties.This peptide forms complexes with metal ions and seems to disrupt ion transport across the cell membranesof other bacterial species,killing them. The structure of the peptide has been determinedfrom the following observations. (a) Complete acid hydrolysis of the peptide followed by amino acid analysisyielded equimolar amounts of Leu, Orn, Phe, Pro, and Val. Orn is ornithine, an amino acid not present in proteins but present in some peptides. It has the structure

-f

H3N-CH2-CH2-CH2-C-COO-

.rfH" (b) The molecularweight of the peptide was estimatedas about 1,200. (c) The peptide failed to undergohydrolysiswhen treated with the enzyrnecarboxypeptidase.This enzyrnecatalyzesthe hydrolysis of the carboxyl-terminalresidue of a polypeptide unlessthe residueis Pro or, for somereason,doesnot contain a free carboxylgroup. (d) Tfeatment of the intact peptide with l-fluoro-2,4dinitrobenzene,followed by complete hydrolysis and chromatography, flelded or[y free amino acids and the followrng derivative:

o.N-( -\/

NO" -J-H // \ \FNH-CH2-CH2-CH2-C-COO|

.-

*frn,

(Hint: The 2,4-dinitrophenyl derivative involves the amino group of a side chain rather than the a-amino group.) (e) Partial hydrolysis of the peptide followed by chromatographicseparationand sequenceanalysisyreldedthe following di- and tripeptides (the amino-terminalamino acid is alwaysat the left): Leu-Phe Val-Orn-Leu

Phe-Pro

Orn-Leu

Phe-Pro-Val

Val-Orn

Pro-Val-Orn

probtems I rrr-l Giventhe aboveinformation,deducethe aminoacid sequence of the peptide antibiotic. Showyour reasoning.Whenyou have arrived at a structure, demonstratethat it is consistentwith each experimentalobservation. 20. Efficiency in Peptide Sequencing A peptide with the primary structure Lys-Arg-Pro-Leu-Ile-Asp-Gly-Ala is sequencedby the Edmanprocedure.If eachEdmancycle is 96% efficient, what percentageof the amino acids liberated in the fourth cycle will be leucine?Do the calculationa secondtime, but assumea 99% efflciencyfor eachcycle. 21. Sequence Comparisons Proteins called molecular chaperones(describedin Chapter 4) assistin the processof protein folding. One class of chaperonefound in organisms from bacteria to mammalsis heat shock protein 90 (Hsp90). All Hsp90 chaperonescontain a 10 amino acid "signaturesequence," which allows for ready identiflcation of these proteins in sequencedatabases.TWo representationsof this signaturesequenceare shownbelow. Y-x-[NQHD]-tKHRl- tDEl- tIVAl-F-tLMl -R-tEDl. 4 oo

f2

1

0

1 N

3

4

5

6

7

8

910

c

(a) In this sequence,which aminoacid residuesare invariant (conservedacrossall species)? (b) At which position(s) are amino acids limited to those with positively chargedside chains?For each position, which amino acid is more commonlyfound? (c) At which positions are substitutionsrestricted to amino acidswith negativelychargedside chains?For eachposition, which amino acid predominates? (d) There is one position that can be any amho acid, although one amino acid appearsmuch more often than any other. What position is this, and which amino acid appearsmost often? 22. Biochemistry Protocols: Your First Protein Purification As the newest and least experiencedstudent in a biochemistryresearchlab, your flrst few weeksare spentwashing glasswareand labelingtest tubes.Youthen graduateto making buffers and stock solutionsfor usein variouslaboratoryprocedures.Finally,you are given responsibilityfor purifying a proof the citric acid cycle, tein. It is citrate slmthase(an enz].ryne to be discussedin Chapter 16), which is located in the mitochondrialmatrix. Followinga protocol for the puriication, you proceedthrough the stepsbelow.As you work, a more experienced student questions you about the rationale for each procedure.Supplythe answers.(Hint: SeeChapter2 for hJormation about osmolarity;seep. 7 for informationon separation of organellesfrom cells.) (a) Youpick up 20 kg ofbeefheartsfrom a nearbyslaughterhouse (muscle cells are rich in mitochondria,which supply energy for muscle contraction). You transport the hearts on ice, and perform each step of the puriflcation on ice or in a walk-in cold room. You homogenizethe beef heart tissue in a high-speedblender in a medium containing0.2 m sucrose,

buffered to a pH of 7.2. Why do gou use beeJheart t'i'ssue, and i,n such large quanti'tg? What is the purpose oJkeepi'ng the ti,ssuecold and suspending it i'n 0.2 u sucrose, at pH 7.2? What happens to the ti'ssuewhen'it i,s homogenized? (b) You subject the resulting heart homogenate,which is dense and opaque, to a series of differential centrifugation sleps.What does this accom'plish'? (c) You proceed with the purification using the supernatant fraction that containsmostly intact mitochondria.Next you osmoticallylyse the mitochondria. The lysate, which is lessdensethan the homogenate,but still opaque,consistsprimarily of mitochondrialmembranesand internal mitochondrial contents.To this lysateyou add ammoniumsulfate,a highly solublesalt, to a speci-f,cconcentration.You centrifugethe solution, decant the supernatant,and discard the pellet. To the supernatant,which is clearer than the lysate, you add more ammoniumsulfate.Onceagain,you centrifugethe sample,but this time you save the pellet becauseit contains the citrate sy'nthase.What 'is the rationale Jor the two-step addi'ti'on of tlrc salt? (d) You solubilizethe ammoniumsulfatepellet containing the mitochondrial proteins and dialyze it overnight against largevolumesof buffered (pHT.2) solution.V7t'gisn't arnn'Lonium sulJate i'ncluded i,n the di'alysi's buffer? Iilhg do gou use the buffer soluti'on i,nsteadof water? (e) You run the dialyzed solution over a size-exclusion chromatographiccolumn Following the protocol, you collect thefi,rst protein fraction that exits the column and discardthe fractions that elute from the column later. You detect the protein by measuringW absorbance(at 280nm) by the fractions. What d,oesthe i,nstruct'ion to collect the fi,rst Jracti'on tell gou about the protein? Whg i's UV absorbanceat 280 nm a good, way to mon'itor for the presence of protei'n i'n the elutedfracti'ons? (f) You place the fraction collected in (e) on a cationexchangechromatographiccolumn.After discardingthe initial solution that exits the column (the flowthrough), you add a washing solution of higher pH to the column and collect the protein fraction that immediately elutes.Erplai'n whclt Aou are d,oi,ng. (g) You run a small sampleof your fraction, now very reduced in volume and quite clear (though tinged pink), on an isoelectric focusing gel. When stained, the gel shows three sharpbands.Accordingto the protocol, the citrate synthaseis the protein with a pI of 5.6, but you decideto do one more assayof the protein'spurity. Youcut out the pI 5'6 band and subject it to SDSpolyacrylamidegel electrophoresis.The protein resolvesas a singleband. Why were aou uncorni'nced of the puritg oJ the "singte" prote'i,n band on gour i'soelectricJocusi,ng gel? What did the results of the SDSgel teLLyou? Wltg i,s i,t inlportclnt to do tlte SDSgel electrophoresasafter the i,soelectricJocusi'ng?

Problem DataAnalysis 23. Determining the Amino Acid Sequence of Insulin Figure 3-24 showsthe aminoacid sequenceof the hormoneinsr.rlin.This structure was determinedby Frederick Sangerand

rt

!

]

A m i nA o c i d sP,e p t i d ea sn,dp r o t e i n s

his coworkers.Most of this work is describedin a seriesof articlespublishednthe Bi,ochemicalJouynal from 194bto 19bb. When Sangerand colleaguesbegantheir work in 194b,it wasknown that insulin was a smailprotein consistingof two or four polypeptide chainslinked by disulfldebonds. Sangerand his coworkershad developeda few simplemethodsfor studying protein sequences Treatnnnt wi,th FDNB. FDNB (1-fluoro-2,4-dinitrobenzene) reacted with free amino (but not amido or guanidino) groupsin proteins to producedinitrophenyl (DNP) derivatives of amino acids: or\

-/

R-NH2 + F-( Amine

\_\

\:/

orN.

\

)-NO, -

R-N{

FDNB

l--\

tl \:/

}NO,

+ Hr

DNp-amine

Acid Hgdrolgsis. Boiling a protein with 10%HCI for several hours hydrolyzedall of its peptide and amidebonds.Short treatmentsproduced short polypeptides;the longer the treatment, the more completethe breakdownof the protein into its amino acids. Onidati,on of Cystei,nesTleatment of a protein with performic acid cleavedall the disulflde bonds and converted all Cysresiduesto cysteicacid residues(Fig.3-26). Paper Chromntography. This more primitive version of thin-layer chromatography(see Fig. 10-24) separatedcompounds based on their chemicalproperties, allowing identiflcation of single amho acids and, in some cases,dipeptides. Thin-layer chromatographyalso separateslarger peptides. As reported in his first paper (194b), Sangerreacted insulin with FDNB and hydrolyzed the resulting protein. He found many free amino acids, but oniy three DNp-amino acids: a-DNP-glycine (DNP group attached to the a-amino group); a-DNP-phenylalanine; and e-DNpJysine (DNp attachedto the e-aminogroup) Sangerinterpreted theseresults as showingthat insulin had two protein chains:one with GIy at its amino terminus and one with Phe at its amino terminus. One of the two chainsalso containeda Lys residue,not at the amino terminus. He named the chain beginning with a GIy residue"A" and the chain beginningwith phe ,,B." (a) Explain how Sanger'sresultssupporthis conclusions. (b) Are the resuits consistentwith the known strrucure of insulin (Fig. 3-24)? In a later paper (1949), Sangerdescribedhow he used these techniques to determine the first few amino acids (amino-terminalend) of each insulin chain To analyzethe B chain,for example,he carried out the following steps:

6. Isolatedfour of the DNP-peptides,which were named81 through84. 7. StronglyhydrolyzedeachDNP-peptideto give free amino acids. 8. Identiied the amino acidsin eachpeptide with paper chromatography. The results were as follows: 81 : a-DNP-phenylalanineonly 82 : a-DNP-phenylalanine; valine 83: asparticacid; a-DNP-phenylalanine; valine 84: aspartic acid; glutamic acid; a-DNP-phenylalanine; valine (c) Basedon these data, what are the first four (aminoterminal) amino acids of the B chain?Explain your reasoning. (d) Doesthis result match the known sequenceof insulin (Fig. 3-24)? Explain any discrepancies. Sangerand colleaguesusedthese and related methodsto determinethe entire sequenceof the A and B chains.Their sequencefor the A chainwas asfollows (aminoterminus on left): r510

Gly-Ile-Val-Glx-Glx-Cys-Cys-Ala-S 15

er-Val20

Cys-Ser-Leu-Tyr'-Glx-Leu-Glx-Asx-Tyr.-Cys-Asx Becauseacid hydrolysishad converted all Asn to Asp and all GIn to Glu, these residueshad to be designatedAsx and Glx, respectively(exact identity in the peptide unknown). Sanger solved this problem by using protease enzymesthat cleave peptide bonds, but not the amide bonds in Asn and GIn residues,to prepare short peptides.He then determined the number of amidegroupspresentin eachpeptide by measuring the NHj released when the peptide was acid-hydrolyzed. Someof the results for the A chain are shownbelow.The peptides may not have been completely pure, so the numbers were approximate-but good enough for Sanger'spurposes. Peptide nane Ac1 Ap15 Ap14 Ap3 Ap1 Ap5pa1 Ap5

Peptide sequence Cys-Asx T[-GIx-Leu TVr-Glx-Leu-Glx Asx-T)'r-Cys-Asx Glx-Asx-Tlr-Cys-Asx Gly-Ite-Vai-Glx Gly-Iie-Val-Glx-Glx-Cys-CysAla-Ser-Val-Cys-Ser-Leu

Numberof amide groupsin peptide 0.7 0.98 1.06

2.r0 r.94 0.15 1.16

1 . Oxidizedinsulin to separatethe A and B chains. z.

Prepareda sampleof pure B chain with paper chromatography

3 . Reactedthe B chain with FDNB. 4. Gently acid-hydrolyzedthe protein so that somesmall

(e) Based on these data, determine the amino acid sequence of the A chain. Explain how you reached your answer. Compare it with Figure 3-24. Beferenees

peptideswould be produced.

Sanger, F (1945) The free amino groups of insulin.Bi,ochem.J. Bg, 507-515

Separatedthe DNP-peptidesfrom the peptidesthat did not contain DNP groups.

Sanger, F. (1949) The terminal peptides of insujin. Bi,ochem. J. 45, 563-574.

Perhapsthe more remarkablefeaturesof [myoglobin]are its complexity and its lack of symmetry.The arrangementseemsto be almosttot a l l y l a c k i n g i n t h e k i n d o f r e g u l a r i t i e sw h i c h o n e i n s t i n c t i v e l y anticipates,and it is more complicatedthan has been predictedby any theoryof proteinstructure. -lohn Kendrew,articlein Nature,7958

TheThree-Dimensional Structure ofProteins 4 ; l 0verview ofProtein Structure113 4.2 Protein Secondary Structure117 Tertiary Structures123 4.3 Protein andQuaternary 4.4 Protein Denaturation andFolding140 he covalent backboneof a typical protein contains hundredsof individualbonds.Becausefree rotation is possiblearoundmany of thesebonds,the protein can asslunea very large number of conformations.However, each protein has a speciflc chemical or structural function, strongly suggestingthat each has a unique structure (Fig. 4-1). By the late three-dimensional 1920s,severalproteins had been crystallized,including hemoglobin (M, 64,500) and the enzyme urease (M. 483,000).Giventhat, generally,the orderedarrayof molecules in a crystal can form only if the molecular

units are identical, the finding that many proteins could be crystallizedwas evidencethat even very large proteins are discrete chemical entities with unique structures. This conclusionrevolutionizedthinking about proteins and their functions. In this chapter, we examine how a sequenceof amino acids in a polypeptide chain is translated into a protein structure. We emdiscrete,three-dimensional phasizeflve themes.First, the three-dimensionalstructure of a protein is determined by its amino acid sequence.Second,the functionof a protein dependson its structure. Third, an isolatedprotein usually exists in one or a small number of stable structural forms. Fourth, the most important forces stabilizing the specifi.cstructures maintainedby a given protein are noncovalentinteractions.Finally, amid the huge number of uniqueprotein structures,we can recognizesomecommon structural patterns that help to organizeour understandingof protein architecture. These themes should not be taken to imply that proteins have static, unchanging,three-dimensional structures.Protein function often entails an interconversionbetweentwo or more structuralforms.The dynamic aspectsof protein structure will be expioredin Chapters5 and 6. An understandingof all levelsof protein structure is essentialto the discussionof function in later chapters.

Structure ofProtein 4.1 Overview 4-1 Structureof the enzymechymotrypsin, a globularprotein. FIGURE Theknown A moleculeof glycine(blue)is shownfor sizecomparison. structures of oroteinsare archivedin the Protein three-dimensional Data Bank,or PDB (seeBox 4-4).The imageshown herewas made usingdatafromthe PDBentry6CCH.

The spatialarrangementof atomsin a protein is called its conformation. The possibleconformationsof a protein include any structural state it can achievewithout breaking covalent bonds. A change in conformation could occur, for example, by rotation about single bonds. Of the many conformationsthat are theoretically possiblein a protein containinghundreds of single [ttr]

-I

5tructure ofProteins [ 14.] IheThree-Dimensional bonds, one or (more commonly) a few generally predominate under biological conditions. The need for multiple stable conformationsreflects the changesthat must take place in most proteinsas they bind to other moleculesor catalyzereactions.The conformationsexisting under a given set of conditions are usually the onesthat are thermodynamicallythe most stable-that is, havingthe lowestGibbsfree energy(G). Proteinsin any of their functional, folded conformationsare called native proteins. What principles determine the most stable conformations of a protein? An understandingof protein conformation can be built stepwisefrom the discussionof primary structure in Chapter 3 through a consideration of secondarytertiary and quaternarystructures.To this traditional approachwe must add the newer emphasis on corrmon and classiflablefolding patterns, called supersecondarystructuresor motifs, which provide an important organizationalcontext to this complexendeavor. We beginby introducing someguiding principles.

AProtein's Conformation lsStabilized Largely by Weak lnteractions In the context of protein structure, the term stability can be definedas the tendencyto maintain a native conformation. Native proteins are only marginally stable; the AG separatingthe folded and unfolded statesin typical proteins under physiologicalconditions is in the range of only 20 to 65 kJ/mol.A given polypeptidechain can theoretically assumecountlessconformations,and as a result the unfolded state of a protein is characterized by a high degree of conformationalentropy. This entropy, and the hydrogen-bondinginteractions of many groups in the polypeptide chain with the solvent (water), tend to maintain the unfolded state.The chemical interactionsthat counteractthese effectsand stabilize the native conformationinclude disul_fide(covalent) bonds and the weak (noncovalent) interactions described in Chapter 2: hydrogenbonds and hydrophobic and ionic interactions. Many proteins do not have disul_flde bonds.The environment within most cells is highly reducing and thus precludesthe formationof -S-S-bonds. In eukaryotes,disulfidebondsare found primarily in secreted,extracellular proteins (for example,the hormone insulin). Disulfide bonds are also uncorunon in bacterial proteins. However,thermophilic bacteria,as well as the archaea, typically have many proteins with disulfide bonds, which stabilize proteins; this is presumably an adaptationto Iife at high temperatures. For the intracellular proteins of most organisms, weak interactionsare especiallyimportant in the folding of pollpeptide chains into their secondaryand tertiary structures. The associationof multiple po\peptides to form quaternary structures also relies on these weak interactions.

About 200 to 460 kJ/mol are required to break a single covalentbond, whereasweak interactionscan be disrupted by a mere 4 to 30 kJ/mol. Individual covalent bonds, such as disulfrdebonds linking separateparts of a single polypeptide chain, are clearly much stronger than individual weak interactions.Yet,becausethey are so numerous,it is weak interactionsthat predominateas a stabilizingforce in protein structure. In general, the protein conformationwith the lowest free energy (that is, the most stable conformation) is the one with the maximum number of weak interactions. The stability of a protein is not simply the sum of the free energiesof formation of the manyweak interactions within it. For every hydrogen bond formed in a protein during folding, a hydrogen bond (of similar strength) between the samegroup and water was broken. The net stability contributed by a given hydrogen bond, or the di,fference in free energiesof the folded and unfolded states,may be closeto zero. Ionic interactions may be either stabilizingor destabilizing.We must therefore look elsewhereto understandwhy a particular native conformationis favored. On carefully examimngthe contribution of weak interactionsto protein stability,we find that hydrophobic interactions generally predominate. Pure water contains a network of hydrogen-bondedH2Omolecules.No other molecule has the hydrogen-bondingpotential of water, and the presenceof other moleculesin an aqueous solution disrupts the hydrogen bonding of water. When water surroundsa hydrophobicmolecule,the optimal arrangementof hydrogenbondsresults in a highly structured shell, or solvation layer, of water around the molecule(seeFig. 2-7). The increasedorder of the water moleculesin the solvationlayer correlateswith an unfavorabledecreasein the entropy of the water. However,when nonpolargroups cluster together,the extent of the solvationlayer decreases becauseeachgroup no longerpresentsits entire surfaceto the solution.The result is a favorableincreasein entropy. As describedin Chapter2, this increasein entropy is the major thermodynamicdriving force for the associationof hydrophobic groups in aqueoussolution. Hydrophobicamino acid side chains therefore tend to cluster in a protein's interior, awayfrom water. Under physiologicalconditions,the formation of hydrogen bonds in a protein is driven largely by this same entropic effect. Polar groups can generally form hydrogen bonds with water and hence are soluble in water. However,the number of hydrogen bonds per unit mass is generally greater for pure water than for any other liquid or solution, and there are limits to the solubility of even the most polar moleculesas their presencecausesa net decreasein hydrogenbonding per unit mass.Therefore,a solvationlayer also forms to someextent aroundpolar molecules.Even though the energy of formation of an intramolecular hydrogen bond betweentwo polar groupsin a macromoleculeis

Structure 4.10verview ofProtein | 115 |

largelycanceledby the eliminationof suchinteractions betweenthese polar groups and water, the releaseof structured water as intramolecularinteractionsform providesan entropic driving force for folding. Most of the net changein free energyas weak interactionsform within a protein is therefore derived from the increased entropy in the surrounding aqueoussolution resulting from the burial of hydrophobicsurfaces.This more than counterbalancesthe Iarge loss of conformationalentropy as a polypeptide is constrainedinto its folded conformation. Hydrophobic interactions are clearly important in stabilizingconformation;the interior of a protein is generallya denselypackedcore of hydrophobicaminoacid side chains. It is also important that any polar or charged groups in the protein interior have suitable partners for hydrogen bonding or ionic interactions. Onehydrogenbond seemsto contributelittle to the stabiJityof a native structure,but the presenceof hydrogenbonding groups without partners in the hydrophobic core of a protein can be so destabi,Lizi,ngthat conformations containingthese groups are often thermodynamicallyuntenable.The favorablefree-energychange resulting from the combinationof severalsuch groups with partners in the surrounding solution can be greater than the free-energydifference between the folded and unfolded states. In addition, hydrogen bondsbetweengroupsin a protein form cooperatively (formation of one makesthe next one more likely) in repeatingsecondarystructuresthat optimizehydrogen bonding, as described below. In this way, hydrogen bonds often have an important role in guiding the protein-foldingprocess. The interaction of oppositely charged groups that form an ion pair, or salt bridge,can haveeither a stabilizing or destabilizing effect on protein structure. As in the case of hydrogenbonds, chargedamino acid side chainsinteract with water and saltswhen the protein is unfolded, and the loss of those interactionsmust be consideredwhen evaluatingthe effect of a salt bridge on the overall stability of a folded protein. However,the strength of a salt bridge increasesas it movesto an environmentof lower dielectricconstant,e (seep. 46): from the polar aqueoussolvent (e near 80) to the nonpolar proteininterior (e near 4). Saltbridges,especiallythose that are partly or entirely buried, canthus provide significant stabilizationto a protein structure. This trend explainsthe increasedoccurrenceof buried salt bridgesin the proteins of thermophilic organisms.Ionic interactions alsoLimitstructural flexibility and confer a uniqueness to protein structure that nonspecifichydrophobic interactionscannotprovide. Most of the structural patterns outlined in this chapter reflect two simple rules: (1) hydrophobic residues are largely buried in the protein interior, away from water; and (2) the number of hydrogen bondsand ionic interactionswithin the protein is max-

imized, thus reducing the number of hydrogenbonding and ionic groups that are not paired with a suitablepartner.Insolubleproteinsand proteinswithin membranes(which we examinein Chapter l1) follow somewhatdifferent rules, becauseof their particular function or environment, but weak interactions are still critical structural elements.

andPlanar Bond lsRigid ThePeptide Covalent bonds, Structure 0 protelnArchitecture-Primary place important constraintson the conformationof too, a polypeptide. In the late 1930s,Linus Pauling and Robert Corey embarkedon a series of studies that laid the foundationfor our current understandingof protein structure. They beganwith a carefulanalysisof the peptide bond.

1901-1 994 LinusPauling,

RobertCorey,1897-1971

The o carbonsof adjacentaminoacid residuesare separated by three covalent bonds, arranged as X-ray diffraction studies of crystals C,-C-N-C'. and of simple dipeptides and tripepof amino acids peptide C-N bond is somewhat the tides showedthat shorter than the C-N bond in a simpleamineand that the atoms associatedwith the peptide bond are coplanar.This indicateda resonanceor partial sharingof two pairs of electrons between the carbonyl oxygen and the amidenitrogen (Fig. 4-2a). The oxygenhas a partial negativechargeand the nitrogen a partial positive charge,setting up a small electric dipole. The six atomsof the peptide group lie in a singleplane,with the oxygenatom of the carbonylgroup trans to the hydrogen atom of the amide nitrogen. From these findings Pauling and Corey concluded that the peptide C-N bonds, becauseof their partial double-bond character,cannot rotate freely. Rotation is permitted about the N-C, and the Co-C bonds.The backbone of a polypeptidechain can thus be pictured as a series of rigid planes,with consecutiveplanessharinga common point of rotation at C. (Fig. 4-2b). The rigid peptide bonds Iimit the range of conformationspossible for a polypeptidechain.

.l 16

T h eT h r e e - D i m e n sSi ot rnuaclt uor feP r o t e i n s

o

oI

06-

ti

ll

(ti

:a-2":*.-"a\

-l

-

-

-C.-.- I Na+-'Co't C^.

"l

H

-

C -cj---ir

-l

H

H

uCo

The carbonyl oxygen has a partial negative charge and the amide nitrogen a partial positive charge, setting up a small electric dipole. Virtually all peptide bonds in proteins occur in this trans configuration; an exception is noted in Fieure 4-7b.

(a)

Carboxyl trermlnus tE3A

t180"

(c)

(d)

Peptide conformationis defined by three dihedral angles(alsoknown as torsion angles)called@ (phi), ,/ (psi), and c,r(omega),reflectingrotation about eachof the threerepeatingbondsin the peptidebackbone.A dihedral angle is the angle at the intersection of two planes.In the caseof peptides,the planesare definedby bond vectorsin the peptide backbone.TWosuccessive bond vectors describea plane. Three successivebond vectorsdescribetwo planes(the centralbond vector is common to both; Fig. 4-2c), and the angle between thesetwo planesis what we measureto describeprotein conformation. KEY(0NVENTI0N: The important dihedralanglesin a peptide are defined by the three bond vectors connecting four consecutivemain-chain(peptide backbone) atoms(Fig. 4-2c): @involvesthe C-N-C.-C bonds (with the rotation occurring about the N-C. bond), and ry'invoivesthe N-C.-C-N bonds.Both -r Q and r! are defined as 180owhen the polypeptideis fully extendedand all peptide groups are in the same plane (Fig. 4-2d). As one iooks down the centralbond

FIGURE4-2Theplanarpeptidegroup.(a) Eachpeptidebond hassome double-bondcharacterdue to resonance and cannotrotate.(b) Three bondsseparate sequential a carbonsin a polypeptide chain.TheN-C" and C"-C bondscan rotate,describedby dihedralanglesdesignated @ and ry',respectively. ThepeptideC-N bond is not freeto rotate.Other singlebondsin the backbonemay alsobe rotationally hindered, dependingon the size and chargeof the R groups.(c) The atomsand planesdefiningf. (d) By convention, @andry'are180"(or-180') when the firstand fourthatomsarefarthestapartand the peptideis fully extended.As the viewer looksout along the bond undergoingrotation (fromeitherdirection)the @and ry'anglesincreaseas the fourthatom rotatesclockwiserelativeto the first.In a protein,someof the conformationsshownhere(e.9.,0") areprohibitedby stericoverlapof atoms. In (b) through(d),the ballsrepresenting atomsaresmallerthanthe van derWaal'sradiifor thisscale

vector in the direction ofthe vector arrow (as depicted in Fig. 4-2c for ry'),the dihedral anglesincreaseas the distal (fourth) atom is rotated clockwise(FiC. 4-2d). From the r- 180oposition,the dihedralangleincreases from -180" to 0', at which point the first and fourth atoms are eclipsed.The rotation can be continued from 0' to *180" (sameposition as -180') to bring the structure back to the starting point. The third dihedral angle,@,is not often considered.It involvesthe C,-C-N-C. bonds.The centralbond in this case is the peptide bond, where rotation is constrained. The peptide bond is normally (99.60/o of the time) in the trans configuration,constrainingro to a value of -f 180'.For a rare cis peptidebond,c'r: 0o.r In principle, @and ry'can have any value between -180' and f 180', but many valuesare prohibited by steric interferencebetween atoms in the polypeptide backboneand amino acid side chains.The conformation in which both @and ry'are 0" (Fig. 4-2d) is prohibited for this reason;this conformationis merely a reference point for describingthe dihedralangles.Allowedvalues

Srtyr u c t u r Ir e t 4 . 2P r o t eS i ne c o n d a

Structure Secondary 4.2 Protein

+ 180 r20 60 a H

b!n

-60 -t20 -180 - 180

i

0

+180

d (degrees) FIGURE 4-3 Ramachandran plot for r--Alaresidues.Peptideconformationsaredefinedby the valuesof @and ry'.Conformations deemedpossibleare thosethat involvelittleor no stericinterference, basedon calculations usingknownvanderWaalsradiianddihedralangles. The areasshadeddarkbluerepresent conformations that involveno steric overlapand thusarefully allowed;mediumblue indicates conformationsallowedat theextremelimitsfor unfavorable atomiccontacts; the lightest blueindicates conformations thatarepermissible if a littleflexibilityis allowedin thedihedralangles. Theyellowregionsareconformationsthat are not allowed.Theasymmetry of the plot resultsfrom the r stereochemistry of the aminoacid residues. Theplotsfor otherI residues with unbranched sidechainsare nearlyidentical.Allowed rangesfor branchedresidues suchasVal, lle, and Thr are somewhat smallerthanfor Ala.TheCly residue, whichis lesssterically hindered, hasa muchbroaderrangeof allowedconformations. Therangefor Pro is greatlyrestricted residues because{ is limitedby the cyclic side c h a i nt o t h er a n s eo f - 3 5 ' t o - 8 5 " .

for @and ry'becomeevidentwhen * is piotted versusd in a Ramachandran plot (F'ig. 4-3), introducedby G. N. Ramachandran.

S U M M A R4Y. 1 O v e r v i eowf P r o t e i n S t r uc t ur e r

Everyprotein hasa three-dimensional structure that reflectsits function.

r

Proteinstructureis stabilizedby multipleweak interactions.Hydrophobicinteractionsare the major contributors to stabilizingthe globularform of most solubleproteins;hydrogenbondsand ionic interactionsare optimizedin the thermod;mamically most stablestructures.

r

The nature of the covalentbondsin the polypeptide backboneplacesconstraintson structure.The peptidebond hasa partial double-bondcharacter that keepsthe entire six-atompeptidegroupin a rigid planarcon-flguration. The N-Co and C.-C bondscan rotate to definethe dihedralanglesd and ry',respectively.

The term secondary structure refers to any chosen segmentof a polypeptidechain and describesthe local spatialarrangementof its main-chainatoms,without regard to the conformation of its side chains or its relationship to other segments. A regular secondary structureoccurswhen eachdihedralangle,d and ry',remains the sameor nearly the samethroughout the segment. Thereare a few typesof secondarystructurethat are particularlystableand occurwidelyin proteins.The anmostprominentarethe a helix andB conformations; patregular Where a other commont1-peis the B turn. tern is not found,the secondarystructureis sometimes referredto as undefinedor as a random coil. This last designation,however,does not properly describethe structure of these segments.The path of the poll'peptide backbonein almost any protein is not random; rather, it is typically unchangingand highly speciflc to the structure and function of that particular protein. Our discussionhere focuseson the regular, cornmon structures.

Structure Frotein Secondary lsa(omrnon Thetr l-lelix Helix Pauling and Corey were Architecture-a C Protein awareof the importanceof hydrogenbonds in orienting polar chemicalgroupssuchasthe C:O and N-H groups of the peptide bond. They also had the experimentalresults of WilLiamAstbury who in the 1930shad conducted pioneeringx-ray studies of proteins.Astbury demonstratedthat the protein that makesup hair and porcupine quills (the flbrous protein a-keratin) has a regular structurethat repeatsevery5.15to 5.2A. (The angstrom, A, namedafter the physicistAndersJ. Angstrom,is equal to 0.1nm. Althoughnot an SI unit, it is useduniversallyby structural biologiststo describeatomic distances-it is approximatelythe length of a typical C-H bond.) With this informationand their data on the peptide bond, and with the help of precisely constructedmodels,Pauling and Coreyset out to determinethe likely conformations of proteinmolecules. The simplestarrangementthe polypeptidechain can assume,given its rigid peptide bonds (but free rotation around its other, singlebonds), is a helical structure, which Paulingand Coreycalledthe c helix (FiS" 4-4). In this structure the polypeptidebackboneis tightly wound around an imaginary axis drawn Iongitudinally through the middle of the helix, and the R groups of the amino acid residuesprotrude outward from the helical backbone.The repeatingunit is a singleturn of the helix, which extends about 5.4 A along the long axis, slightly greater than the periodicity Astbury observedon x-ray analysisof hair keratin. The amino acid residuesin the prototypical a helix have conformationswith Q = -57" and ry'= -47o, and eachhelical turn includes3.6 amino acid residues.The a-helicalsegmentsin proteinsoften

[t t ol

The Three-Dimensional Structure ofproteins

Amino terminus

5.4A Y

(3.6 residues)1

(a)

Carboxyl terminus

ft)

(c)

(d)

FIGURE 4-4 Modelsof the c helix, showingdifferent aspectsof its structure.(a) Ball-and-stick modelshowingthe intrachainhydrogen bonds.Therepeatunit isa singleturnofthe helix,3.6 residues. (b)The a helix viewedfrom one end, lookingdown the longitudinalaxis (derivedfrom PDB lD 4TNC).Notethe positionsof the R groups,represented by purplespheres. Thisball-and-stick model,whichemphasizesthe helicalarrangement, givesthe falseimpression thatthe helix is hollow,because the ballsdo not represent thevanderWaalsradiiof

the individualatoms.(c) As thisspace-filling modelshows,the atoms in thecenterof thea helixarein veryclosecontact.(d) Helicalwheel projectionof an a helix.Thisrepresentation can be coloredto identify surfaceswith particularproperties. The yellow residues, for example, could be hydrophobicand conformto an interfacebetweenthe helix shownhereandanotherpartof thesameor anotherpolypeptide. Thered and blue residues illustrate the potentialfor interaction of negatively and positivelychargedsidechainsseparated by two residues in the helix.

deviate slightly from these dihedral angles,and even vary somewhatwithin a single contiguous segment to producesubtlebendsor kinks in the helicalaxis. In all proteins, the helical twist of the o helix is right-handed (Box 4-1). The a helix proved to be the predominant structure in a-keratins. More generally, about one-

fourth of all amino acid residuesin proteins are found in a helices, the exact fraction varying greatly from one protein to another. Why doesthe a helix form more readily than many other possibleconformations?The answeris, in part, that an a helix makes optimal use of internal hydrogen

There is a simple method for determining whether a helical structure is right-handedor left-handed.Make flsts of your two hands with thumbs outstretched and pointing away from you. Looking at your right hand, think of a helix spiraling up your right thumb in the direction in which the other four fingers are curled as shown (clockwise).The resultinghelix is right-handed. Your left hand will demonstratea left-handed helix, which rotates in the counterclockwisedirection as it spiralsup your thumb.

Left-handed

Structure tt] 4.2 Protein Secondary [,

bonds. The structure is stabilizedby a hydrogen bond betweenthe hydrogenatom attachedto the electronegativenitrogen atom of a peptide linkage and the electronegativecarbonyloxygenatom of the fourth amino acid on the amino-terminalside of that peptide bond (Fig. 4-aa). Within the a helix, every peptide bond (exceptthose closeto eachend of the helix) participatesin such hydrogenbonding.Each successiveturn of the a helix is held to adjacentturns by three to four hydrogenbonds,conferringsignificantstability on the overall structure. Further model-buildingexperimentshave shown that an a helix canform in polypeptidesconsistingof either l- or l-amino acids.However,all residuesmust be of one stereoisomeric series;a n-aminoacid will disrupt a regularstructureconsistingof l-amino acids,and vice versa. In principle, naturally occurring l-amino acids can form either right- or left-handeda helices,but extendedleft-handeda helicesaretheoreticallyIessstable and havenot beenobservedin oroteins.

I

Amino acid

AAC" (kJ/mol)*

Amino ecid

AAC" (kJ/mol)*

Ala

0

Leu

0.79

tug

0.3

Lys

0.63

Asn

Met

0.88

Asp

Phe

2.0

cvs

Pro

>4

Gln

1.3

Ser

2.2

Giu

r.4

Thr

qA

Glv

4.6

TYr

2.0

His

2.6

Tfp

2.0

IIe

r.4

Val

2.r

proline) Betz,S.E,Lu,H.S.,Suich,DJ.,Zhou, fromBryson,.J.W., Data(except Sources: (1995)Protein Sclence a hierarchic approach. design: H X,o'Neil,KT.,& Decrado,WE C.N.,& Scholtz, J.M.(1997)Helix J.K.,Pace, datafromMyers, 270,935 Proline 36, 10,926 propensities Blochemistry in proteins andpeptides. areidentical *AAG" is thedifference for required relative to thatforalanine, in free-energy change, retlect Larger numbers to takeupthec-helicalconformation. the aminoacidresidue derived from greater Dataarea composite difficulty takingupthea-helicalstructure. systems. andexperimental experiments multiple

W0RKED EXAMPIE4-1SecondaryStructureand Protein Dimensions

What is the length of a polypeptide with B0 amino acid residuesin a singlecontiguousa helix? Solution:An idealizeda helix has 3.6 residuesper turn and the rise alongthe helicalaxisis 5.4A. Thus,the rise alongthe axis for eachaminoacid residueis 1.5A. The length ofthe polypeptideis therefore80 residuesx t 5 A/ residue: 120A.

Amino Acid 5equence Affects Stability ofthea Helix Not all polypeptides can form a stable a helix. Each amino acid residue in a polypeptide has an intrinsic propensity to form an a helix (Table 4-1), reflecting the properties of the R group and how they affect the capacity of the adjoining main-chain atoms to take up the characteristic @ and ry'angles. Alanine shows the greatest tendency to form a helices in most experimental model systems. The position of an amino acid residue relative to its neighbors is also important. Interactions between amino acid side chains can stabilize or destabilize the a-helical structure. For example, if a poll-peptide chain has a long block of Glu residues, this segment of the chain will not form an a helix at pH 7.0. The negatively charged carboxyl groups of adjacent GIu residues repel each other so strongly that they prevent formation of the a helix. For the same reason, if there are many adjacent Lys and./orArg residues,with positively charged R groups at pH 7 0, they also repel each other and prevent formation of the a helix. The bulk and shape of Asn, Ser, Thr, and Cys residues can also destabilize an a helix if they are close together in the chain.

The twist of an a helix ensures that critical interactions occur between an amino acid side chain and the side chain three (and sometimes four) residues away on either side of it. This is clear when the a helix is depicted as a helical wheel (Fig. 4-4d). Positively charged amino acids are often found three residues away from negatively charged amino acids, permitting the formation of an ion pair. TWo aromatic amino acid residues are often similarly spaced, resulting in a hydrophobic interaction. A constraint on the formation of the a helix is the presence of Pro or Gly residues, which have the least proclivity to form a helices. In proline, the nitrogen atom is part of a rigid ring (see Fig. 4-7b), and rotation about the N-C. bond is not possible.Thus, a Pro residue introduces a destabilizing kink in an a helix. In addition, the nitrogen atom of a Pro residue in a peptide linkage has no substituent hydrogen to participate in hydrogen bonds with other residues. For these reasons, proline is only rarely found in an a helix. Glycine occurs infrequently in o helices for a different reason: it has more conformational flexibility than the other amino acid residues. Polymers of glycine tend to take up coiled structures quite different from an a helix. A final factor affecting the stability of an a helix is the identity of the amino acid residues near the ends of the a-helical segment of the polypeptide. A small electric dipole exists in each peptide bond (Fig. 4-2a). These dipoles are aligned through the hydrogen bonds of the helix, resulting in a net dipole along the helical

Frr]

T h eT h r e e - D i m e n sSi ot rnuaclt uor feP r o t e i n s

the backbone of the polypeptide chain is extended into a zrgzagrather than helical structure (Fig. 4-6). The zigzagpolypeptide chains can be arrangedside by side to form a structure resemblinga seriesof pleats. In this arrangement,calleda p sheet, hydrogenbonds form betweenadjacentsegmentsof polypeptidechain. The individual segmentsthat form a B sheet are usually nearby on the polypeptide chain, but can also be

Amino terminus

6+

T

T

+

r) Carboxyl terminus

FIGURE 4-5 Helixdipole.The electricdipole of a peptidebond (seeFig.4-2at is transmittedalong an a-helicalsegment throughthe intrachain hydrogen bonds,res u l t i n gi n a n o v e r a l h l e l i xd i p o l e .I n t h i s illustration, the amino and carbonylconstituentsof each peptidebond are indicated by t and - symbols,respectively. Non-hydrogen-bonded amino and carbonyl constituents of the peptidebonds neareachend of the a-helicalregionare shownin red.

axis that increaseswith helix length (Fig. 4-b). The four amino acid residuesat each end of the helix do not participatefully in the helix hydrogenbonds.The partial positiveand negativechargesof the helix dipolereside on the peptide amino and carbonyl groups near the amino-terminal and carboxyl-terminalends, respectively. For this reason,negatively charged amino acids are often found near the amino terminus of the helical segment,where they have a stabilizinginteraction with the positive charge of the helix dipole; a positively chargedamino acid at the amino-terminalend is destabilizing. The opposite is true at the carboxyl-terminal end of the helicalsegment. In summary,flve types of constraintsaffect the stability of an a helix: (1) the intrinsic propensity of an amino acid residue to form an a helix; (2) the interactions betweenR groups,particularlythosespacedthree (or four) residuesapart; (3) the bulkinessof adjacentR groups;(4) the occurrenceof Pro and Gly residues;and (5) interactions between amino acid residuesat the ends of the helical segmentand the electric dipole inherent to the a helix. The tendencyof a given segment of a polypeptide chain to form an a helix therefore depends on the identity and sequenceof amino acid residueswithin the segment.

Top view

Side view

(b) Parallel Top view

Side view

6 . 5A

TheB Conformation 0rganizes Polypeptide Chains intoSheets $ eroteinArchitecture-BSheetIn 1951, Pauling and Corey predicted a second type of repetitive structure, the B conformation. This is a more extended conformation of polypeptide chains, and its structure has been confirmed by x-ray analysis. In the B conformation,

FIGURE 4-6 Thep conformationof polypeptidechains.Thesetop and sideviewsrevealthe R groupsextendingout from the B sheetand emphasizethe pleatedshapedescribedby the planesof the peptide bonds (An alternativename for this structureis B-pleatedsheet.) Hydrogen-bond cross-links betweenadjacentchainsarealsoshown. The amino-terminal to carboxyl-terminal orientationsof adlacent chains(arrows) can be the sameor opposite, forming(a)an antiparalIelB sheetor (b) a parallelB sheet.

Structure12 4.2Protein Secondary

quite distant from each other in the linear sequence of the polypeptide;they may even be in different polypeptide chains. The R groups of adjacent amino acids protrude from the zigzagstructure in opposite directions,creating the alternatingpattern seen in the side views in Figure 4-6. The adjacentpollpeptide chainsin a € sheet can be either parallel or antiparallel(having the same or oppositeamino-to-carboxylorientations,respectively). The structures are somewhatsimilar, although the repeat periodis shorterfor the parallelconformation(6.5 A, vs. 7 A for antiparaliel) and the hydrogen-bonding patterns are different. The idealizedstructurescorrespondto @ = -119', f : +113" (parallel)and @ : -139o, : +135' (antiparallel);these valuesvary 4l somewhatin real proteins,resultingin structuralvariation, as seenabovefor o helices. Someprotein structures limit the kinds of amino acidsthat can occur in the F sheet.When two or more B sheetsare layeredclosetogetherwithin a protein, the R groupsof the aminoacid residueson the touching surfacesmust be relatively small.B-Keratinssuch as silk fibroin and the fibroin of spider webs have a very high content of Gly and AIa residues,the two amino acidswith the smallestR groups.Indeed,in silk fibroin GIy and AIa alternate over large parts of the sequence.

p Turns Are(ommon inProteins Architecture-B TurnIn globularproteins,which f Protein havea compactfoldedstructure,nearlyone-thirdof the amino acid residuesare in turns or loops where the polypeptidechainreversesdirection(FiS. 4-7). These

are the connectingelementsthat link successiveruns of a helix or B conformation.Particularlycommonare p turns that connect the ends of two adjacent segmentsof an antiparallelB sheet.The structureis a 180' turn involving four amino acid residues,with the carbonyl oxygen of the fi.rst residue forming a hydrogen bond with the amino-grouphydrogen of the fourth. The peptide groups of the central two residuesdo not participatein any inter-residuehydrogenbonding.Gly and Pro residuesoften occurin B turns, the former because it is smalland flexible,the latter becausepeptidebonds involving the imino nitrogen of proline readily assume the cis conflguration (Fig. 4-7b), a form that is particuIarly amenableto a tight turn. Of the severaltypes of B turns, the two shov,rtin Figure 4-7a are the most common. Beta turns are often found near the surfaceof a protein, where the peptide groups of the central two aminoacidresiduesin the turn canhydrogen-bondwith water.Considerablylesscommonis the 7 turn, a threeresidue turn rnetha hydrogenbond between the first and third residues.

Have Characteristic Structures Secondary Common DihedralAnqles The a helix and the B conformation are the major repetitive secondarystructures in a wide variety of proteins, althoughother repetitive structures exist in somespecializedproteins (an exampleis collagen;see FiC. 4-12). Every tlpe of secondarystructure can be completely described by the dihedral angles @ and ry'associatedwith each residue. As shovrn by a Ramachandranplot, the a helix and B conformation fall within a relativelyrestrictedrangeof stericallyallowed

Glycine

/z 1

Proline I

trans

(a) F Ttrrns

Type I

FIGURE4-7Structures of p turns.(a)TypeI andtype ll B turnsaremost common;type I turnsoccur more than twice as frequentlyas type ll Typell B turnsusuallyhaveCly asthethirdresidueNotethehydrogen bond betweenthe peptidegroupsof the firstand fourthresidues of the bends(lndividual aminoacidresidues areframedby largebluecircles)

Type II

cis

(b) Proline isomers

(b)Transand cis isomers of a peptidebond involvingthe iminonitrogen of proline Of the peptidebondsbetweenamino acid residues For otherthan Pro,morethan 99.95%are in the transconfiguration. about peptidebondsinvolvingthe iminonitrogenof proline,however, manyoftheseoccuratB turns' 67oarein the cis configuration;

12')

T h eT h r e e - D i m e n sSi ot rnuaclt uor feP r o t e i n s

Collagen triple heli X

+180

+180

Left-handed a helix

r20 a o o

Right-handed a helix

-60

60

o

0

+

-60 -r20

120 - 180 - 180 (a)

- 180 - 180

0 (b)

d (degrees)

0

+180

d (degrees)

FIGURt4-8 Ramachandran plots showinga varietyof structures. (a)Thevaluesof @and rl for variousallowedsecondary structures are overlaidon the plot from Figure4-3. Althoughleft-handed a helices extendingover severalamino acid residuesare theoretically possible,theyhavenot beenobservedin proteins.(b)Thevaluesof @and

ry'forall the aminoacid residues exceptCly in the enzymepyruvate kinase(isolated from rabbit)areoverlaidon the plot of theoretically (Fig.a-3). The small,flexibleCly residues allowedconformations were excludedbecausethey frequentlyfail outsidethe expected (blue)ranges

structures(Fig. -1-tta).Mostvaluesof @and ry'taken from known protein structures fall into the expected regions,with high concentrationsnear the a helix and B conformationvalues as predicted (Fig. 4-8b). The only amino acid residueoften found in a conformation outsidetheseregionsis giycine.Becauseits sidechain

is small, a Gly residue can take part in many conformations that are stericallv forbidden for other amino acids

25

20 15

10

190

200

210

220

230

240

250

Wavelength (nm) FIGURE 4-9 Circulardichroismspectroscopy. Thesespectrashow polylysineentirelyas a helix,as p conformation, or as a denatured, randomcoil They axisunit is a simplifiedversionof the unitsmost commonlyusedin CD experiments Sincethe curvesaredifferent for a helix,B conformation, andrandomcoii,theCD spectrum for a given proteincan providea roughestimatefor the fractionof the protein madeup of the two mostcommonsecondary structures. TheCD spectrum of the nativeproteincan serveas a benchmarkfor the folded state,usefulfor monitoringdenaturation or conformational changes broughtaboutby changes in solutionconditions.

(ommon (anBeAssessed Secondary Structures by (ireular Dichroisnn Structural asyrnmetryin a moleculegives rise to differencesin absorptionof left-handedversusright-handed plane-polarized light. Measurementof this differenceis calledcircular dichroism (CD) spectroscopy.An orderedstructure,suchasa foldedprotein,givesriseto an absorption spectrum that can have peaks or regions with both positive and negativevalues.For proteins, spectraareobtainedin the far IIV region(190to 250nm). The light-absorbingentity, or chromophore,in this regionis the peptidebond;a signalis obtainedwhen the peptide bond is in a folded environment.The difference in moiar extinction coefflcients(see Box 3-1) for leftand right-handedplane-polarized light (Ae) is plottedas a function of waveiength.The a helix and B conformations have characteristicCD spectra(Fig. 4-9). Using CD spectra,biochemistscan determinewhether proteins are properly folded, estimatethe fraction of the proteinthat is foldedin eitherof the commonsecondary structures,and monitor transitionsbetweenthe folded and unfoldedstates.

S U M M A R4Y. 2 P r o t e iS n e c o n d aSr tyr u c t u r e r

Secondarystructure is the local spatialarrangement of the main-chainatomsin a selectedsegmentof a polypeptidechain.

andQuaternary structures Tertiary 4.3Protein Itrt--] r

The most conrmonregularsecondarystructuresare lhe a helix, the B conformation,and B turns.

r

The secondarystructureof a pollpeptide segment can be completelydefinedif the @and ry'angles are known for all amino acid residuesin that segment.

Globularproteins often contain severaltypes of secondary structure.The two groupsalsodiffer functionally:the structuresthat provide support,shape,and externalprotection to vertebrates are made of flbrous proteins, whereasmost enz;nnesand regulatoryproteins are globular proteins.

r

Circulardichroismspectroscopy is a methodfor assesshgcorunon secondarystructure and monitoringfolding in proteins.

fora Proteins AreAdapted Fibrous Function Structural

4.3 Protein Tertiary andQuaternary Structures Protein Architecture-lntroduction to Tertiary Structure The overall three-dimensional

arrangement

of all atoms in a

protein is referred to as the protein's tertiary structure. Whereasthe term "secondarystructure" refers to the spatial arrangementof amino acid residuesthat are adjacent in a segment of a polypeptide, tertiary structure includes longer-range aspectsof amino acid sequence.Amino acidsthat are far apart in the polypeptide sequenceand are in different t),?esof secondary structure may interact within the completely folded structureof a protein.The locationof bends(including B turns) in the polypeptide chain and the direction and angleof thesebendsare determinedby the numberand Iocation of specificbend-producingresidues,such as Pro, Thr, Ser,and GIy.Interacting segmentsof polrueptide chains are held in their characteristic tertiary positions by severalkinds of weak interactions (and sometimesby covalentbonds such as disulfldecrosslinks) betweenthe segments. Some proteins contain two or more separate polypeptidechains,or subunits,which may be identical or different. The arrangementof these protein subunits in three-dimensionalcomplexesconstitutes quaternary structure. In consideringthese higher levels of structure, it is usefulto classi$'proteinsinto two major groups:fibrous proteins, with polypeptide chains arranged in long strandsor sheets,and globular proteins, with polypeptide chainsfolded into a sphericalor globularshape.The two groups are structurally distinct. Fibrous proteins usuallyconsistlargelyof a singletype of secondarystructure, and their tertiary structure is relatively simple.

I

ProteinArchitecture-TertiaryStructureof Fibrous Proteins

a-Keratin, collagen,and silk flbroin nicely illustrate the relationshipbetween protein structure and biological function (Table 4-2). Fibrous proteins shareproperties that give strength and./orflexibility to the structures in which they occur. In each case,the fundamentalstructural unit is a simple repeating element of secondary structure. All fibrous proteins are insoluble in water, a property conferred by a high concentration of hydrophobicaminoacid residuesboth in the interior of the protein and on its surface.These hydrophobic surfaces are largely buried as many similar polypeptide chains are packed together to form elaborate supramolecular complexes.The underlying structural simplicity of fibrous proteins makesthem particularly useful for illustrating some of the fundamental principles of protein structurediscussedabove. a-Keratin The a-keratins have evolved for strength. Found only in mammals, these proteins constitute almost the entire dry weight of hair, wool, nails, claws, quills, horns, hooves,and much of the outer layer of skin. The a-keratins are part of a broader family of proteins called intermediate f,lament (IF) proteins. Other IF proteins are found in the cytoskeletonsof animal cells.All IF proteinshavea structural function and share the structural featuresexemplifiedby the a-keratins. The o-keratin helix is a right-handed a helix, the samehelix found in many other proteins. Francis Crick and Linus Paulingin the early 1950sindependentlysuggestedthat the a helicesof keratin were arrangedas a coiled coil. Tlro strandsof a-keratin, oriented in parallel (with their aminotermini at the sameend), are wrapped about each other to form a supertwistedcoiled coil. The supertwisting amplifles the strength of the overall structure, just as strands are twisted to make a strong

Structure

Cha,racteristics

of occurrence Exa,mples

a Heiix, cross-linked by disulflde bonds

Tough,insolubleprotective structures of varying hardnessand flexibility

a-Keratin of hair, feathers,and nails

B Conformation

Soft, flexible fllaments

Silk flbroin

Collagen triple helix

High tensile strength,without stretch

Collagenof tendons,bone matrix

'124 The Three-Dimensional Structure | ofProteins rope (Fig. 4-f 0). The twisting of the axis of an a helix to form a coiled coil explainsthe discrepancybetween the 5.4 A per turn predicted for an a helix by Pauling and Coreyand the 5.15to 5.2A repeatingstructureobservedin the x-ray diffraction of hair (p. 117). The helical path of the supertwists is left-handed, opposite in senseto the a helix, The surfaceswhere the two a helices touch are made up of hydrophobic amino acid residues,their R groupsmeshedtogetherin a regularinterlocking pattern. This permits a close packing of the pol;peptide chains within the left-handed supertwist. Not surprisingly, @-keratinis rich in the hydrophobic residuesAla, Val,Leu, Ile, Met, and Phe. Keratin a helix _ T\'yo-chain coiled coil

Protofrlament {

]2oio

_f1 Protofrbrill

A

I

lr (a)

An individual polypeptidein the a-keratin coiled coil has a relatively simple tertiary structure, dominated by an a-helicalsecondarystructurewith its helical axis twisted in a left-handed superhelix. The intertwining of the two a-helicalpolypeptidesis an exampleof quaternarystructure.Coiledcoils of this type are common structural elementsin filamentousproteins and in the muscleprotein myosin (seeFig. 5-27). The quaternary structure of a-keratin can be quite complex.Manycoiledcoilscan be assembledinto large supramolecularcomplexes,suchasthe arrangementof a-keratin to form the intermediate filament of hair (Fig. 4-10b). The strength of flbrous proteins is enhancedby covalent cross-linksbetween polypeptide chains in the multihelical "ropes" and between adjacent chains in a supramolecularassembly.In a-keratins,the cross-links stabilizing quaternary structure are disulfide bonds (Box 4-2).In the hardestand toughesta-keratins,such as those of rhinoceroshorn, up to 18% of the residues are cysteinesinvolvedin disulfldebonds. Collagen Like the a-keratins, collagenhas evolvedto provide strength.It is found in connectivetissuesuch as tendons, cartilage,the organic matrix of bone, and the cornea of the eye. The collagenhelix is a unique secondarystructure (d : -51", r/ : +153") quite distinct from the a helix. It is left-handed and has three amino acid residuesper turn (Fig. 4-11). Collagenis also a

Cells Intermediate frlament Protofibril Protofrlament

TWo-chain coiled coil aHelix

(b) Cross section ofa hair FIGURE4-10 Structure of hair.(a)Haira-keratinisan elongated a helix with somewhat thickerelementsnearthe aminoand carboxyltermini. Pairsof thesehelicesare interwoundin a left-handed senseto formtwo-chaincoiledcoils Thesethencombinein higher-order structurescalled protofilaments and protofibrils.About four protofibrils32 strandsof a-keratinin all-combine to form an intermediate filament. Theindividualtwo-chaincoiledcoilsin the varioussubstructuresalsoseemto be interwound,but the handedness of the interwinding and otherstructuraldetailsare unknown.(b) A hair is an arrayof many a-keratinfilaments,made up of the substructures shown in (a).

(Derivedfrom FIGURE4-11 Structureofcollagen. PDBtD lCCD) (a)The a chainof collagenhasa repeating secondary structure uniqueto thisprotein.TherepeatingtripeptidesequenceCly-X-Proor Cly-X-4-Hyp adopts a left-handed helicalstructure with threeresidues perturn.Therepeating sequence usedto generate thismodelis Cly-Pro-4-Hyp.(b) Space-filling modelof the samea chain (c)Threeof thesehelices(shownherein gray, blue,and purple)wrap aroundone anotherwith a right-handed twist. (d)Thethree-stranded collagensuperhelix shownfromoneend,in a balland-stick representation. Cly residues areshownin red.Clycine,because of itssmallsize,is requiredat thetightjunctionwherethethreechainsare in contact.Theballsin thisillustration do not represent the vanderWaals radiiofthe individualatomsThecenterofthethree-stranded suoerhelix is not hollow,as it appears here,but verytightlypacked.

structures Tertiary andQuaternary 4.3Protein [i rf

Whenhair is exposedto moist heat,it can be stretched. At the molecularlevel, the a helicesin the a-keratin of hair are stretched out until they arrive at the fully extended B conformation.On cooling they spontaneously revert to the a-heLicalconformation.The characteristic "stretchability" of a-keratins, and their numerousdisulflde cross-linkages,are the basis of permanentwaving. The hair to be wavedor curled is first bent arounda form of appropriateshape.A solutionof a reducingagent,usually a compound containing a thiol or sulfhydryl group (-SH), is then applied with heat. The reducing agent cleavesthe cross-linkagesby reducing each disulfide bond to form two Cys residues.The moist heat breaks hydrogenbondsand causesthe a-helicalstructureofthe polypeptide chains to uncoil. After a time the reducing solution is removed,and an oxidizing agent is added to establish new disttlfide bonds between pairs of Cys residuesof adjacent polypeptide chains,but not the same pairs as before the treatment. After the hair is washed and cooled,the pol;,peptidechains revert to

coiled coil, but one with distinct tertiary and quaternary structures:three separatepollpeptides,calleda chains (not to be confusedwith a helices),are supertwisted abouteachother (Fig. 4-1lc). The superhelicaltwisting is right-handedin collagen,oppositein senseto the lefthandedhelix of the a chains. There are many types ofvertebrate collagen.TVpically they contain about 35% GIy, 11% AIa, and2lo/o Pro and 4-Hyp (4-hydroxyproline, an uncommon amino acid; seeFig. 3-8a). The food product gelatinis derived from collagen;it has little nutritional value as a protein, becausecollagenis extremely low in many amino acids that are essentialin the human diet. The unusual amino acid content of collagenis related to structural constraints unique to the collagen helix. The amino acid sequencein collagenis generallya repeatingtripeptide unit, Gly-X-! where X is often Pro, and Y is often 4-Hyp. Only Gly residuescan be accommodatedat the very tight junctions betweenthe individual a chains (FiC. 4-11d). The Pro and 4-Hyp residuespermit the sharp twisting of the collagenhelix. The amino acid sequenceand the supertwisted quaternary structure of collagen allow a very close packing of its three polypeptides. 4-Hydroxyproline has a specialrole in the structure of collagen-and in human history (Box 4-3). The tight rvrapping of the a chains in the collagen triple helix provides tensile strength greater than that of a steel wire of equal cross section.Collagenflbrils (Fig. 4-f2) are supramolecularassembliesconsisting of triple-helicalcollagenmolecules(sometimesreferred in a varietyof to as tropocollagenmolecules)associated ways to provide different degreesof tensile strength.

lheir o-helical conformation.The hair flbers now curl in the desired fashion becausethe new disulfide crosstinkagesexert sometorsion or twist on the bundlesof ahelical coils in the hair fibers. The sameprocesscan be used to straightenhair that is naturally curly. A permanent wave (or hair straightening)is not truly permanent, becausethe hair grows;in the new hair replacingthe old, the c-keratin has the natural pattern of disulfidebonds.

I{Si

reduce

ssl

oxidize

HS -i HSHS _I -^"1

I{SJ

Heads ofcollagen molecules

Cross-striations 640A (64nm)

Section of collagen FIGURE4-12Structureof collagenfibrils. Collagen(M, 300,000)is a molecule,about3,000A longandonly 15 A thick.ltsthree rod-shaped eachchain helicallyintertwineda chainsmayhavedifferentsequences; made up of fibrils are Collagen hasabout1,000aminoacid residues. for fashion and cross-linked in a staggered collagenmoleculesaligned with vary cross-linking degree of and alignment The specific strength. in an electronmicross-striations the tissueand producecharacteristic crograph.In the exampleshownhere,alignmentof the headgroupsof everyfourthmoleculeproducesstriations640A 64 nm)apart.

F'C

T h eT h r e e - D i m e n sSi ot rnuaclt uor feP r o t e i n s

. . . from this misfortune, together with the unhealthiness of the country where there never falls a drop of rain, we were stricken with the "camp-sickness,"which was suchthat the flesh of our limbs all shrivelledup, and the skin of our legs became all blotched with black, mouldy patches, Iike an old jack-boot, and proud flesh caJneupon the gums of those of us who had the sickness,and none escapedfrom this sicknesssavethrough the jaws of death.The signalwasthis: when the nosebegan to bleed, then death was at hand . . . -The Memoirsof the Lordof Joinville,ca. 1300 This excerpt describesthe plight of Louis IX's army toward the end of the Seventh Crusade (1248-1254), when the scurvy-weakened Crusader army was destroyed by the Eg;,ptians.What was the nature of the maladyafflicting thesethirteenth-century soldiers? Scurvy is causedby lack of vitamin C, or ascorbic acid (ascorbate).Vitamin C is required for, amongother things, the hydroxylation of proline and lysine in collagen; scurvy is a deficiency diseasecharacterizedby general degeneration of connective tissue. Manifestations of advancedscurvy include numerous small hemorrhagescausedby fragile blood vessels,tooth loss, poor wound healing and the reopening of old wounds, bone pain and degeneration,and eventually heart failure. Milder casesof vitamin C deficiency are accompanied by fatigue, irritability, and an increasedseverity of respiratory tract infections. Most animals make large amounts of vitamin C, converting glucose to ascorbate in four enz;rmaticsteps.But in the course of evolution, humans and some other animals-gorillas, guineapigs, and fruit bats-have lost the last enzyrmein this pathway and must obtain ascorbatein their diet. Vitamin C is availablein a wide range of fruits and vegetables.Until 1800,however,it was often absentin the dried foods and other food supplies stored for winter or for extended travel. Scurvywas recordedby the Egptians in 1b00scp, and it is describedin the fifth century nco writings of Hippocrates.Yet it did not come to wide public notice until the Europeanvoyagesof discoveryfrom 1b00to 1800.The first circumnavigationof the globe, led by Ferdinand Magellan (1520), was accomplishedonly with the loss of more than 80%oof his crew to scurvy. During Jacques Cartier's second voyage to explore the St. LawrenceRiver (1535-1536),his band was threatened with complete disaster until the native Americans taught the men to make a cedar tea that cured and prevented scurvy (it containedvitamin C). Mnter outbreaksof scurvy in Europe were gradually

eliminated in the nineteenth century as the cultivation of the potato,introducedfrom SouthAmerica,became widespread. ln 1747,JamesLind, a Scottishsurgeonin the Royal Navy, carried out the first controlled clinical study in recordedhistory.Dufing an extendedvoyageon the 50gun warshipHMS Sali,sbury,Lnd selected12 sailorssuffering from scurvy and separatedthem into groups of two. AII 12receivedthe samediet, exceptthat eachgroup was given a different remedy for scurvy from among those recommendedat the time. The sailorsgiven lemons and orangesrecoveredand returned to duty. The sailors given boiled apple juice improvedslightly.The remainder continuedto deteriorate. Lurd'sIYeatzseon th,eScurDAwas published in 1753, but inaction persistedin the RoyaiNavyfor another 40 years.In 1795the British admiralty finally mandated a ra- JamesLind,1716-1794; tion of concentratedlime or lemon navarsurgeon/ juice for all British sailors (hence 1739-1748 the name"Iimeys").Scurvycontinuedto be a problemin someother parts of the world until 1932,when Hungarian scientistAlbert Szent-Gydrgyi, and W.A. Waughand C. G. King at the University of Pittsburgh, isolated and synthesizedascorbicacid. t -Ascorbic acid (vitamin C) is a white, odorless, crystallinepowder.It is freely solublein water and relatively insolublein organic solvents.In a dry state, away from light, it is stable for a considerablelength of time. The appropriate daily intake of this vitamin is still in dispute. The recommended daily allowance in the United States is 60 mg (Australia and the United Kingdom recommend30 to 40 mg; Russiarecommends 100 mg). Along with citrus fruits and almost all other fresh fruits, other good sources of vitamin C include peppers,tomatoes,potatoes,and broccoli.The vitamin C of fruits and vegetablesis destroyedby overcookingor prolongedstorage. Sowhy is ascorbateso necessaryto good health?Of particular interest to us here is its role in the formation of collagen.As noted in the text, collagenis constructed of the repeatingtripeptide unit Gly-X-Y, where X and Y are generallyPro or 4-Hyp-the proline derivative (48)t -hydroxy'proline,which plays an essentialrole in the folding of collagenand in maintainingits structure. The proline ring is normally found as a mixture of two puckered conformations,calledC"-endoand C;exo (Flg, 1). The collagenhelix structure requires the Pro residuein

3r4

andQuaternary Structures 4,3 Protein Tertiary

In the normalpro$ 4-hydroxylasereaction(Fig.2a), one molecule of a-ketoglutarateand one of 02 bind to the enz}rme.The a-ketoglutarate is oxidatively decarboxylated to form CO2 and succinate. The remaining oxygenatom is then usedto hydroxylate an appropriate Pro residue in procollagen.No ascorbateis needed in this reaction. Howevet, prolyl 4-hydroxylasealso catalyzesan oxidative decarboxylationof a-keto$utarate that is not coupled to proline hydroxylation (Fig. 2b). During this reaction the heme Fe2+ becomesoxidized, inactivating the enzynneand preventing the proline hydroxylation. The ascorbateconsumedin the reaction is needed to restore enzyme activity-by reducing the heme iron. Scuny remainsa problem today,not only in remote regions where nutritious food is scarce but, surprisingly, on U.S. collegecampuses.The only vegetables consumedby somestudentsare thosein tossedsalads, and days go by without these young adults consuming fruit. A 1998study of 230 studentsat ArizonaStateUni versity revealed that 10% had serious vitamin C deflciencies,and 2 studentshad vitamin C levels so low that they probably had scurvy.Only half the students in the study consumedthe recommendeddaily allowance of vitamin C. Eat your fresh fruit and vegetables.

FIGURE 1 The C"-endo conformation of proline and the Cr-exo conformationof 4-hydroxyproline.

the Y positionsto be in the Crrexo conformation,and it is this conformation that is enforced by the hydroxyl substitution at C-4 in 4-Hyp. The collagenstructure also requiresthat the Pro residuein the X positionshavethe C"-endo conformation,and introduction of 4-Hyp here can destabilizethe helix. In the absenceof vitamin C, cells carnot hydroxylate the Pro at the Y positions.This leads to collagen instability and the cormectivetissue problemsseenin scuny. The hydroxylationof speciflcPro residuesin procollagen, the precursor of collagen,requires the action of the enzyme prolyl 4-hydroxylase. This enzyrne (M, 240,000)is an a2B2tetramer in all vertebratesources. The proline-hydroxylatingactivity is found in the o subunits. Each o subunit contains one atom of nonheme iron (Fe2+),and the enzlme is one of a classof hydroxylases that require a-ketoglutarate in their reactions.

(a) |

o:c

lHo HC-C-

It /

tcn"

N-C

lu" II

Pro residue

cooH I CHo t-

+ CH"

l'

I

o:c

I

cooH I

HC+O,

Fe2*

|

-

c:o I

COOH o-Ketoglutarate

+ CHo

| N_

tCHo t-

II

I 4-Hyp residue

+ COo

COOH Succinate H2COH HCOH

COOH

I

CH,

tt-

+ COo

CHo

I

COOH a-Ketoglutarate

HO OH Ascorbate

COOH Succinate

*

| ,or

".ic-c).:o llll

oo Dehydroascorbate

(a) The normal reaction,coupledto prolinehyFIGURE 2 Reactionscatalyzedby prolyl 4-hydroxylase. Thefateof the two oxygenatomsfrom 02 is shownin red' droxylation,which doesnot requireascorbate. without hydroxylation (b) The uncoupledreaction,in which a-ketoglutarate is oxidativelydecarboxylated in this processas it is convertedto dehydroascorbate. of proline.Ascorbateis consumedstoichiometrically

Three-Dimensional Structure ofProteins -128) The The a chains of collagenmoleculesand the collagen moleculesof f,brils are cross-linkedby unusualt;pes of covalent bonds involr,rngLys, Hylys (5-hydroxylysine; seeFig. 3-8a), or His residuesthat are presentat a few of the X and Y positions.Theselinks createuncofirmon aminoacid residuessuch as dehydrohydroxylysinonorleucine.The increasinglyrigid and brittle characterof agingconrLective tissueresultsfrom accumulatedcovalent cross-linksin collagenflbrils. H-N:

tN-H

\_-/---

^ ^/CH-CH2-CH2-CH2-CH:N-CH: o:c...

Polypeptide chain

Lys residue minus e-amino group (norleucine)

CH

CH,

CHr-CH

oH Hylys residue

dromeis characterizedby loosejoints. Both conditions can be lethal, and both result from the substitutionof an amino acid residuewith a larger R group (such as Cys or Ser) for a singleGly residuein eacha chain (a different GIy residue in each disorder). These singleresiduesubstitutionshavea catastrophiceffect on colIagen function because they disrupt the Gly-X-Y repeat that givescollagenits unique helical structure. Givenits role in the collagentriple helix (Fig. 4-1ld), Gly cannotbe replacedby another amino acid residue without substantial deleterious effects on collagen structure. I

,."-o Polypeptide chain

Dehydrohydroxylysinonorleucine

A typical mammalhas more than 30 structural variantsof collagen,particularto certaintissues and each somewhatdifferent in sequenceand function. Somehumangeneticdefectsin collagenstructure illustratethe closerelationshipbetweenaminoacid sequence and three-dimensionalstructure in this protein. Osteogenesisimperfecta is characterizedby abnormalbone formationin babies;Ehlers-Danlossyn-

Silk Fibroin Fibroin,the protein of silk,is producedby insectsand spiders.Its polypeptidechainsare predominantly in the B conformation.Fibroin is rich in Ala and GIy residues,permittinga closepackingof B sheetsand an interlockingarrangementof R groups(l'ig. 4-13). The overall structure is stabilizedby extensivehydrogenbonding betweenall peptidelinkagesin the polypeptidesof eachB sheetandby the optimizationof van der Waalsinteractions betweensheets.Silk doesnot stretch,becausethe B conformationis alreadyhighlyextended(Fig. 4-6). However, the structure is flexible becausethe sheetsare held togetherby numerousweakinteractionsrather than by covalentbondssuchasthe disulfrdebondsin a-keratins.

Antiparallel

Ala side chai

Gly side chains

(b)

FIGURE4-13 Structureof silk.Thefibersin silkclothand in a spider web are madeup of the proteinfibroin.(a) Fibroinconsistsof layersof antiparallel rich in Ala andCly residues. Thesmallsidechains B sheets interdigitate andallowclosepackingof thesheets, asshownin theball andstickview.(b) Strands of fibroin(blue)emergefromthe spinnerets of a spiderin thiscolorizedelectronmicrograph. r--/u llm

Structures[rrr] Tertiary andQuaternary 4.3 Protein

The number of known three-dimensionalprotein structures is now in the tens of thousands and more than doubles every couple of years. This wealth of information is revolutionizingour understandingof protein structure, the relation of structure to function, and the evolutionary paths by which proteins arrived at their present state, which can be seen in the family resemblances that come to light as protein databasesare sifted and sorted. One of the most important resources available to biochemists is the Protein Data Bank (PDB; www.rcsb.org). The PDB is an archive of experimentally determined three-dimensionalstructures of biological macromolecules,containingvirtually all of the macromolecular structures (proteins, RNAs, DNAs, etc.) elucidatedto date. Each structure is assignedan identifying label (a four-characteridentifier called the PDB

ID). Suchlabelsare providedin the figure legendsfor every PDB-derived structure illustrated in this text so that students and instructors can explore the same structures on their own. The data files in the PDB describethe spatial coordinatesof each atom whose position has been determined (many of the cataloged structures are not complete). Additional data files provide information on how the structure was determined and its accuracy.The atomic coordinatescan be convertedinto an image of the macromoleculeusing structure visualizationsoftware.Studentsare encouraged to accessthe PDB and explore structures using visualization software linked to the database.Macromolecularstructure files can also be downloadedand explored on the desktop using free software such as RasMol,ProteinExplorer,or FirstGlancein Jmol,availableat wwwumass.edu/microbio/rasmol.

Functional Diversity in Structural Diversity Reflects Proteins Globular

protein substructureand comparativecategorization. Such discussionsare possibleonly becauseof the vast amount of information availableover the Internet from publicly accessibledatabases,particularly the Protein DataBank (Box 4-4).

In a globularprotein, different segmentsof the pollpeptide chain (or multiple polypeptidechains) fold back on each other, generatinga more compactshapethan is seen in the fibrous proteins (Fig. 4-14). The folding also providesthe structural diversity necessaryfor proteins to carry out a wide array of biological functions. Globularproteinsinclude enzlrnes,transport proteins, motor proteins,regulatoryproteins,immunoglobulins, and proteins with many other functions. Our discussionof globular proteins beginswith the principlesgleanedfrom the first protein structuresto be elucidated.This is followed bv a detailed description of

B Conformgtion 2,000x 5 A

a Helix

gooxtrA

ffi Native glolular form 1 0 0x 6 0 4 are compactand varied. 4-14 Clobularproteinstructures FIGURE in a singlechain. Humanserumalbumin(M,64,500)has585 residues Civen here are the approximatedimensionsits singlepolypeptide or chainwouldhaveif it occurredentirelyin extended B conformation asan a helix.Alsoshownis the sizeof the proteinin its nativeglobuthe polypeptidechain larform,asdeterminedby x-raycrystallography; mustbe very compactlyfoldedto fit into thesedimensions.

of theComplexity about Early Clues Provided Myoglobin Structure Protein 6lobular Protein Architecture-Tertiary Structure of Small Globular teins, ll. Myoglobin The first breakthrough in under-

standing the three-dimensionalstructure of a globular protein camefrom x-ray diffraction studiesof myoglobin carried out by John Kendrew and his colleaguesin the 1950s. Myoglobin is a relatively small (M, 16,700), oxygen-bindingprotein of musclecells.It functionsboth to store oxygenand to facilitate oxygendiffusion in rapidly contractingmuscletissue.Myoglobincontainsa single polypeptide chain of 153 amino acid residues of known sequenceand a single iron protoporphyrin, or heme, group. The sameheme group that is found in myoglobin is found in hemoglobin,the oxygen-binding protein of erythrocytes,and is responsiblefor the deep red-brown color of both myoglobin and hemoglobin. Myoglobinis particularly abundantin the musclesof diving mammalssuch as the whale, seal,and porpoise-so abundantthat the musclesof these animalsare brown. Storageand distribution of oxygenby musclemyoglobin permits di\,mg mammalsto remain submergedfor long periods.The activities of myoglobinand other globin moleculesare investigatedin greaterdetail in Chapter5. Figure 4-15 shows severalstructural representations of myoglobin,illustrating how the polypeptide chain is folded in three dimensions-its tertiary structure. The red group surroundedby proteinis heme.The

["']

T h eT h r e e - D i m e n sSi ot rnuaclt uor feP r o t e i n s

FIGURE 4-15 Tertiarystructureof spermwhale myoglobin.(pDB tD IMBO) Orientationof the proteinis similarin (a) through(d); the hemegroupis shownin red.In additionto illustrating the myoglobin structure, this figureprovidesexamplesof severaldifferentwaysto display proteinstructure.(a) The polypeptidebackbonein a ribbon representation of a type introducedby jane Richardson, which highlightsregionsof secondarystructure. Thea-helicalregionsareevident.

backbone of the myoglobin molecule consists of eight relatively straight segmentsof a helix interrupted by bends,someof which are B turns. The longesto helix has 23 amino acid residuesand the shortest only Z; all helicesare right-handed.MorethanT0o/o of the residues in myoglobinare in these a-helical regions.X-ray analysis has revealed the precise position of each of the R groups, which occupy nearly all the spacewithin the folded chain. Many important conclusionswere drawn from the structure of myoglobin. The positioning of amino acid side chainsreflects a structure that derivesmuch of its stability from hydrophobicinteractions.Most of the hydrophobic R groups are in the interior of the molecule, hidden from exposureto water. All but two of the polar R groupsare locatedon the outer surfaceof the molecule, and all are hydrated. The myoglobinmoleculeis so compact that its interior has room for only four molecules ofwater. This densehydrophobiccore is typical of globular proteins. The fraction of space occupied by atoms in an organicliquid is 0.4 to 0.6. In a globular protein the fractionis about 0.75,comparableto that in a crystal(in a typical crystalthe fractionis 0.20to 0.28, near the theoretical maximum). In this packedenvironment, weak interactions strengthen and reinforce each other. For example,the nonpolarside chainsin the core are so closetogether that short-rangevan der Waalsinteractions make a signiflcant contribution to stabilizing hydrophobicinteractions. Deduction of the structure of myoglobin con_flrmed someexpectationsand introducedsomenew elements of secondarystructure. As predicted by pauling and Corey,all the peptide bonds are in the planar trans conflguration. The a helicesin myoglobinprovided the first direct experimental evidencefor the existence of this type of secondarystructure. Three of the four pro residuesare found at bends.The fourth pro residueoccurs within an a helix, where it createsa kink necessary for tight helix packing.

(b) Surfacecontourimage;this is usefulfor visualizingpocketsin the proteinwhereother moleculesmight bind. (c) Ribbonrepresentation includingsidechains(blue)for the hydrophobic residues Leu,lle,Val, and Phe.(d) Space-filling modelwith all aminoacidsidechains.Each atom is representedby a sphereencompassingits van der Waals radius.The hydrophobicresiduesare againshown in blue; most are buriedin the interiorof the proteinand thusnotvisible.

The flat heme group rests in a crevice,or pocket, in the myoglobinmolecule.The iron atom in the center of the heme group has two bonding (coordination) positions perpendicular to the plane of the heme (Fig. 4-f6). One of these is bound to the R group of the His residueat position 93; the other is the site at which an 02 molecule binds. Within this pocket, the accessibilityof the heme group to solvent is highly restricted.This is important for function, becausefree heme groups in an oxygenatedsolution are rapidly oxidizedfrom the ferrous (Fe'*) form, which is active in the reversible binding of 02, to the ferric (Fe3+) form, which doesnot bind Oz.

(b)

02

FIGURE 4-16 Thehemegroup.Thisgroupis presentin myoglobin,hemoglobin,cytochromes, and manyotherproteins(thehemeproteins). (a) Hemeconsistsof a complexorganicring structure,protoporphyrin, which bindsan iron atom in its ferrous(Fe2*)state.The iron atom has six coordinationbonds,four in the plane of, and bondedto, the flat porphyrinmoleculeandtwo perpendicular to it. (b) In myoglobinand hemoglobin, oneof the perpendicular coordination bondsis boundto a nitrogenatomof a His residue.Theother is ,,open,,and servesasthe bindingsitefor an 02 molecule.

31 Structures andQuaternary Tertiary 4.3Protein Ll As many different myoglobin structures were resolved,investigatorswere able to observethe structural changesthat accompanythe binding of oxygenor other moleculesand thus, for the first time, to understandthe correlation between protein structure and function. Hundreds of proteins have now been subjectedto similar analysis.Today,nuclear magneticresonance(NMR) spectroscopyand other techniquessupplementx-ray diffraction data, providing more information on a protein's structure (Box 4-5, p. 132). In addition,the sequencing of the genomic DNA of many organisms (Chapter 9) has identified thousandsof genesthat encode proteins of known sequencebut, as yet, unknown function; this work continuesapace.

of Proteins Have aVariety Globular Structures Tertiary From what we now know aboutthe tertiary structuresof hundreds of globularprotehs, it is clear that myoglobin illustratesjust one of many waysin which a polypeptide chain can be folded. Table4-3 showsthe proportions of o helix and B conformations(expressedas percentage of residuesin each type) in severalsmall, single-chain, globular proteins. Each of these proteins has a distinct structure, adaptedfor its particular biologicalfurction, but together they share severalimportant properties with myoglobin.Each is folded compactly,and in each case the hydrophobic amino acid side chains are orientedtoward the interior (awayfrom water) and the hydrophilic side chainsare on the surface.The structures are alsostabilizedby a multitude of hydrogenbonds and someionic interactions. For the beginning student, the very complex tertiary structures of globular proteins-some much larger than myoglobin-are best approachedby focusing on corunon structural patterns, recurring in different and

often unrelated proteins. The three-dimensionalstructure of a typical globular protein can be consideredan assemblageof polypeptidesegmentsin the a-helicaland p-sheet conformations,linked by connectingsegments. The structure can then be defined by how these segments stack on one another and how the segmentsthat connectthem are arranged. To understanda completethree-dimensionalstructure, we need to analyzeits folding patterns. We begin by defining two important terms that describe protein structural patterns or elementsin a polypeptide chain, and then turn to the folding rules. The first term is motif, also called a superseeondary structure or fold. A motif is simply a recognizable folding pattern involving two or more elements of secondarystructureand the connection(s)between them. Although there is some confusing application of these three terms in the literature, they are generally used interchangeably.A motif can be very simple, such as two elements of secondarystructure folded against eachother, and represent only a smallpart of a protein. An exampleis a B-a-B loop (Fig. 4-l7a). A motif can also be a very elaborate structure involving scores of protein segmentsfolded together, such as the B barrel (FiS. 4-17b). In some cases,a singlelarge motif may comprisethe entire protein. The term encompassesany advantageousfolding pattern and is useful for describing such patterns. The segmentdefined as a motif may or may not be independently stable. We have already encountered one well-studied motif, the coiled coil of a-keratin, which is also found in some other proteins. Note that a motif is not a hierarchical structural element falling between secondaryand tertiary structure' It is a folding pattern that can describea small part of a protein or an entire polypeptide chain. The synonymous term "supersecondarystructure" is thus somewhat misleadingbecauseit suggestshierarchy.

Residues(%)* Protein (totalresidues) Chymotrrpsin (247) Ribonuclease(124) Carboxypeptidase(307) Cytochromec (10a) Lysozyme(129) Myoglobin(153)

a Helix

B Conformation

T4 26 38 39 40 78

45 .JO

T7 0 12 0

Patll: Chemistty, PR.(1980) Biophysical C.R.& Schimmel, Source:DatafromCantor, p. 100,w. H.Freeman andCompany, TheConfomationof Biolo*icalMacromolecules, NewYork *Portionsof the polypeptide for by a helixor B conformation chainsnotaccounted of d helixand stretches. Segments coiledor extended of bendsandirregularly consist andgeometry deviateslightlyfromtheirnormaldimensions sometimes B conformation

(a)

B-a-BLoop

(b)

B Barrel

4-17 Motifs. (a) A simplemotii the B-a-B loop' (b) A more FIGURE elaboratemotif, the F barrel.This B barrel is a singledomain of a-hemolysin(a toxin that kills a cell by creatinga hole in its aurcus (derived membrane)from the bacteriumStaphylococcus from PDBlD TAHL).

F t{

The Three-Dimensional 5tructure ofproreins

(a)

X-RayDiftaction The spacingof atoms in a crystal lattice can be deter_ mined by measuringthe locationsand intensitiesof spots produced on photographicfllrn by a beam of x rays of given wavelength,after the beamhas been diffracted by the electronsof the atoms.For example,x-ray analysisof sodiumchloridecrystalsshowsthat Na+ and Cl- ions are arrangedin a simplecubic lattice. The spacingof the dif_ ferent kinds of atomsin complexorganicmolecules,even very large onessuchas proteins,can alsobe analyzedby x-ray diffractionmethods.However,the techniquefor an_ alyzingcrystalsof complexmoleculesis far more labori_ ous than for simple salt crystals. When the repeating pattem of the crystalis a moleculeas large as,say,a pro_ tein, the nurnerousatomsin the moleculefleld thousands of diffraction spots that must be analyzed, by computer. Consider how images are generated in a light mi_ croscope.Light from a point sourceis focusedon an ob_ ject. The object scatters the light waves, and these scatteredwavesare recombinedby a seriesof lensesto generatean enlargedimage of the object. The smallest object whose structure can be determinedby such a system-that is, the resolving power of the micro_ scope-is determined by the wavelengthof the light, in this casevisible light, with wavelengthsin the range of 400 to 700 nm. Objects smaller than half the wave_ length of the incident light cannot be resolved.To re_ solve objectsas small as proteinswe must use x rays, with wavelengthsin the range of 0.2 to l.b A (0.02 to 0.15nm). However,there are no lensesthat can recom_ bine x rays to form an image; instead, the pattern of diffracted x rays is collected directly and an imageis re_ constructedby mathematicaltechniques. The amount of information obtained from x_ray crystallographydependson the degreeof structural or_ der in the sample.Some important structural parame_

ters were obtainedfrom early studies of the diffraction patterns of the flbrous proteins arrangedin regular arrays in hair and wool. However, the orderly bundles formed by flbrous proteins are not crystals-the molecules are alignedside by side,but not all are oriented in the same direction. More detailed three-dimensional structural information about proteins requires a higlLly orderedprotein crystal.The structuresof many proteins are not yet known, simply becausethey haveproved difficult to crystallize.Practitionershavecomparedmaking protein crystals to holding together a stack of bowling balls with cellophanetape. Operationally,there are severalstepsin x-ray structural analysis (Fig. 1). A crystal is placed in an x-ray beam between the x-ray source and a detector, and a regular array of spots called reflections is generated. The spots are createdby the diffracted x-ray beam,and each atom in a molecule makes a contribution to each spot. An electron-densitymap of the protein is recon_ structed from the overall diffraction pattern of spots by a mathematicaltechnique calleda Fourier transform. In effect, the computer acts as a "computationallens." A model for the structure is then built that is consistent with the electron-densitymap. John Kendrew found that the x-ray diffraction pattern of crystallinemyoglobin (isolatedfrom muscles of the spermwhale) is very complex,with nearly 25,000 reflections.Computer analysisof these reflectionstook place in stages. The resolution improved at each stage,until in 1959the positionsof virtually all the non_ hydrogen atoms in the protein had been determined. The amino acid sequenceof the protein, obtained by chemical analysis,was consistent with the molecular model.The structures of thousandsof proteins,many of them much more complex than myoglobin,have since been determinedto a similar level of resolution.

Structures andQuaternary Tertiary 4.3 Protein ["']

1 Stepsin determiningthe structureof spermwhale myogloFIGURE (a) X-raydiffractionpatternsaregenerated bin by x-raycrystallography. from a crystalof the protein.(b) Data extractedfrom the diffraction electron-density oatternsare used to calculatea three-dimensional map.The electrondensityof only part of the structure,the heme, is shown.(c) Regionsof greatestelectrondensityrevealthe locationof atomicnuclei,and this informationis usedto piecetogetherthe final structure.Here,the hemestructureis modeledinto itselectron-density map. (d) The completedstructureof spermwhale myoglobin,includingthe heme(PDBlD 2MBW).

(d)

The physical environmentin a crystal, of course,is not identical to that in solution or in a IMng cell. A crystal imposesa spaceand time averageon the structure deducedfrom its analysis,and x-ray diffraction studies provide little information aboutmolecularmotion within the protein. The conformation of proteins in a crystal could in principle also be affected by nonphysiological factors such as incidental protein-protein contacts within the crystal. However, when structures derived from the analysisof crystals are comparedwith structural information obtained by other means (such as NMR,as describedbelow), the crystal-derivedstructure almost always representsa functional conformation of the protein. X-ray crystallographycan be applied successfully to proteins too large to be structurally analyzed by NMR. Nuclea,rMagneticResonance An advantage of nuclear magnetic resonance (NMR) studies is that they are carried out on macromolecules in solution, whereasx-ray crystallographyis limited to moleculesthat can be crystallized.NMR can also illuminate the dl.namic side of protein structure, including conformational changes,protein folding, and interactions with other molecules. NMR is a manifestation of nuclear spin angular momentum, a quantum mechanicalproperty of atomic tH, ttC, ttN, tnq nuclei. Only certain atoms,including 31P, have the kind of nuclear spin that gives rise to and an NMR signal. Nuclear spin generates a magnetic dipole. Whena strong,staticmagneticfleld is appliedto a solution containing a single type of macromolecule,the magnetic dipoles are aligned in the field in one of two orientations, parallel (low energy) or antiparallel (high energy). A short (-10 p,s)puise of electromagneticenergr of suitablefrequency (the resonantfrequency,which is in

the radio frequencyrange) is applied at right anglesto the nuclei aligned in the magnetic fleld. Some energy is absorbedas nuclei switch to the high-energl state,and the absorption spectrum that results contains information about the identity of the nuclei and their immediate chemical environment. The data ftom many such experiments on a sampleare averaged,increasingthe signal-tonoise ratio, and an NMR spectrum such asthat in Figure 2 is generated. lH is particr-rlarlyimportant in NMR experiments becauseof its high sensitivity and natural abundance.For 'H macromolecules, NMRspectracanbecomequite com'H atoms, plicated.Even a smallprotein hashundredsof typically resulting in a one-dimensionalNMR spectrum too complex for analysis.Structural arLalysisof proteins became possible with the advent of two-dimensional NMRtechniques(Frg.3). Thesemethodsallow measurecouplirg of nuclear spinsin ment of distance-dependent nearbyatomsthrough space(the nuclearOverhausereffect Q.JOE),in a methoddubbedNOESY)or the coupling of nuclear spins in atoms cormectedby covalentbonds (total correlationspectroscopy,or TOCSY). (continued on nent Page)

10.0

8.0

6.0

4.0

2.0

0.0

-2.0

rH chemical shift (ppm) of a globinfrom a marine NMR spectrum 2 One-dimensional FIGURE blood worm. This proteinand spermwhale myoglobinare very close structuralanalogs,belongingto the sameproteinstructuralfamilyand function. sharingan oxygen-transport

["4

I h eT h r e e - D i m e n sSi ot rnuaclt uor feP r o t e i n s

Ttanslatinga two-dimensionalNMR spectruminto a complete three-dimensionalstructure can be a laborious process.The NOE signalsprovide someinformation about the distancesbetween individual atoms, but for these distanceconstraintsto be usefiil, the atomsgiving rise to each signal must be identified. Complementary TOCSYexperiments can help identify which NOE signals reflect atoms that are linked by covalent bonds. Certain patterns of NOE signalshave been associated with secondarystructuressuch as a helices.Moderngenetic engineering (Chapter 9) can be used to prepare proteins that contain the rare isotopes t3C or 15N.The new NMR signalsproduced by these atoms, and the coupling with lH signalsresulting from these substitutions, help in the assignmentof individual lH NOE signals. The process is also aided by a knowledge of the amino acid sequenceof the polypeptide. To generate a three-dimensionalstructure, researchersfeed the distanceconstraintsinto a com-

6.1 I

Y= N5 c)u

Y;

@

8.0

6.0 4.0 2.0 0.0 -2.0 lH chemicalshift (ppm)

puter along with known geometric constraints such as chirality, van der Waals radii, and bond lengths and angles. The computer generates a family of closely related structures that represent the range of conformations consistent with the NOE distance constraints(Fig. 3c). The uncertaintyin structures generated by NMR is in part a reflection of the molecular vibrations (known as breathing) within a protein structure in solution, discussedin more detail in Chapter 5. Normal experimental uncertainty can also play a role. Protein structuresdeterminedbyboth x-ray crystallography and NMR generallyagreewell In some cases, the preciselocationsof particr_rlar aminoacid side chains on the protein exterior are different, often becauseof effects related to the packing of adjacent protein moleculesin a crystal.The two techniquestogetherare at the heart of the rapid increasein the availabilityof structural information about the macromoleculesof living cells.

FIGURE 3 Useof wvo-dimensional NMRto generate a three-di mensional structureof a globin,the sameproteinusedto generate the data in Figure2. ThediagonaI in a two-dimensional NMRspectrumisequivalentto a one-dimensional spectrum.The off-diagonalpeaksare NOE signals generatedby close+angeinteractionsof 1H atomsthat maygeneratesignalsquite distantin the one-dimensional spectrum.Two such interactionsare identifiedin (a),and their identitiesareshownwith blue lines in (b) (PDBlD l VRF).Threelinesaredrawnfor interaction 2 betweena methylgroup in the proteinand a hydrogenon the heme.The methyl group rotatesrapidlysuchthat eachof its threehydrogens contributes equallyto the interaction andthe NMR signal.Suchinformationis used to determinethe completethree-dimensional structure(pDB lD IVRE), asin (c).Themultiplelinesshownfor the proteinbackbonein (c) representthe familyof structures consistent with the distanceconstraints in the NMR data.The structuralsimilaritywith myoglobin(Fig.1) is evident.Theproteinsareorientedin the sameway in both figures.

Frr]

Structures andQuaternary Tertiary 4.3 Protein

handedsense.This influencesboth the arrangement of B sheetsrelative to one another and the path of the polypeptidecormectionsbetweenthem. TWoparallelB strands,for example,must be cormectedby a crossoverstrand (Fig. 4-19b). In principle, this crossovercould have a right- or left-handedconformation,but in proteins it is almost alwaysright-handed.Right-handed connectionstend to be shorter than left-handed connectionsand tend to bend through smaller angles,making them easierto form. The twisting ofB sheetsalsoleadsto a characteristictwisting of the structure formed by many such segments together,as seenin the B barrel (4-17b) and twistedB sheet(Fig. -19c), which form the core of many larger structures.

FIGURE 4-18 Structuraldomainsin the polypeptidetroponinC. (PDB with musclehas proteinassociated lD 4TNC)This calcium-binding calcium-binding domains,indicatedin blueand purple. two separate

The secondterm for describingstructuralpatternsis domain. A domain, as defined by Jane Richardsonin 1981,is a part of a polypeptidechain that is independently stableor could undergomovementsas a singleentity with respectto the entire protein. Polypeptideswith more than a few hundred amino acid residuesoften fold into two or more domains,sometimeswith di-fferentfunctions. In many cases,a domain from a large protein will retain its native three-dimensionalstructure even when separated(for example,by proteolyticcleavage)from the remainder of the pollpeptide chain. In a protein with multiple domains,eachdomainmay appearas a distinct globularlobe (Fig. 4-18); more corunonly,extensive contactsbetweendomainsmakeindividualdomainshard to discern. Different domainsoften have distinct functions, such as the binding of small moleculesor interaction with other proteins.Smallproteinsusuallyhaveonly one domain(the domainzsthe protein). Foldingof polypeptidesis subjectto an arrayof physical and chemical constraints, and several rules have emergedfrom studiesof commonproteinfoldingpatterns. 1. Hydrophobicinteractionsmake a large contribution to the stability of protein structures.Burial of hydrophobicamino acid R groups so as to exclude water requires at least two layers of secondary structure.Simplemotifs,suchas the B-a-B loop (Fig. 4-17a), createtwo suchlayers. Z.

D.

A a.

Wherethey occurtogetherin a protein,a helices and B sheetsgenerallyare found in different structural layers.This is becausethe backboneof a polypeptide segmentin the B conformation (FiC.4-6) carnot readily hydrogen-bondto an a helix alignedwith it. Segmentsadjacentto eachother in the amino acid sequenceare usuallystackedadjacentto each other in the folded structure. Distant segmentsof a polypeptidemay cometogether in the tertiary structure,but this is not the norm. Connectionsbetweenconunonelementsof secondarystructure carmotcrossor form knots (Fig. 4-19a). The B conformationis most stablewhen the individual segmentsare twisted slightly in a right-

Following these rules, complex motifs can be built up from simpleones.For example,a seriesof B-a-B loops arrangedso that the B strands form a barrel creates a particularly stable and common motif, the alp bartel

(a)

Typicalconnections in an all-6 motif

(b) Right-handed connection between 6 strands

(c)

Crossover connection (not observed)

Left-handed connection between 6 strands (very rare)

Ttvisted 6 sheet

4-19 Stablefolding Patternsin proteins. (a) Connections FIGURE betweenB strandsin layeredB sheets'The strandshere are viewed from one end, with no twisting.Thick lines representconnectionsat the endsnearestthe viewer;thin linesare connectionsat the far ends Theconnectionsat a givenend (e'8.,nearthe viewer) of the B strands. twist in other.(b) Becauseof the right-handed one do not cross Leftright-handed' generally are strands between connections B strands, handedconnectionsmust traversesharperanglesand are harderto (a protein form. (c)ThistwistedB sheetis from a domainof photolyase that repairscertaintypesof DNA damage)lrom E. coli (derivedfrom PDB lD lDNP). Connectingloops havebeen removedso as to focus on the foldingof theB sheet'

["4

T h eT h r e e - D i m e n sSi ot rnuaclt uor feP r o t e i n s

l3-a-p Loop B-a-B

idea further, researihershave organizedthe complete contentsofprotein databasesaccordingto hierarchical Ievelsof structure.All of these databasesrely on data and information depositedin the Protein Data Bank. The Structural Classificationof Proteins (SCOP)databaseis a good exampleof this important trend in biochemistry. At the highest level of classification,the SCOP database(http ://scop.mrc-lmb. cam.ac.uk/scop) borrows a schemealready in common use, with four classesof protein structure: all c, all F, qlg (with a and B segmentsinterspersedor alternating),and c + p (with a and B regionssomewhatsegregated).Each class includes tens to hundreds of different folding arrangements (motifs), built up from increasingly identifiable substructures.Some of the substructure arrangementsare very common, others have been found in just one protein.Figure 4-21 showsa variety of motifs arrayed among the four classesof protein structure;this is just a minute sampleof the hundreds of known motifs. The number of folding patternsis not infinite, however. As the rate at which new protein structuresare elucidatedhas increased,the fraction of those structures containinga new motif has steadily declined. Fewer than 1,000 different folds or motifs may exist in all. Figure 4-21 also showshow proteins can be organizedbasedon the presenceof the various motifs. The top two levels of organization,class and fold, are purely structural. Below the fold level (see color key in Fig. 4-21), categorizationis basedon evolutionary relationships. Manyexamplesof recurringdomainor motif structures are available,and theserevealthat protein tertiary structure is more reliably conservedthan amino acid sequence.The comparisonof protein structures can thus provide much information about evolution.

alp Banel

FIGURE 4-20 Constructinglarge motifs from smallerones.The o/B barrelis a commonlyoccurringmotifconstructed from repetitions of the B-a-B loop motif fhis a/B barrelis a domainof pyruvatekinase(a glycolyticenzyme)from rabbit(derived from pDB lD l pKN).

(Fig. 4-20). In ttus structure,eachparallelB segment is attachedto its neighborby an a-helicalsegment.All connectionsare right-handed.The o,lpbarrelisfoundin many enzyrnes,often with a binding site (for a cofactor or substrate)in the form of a pocketnearone end of the barrel. Note that domainswith similar folding patterns are said to have the samemotif even though their constituent a helicesand B sheetsmay differ in length.

Protein Motifs AretheBasis forProtein Structura I Classifi cation Structureof LargeGlobularpro$ ProteinArchitecture-Tertiary teins, lV. StructuralClassificationof proteinsAs we have seen,

understandingthe complexitiesof tertiary structure is made easierby consideringsubstructures.Takingthis

:

1AO6

w Serum albumin ia:

t

Serum albumin Serum albumin Serum alburnin Hutnan (Homo sapiens)

I

lBCF

wFerritin-like Ferritinlike H

Ferritin Bacterioferritin (cpochrome 6, I Escherichia coli

FIGURE 4-21 Organizationof proteinsbasedon motifs. Shown herearejust a few of the hundredsof knownstablemotifs.Theyare divided into four classes:all a, all B, a/B, and a * p. Structural classification data from the SCOp(Structural Classification of pro_ teins)database(http://scop mrc-lmbcam.acuk/scop)are also pro_

,

lGAI

ffi a/a toroid

:1-

il

Six-hairpin glycosyltransferase Glucoamylase Glucoamylase Aspergillus awamori, variant x100

I

fi '=

lENH DNA,/RNA-binding 3-helical bundle Homeodomain-like Homeodomain engrailed Homeodomain Dro sophila melanogaster

vided (seethe color key).The PDB identifier(listedfirst for each structure)is the unique accessioncode given to each structure archivedin the ProteinData Bank (www.rcsb.org). fhe a/B bArrel (seeFig.4-2O)is anotherparticularlycommona/B motil. (Figurecontinueson facingpage.)

-

--l

Structures andQuaternary Tertiary 4.3 Protein | 137 |

I

lI'(A Single-stranded left -handed B helix Tlimeric LpxA-Iike enzymes UDPN-acetylglucosamine acyltransferase UDP N-acetylglucosamine acyltransferase Escherichia coli

lDEH NAD(Plbinding Rossmann-fold domains NAD(Plbinding Rossmann-fold domains AlcohoVglucosedehydrogenases, carboxyl-terminal domain Alcohol dehydrogenase Human (Ilonro sapiens)

PDB identifier Fold Superfamily Family Z Prctein I Species

lPEX Four-bladed B propeller Hemopexin-like domain Hemopexin-like domain '=n Collagenase-3 (MMP-13), carboxyl-terminal domain Hrman(Homo sapiens)

1CD8 ImmunoglobulinJike B sandwich Immunoglobulin V set domains (antibody variable domain-like)

cD8 Human(Homo sapiens)

I

lDUB ClpP/crotonase ClpP/crotonase Crotonase-like Enoyl-CoA hydratase (crotonase) Rat (Rat t us noruegicu s)

7

lSYN Thymidylate Thymidylate Thymidylate Thymidylate Escherichia

synthase/dCMP hydroxymethylase synthase/dCMP hydroxymethylase synthase/dCMP hydroxymethylase synthase coli

'll

Escherichia coli

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I h eT h r e e - D i m e n sSi ot rnuaclt uor feP r o t e i n s

Proteins with significant similarity in primary structure and/or with similar tertiary structure and function are saidto be in the sameprotein family. A strongevolutionary relationship is usually evident within a protein family. For example,the globin family has many different proteinswith both structuraland sequencesimilarity to myoglobin (as seen in the proteins used as examplesin Box 4-5 and in Chapterb). T\vo or more families with little similarity in amino acid sequence sometimesmake use of the samemajor structural motif and have functional similarities;these families are groupedas superfamilies. An evolutionaryrelationship amongfamiliesin a superfamilyis consideredprobable, even though time and functional distinctions-that is, different adaptivepressures-may have erasedmany of the telltale sequencerelationships.A protein family may be widespreadin all three domainsof cellularlife, the Bacteria,Archaea,and Eukarya,suggestinga very ancient origin. Other familiesmay be presentin only a smallgroup of organisms,indicatingthat the structure arose more recently. Tfacing the natural history of structural motifs, using structural classificationsin databasessuch as SCORprovidesa powerful complement to sequenceanalysesin tracing evolutionaryrelationships.The SCOPdatabaseis curated manually, with the objective of placing proteins in the correct evolutionary framework based on conserved structural features. Structural motifs become especiallyimportant in defining protein families and superfamilies.Improved classiflcationand comparisonsystemsfor proteins lead inevitably to the elucidation of new functional relationships. Given the central role of proteins in living systems,these structuralcomparisonscan help illuminate every aspectof biochemistry,from the evolutionof individual proteins to the evolutionaryhistory of complete metabolicpathways. Several online databasesand resourcescomplement the SCOP database for analysis of protein structure. The CATH (Class,Architecture, Topology, and HomologousSuperfamily)databasearrangesthe proteins in the PDB in a four-levelhierarchy.Other programs allow the user to input the structure of a protein of interest and then find all the proteins in the pDB that are structurally similar to this protein or somepart of it. These programs include VAST (Vector Alignment SearchTool), CE (CombinatorialExtensionof the Optimal Paths), and FSSP (Fold ClassificationBased on Structure-Structure Alignmentof proteins).

affect the interaction between subunits, causing large changesin the protein's activity in response to small changesin the concentrationof substrateor regulatory molecules(Chapter6). In other cases,separatesubunits take on separatebut related functions,such as catalysis and regulation. Some associations,such as the fibrous proteins consideredearlier in this chapter and the coat proteins of viruses, serve primarily structural roles. Somevery large protein assembliesare the site of complex, multistep reactions.For example,each ribosome, the site of protein synthesis,incorporatesdozens of protein subunitsalongwith a number of RNA molecules. A multisubunit protein is alsoreferred to as a multimer. A multimer with just a few subunitsis often called an oligomer. If a multimer has nonidentical subunits, the overall structure of the protein can be asy.mmetric and quite complicated.However,most multimers have identical subunits or repeating groups of nonidentical subunits, usually in symmetric arrangements.As noted in Chapter3, the repeatingstructural unit in such a multimeric protein, whether a single subunit or a group of subunits,is called a protomer. Greekletters are sometimes used to distinguish the individual subunits that makeup a protomer. The first oligomeric protein to have its threedimensional structure determined was hemoglobin (M, 64,500), which contains four polypeptide chains and four heme prosthetic groups, in which the iron atomsare in the ferrous (Fe'") state (Fig. 4-16). The protein portion, the globin, consistsof two a chains (141 residueseach) and two B chains(146 residues each). Note that in this case,cyand B do not refer to secondary structures. Because hemoglobin is four times as large as myoglobin, much more time and effort were requiredto solveits three-dimensional structure by x-ray analysis,flnally achievedby Max perutz, John Kendrew,and their colleaguesin 19b9.The subunits of hemoglobin are arranged in symmetric pairs (Fig. 4-22), eachpair havingone a and oneB subunit. Hemoglobin can therefore be described either as a tetramer or as a dimer of aB protomers.

Protein Structures Quaternary Range fromSimple Dimers tolarge(omplexes ii ProteinArchitecture-euaternary Structure Many proteins

have multiple polypeptide subunits (from two to hundreds) . The association of polypeptide chains can serve a variety of functions. Many multisubunit proteins have regulatory roles; the binding of small molecules may

Max Perutz,1914-2OO2 (lefr) JohnKendrew,t9j7-1997 (tisht)

4 . 3 P r o t eT i ne r t i aar yn dQ u a t e r n S a rt yr u c t u r e s

[tr{

FIGURE 4-22 Quaternarystructure of de(PDBlD 2HHB) X-raydifoxyhemoglobin. fraction analysis of deoxyhemoglobin (hemoglobinwithout oxygen molecules bound to the hemegroups)showshow the four polypeptidesubunitsare packedto(b)A surgether.(a)A ribbonrepresentation. face contour model. The a subunitsare shown in shadesof gray;the B subunitsin shadesof blue Note that the heme groups (red)are relativelyfar apart.

Identical subunitsof multimeric proteins are generally arranged in one or a limited set of s1'rnmetricpatterns. A descriptionof the structure of these proteins requiresan introductionto conventionsused to define symmetries. Oligomers can have either rotational symmetry or helical symmetry; that is, individual subunitscanbe superimposed on others(broughtto coincidence)by rotation about one or more rotationalaxes or by a helical rotation. In proteins wrth rotational q.mmetry the subunits pack about the rotational axes to form closed structures. Proteins with helical syrnmetry tend to form moreopen-endedstructures,with subunits addedin a spiralingarray. There are severalforms of rotational s;'rnmetry.The stmplest is cyclic symrnetry, involving rotation about a sfigle axis(Fig. 4-23a). If subunitscanbe superimposed by rotationabouta singleaxis,the protein hasa s;rmmetry Twofold

4-23 Rotationalsymmetryin proteins.(a) In cyclic symmetry, FIGURE subunitsare relatedby rotationabouta singlen-fold axis,wheren is the numberof subunits so relatedTheaxesareshownas blacklines; the numbersare valuesof n. Only two of manypossibleCn arrangementsareshown.(b) In dihedralsymmetry,all subunitscan be related by rotationaboutone or both of two axes,one of which is twofold.D, symmetry.Relatingall 20 symmetryis mostcommon.(c) lcosahedral requires rotationaboutoneor more triangular facesof an icosahedron of three separaterotationalaxes:twofold, threefold,and fivefold.An end-onview of eachof theseaxesis shownat the right.

Threefold

v2

C3

Ttvo types of cyclic symmetry (a)

TWofold

definedas Cz (C for cyclic,n for the nlmber of suburLits related by the axis). The axis itself is describedas anrlfold rotationalaxis.The aB protomersof hemoglobin(Fig. 4-22) arerelatedby C2syrnmetry.A somewhatmorecomplicated rotational symmetry is dihedral s5rmmetr5r,in which a twofold rotational axis intersects an n-fold axis at right an$es; this symmetry is defined as Dz (Fig. 4-23b). A protetnwith djhedraisyrnmetryhas2n protomers. Proteins with cyclic or dihedral symmetry are particularly corunon. More complex rotational symmetries are possible,but only a few are regularly encounteredin proteins. One example is icosahedral symmetry. An icosahedronis a regular l2-cornered polyhedronwith 20 equilateraltriangularfaces (Fig. 4-23c). Each face can be brought to coincidencewith another face by rotation about one or more of three axes.This is a common structure in virus coats. or cansids.The human

Fourfold

Ttvofold

Twofold T\ryofold T$o types of dihedral symmetry (b)

Icosahedral symmetry (c)

F-'l

I h eT h r e e - D i m e n sSi ot rnuaclt uor feP r o t e i n s

Globularproteins havemore complicatedtertiary structures,often containingseveraltypes of secondarystructure in the samepo$peptide chain. The fust globularprotein structure to be determined,by x-ray diffraction methods,was that of myoglobin. The complexstructures of globularproteins can be analyzedby examiningfolding patterns called motifs, supersecondarystructures,or folds. The thousandsof knov,rrprotein structures are generallyassembledfrom a repertoire of only a few hundred motifs. Domainsare regionsof a polypeptide chain that can fold stably and independently. Quaternarystructure results from interactions betweenthe subunitsof multisubunit (multimeric) proteins or large protein assemblies.Some multimeric proteins have a repeatedunit consisting of a singlesubunit or a group of subunits,or protomer. Protomersare usually related by rotational or helical s;'rnmetry. (b) FIGURE4-24Viral capsids.(a) Poliovirus(derivedfrom PDBlD 2PLV) as renderedin theVlPERrelationaldatabase for structuralvirology.The coatproteinsof poliovirusassemble into an icosahedron 300 A in diameter.lcosahedral symmetryis a typeof rotationalsymmetry(seeFig. 4-23c). On the left is a surfacecontourimageof the polioviruscapsid. The imageat rightwas renderedat lower resolution,and the coatprotein subunitswere coloredto show the icosahedral symmetry.(b) Tobacco mosaicvirus (derivedfrom PDB lD lVTM). This rod-shaped virus(asshownin theelectronmicrograph) is 3,000A longand 180A in diameter; it hashelicalsymmetry.

poliovirushasan icosahedralcapsid(Fig. 4-24a). Each triangular face is madeup of three protomers,eachcontaining single copies of four different polrueptide chains, three of which are accessibleat the outer surface.Sixty protomersform the 20 facesof the icosahedral shell, which enclosesthe genetic material (RNA). The othermajortype of q'rnmetryfoundin oligomers, helicalsl.rnmetryalsooccursin capsids.Tobaccomosaic virus is a right-handedhelicalfilamentmade up of 2,130 identicalsubunits(Frg.4-24b). This cylindricalstructu-re enclosesthe viral RNA. Proteinswith subunitsarrangedin helical fllaments can also form long, flbrous structures suchasthe actin fllamentsof muscle(seeFig. 5-28).

5UMM AR Y4 . 3 P r o t e T i ne r t i a a r yn d Q u a t e r n aSrtyr u c t u r e s r

Tertiary structure is the completethree-dimensional structure of a polypeptidechain.There are two generalclassesof proteinsbasedon tertiary structure: flbrousand globular.

r

Fibrousproteins,which servemainly structuralroles, havesimplerepeatingelementsof secondarystructure.

andFolding 4.4 Protein Denaturation All proteins begin their existenceon a ribosomeas a linear sequenceof amino acid residues(Chapter 27). This polypeptidemust fold during and following synthesisto take up its native conformation.As we have seen,a native protein conformationis only marginallystable.Modest changes in the protein's environment can bring about structural changesthat can affect function. We now explore the transition that occurs between the folded and unfolded states.

Results inLoss ofFunction Loss ofProtein Structure Protein structures have evolved to function in particular cellular environments.Conditions different from those in the cell can result in protein structural changes,large and small. A loss of three-dimensionalstructure sufflcient to causelossoffunction is calleddenaturation. The denatured statedoesnot necessarilyequatewith completeunfolding of the protein and randomizationof conformation. Undermostconditions,denaturedproteinsexisth a set of partiallyfoldedstates,which asyet arepoorlyunderstood. Most proteins can be denaturedby heat, which has complex effects on the weak interactions in a protein (primarily hydrogen bonds). If the temperature is increasedslowly,a protein's conformationgenerallyremains intact until an abrupt loss of structure (and function) occursovera narrowtemperaturerange(Fig. 4-25). The abruptnessof the changesuggeststhat mfolding is a cooperativeprocess:lossof structurein onepart of the protein destabilizesother parts. The effects of heat on proteinsare not readilypredictable.The very heat-stable proteins of thermophilic bacteria and archaea have evolved to function at the temperature of hot springs (-100 'C). Yet the structuresof these proteins often

4.4Protein Denaturation andFoldinq - ) 41 I 100 tr

Smino Aeld $equence Determlnes Tertiary Structure RibonucleaseA

x{l --

b!

:60 X

T^f

d

I

T^

;40 Apomyoglobin

Hzo

n-

20 (a)

40 60 Temperature('C)

100 80

860 640

(b)

The tertiary structure of a globularprotein is determined by its amino acid sequence.The most important proof of this camefrom experimentsshowingthat denaturationof someproteinsis reversible.Certainglobularproteins denaturedby heat, extremesof pH, or denaturingreagents will regaintheir native structure and their biologicalactinty if returned to conditionsin which the nativecon-formation is stabie.This processis calledrenaturation. A classicexample is the denaturation and renaturation of ribonuclease A, demonstrated by Christian Anfinsenin the 1950s.Pun-fledribonucleaseA denatures completelyin a concentratedurea solution in the presenceof a reducingagent.The reducmgagentcleavesthe four disutfrdebonds to yield eight Cys residues,and the urea disrupts the stabilizing hydrophobic interactions, thus freeing the entire pollpeptide from its folded confomation. Denaturationof ribonucleaseis accompanied by a completelossof catalyticactivity.Whenthe urea and the reducingagentare removed,the randomlycoiled,denatured ribonucleasespontaneouslyrefolds into its correct tertiary structure,with full restorationof its catalltic activity (l-ig. 4-26). The refolding of ribonucleaseis so

23 (m) [GdnHCl]

FIGURt 4-25 Proteindenaturation. Results areshownfor proteins denaturedby two differentenvironmental changesIn each case,the transition fromthefoldedto theunfoldedstateisabrupt,suggesting cooperativity in the unfoldingprocess. (a)Thermaldenaturation of horse (myoglobin apomyoglobin withoutthe hemeprosthetic group)and ri(with bonuclease A itsdisulfidebondsintact;seeFig.4-26).Themidpoint of the temperature rangeover which denaturation occursis calledthe meltingtemperature, or f-. Denaturation of apomyoglobin was monitoredby circulardichroism(seeFig.4-9), which measures the amountof helicalstructure in the protein.Denaturation of ribonucleaseA was trackedby monitoringchangesin the intrinsicfluorescenceof the protein,which is affected by changes in the environment of Trp residues(b) Denaturation of disulfide-intact ribonuclease A by (CdnHCl),monitoredby circulardichroism. guanidinehydrochloride

differ only slightlyfrom thoseof homologousproteinsderived from bacteriasuch asEscheri,chi,acole How these smalldifferencespromotestructuralstabilityat high temperaturesis not yet understood. Proteinscan aisobe denaturedby extremesof pH, by certain miscible organic soiventssuch as alcohoi or acetone,by certainsolutessuch as urea and guanidine hydrochloride,or by detergents.Each of thesedenaturng agentsrepresentsa relatively mild treatment in the sensethat no covalentbonds in the potlpeptide chain are broken. Organicsolvents,urea, and detergentsact primarily by disrupting the hydrophobicinteractions that make up the stable core of globular proteins; extremesof pH alter the net chargeon the protein,causing electrostaticrepulsionandthe disruptionof somehydrogen bonding.The denaturedstructuresobtainedwith thesevarioustreatmentsare not necessarilythe same.

Native state; catalytically active

addition of urea and mercaptoethanol

Unfolded state; inactive. Disulfide cross-links reduced to yield Cys residues.

removal ofurea and mercaptoethanol

Native, catalytically active state. Disulfide cross-links correctly re-formed.

4-26 Renaturationof unfolded, denatured ribonuclease. FIGURE (HOCHzCHzSH) Ureadenatures the ribonuclease, and mercaptoethanol reduces andthuscleaves thedisulfide bondsto yieldeightCysresidues involvesreestablishins Renaturation the correctdisulfidecross-links

711 I h eT h r e e - D i m e n sSi ot rnuaclt uor feP r o t e i n s accuratethat the four intrachain disulfldebonds are reformed in the samepositionsin the renatured molecule as in the native ribonuclease.Calculatedmathematically, the eight Cys residues could recombine at random to form up to four disulfidebonds in 105 different ways.In fact, an essentiallyrandomdistributionof disulfldebonds is obtainedwhen the disulfidesare allowedto re-form in the presenceof denaturant(without reducingagent),indicating that weak bonding interactions are required for correct positioningof disulfidebonds and restorationof the native conformation.Later, similar results were obtained using chemicallysynthesized,catalyticallyactive ribonucleaseA. This eliminatedthe possibilitythat some minor contaminant in Anfinsen'spuri-fiedribonuclease preparationmight havecontributedto the renaturationof the enz5.tne,thus dispelling any remaining doubt that this enzyrnefolds spontaneously. The Anfinsen experiment provided the first evidence that the amino acid sequenceof a polypeptide chain contains all the information required to fold the chain into its native, three-dimensionalstructure. Subsequentwork has shown that this is true only for a mrnority of proteins, many of them small and inherently stable. Even though all proteins have the potential to fold into their native structure, many require some assistance, as we shallsee.

Polypeptides Fold Rapidly bya Stepwise Process In living cells, proteins are assembledfrom amino acids at a very high rate. For example,E. coli,cells can makea complete,biologicallyactiveprotein moleculecontaining 100aminoacid residuesin about 5 secondsat 37 "C.How doesthe pollpeptide chain arrive at its native conformation? Let's assurneconservativelythat eachof the amino acid residuesconld take un l0 different conformations

FIGURE 4-27 A simulatedfolding pathway.The folding pathwayof a 36-residuesegmentof the proteinvillin (an actin-bindingprotein foundprincipallyin the microvilliliningthe intestine) wassimulated by computer.Theprocessstartedwith the randomlycoiledpeptideand 3,000 surroundingwater moleculesin a virtual "waterbox."The mo-

on average,givjng 10i00different conformationsfor the polypeptide.Let's also assumethat the protein folds spontaneouslyby a randomprocessin which it tries out all possibleconformationsaroundeverysinglebond in its backboneuntil it finds its native,biologicallyactiveform. If eachconformationwere sampledin the shortestpossible time (-10-tt second,or the time requiredfor a single molecularvibration), it would take about 1077years to sample all possible conformations. Clearly, protein folding is not a completely random, trial-and-error process.There must be shortcuts.This problemwasfirst pointed out by CyrusLevinthalin 1968and is sometimes calledLevinthal'sparadox. The foldirg pathway of a large pollpeptide chain is unquestionablycomplicated,and not all the principles that guide the processhavebeen worked out. However, there are severalplausible models. In one, the folding processis hierarchical.Local secondarystructuresform first. Certainaminoacid sequencesfold readilyinto a helices or B sheets,guidedby constraintssuch as thosereviewed in our discussionof secondarystructure. Ionic interactions,involvingchargedgroupsthat are often near one another in the linear sequenceof the po\peptide chain, can play an important role in guiding these early folding steps.Assemblyof local structuresis followedby longer-rangeinteractionsbetween,say,two a helicesthat cometogetherto form stablesupersecondarystructures. The processcontinuesuntil completedomainsform and the entire polypeptide is folded (Fig. 4-27). Notably, proteinsdominatedby close-rangeinteractions(between pairs of residuesgenerallylocatednear eachother in the polypeptidesequence)tend to fold faster than proteins with more complexfoldingpatternsand manylong-range interactionsbetweendifferent segments. In an alternative model, folding is initiated by a spontaneouscollapseof the po$peptide into a compact

lecularmotionsof the peptideand the effectsof the water molecules weretakeninto accountin mappingthe mostlikelypathsto the final Thesimulatedfoldingtook structureamongthe countlessalternatives. placein a theoretical time spanof 1 ms.

4.4Protein Denaturation andFoldino ' ) 43 state, mediated by hydrophobic interactions among nonpolar residues. The state resulting from this ,,hydrophobic collapse" may have a high content of secondary structure, but many amino acid side chains are not entirely fixed. The collapsed state is often referred to as a molten globule. Most proteins probably fold by a process that incorporates features of both models. Instead of following a single pathway, a population of peptide molecules may take a variety of routes to the same end point, with the number of different partly folded conformational species decreasing as folding nears completion. Thermodynamically, the folding process can be viewed as a kind of free-energy funnel {[-ig. ,{-Zii }. The unfolded states are characterized by a high degree of conformational entropy and relatively high free energy.

Beginning

of helix formation

and collapse

Entropy

As folding proceeds,the narrowing of the funnel represents a decreasein the number of conformational speciespresent.Smalldepressions alongthe sidesof the free-energyfunnel represent semistableintermediates that can briefly slow the folding process.At the bottom of the funnel, an ensembleof folding intermediateshas been reducedto a singlenative conformation(or one of a small set of native conformations). Thermod;mamicstability is not evenly distributed overthe structure of a protein-the moleculehasregions of high and low stability.For example,a protein may have two stabledomarnsjoined by a segmentwith lower structural stability, or one small part of a domain may have a lower stabilitythan the remainder.The regionsof low stability allow a protein to alter its conformationbetween two or more states.As we shall seein the next two chapters, variationsin the stability of regionswithin a protein are often essentialto protein function. As our understandingof protein folding and protein structure improves, increasingly sophisticatedcomputer programsfor predicting the structure of proteins from their amino acid sequenceare being developed. Prediction of protein structure is a specialtyfield of bioinformatics, and progress in this area is monitored with a biennialtest calledthe CASP(Critical Assessment of Structural Prediction) competition. Entrants from around the world vie to predict the structure of an assignedprotein (whosestructurehasbeendeterminedbut not yet published).The most successfulteamsare invited to presenttheir resultsat a CASPconference.Completely reliable solutionsto the complex problem of predicting protein structure are not yet available,but the success and rigor of new approachesis being enrichedby CASP.

OF

Fr*tplns $onre Lfndergo Assisted Foiding o> oa

tr ^o p

Native structure

100

FIGURE 4-2S The thermodynamicsof protein folding depictedas a free-energyfunnel. At the top, the number of conformations,and hencetheconformational entropy,is large.Only a smallfractionof the intramolecular interactions that will existin the nativeconformation are presentAs foldingprogresses, the thermodynamicpath down the funnel reducesthe numberof statespresent(decreases entropy),increases the amountof proteinin the nativeconformation, and decreasesthe free energy.Depressions on the sidesof the funnel represent semistable foldingintermediates, which in somecasesmay slowthe foldingprocess

Not all proteins fold spontaneouslyas they are slmthesized in the cell. Folding for many proteins requires molecular chaperones, proteins that interact with partially folded or improperly folded polypeptides, facilitating correct folding pathways or providing microenvironments in which folding can occur. TWo classesof molecularchaperoneshavebeen well studied. Both are found in orgarlismsrangingfrom bacteriato humans.The first classis a family of proteins calledHsp70 (seeFig. 3-30) , which generallyhavea molecularweight near 70,000and are more abundantin cells stressedby elevatedtemperatures(hence,heatshockproteinsof M, 70,000,or Hsp70).Hsp70proteinsbind to regionsof unfolded polypeptides that are rich in hydrophobic residues,preventinginappropriateaggregation.These chaperonesthus "protect" both proteins subject to denaturationby heat and new peptide moleculesbeingsynthesized(and not yet folded). Hsp70proteins alsoblock the folding of certainproteinsthat must remainunfolded until they have been translocated across a membrane (as describedin Chapter 27). Some chaperonesalso

144

I h eT h r e e - D i m e n sSi ot rnuaclt uor feP r o t e i n s

@ nna.l stimulates ATP hydrolysis by DnaK. DnaK-ADP binds tightly to the unfolded protein.

@ DnaJ binds to the unfolded or partially folded protein and then to DnaK.

ATPi f

FoIded protern (natrve conformation)

@ere bindsto DnaK and the proteindissociates.

+ Arp:

ADP + GrpE (+ DnaJ ?)

[j :

.,

"

@ I" bacteria,the

l":::"f8ff;::*lii", release ofADP.

FIGURE 4-29 Chaperonesin protein folding.The cyclic pathwayby whichchaperones for theE. bindandrelease polypeptides is illustrated coli chaperoneproteinsDnaK and DnaJ,homologsof the eukaryotic chaperones Hsp70and Hsp40.The chaperones do not activelypromotethe foldingof the substrate protein,but insteadpreventaggregation of unfoldedpeptidesFora population of polypeptide molecules,

somefractionof the moleculesreleasedat the end of the cycle are in The remainder are reboundby DnaKor dithe nativeconformation. vertedto the chaperoninsystem(CroEL;seeFig.4-30). In bacteria,a proteincalledCrpE interacts with DnaK late in the cycle transiently (step@),promotingdissociation of ADP and possiblyDnaJ.No eukaryoticanalogof CrpEis known.

facilitatethe quaternaryassemblyof oligomericproteins. The Hsp70proteinsbind to and releasepollpeptides in a cycle that usesenergyfrom ATP hydrolysisand involves severalother proteins (including a classcalled Hsp40). -l'igure 4-2.9 illustrateschaperone-assisted folding as elucidatedfor the chaperonesDnaK and DnaJ inE. coli,, homologsof the eukaryoticHsp70 and Hsp40 chaperones. DnaK and DnaJ were first identifled as proteins required for in vitro replication of certain viral DNA molecules(hencethe "Dna" designation). The secondclassof chaperones is the chaperonins. Theseare elaborateprotein complexesrequired for the folding of some cellular proteins that do not fold spontaneously. In E coLian estimated70o/o to 15%of celIular proteins require the resident chaperonir system, calledGroEUGroES,for folding under normal conditions (up to 30%requirethis assistancewhen the cellsare heat stressed).The chaperoninsfirst becameknown when they were found to be necessaryfor the growth of certain bacterialviruses(hencethe designation"Gro"). Unfolded proteinsare boundwithin pocketsin the GroELcomplex,

and the pocketsare cappedtransientlyby the GroES"lid" (F'iS. 4-30). GroEL undergoessubstantialconformational changes,coupledto ATP hydrolysisand the binding and releaseof GroES,which promote folding of the bound pobpeptide. The mechanism by which the GroEL/GroESchaperoninfacilitatesfolding is not known in detail, but it dependson the size and interior surface propertiesof the cavitywhere folding occurs. Finally, the folding pathways of some proteins require two enzyrnes that catalyze isomerization reactions. Protein disulfide isomerase (PDI) is a widely distributed enzyrne that catalyzesthe interchange, or shuffling,of disulfldebondsuntil the bonds of the native conformation are formed. Among its functions, PDI catalyzesthe elimrnation of folding intermediateswith inappropriatedisulfide cross-links.Peptide prolyl cistrans isomera"se (PPI) catalyzesthe interconversion of the cis and trans isomers of Pro residue peptide bonds (Fig. 4-7b), which can be a slow stepin the folding of proteinsthat containsomePro peptidebondsin the cis conformation.

4 . 4P r o t e D i ne n a t u r a tai on ndF o l d i n o

['-{

@ unfolded protein binds to the GroEL pocket not blocked by GroES.

-Unfolded

protein

GroEL TADP GroES 7 ATP

@etp binds to each subunit ofthe GroEL heptamer.

@ Proteins not folded when released are rapidly bound agarn.

-7ATP

7 ADP

hydrolysis @eff leads to release of 14 ADP and GroES.

7 Pi, TADP TADP GroES

rygen.At COIIblevelsof 30%to 5\o/o,theneurologicalsymptomsbecome more severe,and at levels near 50%, the individual and can sink into coma.Respiratory losesconsciousness failure may follow.With prolongedexposure,somedamage becomespermanent.Death normally occurs when COFIb levels rise above 60%0.Autopsy on the boys who died at and\2o/o' LakePowellrevealedCOIIblevelsof 59o/o Binding of CO to hemoglobin is affected by many factors,including exercise(Fig. i) and changesin air pressure related to altitude. Becauseof their higher base levels of COHb,smokersexposedto a sourceof CO often developsymptomsfaster than nonsmokers. Individuals with heart, lung, or blood diseasesthat reduce the availability of oxygen to tissues may also experience symptoms at lower Ievels of CO exposure. Fetuses are at particular risk for CO poisoning,becausefetal hemoglobinhas a somewhathigher affinity for CO than adult hemoglobin.Casesof CO exposure have been recorded in which the fetus died but the mother recovered. (conti'rrued on nent PaSe)

gto 8a E pb

()4

'o 0

20

80 60 40 Carbon monoxide (PPm)

1oo

betweenlevelsof COHb in blood and concenI Relationship FIGURE trationof CO in the surroundingair. Fourdifferentconditionsof exposure are shown, comparing the effects of short versusextended exposure,and exposureat restversusexposureduring light exercise'

i

164

]

ProteF i nu n c t i o n

It may seem surprising that the loss of half of one,s hemoglobin to COHb can prove fatal-we know that people with any of several anemic conditions manage [o function reasonably well with half the usual complement of active hemoglobin. However, the binding of CO to hemoglobin does more than remove protein from the pool available to bind oxygen It also affects the affinity of the remaining hemoglobin subunits for oxygen. As CO binds to one or two subunits of a hemoglobin tetramer, the affinity for 02 is increased substantially in the remaining subunits (Fig 2) Thus, a hemoglobin tetramer wrth two bound CO molecules can efficiently bind 02 in the lungs-but it releases very little of it in the tissues. Oxygen deprivation in the tissues rapidly becomes severe. To add to the problem, the effects of CO are not limited to interference with hemoglobin function. CO binds to other heme proteins and a variety of metalloproteins. The effects of these interactions are not yet well understood, but they may be responsible for some of the longer-term effects of acute but nonfatal CO poisoning. When CO poisoning is suspected, rapid evacuation of the person away from the CO source is essential,but this does not always result in rapid recovery. When an individual is moved from the CO-polluted site to a nor_ mal, outdoor atmosphere, 02 begins to replace the CO in hemoglobin-but the COHb levels drop only slowly. The half-time is 2 to 6 5 hours, depending on individual and environmental factors. If 100% oxygen is administered with a mask, the rate of exchange can be increased about fourfold; the half-time for O2-CO exchange can be reduced to tens of minutes if 100% oxygen at a pressure

The expressionfor 0 (seeEqn 5-8) is o_

[L]" lLl" + Ku

3 (5-14)

Rearranging,then taking the log of both sides, yields

e _ ILl" r-0 Kd h-(*)

:nrogtlJ -rogKd

(5-15)

$ ts-rol

wnereKa: [L]6s. Equation 5-16 is the Hill equation, and a plot of log W/Q 0)l versus log [L] is called a Hill plot. Based on the equation, the Hill plot should have a slope of n However, the experimentally determined slope actually reflects not the number of binding sites but the degree of interaction between them. The slope of a Hill plot is therefore de_ noted by ns, the Hill coefficient, which is a measure of the degree of cooperativity. If nsequals 1, ligand binding is not cooperative, a situation that can arise even in a mul_ tisubunit protein if the subunits do not communicate. An

48I2 pO2(kPa) FIGURE2 Several oxygen-binding curves:for normalhemoglobin, hemoglobinfrom an anemicindividualwith only 50% of her hemoglobin functional,and hemoglobin from an individualwith 50% of his hemoglobinsubunitscomplexedwith CO. The pO2 in humanlungs andtissues is indicated

of 3 atm (303kPa) is supplied.Thus,rapid treatmentby a properly equippedmedicalteam is critical. Carbonmonoxide detectorsin all homesare highly recommended.This is a simple and inexpensivemeasure to avoid possibletragedy.After completingthe research for this box, we immediately purchasedseveral new CO detectorsfor our homes.

n11of greater than 1 indicates positive cooperativity in ligand binding. This is the situation observed in hemoglobin, in which the binding of one molecule of ligand facilitates the binding of others. The theoretical upper limit for ns is reached when ns : ?L In this case the binding would be completely cooperative: all bindiry sites on the protein would bind ligand simultaneously, and no protein mole_ cules partially saturated with ligand wor:ld be present under any conditions. This limit is never reached in practice, and the measured value of ns is always less than the actual number of ligand-binding sites in the protein. Annll of less than I indicates negative cooperativ_ ity, in which the binding of one molecule of ligand i,mpedes the binding of others. Well-documented cases of negative cooperativity are rare. To adapt the Hill equation to the binding of oxygen to hemoglobin we must again substitute pO2 for [L] and P{o for Ka: .-(*)

: nbspoz - nlogp5s

(b-17)

n a L i g a n d : 0 x y g e n - B iPnrdoitnegi n s["t] n d i nogfa P r o t e ti o b li e 5 . 1R e v e r s iB

1 s

Hemoglobin high'a{Iinity state \,, nu: | .u

l*0

l'');",'

-1

Iow-affrnity

-2

_J

-2

-1 log p02

FIGURE 5-14 Hill plots for oxygenbindingto myoglobinand hemo-1, The maximum globin.When nr1: thereis no evidentcooperativity. approxiobservedfor hemoglobincorresponds degreeof cooperativity a highlevelof coopermatelyto ng : 3. Notethatwhilethisindicates sitesin hemoglobin. ativity,ns is lessthann, the numberof O2-binding Thisis normalfor a proteinthat exhibitsallostericbindingbehavior

Hill plots for myoglobin

and hemoglobin

subunitsof a cooperativelybinding protein are functionally identical,that eachsubrnit can exist in (at least) two conformations,and that all subunitsundergothe transition from one conformationto the other simultaneously. In this model, no protein has individual subunits in different conformations. The two conformations are in equilibrium.The hgandcan bind to either conformation, but binds eachwith different affinity.Successivebinding of ligand molecules to the low-affinity conformation (which is more stablein the absenceof ligand) makesa transition to the high-affinity conformationmore likely. In the second model, the sequential model (Fig. 5-15b), proposedin 1966by DanielKoshlandand colleagues,ligandbinding can induce a changeof conformation in an individual subunit. A conformational change in one subunit makes a similar change in an adjacentsubunit, as well as the binding of a secondligand molecule,more likely. There are more potential intermediatestatesin this model than in the concerted model. The two models are not mutually exclusive;the concertedmodelmaybe viewedasthe "all-or-none"limiting caseof the sequentialmodel.In Chapter6 we use thesemodelsto investigateallostericenzyrnes'

H- and(0t Also Transports hlernoglobin

are given in

Figure 5-14.

for TwoModels Suggest Mechanisms Binding [ooperative Biochemistsnow know a great deal about the T and R statesof hemoglobin,but much remainsto be learned about how the T -+ R transition occurs.T\.vomodelsfor the cooperativebinding of ligandsto proteins with multiple binding sites have greatly influenced thinking aboutthis problem. The flrst model was proposedby JacquesMonod, JeffriesW;rman,and Jean-PierreChangeuxin 1965,and is called the MWC model or the concerted model (Fig. 5-15a). The concertedmodel assumesthat the

In addition to carryingnearly all the oxygenrequired by cells from the lungs to the tissues,hemoglobincarries two end products of cellularrespiration-H+ and COzfrom the tissuesto the Iungs and the kidneys,where they are excreted.The CO2,producedby oxidationof organicfuels in mitochondria,is hydrated to form bicarbonate: CO2+ H2Oi-

H* + HCOt

This reaction is catalyzedby carbonic anhydrase, an enzyrneparticularly abundant in erythrocytes. Carbon dioxideis not very solublein aqueoussolution,and bubblesof CO2wouldform in the tissuesandbloodif it were not convertedto bicarbonate.As you can seefrom the

A1lE

AllO

ETI

1l

1l ul 1l

l=o-E-E-m oo-oo-m-re=1l\ 11 1t 1t lr\tvtv G(])-Im-trt-m-ul

6XD

fl,lLl

GG)-EO-m-m-ffi

r-Y-) XX

1l on (J.,

tTl

IY]

1l

1l trI 1l

6YD

tilL]

aJ

1l

lxlxt

GXD

m

6)0

(a)

ll 1t

OO=-OO.-@-I=-I

I

1f 1t 1r 1r\11 oo-oo-er.-[I)=-ul 1l 1t 1t 1t\1t (n{) -nlD -tTlt

-ttTil-EItl

60:60:m-re-ETt

1t 1t 1t 1r\1r

cYD .mD -Etil -tr|il-trit m:EGj=- l-Lft 66:66: ft)

5-15 Two generalmodelsfor the interconFIGURE versionof inactiveand activeforms of a proteinduring cooperativeligandbinding'Althoughthe models may be appliedto any protein-includingany enbinding, 6)-that exhibitscooperative zyme(Chapter we show herefour subunitsbecausethe modelwas originallyproposedfor hemoglobin.(a) In the conmodel(MWC model),all subcerted,or all-or-none, postulated to be in the sameconformation, unitsare (low or inactive)or all tr (high affinity eitherall O K1, on the equilibrium, affinityor active).Depending betweenO and D forms,the bindingof one or more (L)will pull the equilibriumtoward ligandmolecules with boundL areshaded.(b) ln the ! form.Subunits model,eachindividualsubunitcan be the sequential in eitherthe O or n form.A very largenumberol is thusPossible. conformations

!"1 P r o t e Fi nu n c t i o n reaction catalyzedbycarbonicanhydrase,the hydration of CO2resultsin an increasein the H+ concentration(a decreasein pH) in the tissues.The bindingof oxygenby hemoglobinis profoundly influenced by pH and CO2 concentration,so the interconversionof CO2and bicarbonate is of great importance to the regulation of oxygenbinding and releasein the blood. Hemoglobintransports abottt 400/o of the total H+ and 15% to 20o/oof the CO2formed in the tissues [o the lungs and kidneys.(The remainderof the H+ is absorbedby the plasma'sbicarbonatebuffer; the remainder of the CO2is transportedas dissolvedHCO| and CO2.)The binding of H* and CO2is inverselyrelated to the binding of oxygen.At the relativelyIow pH and high CO2 concentrationof peripheral tissues, the affinity of hemoglobinfor oxygendecreasesas H+ and CO2are bound,and 02 is releasedto the tissues.Conversely, in the capillaries of the lung, as CO2 is excreted and the blood pH consequentlyrises, the affinity of hemoglobinfor oxygen increasesand the protein binds more 02 for transport to the peripheral tissues.This effect of pH and CO2 concentrationon the binding and release of oxygen by hemoglobinis calledthe Bohr effect, after ChristianBohr, the Danish physiologist (and father of physicist Niels Bohr) who discoveredit in 1904. The binding equilibrium for hemoglobinand one molecule of oxygen can be designatedby the reaction Hb + 02 -+

HbO2

but this is not a completestatement.To accountfor the effect of H+ concentrationon this binding equilibrium, we rewrite the reaction as HHb*+Oz#HbOr+H+ where HlIb+ denotesa protonatedform of hemoglobin. This equationtells us that the O2-saturationcurveof hemo$obin is influencedby the H* concentration(Fig. b-f 6). Both 02 and H* are boundby hemoglobin,but with inverse afnniff. When the oxygen concentrationis high, as in the lungs, hemo$obin binds 02 and releasesprotons.When the oxygen concentrationis low; as in the peripheral tissues,H* is boundand 02 is released. Oxygenand H+ are not bound at the samesites in hemoglobin.Oxygen binds to the iron atoms of the hemes,whereasH* binds to any of severalamino acid residuesin the protein. A major contribution to the Bohr effect is made by Hisra6(His HC3) of the B subunits. When protonated,this residueforms one of the ion pairs-to Aspea(Asp FGI)-that helps stabilize deoxyhemoglobinin the T state (Fig. 5-9). The ion pair stabilizesthe protonated form of His HCB,giving this residue an abnormally high pK, in the T state. The pK. falls to its normal value of 6.0 in the R state becausethe ion pair cannot form, and this residue is largely unprotonatedin oxyhemoglobinat pH 2.6, the blood pH in the lungs. As the concentrationof H+

o o.s

0246810 pO2 ftPa) FIGURE 5-16 Effectof pH on oxygenbindingto hemoglobin.ThepH o f b l o o di s 7 . 6 i n t h e l u n g sa n d 7 . 2i n t h e t i s s u e E s .x p e r i m e n tmael a surements on hemoglobinbindingare oftenperformedat pH 7.4.

rises, protonation of His HC3 promotes release of oxygen by favoring a transition to the T state. protonation of the amino-terminalresidues of the a subunits, certain other His residues,and perhaps other groups has a similar effect. Thus we seethat the four polypeptide chainsof hemoglobin communicatewith each other not only about 02 binding to their hemegroupsbut alsoabout H+ brnding to specificamino acid residues.And there is still more to the story.Hemoglobinalsobinds CO2,againin a manner inverselyrelated to the binding of oxygen.Carbon dioxide binds as a carbamategroup to the a-amino group at the amino-terminalend of each globin chain, forming carbaminohemoglobin:

o C+

o

TT-

H2N-C-C-

--l--)

RO

Amino-terminal residue

Oi TT

C-N-C-C-

ltttl o Ro Carbamino-terminal residue

This reaction produces H+, contributing to the Bohr effect. The bound carbamates also form additional salt bridges (not shown in Frg 5-9) that help to stabilize the T state and promote the release of oxygen. When the concentration of carbon dioxide is high, as in peripheral tissues, some CO2 binds to hemoglobin and the affinity for 02 decreases, causing its release. Conversely, when hemoglobin reaches the lungs, the high oxygen concentration promotes binding of 02 and release of CO2. It is the capacity to communicate ligandbinding information from one potypeptide subunit to the others that makes the hemoglobin molecule so beautifully adapted to integrating the transport of 02, CO2, .--+. and .t-l by en'throcytes.

Proteins 0xygen-Binding t0a Ligand: ofa Protein Binding 5.1Reversible Lt ufl

Einding taFlnmoglohin lsfteEuiated 0xygen hy2,3-$isphosphoglycenate

pO2in pO2 in lungs tissues (4,500m)

YV+

The interaction of 2,3-bisphosphoglycerate (BPG) with hemoglobinmoleculesfurther refinesthe function of hemoglobin,and providesan exampleof heterotropic allostericmodulation.

-oo tc"

pO2 in lungs (sealevel)

o

H-C-O-P-O H-C-H

O_

BPG:

6I

o-T:o o2,3-Bisphosphoglycerate

BPG is present in relatively high concentrations in erythrocytes. When hemoglobin is isolated, it contains substantial amounts of bound BPG, which can be diff,cult to remove completely. In fact, the O2-bindingcurves for hemoglobin that we have examined to this point were obtained in the presence of bound BPG. 2,3-Bisphosphoglycerate is known to greatly reduce the affinity of hemoglobin for oxygen-there is an inverse relationship between the binding of 02 and the binding of BPG. We can therefore describe another binding process for hemoglobin: HbBPG+02+HbO2+BPG BPG binds at a site distant from the oxygen-binding site and regulates the O2-binding afflnity of hemoglobin in relation to the pO2 in the lungs. BPG is important in the physiological adaptation to the lower pO2 at high altitudes. For a healthy human at sea level, the binding of 02 to hemoglobin is regulated such that the amount of 02 delivered to the tissues is nearly 40o/oof the maximum that could be carried by the blood (Fig. rr-"1?l Imagine that this person is suddenly transported from sea level to an altitude of 4,500 meters, where the pO2 i.s considerably lower. The delivery of 02 to the tissues is now reduced. However, after just a few hours at the higher altitude, the BPG concentration in the blood has begun to rise, leading to a decrease in the afflnity ofhemoglobin for oxygen. This adjustment in the BPG level has only a small effect on the binding of 02 in the lungs but a considerable effect on the release of 02 in the tissues. As a result, the delivery of oxygen to the tissues is restored to nearly 400/oof the 02 that can be transported by the blood. The situation is reversed when the person returns to sea level. The BPG concentration in erythrocytes also increases in people suffering from hypoxia, lowered oxygenation of peripheral tissues due to inadequate functioning of the lungs or circulatory system.

oTa

12

16

pOz kPa) f,lfitiRE5-17 Effectof BPG on oxygenbinding to hemoglobin'The in normalhumanbloodis about5 mM at sealevel BPCconcentration high altitudesNote that hemoglobinbindsto mM at B and about oxygenquite tightlywhen BPC is entirelyabsent,and the binding Hill coefficient In reality,the measured curveseemsto be hyperbolic. only slightly(from3 to about decreases cooperativity for O2-binding but the risingpartof the 2.5)when BPCis removedfrom hemoglobin, sigmoidcurveis confinedto a verysmallregioncloseto the origin.At with 02 in the lungs,butjust is nearlysaturated sealevel,hemoglobin over 607osaturatedin the tissues,so the amountof 02 releasedin the is about3B%of the maximumthatcan be carriedin the blood' tissues to 30% of 02 deliverydeclinesby aboutone-fourth, At highaltitudes, the decreases however, concentration, in BPC increase maximum.An what can be of 377o approximately for 02, so hemoglobin of affinity to thetissues. carriedis againdelivered

The site of BPGbinding to hemoglobinis the cavity betweenthe B subunitsin the T state(Fig. 5-18)' This cavity is lined with positively charged amino acid residues that interact with the negatively charged groupsof BPG.Unlike 02, only one moleculeof BPGis bounclto eachhemoglobintetramer'BPGlowershemoglobin'saffirity for oxygenby stabilizingthe T state' The transition to the R state narrows the binding pocket for BPG,precludingBPG binding.In the absenceof BPG, hemoglobinis convertedto the R statemore easily. Regulationof oxygenbinding to hemoglobinby BPG has an important role in fetal development'Becausea fetus must extract oxygenfrom its mother'sblood, fetal hemoglobinmust havegreateraffinity than the maternal hemoglobinfor 02. The fetus synthesizes7 subunits rather than F subunits, formlng a2y2hemoglobin.This tetramer has a much lower affinity for BPGthan normal adult hemoglobin,and a correspondinglyhigher affinity Proteins-Hemoglobinls Susceptible for 02. ff Oxygen-Binding to AllostericRegulation

J68 l

Protein Function

FIGURE 5-18 Bindingof BpG to deoxyhemoglobin. (a) BpC binding stabilizes theT stateof deoxyhemoglobin (pDBlD j HCA),shownhere asa meshsurfaceimage (b)Thenegativechargesof BpC interactwith severalpositivelychargedgroups(shown in blue in this surface

5ickle"(ell Anemia lsa Molecular Disease ofHemoglobin The hereditary human disease sickie-cell anemia demonstrates strikingly the importance of anuno acid sequence in determining the secondary tertiary, and quaternary stmctures of globular proteins, and thus their biological functions. Almost 500 genetic variants of hemoglobin are known to occur in the human population; all but a few are quite rare. Most variations consist of differences in a single amino acid residue. The effects on hemoglobin structure and function are often minor but can sometimes be extraordinary. Each hemogiobin variation is the product of an altered gene. The variant genes are called alleies. Because humans generally have two copies of each gene, an individual may have two copies of one allele (thus being homozygous for that gene) or one copy of each of two different alleles (thus heterozygous). Sickle-cell anemia occurs in individuals who inherit the allele for sickle-cell hemoglobin from both parents. The er;4hrocytes of these individuals are fewer and also abnormal. In addition to an unusually large number of immature cells, the blood contains many long, thin, sickle_ shaped erythroc;,tes (Fig. b-19). When hemoglobin from sickle cells (called hemoglobin S) is deoxygenated, it becomes insoluble and forms polyners that aggregate into tubular flbers (Fig. 5-20). Normal hemoglobin (hemoglobin A) remains soiuble on deoxygenation. The ursotuble flbers of deoxygenated hemoglobin S cause the deformed, sickle shape of the erythroc).tes, and the proportion of sickled cells increasesgreatly as blood is deoxygenated. The altered properties of hemoglobin S result from a single amino acid substitution, a Val instead of a Glu residue at position 6 in the two B chains. The R group of valine has no electric charge, whereas glutamate has a negative charge at pH 7.4. Hemoglobin S therefore has two fewer negative charges than hemoglobin A (one fewer on each B chain). Replacement of the GIu resiclue by Val creates a "sticky" hydrophobic contact point at position 6 of the B chain, which is on the outer surface of

contourimage)thatsurround thepocketbetweentheB subunitsin the T state (c) The binding pocketfor BPC disappearson oxygenation, followingtransition to the R state(pDB lD l BBB).(Compare(b) and (c) with Fig.5-10 )

the molecule.Thesesticky spots causedeoxyhemoglobin S moleculesto associateabnormallywith eachother, forming the long, fibrous aggregatescharacteristicof this disorder. f Oxygen-Bindingproteins-Defects in Hb Lead to SeriousGeneticDisease Sickle-cell anemia, as we have noted, occurs in indi-

viduals homozygousfor the sickle-cellallele of the gene encodingthe B subunitof hemoglobin.Individualswho receive the sickle-cellallele from only one parent and are thus heterozygousexperiencea milder condition called sickle-celltrait; only abot;rtlo/oof their erythrocytesbecome sickled on deoxygenation.These individualsmay live completelynormallivesff they avoidvigorousexercise and other stresseson the circulatorysystem. Sickle-cellanemiais Life-threatening and painful.people with this diseasesufferrepeatedcrisesbrought on by physicalexertion.They becomeweak,dtzzy,andshort of breath,andthey alsoexperienceheartmurmursandan increasedpulserate. The hemoglobincontentof their blood is only about half the normal value of 15 to 16 9100 mL,

(a)

'r.-*

ft)

FIGURE 5-19 A comparison of (a) uniform,cup-shaped, normarerythrocyteswith (b) the variablyshapederythrocytes seenin sickle-cell anemia,whichrangefrom normalto spinyor sickle-shaped.

o a L i g a n d : 0 x y g e n - B iPnrdoitnegi n [st * l n d i nogfa P r o t e ti n b li e 5 . 1R e v e r s iB

Hemoglobin A

Hemoglobin

S

ally high in certain pafts of Africa. Investigationinto this matter led to the flnding that in heterozygousindividuals,the alleleconfersa smallbut signiflcantresistanceto Iethal forms of malaria.Natural selectionhas resulted in an allelepopulationthat balancesthe deleteriouseffects of the homozygouscondition againstthe resistanceto malariaaffordedby the heterozygouscondition.I

b li en d i n g 5Y . 1 R e v e r s iB SUMMAR o fa P r o t e it no a L i g a n d : Oxygen-BinP d irnogt e i n s r

Interaction between molecules

II

+

Strand formation

I

Protein function often entails interactionswith other molecules.A protein binds a molecule,knovm as a ligand, at its binding site. Proteinsmay undergoconformationalchangeswhen a ligand binds,a processcalledinducedflt. In a muttisubunit protein, the binding of a ligand to one subunit may affect ligand binding to other subunits.Ligand binding can be regulated. Myoglobincontainsa heme prosthetic group, which binds oxygen.Heme consistsof a singleatom of Fe2* coordinatedwithin a porphyrin.Oxygenbinds to myoglobinreversibly;this simplereversible binding can be describedby an associationconstant Kuot a dissociationconstantK6. For a monomeric protein such as myoglobin,the fraction of binding sites occupiedby a ligand is a hyperbolicfunction of ligandconcentration. Normal adult hemoglobinhas four heme-containing subunits,two a and two B, similar in structure to eachother and to myoglobin.Hemoglobinexists in two interchangeablestructural states,T and R. The T state is most stablewhen oxygenis not bound. Oxygenbinding promotestransition to the R state.

FIGURE 5-20 Normal and sickle-cellhemoglobin.(a) Subtledifferences S resultfrom A andhemoglobin of hemoglobin theconformations betvveen a singleaminoacidchangein thep chains.(b)As a resultof thischange, whichcauses patchon itssurface, S hasa hydrophobic deoxyhemoglobin fibers. insoluble into that align into strands the moleculesto aSSre8ate

Oxygenbinding to hemoglobinis both allosteric and cooperative.As 02 binds to onebindingsite, the hemoglobinundergoesconformationalchanges that affect the other binding sites-an example of allostericbehavior.Conformationalchanges betweenthe T and R states,mediatedby subunit-subunitinteractions,result in cooperative binding; this is describedby a sigmoidbinding curve and can be analyzedby a Hill plot.

becausesicktedcells are very fragile and rupture easily; this resultsin anemia("lack of blood"). An evenmore serious consequenceis that capillariesbecomeblockedby the long, abnormally shaped cells, causingsevereparn and interferingwith normal organfunction-a major factor in the early deathof many peoplewith the disease. Without medicaltreatment,peoplewith sickle-cell anemia usually die in childhood. Curiously,the frequency of the sickle-cellallele in populationsis unusu-

TWomajormodelshavebeenproposedto explain the cooperativebinding of ligandsto multisubunit proteins:the concertedmodeland the sequential model. HemoglobinalsobindsH* and CO2,resultingin the formation of ion pairs that stabilizethe T state and Iessenthe protein'saffinityfor 02 (the Bohr effect). Oxygenbinding to hemoglobinis alsomodulated which bindsto and by 2,3-bisphosphoglycerate, stabilizesthe T state.

AJigrrment and crystallization (frber formation) (b)

!tol r

p r o t eF i nu n c t i o n

Sickle-cell anemia is a genetic diseasecaused by a single amino acid substitution (Glub to Val6) in each B chain of hemoglobin. The change produces a hydrophobic patch on the surface of the hemoglobin that causesthe molecules to aggregate into bundles of fibers. This homozygous condition results in serious medical comolications.

5,2 Complementary Interactions between Proteins andLigands:The lmmune System andlmmunoglobulins We have seen how the conformations of oxygen-binding proteins affect and are affected by the binding of small ligands (O2 or CO) to the heme group. However, most proteinJigand interactions do not involve a prosthetic group. Instead, the binding site for a Iigand is more often Iike the hemoglobin binding site for BpG-a cleft in the protein lined with amino acid residues,arranged to make the binding interaction highly specific. Effective discrimination between ligands is the norm at binding sites, even when the hgands have only minor structural differences. All vertebrates have an immune system capable of distinguishing molecular "self" from ,,nonself',and then destroflng what is identified as nonself. In this way, the immune syslem eliminates viruses, bacteria, and other pathogens and molecules that may pose a threat to the organism. On a physiological level, the immune re_ sponseis an intricate and coordinated set of interactions among many classes of proteins, molecules, and cell types. At the level of individual proteins, the immune re_ sponse demonstrates how an acutely sensitive and spe_ cific biochemical system is built upon the reversible binding of ligands to proteins.

Thelmrmune Response Features aSpecialized Array nf[ellsandProteins Immunity is brought about by a variety of leukoc5rtes (white blood cells), including macrophages and lymphocytes, all of which develop from undifferentiated stem cells in the bone marrow. Leukocytes can leave the bloodstream and patrol the tissues, each cell producing one or more proterns capable of recognizing and binding to molecules that might signal an infection. The immune response consists of two complementary systems,the humoral and cellular immune systems. The humoral immune system (Latin humor, ,,fluid") is directed at bacterial infections and extracellular viruses (those found in the body fluids), but can also respond to individual foreign proteins. The cellular immune system destroys host cells infected by viruses and also destroys some parasites and foreign tissues. At the heart of the humoral immune response are sol_ uble proteins called antibodies or immunoglobulins, often abbreviated Ig. Immunoglobulins bind bacteria,

viruses,or largemoleculesidentifiedasforeignand target them for destruction.Making up 200/oof blood protein, the immunoglobulinsare produced by B lymphocytes, or B cells, so namedbecausethey completetheir developmentin the bonemarrow. The agentsat the heart of the celluiarimmune responseare a classof T lymphocytes, or T cells (so called becausethe latter stagesof their development occur in the fhymus),known as cytotoxic T cells (Ts cells, alsocalledkiller T cells).Recognitionof infected cellsor parasitesinvolvesproteinscalledT-cell receptors on the surfaceof T6 cells.Receptorsare proteins, usuallyfound on the outer surfaceof cellsand extending through the plasmamembrane;they recognizeand bind extracellular ligands, triggering changesinside the cell. In additionto cltotoxic T cells,there are helper T cells (T11cells), whosefunction it is to producesoluble signalingproteins called cytokines,which include the interleukins.T6 cells interact with macrophages. The Ts cells participateonly indirectly in the destruction of infected cells and pathogens,stimulating the selectiveproliferationof those Ts and B cells that can bind to a particularantigen.This process,calledclonal selection, increasesthe number of immune system cellsthat can respondto a particularpathogen.The importance of Ts cells is dramaticallyillustrated by the epidemicproducedby HIV (human immunodeflciency virus), the virus that causesAIDS (acquiredimmune deficiency syndrome). The primary targets of HIV infection are Ts cells. Elimination of these cells progressively incapacitatesthe entire immune system. Table5-2 summarizesthe functionsof someleukocvles of the immunesystem. Each recognitionprotein of the immune system,either a T-cellreceptoror an antibodyproducedby a B cell, speciflcallybinds some particular chemicalstructure.

Celltpe

Funetion

Macrophages

Ingestlarge particles and cellsby phagocytosis

B lymphocytes (B cells)

Produceand secrete antibodies

T lyrnphocytes (T cells) Cytotoxic (killer) T cells (T6) Helper T cells (Ti1)

Interact with infected host cellsthroughreceptors on T-cell surface Interact with macrophages and secretecytokines (interleukins) that stimulateT6, Tg, and B cells to proliferate.

5 . 2C o m p l e m e nItnatreyr a c t i 0bnest w e ePnr o t e i a nn s dL i g a n d s : Tl m he m u nSey s t eamn dl m m u n o g l o b u l!i7n1t _ _ l

distinguishing it from virtually all others. Humans are capableof producingmore than 108different antibodies with distinct binding speciflcities.Given this extraordinary diversity, any chemrcalstructure on the surfaceof a virus or invadingcell will most likely be recognizedand bound by one or more antibodies.Antibody diversity is derived from random reassembly of a set of immunoglobulingene segmentsthrough genetic recombination mechanismsthat are discussedin Chapter 25 (seeFig. 25-26). A specialized lexiconis usedto describethe unique interactionsbetweenantibodiesor T-cellreceptorsand the moleculesthey bind. Any molecule or pathogen capableof eliciting an immune responseis called an antigen. An antigenmay be a virus, a bacterialcell wall, or an individual protein or other macromolecule.A complex antigenmay be bound by severaldifferent antibodies. An indMdual antibody or T-cell receptor binds only a particular molecular structure within the antigen, calledits antigenic determinant or epitope. It would be unproductivefor the immune systemto respondto small moleculesthat are corrrmonintermediatesand productsof cellularmetabolism.Moleculesof M.

A-x + B 13 A + X: + B

This alters the pathway of the reaction, and it results in catalysisonlA when the new pathway has a lower activation energythan the uncatalyzedpathway.Both of the new stepsmust be fasterthan the uncatalyzedreaction. A number of amino acid side chains,including all those in Figure 6-9, and the functional groupsof someenzyrne cofactorscan serve as nucleophilesin the formation of covalentbonds with substrates.These covalent complexes alwaysundergo further reaction to regenerate the free enz).rne.The covalentbond formed betweenthe enz).rneand the substrate can activate a substrate for further reaction in a manner that is usually speciflc to the particular group or coenzlirne. Metal Ion Catalysis Metals,whether tightly bound to the enz;rmeor taken up from solution along with the substrate, can participate in catalysisin severalways.

Fr{

Enzymes

Ionic interactions between an enz),rne-boundmetal and a substrate can help orient the substrate for reaction or stabilze charged reaction transition states. This use of weak bonding interactions between metal and substrate is similar to some of the uses of enzl'rne-substrate binding energy described earlier. Metals can also mediate ondation-reduction reactions by reversible changesin the metal ion's oxrdation state. Near$ a third of all hrown en4rrnes requue one or more metal ions for catalytic activity. Most enzymes combine several catalytic strategies to bring about a rate enhancement. A good example is the use of covalent catalysis,general acid-base catalysis, and transition-state stabilization in the reaction catalyzed by chSrmotrypsin,detailed in Section 6.4.

SUMMAR 6Y . 2 H o wE n z y mW e so r k r

Enzlmes are highly effective catalysts, commonly enhancing reaction rates by a factor of 105to 1017.

r

Enz;.'rne-catalyzed reactions are characterized by the formation of a complex between substrate and enzyrne (an ES complex). Substrate binding occurs in a pocket on the enzlrrnecalled the active site.

r

The function of enz).'mesand other catalysts is to lower the activation energy, AG+,for a reaction and thereby enhance the reaction rate. The equilibrium of a reaction is unaffected by the enzJ..rne. A sigmf,cant part of the energy used for enzyrnatic rate enhancements is derived from weak interactions (hydrogen bonds and hydrophobic and ionic interactions) between substrate and enzyrne. The enz).rne active site is structured so that some of these weak interactions occur preferentially in the reaction transition state, thus stabilizing the transition state. The need for multiple interactions is one reason for the large size of enzSrrnes.The binding energy, AGs, can be used to lower substrate entropy or to cause a conformational change in the enz;'rne (induced flt). Binding energy also accounts for the exquisite specificity of enzSrmes for their substrates. Additional catalytic mechanisms employed by enzyrnesinclude general acid-base catalysis, covalent catalysis,and metal ion catalysis. Catalysis often involves transient covalent interactions between the substrate and the enzyrne, or group transfers to and from the enzyrne, so as to provide a new, Iower-energy reaction path.

6.3 Enzyme Kinetics asanA,pproach to Understanding Mechanism Biochemists commonly use several approaches to study the mechanism of action of purified enzymes. The three-dimensional structure of the protein provides important information, which is enhanced by classical

protein chemistryand modernmethodsof site-directed mutagenesis(changingthe amino acid sequenceof a protein by genetic engineering;see Fig. 9-11). These technologiespermit enzl'rnologists to examinethe role of individual amino acids in enz).rnestructure and action. However,the oldest approachto understanding enzymemechanisms,and the one that remains most important, is to determinethe rate of a reaction and how it changesin responseto changesin experimental parameters,a disciplineknown as enzJrrnekinetics. We provide here a basicintroductionto the kinetics of enzyme-catalyzedreactions. More advanced treatments are availablein the sourcescited at the end of the chapter.

(oncentration Substrate Affects theRate of Inzyme-[ata lyzed Reactions A key factor affecthg the rate ofa reactioncatalyzedby an enzyrneis the concentrationof substrate,[S]. However, studying the effects of substrate concentrationis complicatedby the fact that [S] changesduring the courseof an il vitro reactionassubstrateis convertedto product. One simplifying approach in kinetics experiments is to measurethe initial rate (or initial velocity), designated76 (Fig. 6-10). In a typical reaction,the enzyrnemay be present in nanomolarquantities,whereas [S] may be flve or six orders of magnitudehigher.If only the beginning of the reaction is monitored (often the first 60 secondsor less),changesin [S]canbe limited to a few percent,and [S] can be regardedas constant.7e can then be exploredas a function of [S], which is adjusted by the investigator.The effect on 7s of varying [S] when the enzyme concentrationis held constant is

I

c)

F

lSl = 0.2iuu

Time FIGURE 6-10 lnitial velocitiesof enzyme-catalyzed reactions.A theoreticalenzymecatalyzesthe reactionS 3 P,and rs presenrar a concentration sufficient to catalyzethe reactionat a maximumveloc.l ity, V^n,, of pxalmin The Michaelisconstant,K- (explainedin the text),is 0.5 pv. Progress curvesareshownfor substrate concentrattons below,at, and abovethe K-. Therateof an enzyme-catalyzed reaction declinesas substrate is convertedto product.A tangentto eachcurve takenat time : 0 definesthe initialvelociry V6,of eachreaction.

c sa nA p p r o atcohU n d e r s t a n dMi negc h a n i s lmr { 6 . 3 E n z y mKei n e t i a

x 0)

Leonor Michaelis, 187 5-1 949

Substrate concentration, IS] (mu) FIGURE 6-1 1 Effectof substrate concentration on the initialvelocityof an enzyme-catalyzed reaction.Themaximumvelocity,yma,,is extrapolatedfrom the plot, becauseV6 approachesbut neverquite reaches V-"*. Thesubstrate concentration at which Vois half maximalis K-, the Michaelis constant. Theconcentration of enzymein an experiment such asthisis generally so low thattsl >> tElevenwhen [S]is described as low or relativelylow Theunitsshownaretypicalfor enzyme-catalyzed reactions andaregivenonlyto helpillustrate the meaning of Voand [S]. (Notethatthe curvedescribes part ol a rectangular hyperbola,with one asymptote at V.n". lf the curvewerecontinuedbelow [S] : 0, it would K- ) approacha verticalasymptote at [S] :

shown h Figure 6-11. At relatively Iow concentrations of substrate, Ze increases almost linearly with an increase in [S]. At higher substrate concentrations, 70 in-

creases by smaller and smaller amounts in response to increasesin [S]. Finally, a point is reached beyond which increases in 7e are vanishingly small as [S] increases. This plateauJike Ze region is close to the maximum velocity, Y*a*. The ES complex is the key to understanding this kinetic behavior, just as it was a starting point for our discussion of catalysis. The kinetic pattern in Figure 6-1 1 led Victor Henri, following the lead of Wurtz, to propose in 1903 that the combination of an enz),me with its substrate molecule to form an ES complex is a necessary step in enzyrnatic catalysis.This idea was expanded into a general theory of enzyme action, particularly by Leonor Michaelis and Maud Menten in 1913. They postulated that the enzyrne first combines reversibly with its substrate to form an enzyrne-substrate complex in a relatively fast reversible step: E+S

ES

reacAt any given instant in an enzyme-catalyzed tion, the enzymeexists in two forms, the lree or uncombinedform E and the combinedform ES. At low [S], most of the enzymeis in the uncombinedform E. Here, the rate is proportionalto [S] becausethe equilibrium of Equation 6-7 is pushedtoward formation of more ES as [S] increases.The maximum initial rate of f,he catalyzedreaction (Z-ur) is observedwhen virtually all the enzymeis present as the ES complex and [E] is vanishinglysmall. Under these conditions,the enzymeis "saturated"Ivith its substrate,so that further increasesin [S]haveno effect on rate. This condition existswhen [S] is sufficientlyhigh that essentially all the free enzymehasbeen convertedto the ES form. After the ES complexbreaksdownto yield the product P, the enzymeis free lo catalyzereaction of another moleculeof substrate.The saturationeffect is a distinguishingcharacteristicof enzymaticcatalystsand is responsiblefor the plateauobservedin Figure 6-11. The pattern seenin Figure6-11 is sometimesreferredto as saturationkinetics. Whenthe enzyrneis first mixed with a largeexcessof substrate,there is an initial period, the pre-steady state, during which the concentrationof ES builds up. This periodis usuallytoo short to be easilyobserued,lasting just microseconds,and is not evidentin Figure 6-10. The reaction quickly achievesa steady state in which [ES] (and the concentrationsof any other htermediates) remainsapproximatelyconstantover time. The concept of a steadystatewasintroducedby G. E. Bnggsand Haldane in 1925.The measured76 generallyreflectsthe steadystate,eventhough 7e is limited to the earlypart of the reaction,and analysisof thesernitial rates is referred to as steady-state kinetics.

(6-7)

The ES complex then breaks down in a slower second step to yield the free enzy'rneand the reaction product P:

nsJae+p

Maud Menten,1879-1960

(6-8)

h2

Because the slower second reaction (Eqn 6-8) must Iimit the rate of the overall reaction, the overall rate must be proportional to the concentration of the species that reacts in the second step, that is, ES

(oncentration and 5ubstrate between TheRelationship Rate [anBe[xpressed fteaction Quantitatively The curve expressing the relationship between [S] and 7e (Fig 6-11) has the same general shape for most enzymes (it approaches a rectangular hyperbola), which can be expressed algebraically by the MichaelisMenten equation Michaelis and Menten derived this equation starting from their basic hypothesis that the rate-limiting step in enzymatic reactions is the

-| 196] Enzymes

breakdown of the ES complex to product and free enz;,me. The equation is

s (6-e) The important terms are [S], Vo, V^u,, and a constant called the Michaelisconstant,K-. N these terms are readily measuredexperimentally. Here we developthe basic logic and the algebraic steps in a modern derivationof the Michaelis-Menten equation,which includesthe steady-stateassumption introduced by Briggsand Haldane.The derivation starts with the two basic steps of the formation and breakdov,rrof ES (Eqns 6-7 and 6-8). Early in the reaction, the concentrationof the product, [P], is negligible,and we makethe simplifyingassumptionthat the reversereaction,P+S (describedby k_2), canbe ignored.This assumptionis not critical but it simpli_f,es our task. The overallreactionthen reducesto e+sJ\ES

A',E+p

AltEJtSl- ftrtESltsl: (h_, + ftz)[ES] (6-15) Adding the term kltESl[S]to both sidesof the equation and simplifyinggives eltEJtsl: (ftrlsl+ k-r+ &z)[ES]

(6-16)

We then solvethis equationfor [ES]: ertEtltSl

tES]:

& r l s j+ h _ 7 + k 2

(6-17)

This can now be simplif,edfurther, combining the rate constantsinto one expression: tEJtS] l s l + ( f t - 1+ k ) l k r

tES]:

(6-18)

The term (k t + k)/k1is defined as the Michaelis constant,l{-. Substitutingthis into Equation6-18 simplifies the expressionto (6-19)

(6-10)

h 1

7e is determinedby the breakdownof ES to form product, which is determinedby [ES]: Vo : &zlESl

(6-11)

Because[ES]rn Equation6-1 I is not easilymeasuredexperimentally,we must beginby finding an alternativeexpressionfor this term. First, we introducethe term [E,], representingthe total enzlirneconcentration (the sum of free and substrate-bound enzSrme). Free or unbound enz).rnecan then be representedby [Er] - [ES].Also, because [S] is ordinarily far greater than [Er], the amount of substratebound by the enz).rneat any given time is negligiblecomparedwith the total [S].With these conditionsin mind, the following stepslead us to an expressionfor I/s ln terms of easilymeasurableparameters. Step 1 The rates of formation and breakdown of ES are determinedby the stepsgovernedby the rate constantskr (formation)and k_1 * k2 (breakdownto reactants and products, respectively), according to the expresslons Rateof ESformation: ft1([EJ- tES])tSl rc_n) : & rlESl + ftrlESl (6-19) Rateof ESbreakdown Step 2 We now make an important assumption:that the initial rate of reactionreflectsa steadystatein which [ES] is constant-that is, the rate of formation of ES is equal to the rate of its breakdown.This is called the steady-state assumption. The expressionsin Equations6-12 and 6-13 can be equatedfor the steady state,giving e1(tEJ- tEsl)tsl: ft_rlESl+ &rlESl

Step 4 Wecannow express7e in terms of [ES].Substituting the right side of Equation 6-19 for [ES] in Equation 6-11 gives

(6_14)

Step 3 In a series of algebraicsteps, we now solve Equation6-14 for [ES].First, the left side is multiplied out and the right side simplifledto give

(6-20)

This equation can be further simplified. Becausethe maximumvelocity occurswhen the enzyrneis saturated (that is, with [ES] : tEJ V^u*canbe definedas k2[El. Substitutingthis in Equation6-20 givesEquation6-9: V-u* [S] K- + [s]

uo:

This is the Michaelis-Menten equation, the rate equation for a one-substrateenzyme-catalyzed reaction. It is a statementof the quantitativerelationshipbetween the initial velocity Ze, the maximum velocity 7-u,, and the initial substrate concentration [S], all related through the MichaelisconstantK*. Note that Khas units of concentration.Doesthe equationflt experimental observations?Yes;we can confirm this by consideringthe limiting situationswhere [S] is very high or very low,as shownin Figure 6-12. An important numerical relationship emergesfrom the Michaelis-Mentenequationin the specialcasewhen Z6is exactly one-half I/*a,. (Fig. 6-12). Then V-* 2

-

Y** [S] K-+ [S]

$-2r)

On dividing by V^u,, we obtain 1 2

tsl

$-22)

K* + [S]

Solvingfor K-, we get Km + [S] : 2[S],or K- : [S], when Vo :

|,, 2'^*

(6-23)

Mechanism asanApproach t0Understanding Kinetics 6.3Enzyme ]tr) This is a very useful, practical definition of K-: K- is equivalentto the substrateconcentrationat which 7s is one-halftr/max. The Michaelis-Mentenequation (Eqn 6-9) can be algebraicallytransformedinto versionsthat are useful in the practical determination of Km and l/max@ox 6-1) and, as we describelater, in the analysisof inhibitor action (seeBox6-2 on page202).

E

toCompare AreUsed Parameters Kinetic Artivities Enzyme K^ lSl(mu) FIGURt 5-1 2 Dependence of initialvelocityon substrate concentration. Thisgraphshowsthe kineticparameters thatdefinethe limitsof thecurve at highand low [S].At low [S],K. >> [S]andthe [S]termin thedenominatorof the Michaelis-Menten equation(Eqn6-9) becomesinsignificant Theequationsimplifiesto V6 : V-",[S]/K- and Voexhibitsa lineardependence on [S],asobserved here.At high [S],where[S]>> K., the Kterm in the denominator of the Michaelis-Menten eouationbecomesinandtheequationsimplifies with significant to Vo: V-*, thisisconsistent the plateauobserved at high tSl.TheMichaelis-Menten equationistherewith theobserved foreconsistent dependence of V6on [S],andtheshape ofthecurveisdefinedby thetermsV-u,/K- at low [S]and V-". at high[S].

The Michaelis-Menten eouation uo:

y-u* [s] KJ [sl

can be algebraically transformed into equations that are more useful in plotting experimental data. One common transformation is derived simply by taking the reciprocal of both sides of the Michaelis-Menten equation: 1

K-+[S]

vo

v-"* [s]

Separating the components of the numerator on the right side of the equaLiongives I

%

:

K^

It is important to distinguishbetween the MichaelisMenten equationand the speciflckinetic mechanismon which it was originally based. The equation describes the kinetic behaviorof a great many enzyrnes,and all enzymesthat exhibit a hyperbolic dependenceof 7e on [S] are said to follow Michaelis-Menten kinetics. The practical rule that K* : [S] when 7e : l, V^-, TEqn 6-23) holds for all enzymes that follow MichaelisMenten kinetics. (The most important exceptionsto Michaelis-Mentenkinetics are the regulatory enzyrnes, discussedin Section 6.5.) However, the MichaelisMenten equationdoesnot dependon the relatively simple two-stepreactionmechanismproposedby Michaelis

called a Lineweaver-Burkplot, has the great advantage of allowing a more accurate determination of Zmax, which can only be approri'mnted from a simple plot of 7eversuS[S] (seeFig. 6-12). Other transformations of the Michaelis-Menten equationhave been derived, each with someparticular advantagein analyzingenz).rnekinetic data. (SeeProbIem 14 at the end of this chapter.) plot of enzyrnereactionrates The double-reciprocal is very useful in distinguishingbetween certain types of enzymaticreaction mechanisms(see Fig. 6-14) and in analyzingenz),rneinhibition (see Box 6-2).

tsl

y*JSl * u*. lrl

which simplifi.es to lK^1

vo: v*Lsl

-

v*

This form of the Michaelis-Menten equation is called the Lineweaver-Burk equation. For en4.mes obeying the Michaelis-Menten relationship, a plot of 1/7s versus 1/[S] (the "double reciprocal" of the Ze versus [S] plot we have been using to this point) yields a straight line (Fig. 1) . This line has a slope of K^/V^u,, an intercept of llv^u" on the IlVo axls, and an intercept of -1lK- on the 1/[S] axis. The double-reciprocal presentation, also

plot or Lineweaver-Burk 1 A double-reciprocal FIGURE

F"l

Enzymes

and Menten (Eqn 6-10). Many enz;.'rnesthat follow Michaelis-Menten kinetics have quite different reaction mechanisms, and enzymes that catalyze reactions with six or eight identifiabie steps often exhibit the same steady-state kinetic behavior. Even though Equation 6-23 holds true for many enzymes, both the magnitude and the real meaning of Z^u* and K^ can differ from one enzyme to the next. This is an important limitation of the steady-state approach to enzyrne kinetics. The parameters 7,.u" and K- can be obtained experimentally for any given enzyme, but by themselves they provide little information about the number,

Enz5me

Substrate

Hexokinase(brain)

A TTID

ff- (mu)

o-Glucose o-Fructose Carbonicanhydrase

HCOt

Ch;'rnotrypsin

Glycyltyro sinylglycine N-B eruoyltl'ro sinamide

B-Galactosidase Threoninedehydratase

o-Lactose t -Threonine

rates,or chemicalnature of discretestepsin the reaction. Steady-statekineticsneverthelessis the standard languageby which biochemistscompareand characterize the catal;,ticefficienciesof enzl'rnes. Interpreting Z^* and 1{- Figure 6-12 showsa simple graphicalmethod for obtairungan approximatevalue for K-. A more convenientprocedure,usrnga doublereciprocal plot, is presentedin Box 6-1. The K* can vary greatlyfrom enz;.'rne to enzy.rne, and evenfor different substratesof the sameenz),rne(Table6-6). The term is sometimesused (often rnappropriately)as an indicator of the affinity of an enzyrnefor its substrate.The actual meamngof K- dependson specificaspectsof the reaction mechanismsuch as the number and relativerates of lhe individualsteps.For reactionswith two steps, (6-24)

0.4 0.05 1.5 26 108 25 40 5.0

For example, consider the quite common situation where product release,EP -+ E + R is rateJimiting. Early in the reaction (when [P] is low), the overall reaction can be describedby the scheme ht

h2

E+S:ESr-EPi-E+P kr

h3

(6-25)

k2

In this case,most of the enzyrneis in the EP form at saturation,and 7*u" : ke[Et].It is useful to define a moregeneralrate constant,kgs1,to describethe limiting rate of any enzyrne-catalyzedreaction at saturation. If the reactionhas severalsteps and one is clearly rateIimiting, k"u, is equivalentto the rate constant for that limiting step.For the simplereactionof Equation6-10, k"ut : kz For the reactionof Equation6-25, k"4 : ks. When several steps are partially rate-limiting, k.u, can becomea complexfunction of severalof the rate constants that define each individual reaction step. In the Michaelis-Menten equation,k"us: V^n /fBrl, and Equation 6-9 becomes

When k2 is rate-hmiting, k2 11k_, and K- reduces to k-r/kr, which is defined as the dissociation constant, ft""tlEtllSl (6-26) Ka, of the ES complex. Where these conditions hold, KVo= K- + [S] does represent a measure of the affinity of the erz1rynefor its substrate in the ES complex. However, this scenario The constantk"u,is a flrst-orderrate constantand hence does not apply for most enz;.'rnes. Somettmes k2)) k_1, has units of reciprocal time. It is also called the and then K- : k2/ky In other cases,k2 and k_1 are comturnover number. It is equivalent to the number of parable and K- remains a more complex function of all substratemoleculesconvertedto productin a givenunit three rate constants (Eqn 6-24). The Michaelis-Menten of time on a singleenz).rnemoleculewhen the enz}.'rne is equation and the characteristic saturation behavior ofthe saturatedwith substrate.The turnovernumbersof sevenz),Tnestill apply, but K* cannot be considered a srmple eral enz),Tnes are given in Table6-7. measure of substrate affinity. Even more corunon are casesin which the reaction goes through several steps after formation of ES; K^canthen become a very complex fu-rction Enzyme Subshate fto, (s-t) of many raLeconstanls. Catalase Hzoz 40,000,000 The quantity 7-u" also varies greatly Carbonicanhydrase HCOt 400,000 from one enzyrne to the next If an enz).rne Acetylcholinesterase Acetylcholine 14,000 reacts by the two-step Michaelis-Menten mechanism, V^u,: kz[Et], where k2 is rateBenzylpenicillin 2,000 B-Lactamase Iimiting. However, the number of reaction Fumarase Fumarate 800 steps and the identity of the rate-limiting RecA protein (an MPase) ATP 0.5 step(s) can vary from enzyme to enzlrne.

m c sa nA p p r o atcohU n d e r s t a n dMi negc h a n i s ["{ 6 . 3 E n z y mKei n e t i a

Comparing Catalytic Mechanisms and Effi ciencies The kinetic parametersk"u, and K^ are useful for the study and comparisonof different enzymes,whether their reactionmechanismsare simpleor complex.Each enzlirnehasvaluesof k"u,and K- that reflect the cellular environment, the concentration of substrate normally encounteredin vivo by the enzlirne,and the chemistry of the reaction being catalyzed. The parametersk.u, and K* also allow us to evaluate the kinetic efflciency of enzy'rnes,but either parameter alone is insufficient for this task. TWo enz}rmes catalyzingdifferent reactions may have the same k"u, (turnovernumber),yet the ratesof the uncatalyzedreactions may be different and thus the rate enhancements brought about by the enz;,'rnes may differ greatly. Experimentally,the K* for an enzyrnetends to be simiIar to the cellularconcentrationof its substrate.An enzyme that acts on a substratepresent at a very low concentrationin the cell usually has a lower K- than an enzyrnethat acts on a substratethat is more abundant. The best way to comparethe catalytic efficienciesof different enzymes or the turnover of different substrates by the same enzymeis to comparethe ratio k.ut/K^ for the two reactions.This parameter,sometimes called the specificity constant, is the rate constantfor the conversionof E + S to E f P. When [S] O > N > C = S > P = H. For example,the two electronpairs making up a C: O (carbonyl) bond are not sharedequally;the carbonis relatively electrondeficient as the oxygendraws awaythe electrons.Many reactions involve an electron-richatom (a nucleophile)reactingwith an electron-deficientatom (an electrophile).Somecofiimon nucleophilesand electrophilesin biochemistryare shownat right. In general,a reaction mechanismis initiated at an unshared electron pair of a nucleophile.In mechanismdiagrams,the base of the electron-pushingarrow originatesnear the electron-pair dots, and the head of the arrow points direcily at the electrophilic center being attacked.Where the unsharedelectron pair confersa formal negativechargeon the nucleophile,the negative chargesy'rnbolitself can representthe unshareclelectron pair and servesas the baseof the arrow. In the chymotrypsinmechanism,the nucleophilicelectronpair in the ES complexbetween steps@ and @ is provided by the oxygenof the Serr95hyciroxyl group.This electronpat (2 ofthe 8 valenceelectronsofthe hydroxyl oxygen) providesthe baseofthe curved arrow.The eiectrophilic center under attack is the carbonyl carbonof the peptide bond to be cleaved.The C, O, and N atomshave a maximum of 8 valenceelectrons,and H has a madmum of 2 These atomsare occasionallyfound in unstablestateswith less than their maximum allotment of electrons,but C, O, and N cannot havemore than 8. Thus, when the electron pair from ch].'rnotrypsin'sSer195attacks the substrate'scarbonyicarbon,an electron pair is displacedfrom the carbonvalenceshell (you cannot have 5 bonds to carbon!).Theseelectronsmove towarcl the more electronegativecarbonyl oxygen.The oxygenhas g valenceeiectronsboth before and after this chemicalprocess,but the number sharedwith the carbonis reducedfrom 4 to 2, and the carbonylorygen acquiresa negativecharge.In the next step, the electron pair conferringthe negativechargeon the oxygen movesback to re-form a bond with carbonand reestablishthe carbonyllinkage.Again,an electron pair must be displacedfrom the carbon,and this time it is the electron pair sharedwith the amino group of the peptide linkage.This breaksthe pepticte bond. The remainingstepsfollow a similar pattem.

\

H -NSerle5

When substrate binds, the side chain ofthe residue adjacent to the peptide bond to be cleaved nestles in a hydrophobic pocket on the enzyme, positioning the peptide bond for attack.

Enzyrne-product complex

2

1His57

H..Aa N Diffusion of the secondproduct from the active site regenerates free enz;'rne.

Nucleophiles

\zN Hof

Ser1s5

Electrophiles :R

-(, Negatively charged oxygen (as in an unprotonated hydroxyl group or an ionized carboxylic acid)

-) Negatively charged sulfhydryl

-co Carbonatom of a carbonyl group (the more electronegative oxygen of the carbonyl group pulls electrons away from the carbon) :R \+

/ ,c:Nl

- \ ,I I Carbanion -N-

I

H Pronated imine group (activated for nucleophilic attack at the carbon by protonation of the imine)

Uncharged amme group

\'--_ /-\ ff fri*-rzN midazole

O-

t"'

o-

Phosphorus of a phosphate group :R

II-O_ Hydroxide

ion

,p

-O-P:O I

TI+ Proton

Reactions of Enzymatic 6.4 Examples | 209 |

ES complex

Short-lived interrnediate* (acylation)

MECHAiIISM FIGURE 5-21 Hydrolytic cteavageof a peptide bond by chymotrypsin.The reactionhas two phases.In the acylationphase (steps@ to @), formationof a covalentacyl-enzymeintermediate is coupled to cleavageof the peptidebond. In the deacylationphase the free enzyme;this is eslsteps@ to @), deacylationregenerates sentiallythe reverseof the acylationphase,with watermirroring,in rerse,the role of the aminecomponentof the substrate. Chymotrypsin Mechanism

Acyl-enz5rme interrnediate

*The tetrahedralintermediatein the chymotrypsinreactionpathway, intermediate thatformslater,aresometimes and the secondtetrahedral referredto as transitionstates,which can lead to confusion.An lntermediateis any chemicalspecieswith a finitelifetime,"finite"beingdefinedaslongerthanthe time requiredfor a molecularvibration(-10-13 seconds).A transitionstate is simply the maximum-energyspecies formedon the reactioncoordinateand doesnot havea finite lifetime. The tetrahedralintermediatesformed in the chymotrypsinreaction closely resemble,both energeticallyand structurally,the transition statesleadingto their formationand breakdown.However,the intera commiftedstageof completedbond formation, mediaterepresents

Acyl-enzyme internrediate

whereasthe transitionstateis part of the processof reaction.In the case betweenthe intermediate giventhe closerelationship of chymotrypsin, and the actualtransitionstatethe distinctionbetweenthem is routinely the interactionof the negativelycharged glossedover. Furthermore, oxygenwith the amidenitrogensin the oxyanionhole,oftenreferredto alsoservesto stabilizethe intermediate stabilization, asiransition-state areso short-livedthat they resemble in this case.Not all intermediates transitionstates.The chymotrypsinacyl-enzymeintermediateis much morestableand morereadilydetectedand studied,and it is neverconfusedwith a transitionstate.

[rtd

Enzymes

The transition state of a reaction is difflcult to study because it is so short-lived. To understand enzymatic catalysis,however,we must understandwhat occurs during this fleetingmomentin the courseof a reaction. Complementaritybetweenan enzyrneand the transition state is virtually a requirementfor catalysis,becausethe energy hill upon which the transition state sits is what the enz;.'rnemust lower if catalysisis to occur. How can we obtain evidencefor enzy'rne-transitionstate complementarity? Fortunately, we have a variety of approaches,old and new, to addressthis problem, each providing compellingevidencein support of this general principle of en4.'rneaction. Shucture-ActivityConelations If enzymesare complementaryto reaction transition states,then some functional groups in both the substrate and the enzyme must interact preferentially in the transition state rather than in the ES complex. Changingthese groups should have little effect on formation of the ES complexand hence shouldnot affect kinetic parameters (the dissociationconstant,K6; or sometimes K^,1f Kd: K-) that reflectthe E * S=ES equilibrium. Changingthese samegroups shouldhave a largeeffecton the overallrate (k"u,or k"urlK^) of the reaction,however,becausethe bound substratelackspotential binding interactions needed to lower the activation energy. An excellent exampleof this effect is seenin the kinetics associatedwith a seriesof related substrates for the enzymechymotrypsin (Fig. 1). Chymotrypsin normally catalyzesthe hydrolysisof peptide bonds next to aromaticaminoacids The substratesshownin

Figure I are convenientsmallermodelsfor the natural substrates(long polypeptidesand proteins). The additional chemicalgroupsaddedin eachsubstrate(A to B to C) are shaded.As the table shows,the interaction between the enzyme and these added functional groupshas a minimal effect on K- (taken here as a reflection of K) but a large, positive effect on k"u1and k.at/K^. This is what we would expect if the interaction contributed largely to stabilizationof the transition state. The results also demonstratethat the rate of a reaction can be affected greatly by enzyme-substrate interactionsthat are physicallyremote from the covalent bonds that are altered in the enzyme-catalyzed reaction. Chymotrypsin is described in more detail in the text. A complementary experimental approach is to modify the enzyme, eliminating certain enzyme-substrate interactions by replacing specific amino acid residuesthrough site-directedmutagenesis(see Fig. 9-11). Resultsfrom such experimentsagain demonstrate the importance of binding energy in stabilizing the transitionstate. Tlansition-StateAnalogs Even though transition states cannot be obserueddirectly, chemists can often predict the approximate structure of a transition state based on accumulated knowledge about reaction mechanisms.The transition state is by definition transient and so unstable that direct measurementof the binding interaction between this speciesand the enzyme is impossible.In some cases,however,stablemoleculescan be designedthat resembletransition states.Theseare calledtransition-

h.^t

K^

(s -)

(mM)

Substrate A

006

JI

Substrate B

0.14

h.^t (M-

FIGURE 1 Effects of smallsrructural changesin the substrate Substrate C

2.8

It4

on kinetic parameters for chymotrypsin-catalyzed amide hydrolysis.

6.4Examples ofEnzymatic Reactions [tt f

state analogs. In principle, they should bind to an enzymemore tightly than doesthe substratein the ES complex,becausethey shouldfit the active site better (that is, form a greater number of weak interactions) than the substrateitself. The idea of transition-state analogswas suggestedby Paulingin the 1940s,and it has been exploredusing a number of enzymes.These experimentshave the limitation that a transition-state analogcannotperfectly mimic a transition state.Some analogs,however,bind an enzyme 102 to 106 times more tightly than doesthe normal substrate,providing good evidence that enzyme active sites are indeed complementaryto transition states.The sameprinciple is used in the pharmaceuticalindustry to design new drugs. The powerful anti-HIV drugs called proteaseinhibitors were designedin part as tight-binding transition-stateanalogsdirected at the active site of HIV protease. CatalyticAntibodies If a transition-stateanalogcanbe designedfor the reaction S -+ P then an antibody that binds tighily to this analogmight be expectedto catalyzeS -+ p. Antibodies (immunoglobulins;see Fig. 5-21) are key components of the immuneresponse.Whena transition-stateanalog is usedas a protein-boundepitopeto stimulatethe productionof antibodies,the antibodiesthat bind it are potential catalystsof the correspondingreaction.This use of "catalytic antibodies,"first suggestedby William p. Jencksin 1969,has becomepracticalwith the development of laboratorytechniquesto producequantitiesof identical antibodiesthat bind one speciflcantigen (monoclonalantibodies, p. 173). Pioneering work in the laboratories of Richard Lerner and Peter Schultzhasresultedin the isolationof a number of monoclonaiantibodiesthat catalyzethe hydrolysisof estersor carbonates(Fig. 2). In thesereactions,the attackby water (OH-) on the carbonylcarbon producesa tetrahedraltransition state in which a partial negativechargehas developedon the carbonyloxygen. Phosphonateestercompoundsmimic the structureand charge distribution of this transition state in ester hydrolysis, making them good transition-stateanalogs; phosphateester compoundsare usedfor carbonatehydrolysis reactions.Antibodiesthat bind the phosphonate or phosphatecompoundtightly havebeenfound to acceleratethe correspondingesteror carbonatehydrolysisreactionby factorsof 103to lOa Structuralanalyses of a few of these catalytic antibodieshave shown that some catalytic amino acid side chains are arranged such that they could interact with the substratein the transitionstate.

Catalytic antibodies generally do not approach the catalylic efflciency of enzSrmes,but medical and industrial uses for them are nevertheless emerging. For example, catal;'tic antibodies designed to degrade cocaine are being investigated as a potential aid in the treatment of cocaine addiction.

Ester hydrolysis R\

+ u-gH R-ir i o-

Several

stePs -gz

> Products

lr o6 Transition .C)5

R -' n p-

O

state

'R,

05(phosphonate Analog ester) Carbonate hydrolysis

,li,-'-'--o.t

+ Several SICDS j

)

Products

NO^

Transition state

Analog (phosphate ester) FIGURE 2 Theexpectedtransitionstatesfor esteror carbonatehydrolysis reactionsPhosphonate esterand phosphateestercompounds,respectively, makegoodtransition-state analogsfor thesereactions.

? r1

E n z y m es

Hexokinase Undergoes Induced tit on 5ubstrate Binding Yeasthexokinase(M. I07,862) is a bisubstrate enzy.rne that catalyzesthe reversiblereaction Mg.ATP

p-o-Glucose

Glucose 6-phosphate

but in a position where it cannot be phosphorylated. additionof xyloseto the reactionmixture Nevertheless, increasesthe rate of ATP hydrolysis.Evidently, the binding of xyloseis sufflcient to induce a changein hexokinase to its active conformation, and the enzyme is thereby "tricked" into phosphorylatingwater. The hexokinasereaction also illustrates that enzyrnespeciflcity is not alwaysa simple matter of binding one compound but not another.In the caseof hexokinase,speciflcityis observednot in the formation of the ES complexbut in the relative rates of subsequentcatalytic steps.Wateris not excluded from the active site, but reaction rates increase greatly in the presence of the functional phosphorylgroup acceptor(glucose).

ATP and ADP alwaysbind to enz).rnesas a complexwith the metalion Mgz+. The hydroxyl at C-6 of glucose(to which the 7-phosphoryl of ATP is transferredin the hexokinasereaction) is similarin chemicalreactMty to water,and water freely entersthe en4.'rneactive site. Yet hexokinasefavorsthe reactionwith glucoseby a factorof 106.The enzyrnecan discriminatebetween glucoseand water becauseof a conformationalchangein the enz;,rnewhen the correct substratesbinds (Fig. 6-22). Hexokinasethus provides a good example of induced fit. When glucoseis not present,the enz5.'rne is in an inactive conformationwith the active-siteamino acid side chainsout of position for reaction. When glucose (but not water) and Mg . ATP bind, the binding energy derived from this interaction induces a conformationalchangein hexokinaseto the catalfiically activeform. This model has been reinforcedby kinetic studies. The flve-carbon sugar xylose, stereochemicallysimilar to glucosebut one carbonshorter,binds to hexokinase

Induced flt is only one aspect of the cataly'tic mechanism of hexokinase-like chymotrypsin, hexokinase uses several cataly'tic strategies. For example, the active-site amino acid residues (those brought into position by the conformational change that follows substrate binding) participate in general acid-base catalysis and transition-state stabilization.

(a)Hexokinasehasa U-shaped FIGURE 6-22 Inducedfit in hexokinase. (PDBlD 2YHX)(b)Theendspinchtowardeachotherin a constructure

formationalchangeinducedby bindingof o-glucose(red)(derivedfrom .l .l PDBlD HKC and PDBlD CLO.

H" zzo C I H-C-OH I HO-C-H I

H,O \// C

I

H-C-OH I HO-C-H

I I

H-C-OH I

H-C-OH

XyIose

Glucose

CH2OH

H-C-OH I

CH2OH

6.4Examples 0fEnzymatic Reactions Et tl

facilitates Glu211

-o\ \oHo H_NI_H --l'

\_//

o

c

^ | Gluzt I Enolic intermediate

16\

\e

Pa? ,,H

,rc-c:c,,

OH

Lvsa4b -r -

(a)

2-Phosphoglycerate bound to enzyme

Phosphoenolpyruvate

(b) MICHANISM FIGURE 6-23 Two-stepreaction catatyzedby enolase. (a) The mechanismby which enolaseconverts2-phosphoglycerate (2PCA)to phosphoenolpyruvate. Thecarboxylgroupof 2-PCAis coordinatedby two magnesium ionsat theactivesite.(b)Thesubstrate, 2-pCA,

in relationto the Mg2+ions,Lys3as, and Clu2rl in the enolaseactivesite. Nitrogenis shownin blue, phosphorusin orange;hydrogenatomsare not shown(PDBlD lONE).

TheInulast,$leartinn fo4r,chanirrn !{cq*ire:f,4*t-ll$rns

However,in the enzymeactive site, 2-phosphoglycerate undergoes strong ionic interactions with two boundMg2* ions (Fig. 6-23b), makingthe C-2proton more acidic (loweringthe pKr) and easierto abstract. Hydrogen bonding to other active-site amino acid residues also contributes to the overall mechanism. The various interactionseffectivelystabilizeboth the enolateintermediateand the transition state preceding its formation.

Another glycolytic enzFne, enolase,catalyzesthe reversible dehydration of 2-phosphoglycerateto phosphoenolpyruvate:

o*

,ro-

CO

tI tl

H-C-O-P-OHO-CH2

O

2-Phosphoglycerate

ox .ro CO ttl c-o-P-olll cH,

+ HrO

o-

Phosphoenolpytuvate

Yeast enolase(M. 93,316) is a dimer with 436 amino acid residuesper subunit. The enolasereaction illustrates one type of metal ion catalysis and provides an additional example of general acid-base catalysisand transition-statestabilization.The reaction occursin two steps (Fig. 6-2:Ja). First, Lys3ab acts as a general base catalyst, abstracting a proton from C-2 of 2-phosphoglycerate; then GIu211 acts as a general acid catalyst, donating a proton to the -OH leavinggroup. The proton at C-2 of 2-phosphoglycerate is not very acidic and thus is not readily removed.

[hesTwoSuccessive Lysurynre Nucleophilic fieactions [}isplacement Lysozymeis a natural antibacterialagent found in tears and eggwhites. The hen eggwhite lysozyme(M,14,296) is a monomerwith 129aminoacid residues.This was the first erzyme to have its three-dimensionalstructure determined, by David Phillips and colleaguesin 1965.The structure revealed four stabilizing disul-fldebonds and a cleft containingthe active site (FiS. 6-24a). More than flve decadesof investigationshave provided a detailed picture of the structure and activity of the enz5.'rne, and an interesting story of how biochemical science progresses.

?ra

Enzymes

I o I

GlcNAc

RO = CHgCHCOO-

NAc\

NAdAcN = -NH-C-CH3 tl

o

\* Mur2Ac Hydrogen bonds

\\ 1'Mur2Ac

FIGURE 6-24 Hen egg white lysozymeand the reaction it catalyzes. (a) Ribbondiagramof theenzymewith the active-site residues Clursand Asps2shownas blue stickstructures shownin red and boundsubstrate (PDBlD l LZE).(b) Reaction A segcatalyzedby heneggwhitelysozyme. mentof a peptidoglycan polymeris shown,with the lysozymebinding sitesA throughF shaded.The glycosidicC--{ bond betweensugar residues boundto sitesD and E is cleaved,asindicatedby the redanow. Thehydrolyticreactionis shownin the inse! with the fateof the oxygen in the HrO tracedin red.Mur2Acis N-acetylmuramic acid;ClcNAc,Nacetylglucosamine. RO- represents a lactyl(lacticacid)group;-NAc and AcN-, an N-acetylgroup(seekey).

The substrate of lysozyme is peptidoglycan,a carbohydratefound in many bacterial cell walls (see Fig. 20-31). Lysozymecleavesthe (B1-+4) glycosidic C---O bond (see p. 243) between the two types of sugar residue in the molecule,N-acetylmuramicacid (Mur2Ac) and N-acetylglucosamine(GlcNAc), often referred to as NAM and NAG, respectively,in the research literature on enzymology(Fig. 6-24b). Six residues of the alternating Mur2Ac and GIcNAc in peptidoglycanbind in the active site, in binding sites Iabeled A through F. Model building has shown that the lactyl side chain of Mur2Ac cannot be accommodated in sites C and E, restricting Mur2Ac binding to sites B, D, and F. Only one of the bound glycosidic bonds is cleaved,that between a Mur2Ac residue in site D and a GlcNAc residue in site E. The key catalytic amino acid residuesin the active site are Glu35 and Asp52(Fig. 6-25a). The reaction is a nucleophilic substitution, with -OH from water replacing the GlcNAcat C-1 of Mur2Ac. With the activesite residuesidentifiedand a detailed structure of the enz).rneavailable,the path to understanding the reaction mechanismseemedopen in the 1960s.However, definitive evidence for a particular mechanismeludedinvestigatorsfor nearly four decades.

,oH$

Mur2Ac

+

There are two chemicallyreasonablemechanismsthat could generatethe observedproduct of lysozlmre-mediated cleavageof the glycosidic bond. Phillips and colIeaguesproposeda dissociative(Spl-type) mechanism (Fig. 6-25a, left), in which the GIcNAcinitially dissociates in step @ to leavebehind a glycosylcation (a carbocation)intermediate.In this mechanism,the departing GlcNAcis protonatedby generalacid catalysisby Glu35, located in a hydrophobicpocket that gives its carboxyl group an unusuallyhigh pK,. The carbocationis stabilized by resonanceinvolvingthe adjacentring oxygen,as well asby electrostaticinteractionwith the negativechargeon the nearby A.pu'. In step @, water attacksat C-1 of Mur2Ac to yield the product. The alternativemechanism (Fig. 6-25a, right) involves two consecutive directdisplacement(Sp2-t1pe)steps.In step @, Asp52attacks C-l of Mur2Ac to displacethe GIcNAc.As in the first mechanism,Glu35acts as a generalacid to protonatethe departingGlcNAc.In step@, water attacks at C-l of Mur2Ac to displacethe Asp52and generateproduct. was widely accepted The Phillips mechanism(Sr.11), for more than three decades.However,somecontroversy persistedand tests continued.The scientiflcmethod sometimesadvancesan issueslowly,and a truly tnsightfitl experiment can be difficult to design. Some early

6 . 4E x a m p loefsE n z y m aRt ieca c t i o n[ st t u ]

w

Peptidoglycan binds in the active site of lysozyme

mechanism

Sr2 mechanism

Glu35 A rearrangement produces a glycosyl carbocation. General acid catalysis by Glu52 protonates the displaced GlcNAc oxygen and facilitates its departure.

Asp52 acts as a covalent catalyst, directly displacing the GlcNAc via an Sy2 mechanism. Glu35 protonates the GlcNAc to facilitate its departure.

AcN

-otro H Lysozyme I Asp52

cE

Glu35

Glu35

I ("o-

I (to-

Glycosyl carbocation interrnediate

-o'.zro T Aspsz

Arp52

6strH,O :l

6:V-H'O :I

Glu35

Glu35

General base catalysis by Gls35 facilitates the attack of water on the glycosyl carbocation to form product.

Glu35 acts as a general base catalyst to facilitate the 5112 attack ofwater, displacing Asp52 and generating product.

AcN Y Aspsz

Asp52

Glu 35

I (to I

H

"Y"

Covalent intermediate bound in the active site

Asnsz (a) l,lE(HANlSM FIGURE6-25 Lysozyme reaction.In this reaction(described in the text),the waterintroducedintothe productat C-.1of Mur2Acis in the sameconfiguration as the originalglycosidicbond.The reactionis (a)Twoprothusa molecularsubstitution with retention of configuration. posedpathways potentially explaintheoverallreactionand itsproperties. The 511 pathway(left)is the originalPhillipsmechanism. The S*2

(b) with cunentdata.(b)A pathway(righ0is the mechanism mostconsistent of the lysozymeactivesitewith the covalentenzymesurfacerendering structureSidechainsof intermediate shownasa ball-and-stick substrate protruding from structures residues areshownasball-and-stick active-site ribbons(PDBlD 1H6M).

itr4

Enzymes

argumentsagainstthe Phillips mechanismweresuggestive but not completelypersuasive.For example,the half-Jife of the proposedglycosylcationwasestimatedto be 10-12 seconds,just longer than a molecularvibration and not long enoughfor the neededdjffusionof other molecules. More important, lysoz;rmeis a memberof a family of enzyrnescalled "retaining glycosidases,"all of wluch catalyze reactions in which the product has the same anomericconfigurationas the substrate(anomericconflgurationsof carbohydratesare examinedin Chapter7), and all of which are known to have reactivecovalentintermediateslike that envisionedin the alternative(Sy2) pathway.Hence,the Phillips mechanismran counter to experimentalfindingsfor closelyrelated enz).rnes. A compellingexperimenttipped the scalesdecidedly in favor of the Sy2 pathway, as reported by StephenWithersand colleaguesin 2001.Makinguse of a mutant enzyme(with residue 35 changedfrom GIu to Gln) and artif,cial substrates,which combinedto slow the rate of key steps in the reaction, these workers were ableto stabilizethe elusivecovalentintermediate. This in turn allowedthem to observethe intermediate directly, using both mass spectrometry and x-ray crystallography(Fig 6-25b). Is the lyso4rmemechanismnow proven?No. A key featureof the scientificmethod,asAlbert Einsteinonce summarizedit, is "No amount of experimentation can ever prove me right; a single experiment can prove me wrong." In the case of the lysozymemechanism,one might argue (and some have) that the artificial substrates, with fluorine substitutions at C-1 and C-2, that were used to stabilizethe covalentintermediate might have altered the reaction pathway. The highly electronegativefluorine could destabilizean already electron-deficientoxocarbeniumion in the glycosyl cation intermediatethat might occur in an SN1pathway. However,the SN2pathway is now the mechanismmost in concert with availabledata.

)"... t--Ala

H"N-(GlvL-N'" ' H

l-

c:o

Peptidoglycan chain 1

l--aa

L-ru4

o-Glu

n-Glu

tl ll

c:o I

Ser

)n

..

.

)o'..

L-AIA

l--Ala

o-Glu

o-Glu

I

AnUnderstanding of Enzyme Mechanism lmportant Drives Advances inMedicine

t,

I

{Gly)5-(t--Lys) -(o-AJa) -(GIy)5-

(t--L, ys)I o-Ala

I

The drugs used to treat maladies ranging from headacheto HIV infection are almost always inlubitors of an enz5,.rne. TWo examples are explored here: the antibiotic penicilJrn (and its derivatives) and the protease inhibitors used to treat HIV mfections, all of which are irreversible hhibitors. Penicillin was discovered in 1928 by Alexander Fleming, but it took another 15 years before this relatively unstable compound was understood well enough to use it as a pharmaceutical agent to treat bacterial infections. Penicillin interferes with the synthesis of peptidoglycan (described in Chapter 20, Fig. 20-32), the major component of the rigid cell wall that protects bacteria from osmotic lysis. Peptidoglycan consists of polysaccharides and peptides cross-linked in several steps that include a transpeptidase reaction (Fig. 6-26). It is

I I (l-Lvs) o-Glu

o-Ala Cross-linked peptidoglycan

O

N-Acetylglucosamine (} (GlcNAc)

N-Acetylmuramic acid (Mur2NAc)

FIGURE 6-26 The transpeptidase reaction.This reaction,which links precursors two peptidoglycan into a largerpolymer,is facilitatedby an active-site Seranda covalentcatalysis mechanism similarto thatof chymotrypsin.Note that peptidoglycanis one of the few placesin nature where o-aminoacid residuesarefound.The active-site Serattacksthe carbonylof the peptidebond betweenthe two o-Ala residues, creating a covalentesterlinkagebetweenthe substrate and the enzymewith releaseof the terminalo-Ala residueAn amino groupfrom the second peptidoglycanprecursorthen aftacksthe esterlinkage,displacingthe enzymeand cross-linking the two precursors.

?,4

6 . 4 E x a m p loefsE n z y m aRt iec a c t i o n s

(

\/ '

Side chain

Thiazolidine

,FCH"-

Penicillin G (benzylpenicillin)

ring

OHH i

ll

i

n -i-N--c

HIIC c

O/;'"

o-cH2-

e-"t ,cH. N

"-qH

R groups

/'CHs

Penicillin V

i

0-LactamCooH nng

NHz Amoxicillin General structure of penicillins (a)

OHH

n-8-N--C-g-sr zcH' Htp" N-"7t'cHs -s".-o-t\ o cooH Stably derivatized, inactive transpeptidase ft) FIGURE 6-27 Transpeptidase inhibition by B-lactamantibiotics.(a) featurea five-membered thiazolidineringfused B-Lactamantibiotics to a four-membered B-lactamring.The latterring is strainedand includesan amidemoietythatplaysa criticalrole in the inactivation of peptidoglycan synthesis. The R groupvariesin differentpenicillins PenicillinC was the firstto be isolatedand remainsone of the most effective, but it is degraded by stomachacidand mustbe administered

and is acid stable,so it by injection.PenicillinVis nearlyaseffective orally.Amoxicillinhas a broadrangeof effeccan be administered is readilyadministered orally,and is thus the mostwidely tiveness, prescribed B-lactamantibiotic.(b) Attackon the amidemoietyof the active-site Serresultsin a covalent B-lactamring by a transpeptidase product.Thisis hydrolyzed so slowlythatadductformaacyl-enzyme is inactivated. tion is practicallyirreversible, and the transpeptidase

this reaction that is inhibited by penicillin and related compounds(Fig. 6-27a), all of which mrmic one conformation of the n-Ala-D-Ala segmentof the peptidoglycanprecursor.The peptide bond in the precursoris replacedby a highly reactiveB-lactamring. When penicillin binds to the transpeptidase, an active-siteSer attacksthe carbonylof the BJactamring and generatesa covalent adduct between penicillin and the enzyme. However,the leaving group remains attached because it is linked by the remnant of the B-lactam ring (Fig. 6-27b). The covalent complex irreversibly inactivates the enz),rne.This, in turn, blocks syrrthesisof the

bacterial cell wall, and most bacteria die as the fragile inner membraneburstsunder osmoticpressure. Human use of penicillin and its derivativeshas Ied to the evolutionof strains of pathogenicbacteriathat express p-lactamases (Fig. 6-28a), enzymesthat cleaveB-lactam antibiotics,rendering them inactive. The bacteria thereby become resistant to the antibiotics.The genesfor theseenzymeshavespreadrapidly through bacterialpopulationsunder the selectivepressure imposedby the use (and often overuse)of B-lactam antibiotics.Human medicine respondedwith the developmentof compounds such as clavulanic acid, a

[r.r4 Enzymes OH

Clavulanic acid

o

J :-t-'/*

HH

R-C-N--C HI

;ZS

o R-C

N> H

o' Inactive penicillin H\

(a) FIGURE 6-28 p-Lactamasesand p-lactamaseinhibition. (a) PLactamases promotecleavage of theB-lactamring in B-lactamantibiotics, inactivating them. (b) Clavulanicacid is a suicideinhibitor, makinguseof the normalchemicalmechanism of B-lactamases to createa reactivespeciesat the activesite.Thisreactivespeciesis attacked by groupsin the activesiteto irreversibly acylatethe enzyme. suicide inactivator, which irreversibly inactivates the Blactamases (Fig. 6-28b). Clavulanic acid mimics the

structure of a B-lactam antibiotic, and forms a covalent adduct with a Ser in the B-lactamase active site. This leads to a rearrangement that creates a much more reactive derivative, which is subsequently attacked by another nucleophile in the active site to irreversibly acylate the en4.rne and inactivate it. Amoxicillin and clamlanic acid are combined in a widely used pharmaceutical formulation with the trade name Augmentin. The cycle of chemical warfare between humans and bacteria continues unabated. Strains of disease-causing bacteria that are resistant to both amoxicillin and clavulanic acid (reflecting mutations in B-lactamase that render it unreactive to clamlanic acid) have been discovered. The development of new antibiotics promises to be a growth industry for the foreseeable future. Antiviral agents provide another example of modern drug development. The human immunodeflciency virus (HIV) is the causative agent of acquired immune defi.ciency sy.ndrome,or AIDS. In 2005, an estimated 37 to 45 million people worldwide were living with HIV infections, with 3.9 to 6.6 million new infections that year and more than 2.4 million fatalities. AIDS flrst surfaced as a world epidemic in the 1980s; HIV was discovered soon after and identified as a retrovirus. Retroviruses Dos-

-H

ser-o

O.

.CH2OH

\_i" t,-L.H

NT /,,N-Cfi

\H

\--

i

cooH

Inactive B-lactamase

ft)

sessan RNA genomeand an enzyrne,reversetranscriptase,capableof using RNA to direct the synthesisof a complementaryDNA. Efforts to understandHIV and develop therapies for HIV infection benefited from decadesofbasicresearchon other retroviruses.A retrovirus such as HIV has a relatively simple life cycle (see Fig. 26-33). Its RNA genomeis convertedto duplex DNA in severalsteps catalyzedby a reverse transcriptase (describedin Chapter26). The duplexDNA is then insertedinto a chromosomein the nucleusof the host cell by the enz;'rneintegrase(describedin Chapter25). The integratedcopy of the viral genomecan remain dormant indefinitely. Alternatively, it can be transcribed back into RNA, which can then be translated into proteins to construct new virus particles. Most of the viral genesare translated into large polyproteins,which are cut by the HIV proteaseinto the individual proteins needed to make the virus (see Fig. 26-34). There are only three key en4,rnesin this cycle-the reversetranscriptase,the integrase,and the protease-which thus are the potentialdrug targets.

6 . 4E x a m p l e s o f E n z y m a t i c[R r tel a c t i o n s Aided by general

The tetrahedral

o=.,/ "1 Peptides

.

ruv

GC-

l,rulgase

FIGURE 6-29 Mechanismof action of HIV protease.Two active-site (fromdifferentsubunits) Asp residues act asgeneralacid-base catalysts,

There are four major subclassesof proteases.Serine proteases,such as chymotrypsinand trypsin, and cysteineproteases(in which Cys servesa catalyticrole similarto that of Serin the activesite) featurecovalent enzyme-substratecomplexes;aspartyl proteasesand metalloproteasesdo not. The HIV proteasers an aspartyl protease.Two active-siteAsp residuesfacilitatea direct attackofwater on the peptidebond to be cleaved (Fig. 6*29). The initial product of the attack of water on the carbonylgroup of the peptidebond to be cleaved is an unstable tetrahedral intermediate,much as we haveseenfor the chymotrypsinreaction.This intermediate is close in structure and energy to the reaction transitionstate.The drugsthat havebeendevelopedas HIV proteaseinhibitors form noncovalentcomplexes with the enzyme,but they bind to it so tightly that they can be consideredirreversible inhibitors. The tight binding is derived in part from their designas transition-stateanalogs(seeBox 6-3). The successof these drugs makesa point worth emphasizing.The catalytic principleswe have studiedin this chapterare not simply abstruseideasto be memorized-their application saveslives. The HIV proteasecleavespeptide bonds between Phe and Pro residues most efficiently. The active site thus has a pocket to bind aromatic groups next to the bondto be cleaved.The structuresof severalHIV protease inlubitorsare shownin Figure 6-30. Althoughthe structures appear varied, they all share a core structure-a main chain with a hydroxyl group positionednext to a branch containing a berzyl group. This arrangementtargetsthe berzyl groupto the aromaticbindrngpocket.The adjacenthydroxyl group mimics the negativelycharged oxygenin the tetrahedralintermediatein the normalreaction, providinga transition-stateanalog.The remainderof eachinhibitor structurewas designedto flt into and bind to variouscrevicesalongthe surfaceof the enz5,'rne, enhancing overall binding. Avaitability of these effective drugshasvastly increasedthe lifespanand quality of life of millionsof peoplewith HIV and AIDS.r

facilitatingthe attackof wateron the peptidebond.The unstabletetra hedralintermediate in the reactionpathwayis highlighted in pink.

'HrSOa

Indinavir

Nelfrnavir

HeC

CHa

Lopinavir

H N

o

H NC(CH3)3

Saquinavir

FIGURE 6-30 HIVprotease inhibitors. Thehydroxyl group(red)actsas a transition-state analog, mimicking theoxygen interof thetetrahedral mediate Theadjacent benzylgroup(blue)helpsto properly position the d r u g i n t h e a c t i v es i t e

Fttl

Enzymes

e fsE n z y m a t i c S U M M A R6Y. 4 E x a m p l o Reactions r

Ch;'rnotrypsinis a serineproteasewith a well-understoodmechanism,featuring general acid-basecatalysis,covalentcatalysis,and transition-statestabilization.

r

Hexokinaseprovidesan excellentexampleof induced flt as a meansof using substratebinding energy.

r

The enolasereactionproceedsvia metal ion catalysis.

r

makesuse of covalentcatalysisand Lysoz;..rne generalacid catalysisas it promotestwo successive nucleophilicdisplacementreactions.

r

mechanismallowsthe Understandingenz),.rne developmentof drugs to inhibit enz),rneaction.

6.5 Regulatory Enzymes work together In cellularmetabolism,groupsof enz5.'rnes in sequentialpathways to carry out a given metabolic process,suchasthe multireactionbreakdov,nof glucose to lactate or the multireactionsynthesisof an aminoacid from simplerprecursors.In such enz).rnesystems,the reactionproduct of one enzyrnebecomesthe substrate of the next. Most of the erzyrnesin eachmetabolicpathwayfolIow the kinetic patternswe havealreadydescribed.Each pathway,however,includes one or more enz),Tnes that have a greatereffect on the rate of the overallsequence These regulatory enzJrrnesexhibit increased or decreasedcatalytic activity in responseto certain signals. Adjustmentsin the rate of reactionscatalyzedby regulatory erz;.rnes,andthereforein the rate of entiremetabolic sequences,allow the cell to meet changingneedsfor enrequrredin growth and repair. ergy and for biomolecr.rles In most multienzSrmesystems,the flrst enz;rmeof the sequenceis a regulatoryenzyme.This is an excellent place to regulate a pathway, because catalysisof eventhe flrst few reactionsof a sequencethat leadsto an unneededproduct diverts energy and metabolites from more important processes.Other enzymesin the sequencemay play subtler roles in modulating the flux througha pathway,as describedin Chapter15. The activities of regulatory enzlrnes are modulated in a variety of ways. Allosteric enzJflnes function through reversible, noncovalent binding of regulatory compoundscalledallosteric modulators or allosteric effectors, which are generally small metabolites or cofactors.Other erz;'rnesare regulatedby reversiblecovalent modification. Both classesof regulatory enzyrnestend to be multisubunit proteins, and in some casesthe regulatorysite(s) and the activesite are on separate subunits.Metabolicsystemshaveat leasttwo other mechanismsof enzyme regulation. Some enz}.'rnesare stimulatedor inhibited when thev are bound bv senarate

regulatory proteins. Others are activated when peptide segmentsare removedby proteolfiic cleavage;unlike effector-mediated regulation, regulation by proteoly[ic cleavageis irreversible. Important examples of both mechanismsare found in physiologicalprocessessuchas digestion,blood clottng, hormoneaction,and vision. Cell growth and survivaldependon efficient use of resources,and this efficiencyis madepossibleby regulatory enzymes.No single rule governs the occurrence of drfferent types of regulation in different systems.To a degree,allosteric (noncovalent)regulation may permit fine-tuning of metabolic pathways that are required continuouslybut at different levels of activity as cellular conditionschange.Regulationby covalentmodificationmay be all or none-usually the case with proteolytic cleavage-or it may allow for subtle changesin activity. Severaltypes of regulation may occur in a singleregulatoryenzyme.The remainder of this chapter is devotedto a discussionof these methodsof enzymeregulation.

(hanges (onformational in Undergo Inzymes Allosteric Binding Response toModulator As we sawin Chapter5, allostericproteinsare thosehaving "other shapes"or conformationsinducedby the bindrLg of modulators.The sameconcept appliesto ceftain as conformationalchangesmduced regulatoryenzliTnes, by one or more modulatorsinterconvefi more-activeand Iess-activeforms of the erzyme. The modulatorsfor alIosteric enz).rnesmay be inhibitory or sttmulatory.Often the modulatoris the substrateitself; regulatoryenzyrnes are identicalare called for which substrateand modr.rlator is to that of 02 binding to The effect similar homotropic. (Chapter of the ligand-or sub5): binding hemoglobin strate, in the case of enz).rnes-causesconformational changesthat affect the subsequentactivity of other sites on the protein. When the modulatoris a moleculeother than the substrate,the enz;rmeis saidto be heterotropic. Note that allostericmodulatorsshould not be confused with uncompetitiveand mixed inhibitors. Although the latter bind at a secondsite on the erz;rme,they do not necessarilymediateconformationalchangesbetweenactive andinactiveforms,andthe kinetic effectsare distinct. The propertiesof allostericenzyrnesare significantly different from those of simple nonregulatoryelz)trnes. Someof the differencesare structural. In addition to active generallyhaveone or more regusites,allostericenz)trnes Iatory or allosteric,sitesfor bindqg the modulator(Fig. 6-31). Just as an enz;.'rne'sactive site is specific siteis spectic for its modfor its substrate,eachregr.ilatory ulator. Enz;rmeswith severalmodulatorsgenerallyhave different specificbindlngsitesfor each.In homotropicensite are the same. zyrnes,the activesite and regr-rlatory Allosteric enzyrnesare generallylarger and more complexthan nonallostericenz;rmes.Most havetwo or more subunits. Aspartate transcarbamoylase,which catalyzesan early reaction in the biosynthesisof pyrim-

6.5Regulatory Enzymes frr:_|

F

Srbstrate

ffi Positivemodulator

-*lf.* E>

(1.

V

Less-active enz;nne

InManyPathways, Regulated Steps AreCatalyzed by Allosteric Inzymes

R More-active enzyme

1l c

R

idine nucleotides(see Fig.22-36), has 12 polypeptide chainsorganizedinto catall'tic and regulatory subunits. Figure 6-32 shows the quaternary structure of this enzyme,deducedfrom x-ray analysis.

Active enzyme-substrate complex

FIGURt6-31 Subunitinteractionsin an allostericenzyme,and interactionswith inhibitorsand activators.In manyallostericenzymesthe substrate bindingsiteandthe modulatorbindingsite(s) areon different subunits,the catalytic(C) and regulatory(R) subunits,respectively. Bindingof the positive(stimulatory) modulator(M) to its specificsite on the regulatory subunitis communicated to the catalyticsubunit througha conformational change.Thischangerendersthe catalytic subunitactiveand capableof bindingthe substrate (S)with higher affinity.On dissociation of the modulatorfromthe regulatory subunit, the enzymerevertsto its inactiveor lessactiveform.

In somemultierzgne systems,the regulatory enzlirnesare specificallyinhibited by the end product of the pathway wheneverthe concentrationof the end product exceeds the cell'srequtements. When the regr.rlatoryenzyrnereactionis slowed,subsequentenzJ,rnes may operateat different ratesastheir substratepoolsare depleted.The rate of production of the pathway'send product is thereby brought into balancewith the cell's needs.This type of regulation is called feedback inhibition. Buildup of the end product nltimately slowsthe entfe pathway. One of the first known examplesof allosteric feedbackinhibition wasthe bacterialenz),rnesystemthat catalyzesthe conversionof r,-threonineto l-isoleucine in five steps (Fig. 6-33). In this system,the first enzyrne, threonine dehydratase,is inhibited by isoleucine,the product of the last reaction of the series.This is an example of heterotropic allosteric inhibition. Isoleucineis quite specific as an inhibitor. No other intermediate in this sequenceinhibits threonine dehydratase,nor is any other enz;..rnein the sequenceinhibited by isoleucine. Isoleucinebinds not to the active site but to anotherspeci-flcsite on the enzyrnemoleclile, the regulatory site. coo-

+l H3N-C-H | H-C-OH

r,-Threonine

I

CHs

/--8 ]' ll;1T,,:;"" A

Il E .

J B

I

lFl

J c I

FIGURE 6-32 Two viewsof the regulatoryenzymeaspartatetranscar(Derivedfrom PDBlD 2AT2.)Thisallostericregulatoryenbamoylase. zyme has two stackedcatalyticclusters,each with three catalytic polypeptide chains(inshades of blueandpurple),andthreeregulatory clusters,eachwith two regulatorypolypeptidechains(in red and yelIow).Theregulatory clusters form the pointsof a trianglesurrounding thecatalyticsubunits. Bindingsitesfor allosteric modulators areon the regulatory subunits. Modulatorbindingproduceslargechanges in enzymeconformation andactivity. Theroleof thisenzymein nucleotide synthesis, and detailsof its regulation, arediscussed in Chapter22.

lo J D

I

lH.

J cooI

Hrfr-c-H \__H_

I

?-""r CH"

t-

CHs

FIGURE 6-33 Feedbackinhibition. The conversionof r-threonine to r-isoleucineis catalyzed by a sequenceof five enzymes(E1 (E1) to E5).Threoninedehydratase is specifically inhibitedallosterically by r-isoleucine,the end productof the sequence,but not by any of the four intermediates (A to D). Feedbackinhibitionis indicatedby the dashedfeedback r.-Isoleucine line and the $ symbol at the threonine dehydratasereaction arrow a device usedthroughout thisbook

'222

Enzymes

This binding is noncovalent and readily reversible; tf the isoleucine concentration decreases,the rate of threonine dehydration increases. Thus threonine dehydratase actMty responds rapidly and reversibly to fluctuations in the cellular concentration of isoleucine. As we shall see in Part II of this book, the patterns of regulation h many other metaboiic pathways are much more complex.

I

from [nzymes $iuerge Fr*perties ofAllcsterir TheKinetic Behavior Miehaeiis-l'{enten Allostericenz).rnesshow relationshipsbetween7o and kinetics.They do [S] that differ from Michaelis-Menten exhibit saturation with the substrate when [S] is sufflplots of 76 ciently high, but for someallostericenzJ,/rnes, versusISI (Fig. 6-3+) producea sigmoidsaturation curve, rather than the hyperbolic curve tlpical of nonregulatory enz).nnes. On the sigmoidsaturationcurvewe can find a value of [S] at which 70 is half-maximal,but we cannotrefer to it with the designationK^, because the enz).ryne does not follow the hyperbolic MichaelisMentenrelationship.Instead,the symbol[S]os or Ko r is often usedto representthe substrateconcentrationgiving half-maximalvelocity of the reaction catalyzedby an allostericenzyrne(Fig. 6-34). Sigmoidkmetic behaviorgenerallyreflects cooperative interactions between protein subunits. In other words, changesrn the structure of one subunit are transIated into structural changesin adjacentsubunits,an effect mediatedby noncovalentinteractionsat the interface betweensuburLits.The principlesare particularlywell ilIustratedby a nonerulrne:O2bindingto hemoglobinStgmoid krretic behavioris explaned by the concertedand sequentialmodelsfor subunitinteractions(seeFig. 5-15). Homotropic allosteric enzymesgenerallyare multisubunitproteinsand,as noted earlier,the samebinding site on eachsubunit functions asboth the active site and the regulatorysite. Most commonly,the substrateacts as a positivemodulator(an activator),becausethe subunits act cooperatively:the binding of one moleculeof substrateto one binding site alters the enzyrne'sconformation and enhancesthe binding of subsequentsubstrate molecules.This accountsfor the sigmoidrather than hyperbolic change in 7e with increasing [S]. One characteristicof sigmoid kinetics is that small changes in the concentrationof a modulatorcan be associated with large changesin activity. As is evident in Figure 6-34a,a relativelysmallincreasein [S]in the steeppart of the curve causesa comparativelylarge increasein 20. For heterotropic allosteric enzymes,those whose modulatorsare metabolitesother than the normal substrate, it is difflcult to generalizeabout the shapeof the substrate-saturationcurve. An activator may causethe curveto becomemore nearlyhyperbolic,with a decrease it Kos but no changein 7-u", resulting in an increased reactionvelocity at a fixed substrateconcentration(70 is higher for any value of [S]; FiC. 6-34b, upper curve). Other heterotropic allosteric enzyrnesrespond to an

Kos lSl(mu) (a)

E

KP5 Kos

KPs lSl (mtu) ft)

E

Kou lsl(mM) (c) FIGURI6-34 Substrate-activity curvesfor representativeallosteric of allostericenzymes enzymes.Threeexamplesof complexresponses (a)Thesigmoidcurveof a homotropicenzyme,in to their modulators. modulator,or alsoservesasa positive(stimulatory) which the substrate curveof heto the oxygen-saturation activator.Note the resemblance moglobin(seeFig.5-12). (b)Theeffectsof a positivemodulator(+) and enzymein which Ko5is almodulator(-) on an allosteric a negative teredwithouta changein V-u*.Thecentralcurveshowsthe substrate(c) A lesscommontype of withouta modulator. activityrelationship in which V-n" is alteredand K65is nearlyconstant. modulation,

activator by an increase in 7^." with Iittle change in Ko s

(Fig. 64ac). A negativemodulator (an inhibitor) may producearnore sigmoidsubstrate-saturationcurve,with an increase in Kor @ig. 6-34b, lower curwe). Heterotropic allosteric enzyrnestherefore show different kinds of responsesin their substrate-activitycurves, becausesome have inhibitory modulators,some have activatingmodulators,and somehaveboth.

6.5Regulatory Enzymes fr{

5ome Inzymes AreReguiated byReversible (ovalent Modifieation In another important ciass of regulatory enz)rmes,activity is modulated by covalent modiflcation of one or more of the amino acid residues in the enzJ..rnemolecule. Over 500 different types of covalent modiflcation have been found in proteins. Common modifying groups include phosphoryl, acetyl, adenylyl, uridylyl, methyl, amide, carboxyl, myristoyl, palmitoyl, prenyl, hydroxyl, sulfate, and adenosine diphosphate ribosyl groups (FiS. 6-Bb). There are even entire proteins that are used as specialized modifying groups, including ubiquitin and sumo. These varied groups are generally linked to and removed from a regulated enz).rne by separate enzymes. When an amino acid residue in an enz}.'rneis modifled, a novel amino acid with altered properties has effectively been introduced into the enzyme. Introduction of a charge can alter the local properties of the enzyme and induce a change in conformation. Introduction of a hydrophobic group can trigger association with a membrane. The changes are often substantial and can be critical to the function of the altered enz).rne. The variety of enzJ,ryne modifications is too great to cover in detail, but some examples can be offered. An example of an enzyme regulated by methylation is the methyl-accepting chemotaxis protein of bacteria. This protein is part of a system that permits a bacterium to swim toward an attractant (such as a sugar) in solution and away from repellent chemicals. The methylating agent is S-adenosylmethionine (adoMet) (see Fig. 18-18). Acetylation is a common modiflcation, with approximately 80% of the soluble proteins in eukaryotes, including many enzyrnes,acetylated at their amino termini. Ubiquitin is added to proteins as a tag that predestines them for proteolytic degradation (see Fig. 27-47). Ubiquitination can also have a regulatory function. Sumo is found attached to many eukaryotic nuclear proteins with roles in the regulation of transcription, chromatin structure, and DNA repair. ADP-ribosylation is an especially interesting reaction, observed in a number of proteins; the ADP-ribose is derived from nicotinamide adenine dinucleotide (NAD) (see Fig. 8-38). This tyLoeof modif,cation occurs for the bacterial enz)..medinitrogenase reductase, resulting in regulation of the important process of biological nitrogen fixation. Diphtheria toxin and cholera toxin are enzlrnes Ihat catalyze the ADP-ribosylation (and inactivation) of key cellular enzyrnes or proteins. Phosphorylation is probably the most important type of regulatory modiflcation. It is estimated that onethird of all proteins in a eukaryotic cell are phosphorylated, and one or (often) many phosphorylation events are part oflertually every regulatory process. Some proteins have only one phosphorylated residue, others have several, and a few have dozens of sites for phosphorylation. This mode of covalent modification is central to a large number of regulatory pathways, and we therefore

Covalent modifrcation (target residues) Phosphorylation (Tyr, Ser, Thr, His) ATP ADP

o

) I

o Adenylylation (Tyr) ATP

PP,

llnz.

o

o"'-l*iy

Acetylation (Lys, a-amino (amino terminus)) Acetyl-CoA

HS-CoA

O

h)nz

linz-C-CH3

Myristoylation (a-amino (amino terminus)) Myristoyl-CoA

HS-CoA -r/

[,]nz

O Enz-C-(CHz)rz-CHs

Ubiquitination (LYs) HS-@ ,O \*

G.t-ca1

o

6)-c-s-@

activation

itrrrated,ruil'oiti'

o

@-.-r-@ ctivatedubiquitin

,'"

\..-

HS-@

--_."

ADP-ribosylation (Arg, Gln, Cys, diphthamide-a NAD

?

, E",_N_A_@ modifred His)

nicotinamide

Methylation (Glu) S-adenosyl- S-adenosylmethionine homocysteine

\) Enz€linz_CH3

FIGURE 6-35 Someenzymemodificationreactions.

t 2 2 4)

Enzymes

discussit in somedetail. It will be discussedat length in Chapter12. AII of these modificationswill be encounteredaqain in this text.

Phosphoryl Groups Affect theStructure and (atalytir Artivity ofEnzymes The attachment of phosphoryl groups to specific amino acid residuesof a protein is catalyzedby protein kinases; removal of phosphoryl groups is catalyzed by protein phosphatases. The addition of a phosphorylgroup to a Ser, Thr, or Tlr residue introduces a bulky, charged group into a region that was only moderately polar. The oxygen atoms of a phosphoryl group can hydrogen-bondwith one or several groupsin a protein, commonlythe amidegroupsof the peptide backbone at the start of an a helix or the chargedguanidiniumgroup of an Arg residue.The two negativechargeson a phosphorylatedside chain can also repel neighboring negatively charged (Asp or Glu) residues.When the modified side chain is located in a region of an enzyme critical to its three-dimensional structure, phosphorylationcan have dramatic effectson enzymeconformationand thus on substrate binding and catalysis. An important example of enzyme regulation by phosphorylationis seen in glycogenphosphorylase(M, 94,500) of muscle and liver (Chapter 15), which catalyzesthe reaction (Glucose), + Pi -----+ (glucose),-r + glucose l-phosphate Glycogen Shortened glycogen

Theglucose t-pt o.ptut'Jl'Jro.^"0canbeusedforATP synthesisin muscle or convertedto free glucosein the liver. Glycogenphosphorylaseoccurs in two forms: the

FIGURE 6-36 Regulation of muscleglycogenphosphorylase activityby multiplemechanisms. in muscle Theactivityof glycogenphosphorylase is subjectedto a multilevelsystemof regulation,involvingcovalent modification(phosphorylation), allostericregulation,and a regulatory cascadesensitive to hormonalstatusthat actson the enzymesinvolved in phosphorylation and dephosphorylation ln the more activeform of the enzyme,phosphorylase a, specificSerresidues, one on eachsubunit,arephosphorylated. Phosphorylase a is convertedto the lessactive phosphorylase b by enzymaticlossof thesephosphorylgroups,promotedby phosphoprotein phosphatase 1 (PP1 b can be ). Phosphorylase (reactivated) reconverted to phosphorylase a by the actionof phosphorylasekinase.The activityof both formsof the enzymeis allosterically regulatedby an activator(AMP)and by inhibitors(glucose6-phosphate and ATP)that bind to separatesiteson the enzyme.The activitiesof phosphorylase kinaseand PP.lare also regulatedvia a shortpathway that responds to the hormonesglucagonand epinephrineWhen blood sugarlevelsare low, the pancreas and adrenalglandssecreteglucagon

moreactivephosphorylasea and the Iessactivephosphorylaseb (FiS. 6-36). Phosphorylaseo has two subunits, eachwith a specificSer residuethat is phosphorylatedat its hydroxyl group. Theseserinephosphateresiduesare required for maximal activity of the erzyme. The phosphoryl groupscanbe hydroly'ticallyremovedby a separate phosphatase: enzJ,rne calledphosphorylase b + 2P, Phosphorylase a + 2H2O-----+phosphorylase (more active)

(less active)

o is convertedto phosphoIn this reaction,phosphorylase serinephosphatecovalent cleavage of two b by the rylase bonds,one on eachsubunitofgJycogenphosphorylase. Phosphorylaseb can in turn be reactivated-covalently transformed back into active phosphorylaseoby another enzyme, phosphorylase kinase, which catalyzesthe transfer of phosphorylgroupsfrom ATP to the hydroxyl groups of the two speciflc Ser residuesin phosphorylase b: o e b ----+2ADP+ phosphorylase 2ATP+ phosphorylas (moreactive) (lessactive) The breakdownof glycogenin skeletalmusclesand the Iiver is regulated by variations in the ratio of the two forms of glycogenphosphorylase.The a and b forms differ in their secondary,tertiary, and quaternary structures; the active site undergoeschangesin structure and, consequently,changesin catalytic activity as the two forms are interconverted. The regulation of glycogenphosphorylaseby phosphorylation illustrates the effects on both structure and catalytic activity of adding a phosphoryl group. In the unphosphorylatedstate, each subunit of this en4rme is folded so as to bring the 20 residuesat its amino terminus, including a mnnber of basic residues,into a region containing severalacidic amino acids;this producesan

in muscleandsome Epinephrine bindsto itsreceptor andepinephrine. other tissues,and activatesthe enzyme adenylylcyclase.Glucagon in the liver.Thisleadsto thesynplaysa similarrole,bindingto receptors thesisof high levelsof the modifiednucleotidecyclicAMP (cAMP;see proteinkinase(also p. 298\, activatingthe enzymecAMP-dependent severaltargetprocalledproteinkinaseA or PKA).PKAphosphorylates phoskinaseand phosphoprotein teins, among them phosphorylase phatase phosphorylase kinaseis inhibitor1 (PPl-l).Thephosphorylated glycogenphosphoryand activates activatedand in turn phosphorylates PPI-1interactswith and inlase.At the sametime, the phosphorylated by inhibiting hibitsPPI. PPI-1alsokeepsitselfactive(phosphorylated) phosphoprotein phosphatase 28 (PP2B), the enzymethatdephosphorylates(inactivates) it. In this way,the equilibriumbetweenthe a and b is shifteddecisivelytowardthe more formsof glycogenphosphorylase a. Notethatthe two formsof phosphoryactiveglycogenphosphorylase lasekinaseareboth activatedto a degreeby Ca2* ion (notshown).This pathwayis discussed in moredetailin Chapters14, 15, and23.

6 . 5 R e g u l a t oErnyz y m e s

[rrr]

electrostaticinteractionthat stabilizesthe conformation. Phosphorylationof Serrainterfereswrth this interaction, forcing the amino-terminaldomainout of the acidic enr,rronment and into a conformationthat allowsinteraction betweenthe @-Ser and severalArg side chains.In this conformation,the enz;.nne is much more active. Phosphorylationof an enzyrnecan affect catalysisin another way: by altering substrate-bindingaffinity. For example,when isocitrate dehydrogenase(an enz;,rneof the citric acidcycle;Chapter16) is phosphorylated, electrostatic repulsionby the phosphorylgroup inhibits the binding of citrate (a tricarboxylic acid) at the active site.

Multiple Phosphorylations Allow Exquisite Regulatory [ontrol The Ser,Thr, or Tlr residuesthat are phosphorylated in regulatedproteinsoccurwithin commonstructuralmotifs, calledconsensussequences, that are recognizedby speciflcprotein kinases(Table6-10) Somekinasesare basophilic,preferringto phosphorylatea residuehaving basicneighbors;othershave different substrateprefer-

ences,such as for a residuenear a Pro residue.Amino acid sequenceis not the only important factor in determining whether a given residue will be phosphorylated, however.Protein folding brings together residues that are distant in the primary sequence;the resulting threedimensionalstructure can determinewhether a protein kinasehasaccessto a givenresidueand canrecognizeit as a substrate.Another factor influencing the substrate specificity of certain protein kinasesis the proximity of other phosphorylatedresidues. Regulationby phosphorylationis often complicated. Someproteinshaveconsensus sequences recognizedby several different protein kinases,each of which can phosphorylatethe protein and alter its enzSrmatic activity. In somecases,phosphorylationis hierarchical:a certain residuecan be phosphorylatedonly if a neighboring residuehas alreadybeenphosphoryIated. For example, glycogensynthase,the enz;'rnethat catalyzesthe condensation of glucose monomers to form glycogen (Chapter15), is inactivatedby phosphorylationof specific Ser residuesand is alsomodulatedby at leastfour other protein kinases that phosphorylatefour other

$ucose are a-D-glucopyranose and B-o-glucopyranose. Aldoses also exist in cyclic forms having fivememberedrings,which,becausethey resemblethe flvememberedring compoundfuran, are calledfuranoses. However,the six-memberedaldopyranosering is much more stable than the aldofuranosering and predominatesin aldohexoseand aldopentosesolutions.Only aldoses having five or more carbon atoms can form pyranosenngs. Isomeric forms of monosaccharidesthat differ only in their con-figurationabout the hemiacetalor hemiketal carbon atom are called anomers. The hemiacetal (or carbonyl) carbon atom is called the anomeric carbon. The a and B anomers of o-glucoseinterconvert in aqueoussolution by a process called mutarotation (Fig. 7-6). Thus,a solutionof a-n-glucoseand a solution of B-n-glucoseeventually form identical equilibrium mixtures having identical optical properties. This mixtwo-thirds ture consistsof aboutone-thirdd-D-glucose,

B-l-glucose, and very small amounts of the linear and flve-memberedring (glucofuranose)forms. Ketohexosesalso occur in a and p anomericforms. In these compoundsthe hydroxyl group at C-5 (or C-6) reactswith the keto groupatC-2,forminga furanose(or pyranose)ring containinga hemiketallinkage(Fig. 7-5). o-Fructose readily forms the furanose ring (Fig. 7-7); the more common anomer of this sugar in combined forms or in derivativesis B-n-fructofuranose. Haworth perspective formulas like those in Figure 7-7 are commonlyusedto showthe stereochemistry of ring forms of monosaccharides.However, the sixmemberedpyl'anosering is not planar,as Haworth perspectivessuggest,but tends to assumeeither of two "chair" conformations(Fig. 7-8). Recallfrom Chapter 1 (p 18) that two conJormnti,onsof a moleculeare interconvertiblewithout the breakageof covalent bonds, whereastwo configurati,ons can be interconvertedonly by breaking a covalent bond. To interconvert a and B confuurations,the bond involvingthe ring oxygenatom would haveto be broken,but interconversionof the two chair forms doesnot require bond breakage'The speci-flc units structuresof the monosaccharide three-dimensional areimportantin determiningthe biologicalpropertiesand aswe shallsee. functionsof somepolysaccharides,

ocHroH

\+

H 4

HO

OH

ax

2

H

eq

eq

)1

OHH

OH

a-o-Glucopyranose

OHH a-o-Fructofuranose

TWo possible chair forms (a) Axis I I

Hi

HOH B-o-Glucopyranose

OHH 6-n-Fructofuranose

OH

rGlucopyranose (b)

FIGURE7-7 Pyranosesand furanoses.The pyranose forms of o-glucoseand the furanoseformsof o-fructoseare shownhereas Haformulas.The edgesof the ring nearestthe reader worth perspective by bold lines.Hydroxylgroupsbelowthe planeof the are represented would appearat the rightsideof a ring in theseHaworthperspectives Fischerprojection(comparewith Fig.7-6). Pyranand furanareshown

7-8 Conformationalformulas of pyranoses.(a) Two chair FIGURE and hydrogenatoms formsof the pyranosering Bondsto substituents on the ring carbonsmay be eitheraxial (ax),projectingparallelto the verticalaxis throughthe ring, or equatorial(eq),projectingroughly sucharethesearenot readily to thisaxis.Twoconformers perpendicular without breakingthe ring. However,when the moleinterconvertible see Box 11-1),an (by atomicforce microscopy; cule is "stretched" forcethe intercan of sugar per mole kJ of energy input of about 46 in the equatorial substituents Cenerally, forms. of chair conversion and substituents, positions hinderedby neighboring are lesssterically in equatorialpositionsarefavored' conformerswith bulky substituents Anotherconformation,the "boat" (not shown),is seenonly in deriva(b)Theprefenedchairconformation tiveswith verybulkysubstituents.

for comoarison.

of a-o-glucopyranose.

HC_O.

r'\

HC' \/ H,C-CH

-CH

Pyran

i-'l

( a r b o h y d r aatnedsG l y c o b i o l o g y

(ontain 0rganisms aVariety ofHexose Derivatives In addition to simple hexoses such as glucose, galactose, and mannose, there are a number of sugar derivatives in which a hydroxyl group in the parent compound is replaced with another substituent, or a carbon atom is oxidized to a carboxyl group (Fig. 7-g). In glucosamine, galactosamine, and mannosamine, the hydroxyl at C-2 of the parent compound is replacecl with an amino group. The amino group is nearly always condensed wrth acetic acid, as in l/-acetylglucosamine. This glucosamine derivative is part of many structural polymers, including those of the bacterial cell wall. Bacterial cell walls also contain a derivative of glucosamrne, N-acetylmuramic acid, in which lactic acid (a threecarbon carboxylic acid) is ether-linked to the oxygen at C-3 of N-acetylglucosamine. The substitution of a hydrogen for the hydroxyl group at C-6 of r,-galactose

or L-mannose produces L-fucose or l-rhamnose, respectively. L-Fucose is found in the complex oligosaccharide components of glycoproteins and glycolipids; l-rhamnose is found in plant polysaccharides Oxidation of the carbonyl (aldehyde) carbon of glucose to the carboxyl level produces gluconic acid; other aldoses yield other aldonic acids. Oxidation of the carbon at the other end of the carbon chain-C-6 of glucose, galactose, or mannose-forms the corresponding uronic acid: glucuronic, galacturonic, or mannuronic acid. Both aldonic and uronic acids form stable intramolecular esters called lactones (Fig. 7-9, Iower left). In addition to these acidic hexose derivatives, one nine-carbon acidic sugar deserves mention: N-acetylneuraminic acid (a sialic acid, but often referred to simply as "sialic acid"), a derivative of l/-acetylmannosamine, is a component of many glycoproteins and glycolipids in animals. The

Glucose farnily

p-o-Glucose Deoxy sugars

OHH

p-o-GIucose 6-phosphate

o\

a-r--Rhamnose

B-r,-Fucose

/o-

oa o

C

./-

D_

H-C-OH

I

H-C-OH HOH B-o-Glucuronate

HOH o-Glucono-6-lactone

FIGURE7-9 Some hexose derivatives important in biology. In amino g r o u p r e p l a c e so n e o f t h e - - - O H g r o u p s i n t h e p a r e n t hexose. Substitution of -H for --OH produces a deoxy sugar; note

CH2OH N-Acetylneuraminic acid (a sialic acid)

s u g a r s ,a n - N H 2

a c i d i c s u g a r sc o n t a i n a c a r b o x y l a t eg r o u p , w h i c h c o n f e r s a n e g a t i v e c h a r g e a t n e u t r a l p H o - C l u c o n o - 6 - l a c t o n er e s u l t sf r o m f o r m a t i o n o f

t h a t t h e d e o x y s u g a r ss h o w n h e r e o c c u r i n n a t u r ea s t h e r i s o m e r s T h e

a n e s t e r l i n k a g e b e t w e e n t h e C - l c a r b o x y l a t eg r o u p a n d t h e C - 5 ( a l s o k n o w n a s t h e 6 c a r b o n )h y d r o x y l g r o u p o f D - g l u c o n a t e .

andDisaccharides 7.1Monosaccharides frni|

carboxylic acid groups of the acidic sugar derivatives are ionized at pH 7, and the compoundsare therefore correctly named as the carboxylates-glucuronate, galacturonate,and so forth. In the synthesisand metabolismof carbohydrates, the intermediates are very often not the sugarsthemselvesbut their phosphorylatedderivatives.Condensation of phosphoricacid with one of the hydroxyl groups of a sugar forms a phosphate ester, as in glucose 6-phosphate(FiS. 7-9). Sugarphosphatesare relatively stableat neutral pH and bear a negativecharge.One effect of sugar phosphorylationwithin cells is to trap the sugar inside the cell; most cells do not have plasma membrane transporters for phosphorylated sugars. Phosphorylationalso activatessugarsfor subsequent chemicaltransformation.Severalimportant phosphorylated derivatives of sugars are components of nucleotides(discussedin the next chapter).

t.'.

ro

1C

r#J-oH

6cH2OH H

H OHH

oH .Jr

'il' :

HojJ-H .- nL ^,, II-C-OH

3cu2*BCu* Complex mixture oI ""i t"

\'

>

B-o-Glucose

2''3-'4''and

6-carbon acids

| "l[-or ucHooH o-Glucose (linear form)

7-10 Sugarsas reducingagents.Oxidationof the anomeric FIGURE carbon (and probablythe neighboringcarbon)of glucoseand other reaction' The is the basisfor Fehling's underalkalineconditions sugars In precipitate. cuprousion (Cu+)producedformsa red cuprousoxide by oxidized be (ring) cannot form, C-l of glucose the hemiacetal complexed Cu2* However,the open-chainform is in equilibrium with the ringform, and eventuallythe oxidationreactiongoesto comoletion.Thereactionwith Cu2* is complex,yieldinga mixtureof productsand reducing3 mol of Cu2* per mole of glucose'

AreReducing Agents Monosaccharides Monosaccharides can be oxidized by relatively mild oxidizing agents such as cupric (Cuz*) ion

(FiS. 7-10). The carbonylcarbonis oxidizedto a carboxyl group. Glucose and other sugars capable of reducing cupric ion are called reducing sugars. They form enediols,which are convertedto aldonic acidsand then to a complexmixture of 2-,3-,4-, and 6-carbon

Glucoseis the principal fuel for the brain. When the amount of glucosereachingthe brain is too loq the consequencescan be dire: lethargy,coma,permanent brain damage, and death (see Fig. 23-25). Animals haveevolvedcomplexhormonalmechanismsto ensure that the concentrationof glucosein the blood remains high enough(about 5 mu) to satisfythe brain'sneeds, but not too high, becauseelevatedblood glucosecan alsohaveseriousphysiologicalconsequences. Individuals with hsulin-dependent diabetes mellitus do not produce sufficient insulin, the hormone that normally servesto reduce blood glucoseconcentration, and if the diabetesis untreated their blood glucoselevels may rise to severalfoldhigher than normal. These high glucoselevels are believedto be at least one cause of the seriouslong-term consequencesof untreated diabetes-kidney failure, cardiovasculardisease,blindness, and impaired wound healing-so one goal of therapy is to provide just enoughinsulin (by injection) to keep blood glucose levels near normal. To maintain the correct balanceof exercise.diet. and insulin for the individual, blood glucose concentrationneeds to be measuredseveraltimes a day,and the amount of insulin inj ected adjustedappropriately.

acids.This is the basisof Fehling'sreaction,a qualitative test for the presenceof reducing sugar.By measuring the amount of oxidizingagentreducedby a solution of a sugar,it is alsopossibleto estimatethe concentrationof that sugar.For many years this test was used to detect and measureelevatedglucoselevels in blood and urine in the diagnosisof diabetesmellitus (Box 7-1). r

The concentrationsof glucose in blood and urine can be determinedby a simple assayfor reducing sugar, such as Fehling's reaction, which for many years was usedas a diagnostictest for diabetes(Fig. 7-10). Modern measurementsrequire just a drop of blood, added to a test strip containing the enzyme glucose oxidase (Fig. 1); a simple photometer measuresthe color producedwhen the H2O2from $ucose oxidationreactswith a dye, and readsout the blood $ucose concentration' Becauseblood glucoselevelschangewith the timing of mealsand exercise,single-timemeasurementsdo not necessarilyreflect the auerageblood glucoseover hours and days, so dangerousincreasesmay go undetected. The average$ucose concentrationcan be assessedby looking at its effect on hemo$obin, the oxygen-carrying protein in erythrocytes(p. 158).Ttansportersin the erythrocyte membrane equilibrate intracellular and plasma o-Glucose f 02

o-Glucono-6-lactone* H2O2

ol 1 The glucoseoxidasereaction,usedin the measurement FIGURE the reaction catalyzes peroxidase, a enzyme, A second blood glucose. of the H2O2with a colorlesscompoundto producea coloredproduc! which is measuredspectrophotometrically.

(conti'nued on nefrt Page)

?-1

Carbohydrates andGlycobiology

glucoseconcentrations,so hemoglobinis constantlyexposedto glucoseat whateverconcentrationis presentin the blood.A nonerz;rmaticreaction occrrrsbetweenglucose and primary amino groups in hemoglobin (either the amino-terminal Val or the e-amino groups of Lys residues;seeFig. 2). The rate of this processis proportional to the concentration of glucose,so the reaction can be usedasthe basisfor estimatingthe averageblood glucoselevel over weeks.The amount of glycatedhemoglobin (GHB) present at any time reflects the average blood glucose concentration over the circulating ,.lifetime" of the erythrocyte (about 120 days), althoughthe concentrationin the last two weeks is the most important in setting the level of GHB. The extent of hemoglobin glycation (so named to distinguish it from glycosylation,the enzymat,ic transfer of glucoseto a protein) is measuredclinically by extracting hemoglobinfrom a small sampleof blood and separatingGHBfrom unmodifiedhemoglobinelectrophoretically, taking advantageof the charge difference resulting from modification of the amino group(s). NormalGHBvaluesare about b% of total hemoglobin (correspondingto blood glucoseof I20 mgl 100 mL). In people with untreated diabetes, however, this value may be as high as 1B%,indicating an averageblood glucoselevel of about 300 mg/100mLdangerouslyhigh. One criterion for successin an individual program of insulin therapy (the timing, frequency,and amount of insulin injected) is maintaining GHB values at about 7o/o. In the hemoglobinglycation reaction, the first step (formation of a Schiff base) is followed by a seriesof rearrangements,oxidations,and dehydrationsof the carbohydrate moiety to produce a heterogeneousmixture ofAGEs,advancedglycationend products.Theseproducts can leavethe erythrocl'te and form covalentcrosslinks between proteins, interfering with normal protein function (Fig 2) The accumulation of relatively high concentrationsof AGEsin peoplewith diabetesmay,by cross-linkingcritical proteins, causethe damageto the kidneys,retinas,and cardiovascularsystemthat characterize the disease.This pathogenicprocessis a potential target for drug action. FIGURE 2 Thenonenzymaticreactionof glucosewith a primaryamino groupin hemoglobinbeginswith @ formationof a Schiffbase,which theAmadorirearrangement @ undergoes to generatea stableproduc! this ketoamine can furthercyclizeto yield CHB.@ Subsequent @ reactionsgenerateadvancedglycationend products(ACEs),such as e-N-carboxymethyllysine and methylglyoxal, compoundsthat@ can damageother proteinsby cross-linking them, causingpathological cnanges.

oHu I

H OH

It ,rC:O

+ H2N-R Hemoglobin

HOH I Glucose

oJ

oH tt I

+/rt OH

HO

H

C:N-R

Schiffbase

t

6)l -l

+/il OH

HO

OH HrH C_N-R

HU ^ | G )l

Ketoamine

H -N-R

I

Glycated hemoglobin

(cHB)

@i

J

AGEs

/@I \ Protein cross-linking

Jr

Jr

Jz

Damageto kidneys, retinas, cardiovascularsystem

andDisaccharides 7.1Monosaccharides f,ol

aGlycosidir Bond Contain Disaccharides Disaccharides(such as maltose,lactose,and sucrose) joined covalentlyby an consistof two monosaccharides when a hydroxyl which is formed bond, O-glycosidic group of one sugarreactswith the anomericcarbonof the other (FiS. 7-11). This reactionrepresentsthe formation of an acetalfrom a hemiacetal(such as glucopyranose)and an alcohol(a hydroxylgroup ofthe second sugarmolecule)(Fig. 7-5), andthe resultingcompound is called a glycoside. Glycosidic bonds are readily hydrolyzedby acid but resist cleavageby base.Thus disaccharidescan be hydrolyzed to yield their free componentsby boilingwith dilute acid. monosaccharide N-glycosylbondsjoin the anomericcarbonof a sugarto a nitrogenatomin glycoproteins(seeFig. 7-29) and nucleotides(seeFig.8-1). The oxidation of a sugarby cupric ion (the reaction that defines a reducing sugar) occurs only with the Iinear form, which exists in equilibrium with the cyclic form(s). When the anomericcarbonis involvedin a glycosidicbond, that sugarresiduecannottake the Iinear form and therefore becomesa nonreducingsugar.In the end of a or polysaccharides, describingdisaccharides chain with a free anomericcarbon (one not involvedin a glycosidicbond) is commonlycalledthe reducing end.

h c n ri u t t t r t L

+

H

OH

nlcr

p-n-Glucose

a-l-Glucose

The disaccharidemaltose(Fig. 7-11) containstwo o-glucoseresiduesjoined by a glycosidiclinkage between C-l (the anomeric carbon) of one glucose residueand C-4 of the other,Becausethe disaccharide retains a free anomeric carbon (C-1 of the glucose residueon the right in Fig. 7-11), maltoseis a reducing sugar.The conflgurationof the anomericcarbonatom in the glycosidiclinkageis a. The glucoseresiduewith the free anomericcarbonis capableof existingin a- and B-pyranoseforms. such To name reducingdisaccharides KEY(0NVENTI0N: more name to especially and unambiguously, as maltose severalrules are followed.By complexoligosaccharides, the compoundwritten describes name the convention, left, and we can "build to the end with its nonreducing (1) Givethe conflgorder. following the up" the namein joining the flrst carbon (a anomeric the uration or B) at second.(2) (on to the left) the monosaccharideunit flve- and to distinguish residue; Name the nonreducing or "furano" insert six-membered ring structures, parentheses the (3) in Indicate "py'ano" into the name. two carbonatomsjoined by the glycosidicbond, with an arrow connectingthe two numbers;for example,(l-+4) showsthat C-l of the flrst-namedsugarresidueis joined to C-4 of the second.(4) Name the secondresidue.If there is a third residue,describethe secondglycosidic bond by the sameconventions.(To shortenthe description of complex polysaccharides,three-letter abbreviations or colored symbols for the monosaccharides are often used, as given in Table 7-1.) Followhg this maltoseis a-oconventionfor naming oligosaccharides, Becausemost glucopyranosyl-(1-+4)-l-glucopl'ranose' sugarsencounteredin this book are the I enantiomers and the pyranoseform of hexosespredominates,we generallyuse a shortenedversion of the formal name of

h c m i i l c e L rl

H

OH:'

4

H HOHHOH Maltose a-l- glucopy'ranosyl-( 1+4)-o- glucopyranose FIGURE7-11 Formation of maltose. A disaccharide is formed from t w o m o n o s a c c h a r i d e s( h e r e , t w o m o l e c u l e s o f o - g l u c o s e ) w h e n a n -OH

( a l c o h o l )o f o n e g l u c o s em o l e c u l e ( r i g h t )c o n d e n s e sw i t h t h e i n -

tramolecular hemiacetal of the other glucose molecule (left),with lf e l i m i n a t i o n o f H 2 O a n d f o r m a t i o n o f a g l y c o s i d i cb o n d . T h e r e v e r s a o t h i s r e a c t i o n i s h y d r o l y s i s - a t t a c k b y H 2 O o n t h e g l y c o s i d i cb o n d T h e m a l t o s e m o l e c u l e , s h o w n h e r e a s a n i l l u s t r a t i o n ,r e t a i n s a r e d u c i n g h e m i a c e t a la t t h e C - . 1 n o t i n v o l v e d i n t h e g l y c o s i d i c b o n d B e c a u s e mutarotation interconverts the a and p forms of the hemiacetal, the b o n d s a t t h i s p o s i t i o n a r e s o m e t i m e sd e p i c t e d w i t h w a v y l i n e s , a s shown here, to indicate that the structure may be either a or p.

Rhamnose

Rha

N-Acetylmuramic acid

C ctce E caw N ctcN D catNAc I clcNAc @ taoe Mur Mur2Ac

Ribose

Rib

Xylose

* XvI

N-Acetylneuraminic acid (a sialic acid)

I NeuSAc

Abequose

Abe

Glucuronic acid

Arabinose

Ara

Galactosamine

Fructose

Fru

A Fuc Galactose O cat O ctc Glucose

Fucose

Mannose

O Man

Glucosamine N-Acetylgalactosamine N-Acetylglucosamine Iduronic acid Muramic acid

N-acetylhexas circles, hexoses arerepresented usedconvention, Note:In a commonly with Allsugars divideddiagonally as squares andhexosamines as squares, osamines the"gluco"configurationareblue,thosewiththe"galacto"configurationareyellow sulfate(S), canbe addedas needed: Othersubstituents "man-no" ategreen. sugats (0me) (oAc),or 0-methyl (P),0-acetyl phosphate

3r1

( a r b o h y d r aatnedsG l y c o b i o l o g y

such compounds, giving the configuration of the anomeric carbon and naming the carbonsjoined by the glycosidicbond. In this abbreviatednomenclature. maltoseis Glc(a1-+4)GIc.r The disaccharide lactose(FiS. Z-12), which yields >galactoseand l-glucose on hydrolysis,occursnaturally in milk. The anomeric carbon of the glucoseresidue is availablefor oxidation,and thus lactoseis a reducingdisaccharide.Its abbrevratedname is Gal(Bl-+4)Glc. Sucrose(table sugar) is a disaccharideof glucoseand fructose. It is formed by plants but not by animals.In contrastto maltoseandlactose,sucrosecontainsno free anomericcarbon atom; the anomericcarbonsof both monosaccharideunits are involvedin the glycosidicbond (Fig. 7-12) Sucroseis thereforea nonreducingsugar. In the abbreviated nomenclature, a double-headed arrow connectsthe s)..rnbols specifyingthe anomeric carbonsand their con_figurations. For example,the abbreviated name of sucroseis either Glc(al28)Fruor Fru(B24) glycosidic bondsbetweenglucoseunits. Most animalscannotuse celluloseas a fuel source,becausethey lack an enz).rne to hydrolyze the (B1-+4) linkages.Termitesreadily digest cellulose(and thereforewood), but only because their intestinal tract harborsa syrnbioticmicroorganism,

OH

@1--+4!Iinked o-glucoseunits (a)

FIGURE 7-15 Cellulose.(a) Two unitsof a cellulosechain;the o-glucoseresidues arein (B1-+4)linkage. Therigidchairstructures can rotate relativeto one another.(b) Scaledrawing of segmentsof two parallelcellulosechains,showingthe conformation of the o-glucose residues and the hydrogen-bond cross-links. In the hexoseunit at the lower left, all hydrogenatomsare shown; in the other three hexose units,the hydrogensattachedto carbonhavebeenomittedfor claritv, as they do not participatein hydrogenbonding.

7.2Polysaccharides frorl 7-16 Cellulosebreakdownby wood fungi. A wood fungus FIGURE growingon an oak log. All wood fungi havethe enzymecellulase, so thatwood whichbreaksthe (F1-+4)glycosidicbondsin cellulose, is a sourceof metabolizablesugar(glucose)for the fungus.The only ableto usecelluloseasfood arecattleand otherruminants vertebrates (sheep,goats, camels, giraffes).The extra stomach compartment (rumen)of a ruminantteemswith bacteriaand protiststhat secrete cellulase.

I C:O I

I I

C:O (a)

CHt

CHt

7-17 Chitin. (a) A shortsegmentof chitin,a homopolymerof FIGURE units in (B1-+4) Iinkage.(b) A spottedJune N-acetyl-o-glucosamine beetle(Pelldnotapunctata),showingits surfacearmor (exoskeleton) of chitin

Influence Bonding andHydrogen Factors Steric Folding Homopolysaccharide

Tfichonympha, that secretes cellulase, which hydrolyzesthe (Bl-+4) linkages.Wood-rotfungi and bacteria alsoproducecellulase(Fig. 7-16). composed Chitin is a linear homopolysaccharide of N-acetylglucosamineresidues in (p1-+4) Iinkage (Fig. 7-17). The oniy chemicaldjfferencefrom cellulose is the replacementof the hydroxyl group at C-2 with an acetylated amino group. Chitin forms extended flbers similar to those of cellulose, and like cellulose cannot be digestedby vertebrates.Chitin is the principalcomponent of the hard exoskeletonsof nearly a million speciesof arthropods-insects,Iobsters,and crabs,for example-and is probably the second most abundant polysaccharide,next to cellulose,in nature; an estimated 1 billion tons of chitin are produced eachyear in the biosnhere!

The folding of polysaccharidesin three dimensionsfollows the sameprinciplesasthose governingpolypeptide structure: subunits with a more-or-lessrigid structure dictated by covalent bonds form three-dimensional macromolecularstructures that are stabilized by weak interactions within or between molecules:hydrogen bonds and hydrophobicand van der Waalsinteractions, and, for pol;rmerswith charged subunits, electrostatic interactions. Becausepolysaccharideshave so many hydroxyl groups, hydrogen bonding has an especially important influenceon their structure. Glycogen,starch, and cellulose are composedof pyranoside subunits (having six-memberedrings), as are the oligosaccharides of glycoproteins and glycolipids to be discussed later. Suchmoleculescan be representedas a seriesof rigid pyranose rings connected by an oxygen atom bridging two carbonatoms (the glycosidicbond). There is, in principle, free rotation about both C-O bonds iirking the residues(Fig. 7-l5a), but as in polypeptides (see Figs 4-2,4-8), rotation about each bond is limited by steric hindrance by substituents.The threedimensional structures of these molecules can be

---l

-

andGtycobiotogy [248=] Carbohydrates describedin terms of the dihedralangles,@and g, about the glycosidicbond (FiS. 7-f8), analogousto angles Q and tlr made by the peptide bond (see Fig. 4-2). The bulkiness of the pyranose ring and its substituents, and electronic effectsat the anomericcarbon, place constrahts on the angles{ and g; thus certain conformationsare much more stablethan others,as can be shown on a map of energy as a function of @and rZ (Fig. 7-19). The most stablethree-dimensionalstructure for the (a1-+4)-linked chainsof starch and glycogenis a tightly coiledhelix (Fig. 7-20), stabilizedby interchain hydrogen bonds.In amylose(with no branches)this structure is regular enoughto allow crystallizationand thus determination of the structure by x-ray diffraction. The averageplane of eachresiduealongthe amylosechain forms a 60o angle with the averageplane of the preceding residue, so the helical structure has six residuesper turn. For amylose,the core of the helix is of precisely the right dimensionsto accornrnodateiodine as complex ions (I3- and I5-), givingan intenselyblue complex.This interaction is a commonqualitativetest for amylose. For cellulose,the most stableconformationis that in which eachchair is tumed 180' relativeto its neighbors, yielding a straight,extendedchain.AII -OH groupsare availablefor hydrogen bonding with neighboring chains. With severalchainslying sideby side,a stabilizingnetwork of interchain and intrachain hydrogen bonds produces straight, stable supramolecularfibers of great tensile strength(Fig. 7-15b). This property of cellulosehasmade it a useful substanceto civilizationsfor millennia.Many manufacturedproducts, including papyltrs, paper, cardboard,rayon,insulatingtiles, and a variety of other useful materials,are derivedfrom cellulose.Thewater contentof

Cellulose (81+4)Glc repeats

Amylose (a1+4)GIc repeats

Dextran (a1+6)Glc repeats (with (a1+3) branches, not shown) FIGURE 7-18 Conformationat the glycosidicbondsof cellulose,amylose,and dextran.The polymersare depictedas rigid pyranoserings joined by glycosidicbonds,with freerotationaboutthesebonds Note that in dextranthereis alsofree rotationaboutthe bond betweenC-5 and C-6 (torsionangler,r(omega)).

thesematerialsis low becauseextensiveinterchainhydrogen bonding between cellulosemoleculessatisflestheir capacityfor hydrogen-bondformation. a @,tP: 30",-40"

(a) FIGURE 7-19 A map of favoredconformationsfor oligosaccharides and polysaccharides. ThetorsionanglesrI"and d (seeFig.Z-1B),which define the spatialrelationshipbetweenadjacentrings,can in principle haveanyvaluefrom 0' to 360'. In fact,someof thetorsionangleswould giveconformations thatarestericallyhindered,whereasothersgiveconformationsthat maximizehydrogenbonding.(a)When the relativeenergy (I) is plottedfor each valueof Q and 1Ir,with isoenergy(,,same energy")contoursdrawnat intervalsof .l kcal/molabovethe minimum

(b)

a a.v:

-170".-170'

energystate,the resultis a map of preferredconformations. Thisis analogous to the Ramachandran plot for peptides(see Figs4-3, 4-B). (b) Two energeticextremesfor the disaccharide Cal(Bl+3)Cal; these valuesfall on theenergydiagram(a)asshownby the redand bluedots The red dot indicatesthe leastfavoredconformation, the blue dot the mostfavoredconformation. Theknownconformations of thethreepolysaccharides shownin Figure7-1B havebeendeterminedDyx-raycrystallography, and all fall within the lowest-energy regionsof the map.

7.2Polysaccharides ?rl

HO 4

Agarose 3)o-Gal(F 1+ 4)3,6-anhydro-r,-Gal2S(a 1 repeating units 7-21 Agarose.The repeatingunit consistsof o-galactose FIGURE (in which an etherbridge (B1-+4)-linked to 3,6-anhydro-r-galactose connectsC-3 and C-6).Theseunitsarejoined by (al-+3) glycosidic long.A smallfractionof the linksto form a polymer600 to 700 residues havea sulfateesterat C-2(asshownhere). residues 3,6-anhydrogalactose (a1+4IIinked o-glucose units (a)

.i

(b)

FIGURI 7-20 Starch(amylose).(a)In themoststableconformation, with adjacentrigidchairs,the polysaccharide chainis curved,ratherthan linearas in cellulose(seeFig.7-15). (b)A modelof a segment of amylose; for clarity,the hydroxylgroupshavebeenomiftedfromall but oneof the glucoseresiduesComparethe two residuesshadedin pink with the chemicalstructures in (a).Theconformation ol (al-+4\ linkagesin amylose,amylopectin, andglycogencauses thesepolymersto assume tightly producethe dense coiled helicalstructuresThesecompactstructures granulesof storedstarchor glycogenseenin manycells(seeFig.20-2).

(ontain Bacterial andAlgal Cetl Walls Heteropolysaccharides 5tructural The rigid componentof bacterial cell walls (peptidoglycan) is a heteropolymerof alternating(B1-+4)-linked N-acetylglucosamine and N-acetylmuramicacid residues (see Fig. 20-31). The linear polyrnerslie side by side in the cell wall, cross-Linkedby short peptides, the exact stmctrre of which dependson the bacterialspecies.The peptide cross-linksweld the polysaccharidechainsinto a strong sheaththat envelopsthe entire cell and prevents cellular swelling and lysis due to the osmotic entry of water. The enzymelysoz}.'rnekills bactena by hydrolyzing the (F1-+4) glycosidicbond betweenN-acetylglucosamine and ly'-acetylmuramic acid (see Fig. 6-24). Lysozymeis notablypresentin tears,presumablyas a defenseagainstbacterialinfectionsof the eye.It is alsoproducedby certainbacterialvirusesto ensuretheir release from the host bacterialcell, an essentialstep of the viral infectioncycle.Penicillinand relatedantibioticskill bacteria by preventingsynthesisof the cross-lirks,leavingthe cellwalltoo weakto resistosmoticlysis(seepp.216-217). Certainmarine red algae,including someof the seaweeds, have cell walls that contain agat, a mixture of made up of o-galactose sulfatedheteropolysaccharides and an l-galactose derivative ether-linkedbetween C-3 and C-6.Agar is a complexmixture of polysaccharides, all with the samebackbonestructure, but substituted to varying degreeswith sulfate and pyruvate. Agarose (M, -150,000) is the agar componentwith the fewest chargedgroups (sulfates,pyruvates)(Fig. 7-21). The remarkablegel-formingproperty of agarosemakes it

useful in the biochemistry laboratory.When a suspension of agarosein water is heated and cooled, the agaroseforms a double helix: two moleculesin parallel orientation twist together with a helix repeat of three residues;water moleculesare trapped in the central canty. These structures in turn associatewith each other to form a gel-a three-dimensionalmatrix that traps Iargeamountsof water.Agarosegels are usedas inert supports for the electrophoretic separationof nucleic acids,an essentialpart of the DNA sequencing process(p.292). Agar is alsousedto form a surfacefor the growth of bacterial colonies.Another commercial use of agar is for the capsulesin which some vitamins and drugs are packaged;the dried agar material dissolvesreadily in the stomachand is metabolicallyinert'

AreHeteropolysaccharides Glycosaminoglycans Matrix oftheExtracellular The extracellular space in the tissues of multicellular animalsis fllled with a gelJike material,the extracellular matrix (ECM), also called ground substance, which holds the cells together and providesa porous pathway for the diffusion of nutrients and oxygen to individual cells.The reticular ECM that surroundsflbroblastsand other connectivetissuecellsis composed of an interlocking meshwork of heteropolysaccharides and flbrous proteins such as flbrillar collagens,elastin, and fibronectin.Basementmembraneis a specialized ECMthat underliesepithelialcells;it consistsof specialized collagens,laminin, and heteropolysaccharides. the glycosaminoglyThese heteropolysaccharides, polymers composedof cans, are a family of linear (Fig. They are 7-22). repeating disaccharideunits found in not unique to animals and bacteria and are is alwayseither plants. One of the two monosaccharides N-acetylglucosamine or N-acetylgalactosamine; the other is in most casesa uronic acid,usuallyD-glucuronic contain esor L-iduronicacid. Someglycosaminoglycans terified sulfate groups. The combination of sulfate groups and the carboxylate groups of the uronic acid a very high density of residuesgivesglycosaminoglycans negativecharge.To minimizethe repulsiveforcesamong neighboringchargedgroups,thesemoleculesassumean

T

l

[250 ]

Carbohydrates andGlycobiotogy

Glycosaminoglycan

Repeating

disaccharide

FIGURE 7-22 Repeatingunits of somecommon glycosaminoglycans of extracellularmatrix. The moleculesare copolymersof alternating (keratansulfateis the exception), uronicacid andaminosugarresidues with sulfateestersin any of severalpositions,exceptin hyaluronan. The ionizedcarboxylate and sulfategroups(redin the perspective formulas) givethesepolymerstheircharacteristic high negativecharge.Therapeutic heparincontainsprimarilyiduronicacid (ldoA)and a smallerproportionof glucuronicacid (GlcA,not shown),and is generally highly sulfatedand heterogeneous in length.Thespace-filling modelshowsa heparinsegmentas itssolutionstructure,asdeterminedby NMR spectroscopy(PDBlD l HPN).Thecarbonsin the iduronicacidsulfateare coloredblue; thosein glucosamine sulfateare green.Oxygenand sulfur atoms are shown in their standardcolors of red and yellow, respectively. The hydrogenatomsare not shown(for clarity).Heparan sulfate(notshown)is similarto heparinbut hasa higherproportionof CIcA and fewersulfategroups,arrangedin a lessregularpattern.

Number of disaccharides per chain

coo

HO)

,o'

Hyaluronan -50,000

HOH GlcA

GlcNAc

Chondroitin 4-sulfate

20-60 H

OH

GlcA

cH2osot

ft\ Keratan sulfate

o

-25

o

ao' H

ipl ,.1)

Gal

o. Heparin

15-90

coo

\

o H

H osot IdoA2S

(rr1+4) GIcNSBS6S

Heparin segment

extended conformation in solution, forming a rodlike helix in which the negatively charged carboxylate groups occur on alternate sides of the helix (as shown for heparin in Fig.7-22). The extended rod form also provides maximum separation between the negatively charged sulfate groups. The specific patterns of sulfated and nonsulfated sugar residues in glycosaminoglycans provide for specffic recognition by a variety of protein ligands that bind electrostatically to these molecules. The sulfated glycosaminoglycans are attached to extracellular proteins to form proteoglycans (Section Z.B).

The glycosaminoglycanhyaluronan (hyaluronic acid) containsalternating residuesof D-glucuronicacid andN-acetylglucosamine(F ig. 7-22) . With up to 50,000 repeatsof the basic disaccharideunit, hyaluronanhas a molecular weight of several million; it forms clear, highly viscous solutions that serve as lubricants in the synovial fluid of joints and give the vitreous humor of the vertebrate eye its jellylike consistency (the Greek hya\os means "glass";hyaluronan can have a glassyor translucent appearance).Hyaluronan is also a component of the extracellular matrix of cartilage and tendons, to which it contributes tensile strength and elasticity as a result of its strong interactionswith other components of the matrix. Hyaluronidase,an enzyme secretedby some pathogenicbacteria,can hydrolyze the glycosidiclinkagesof hyaluronan,rendering tissues more susceptibleto bacterialinvasion.In many species, a similar enzyme in sperm hydrolyzes an outer glycosaminoglycancoat around the ovum, allowing sperrn penetration. Other glycosaminoglycans differ from hyaluronanin three respects:they are generally much shorter polymers, they are covalentlylinked to specific proteins (proteoglycans),and one or both monomeric units differ from those of hyaluronan.Chondroitin sulfate (Greekchondros, "cartilage") contributesto the tensile strength of cartilage,tendons,ligaments,and the walls of the aorta. Dermatan sulfate (Greek der.rna, "skin") contributesto the pliability of skin and is alsopresentin blood vesselsand heart valves.In this pollrner, many of the glucuronateresiduespresent in chondroitin sulfate are replacedby their 5-epimer,l-iduronate. H

coo

+-o HOH a-l-Iduronate (IdoA)

HOH p-o-Glucuronate (GlcA)

7 . 2P o l y s a c c h a r iLDrt] des

Keratarrsulfates(Greekkeras,"horn") haveno uronic acid and ther sulfatecontent is variable.They are present in corrLea, cartilage,bone,and a varietyof horny structures formedof deadcells:horn,hair,hoofs,nails,andclaws.Heparan sulfate (GreekhEp:ar,"liver") is produced by all animal cells and containsvariable arrangementsof sulfated andnonsulfatedsugars.The sulfatedsegmentsof the chain allowit to interactwith a largenumberof proteins,including growthfactorsandECMcomponents,aswell asvarious enzyrnesand factorspresentin plasma.Heparinis a fractionated form of heparan sulfate derived mostly from mast cells (a type of leukocyle).Heparinis a therapeutic agent used to irLhibitcoaglilationttrough its capacityto bind the proteaseinhibitor antithrombin Heparinbindmg causesantittrombin to bind to and inhibit ttrombin, a proteaseessentialto bloodclotting.Theinteractionis strongly electrostatic;heparinhasthe hrghestnegativechargedensity of any known biologicalmacromolecule(Fig. 7-23). Purifled heparin is routinely added to blood samples obtainedfor clinical analysis,and to blood donated for transfusion,to preventclottrlg. Table7-2 summarizesthe composition,properties, roles,and occurrenceof the polysaccharides described in Section7.2.

and its bindtIGURE7-23 Interactionbetweena glycosaminoglycan growthfactor1 (FCF1),itscell surfacereceptor ing protein. Fibroblast (heparin) were (FCFR), and a shortsegmentof a glycosaminoglycan (PDB lD 1E0O).The shownhere to yieldthe structure co-crystallized as surfacecontourimages,with color to repproteinsare represented potential:red, predominantlynegative resentsurfaceelectrostatic positivecharge.Heparinis shownin a predominantly charge;blue, with the negativecharges(-SO: and ball-and-stickrepresentation, -{OO-) attractedto the positive(blue)surfaceof the FCF.Iprotein. that but the glycosaminoglycan Heparinwasusedin thisexperiment, bindsFCFl in vivo is heparansulfateon the cell surface.

Size(numberof monosaccharide RoleVsignfficance units)

Polymer

TIpe.

Repeatinguniti

Starch Amylose Amylopectin

HomoHomo-

(c1--+4)GIc,linear (a1-+4)Glc,with (a1-+6)GIc branchesevery 24-30 residues

50-5,000 Up to 106

Glycogen

Homo-

(a1--+4)Glc, with (a1--+6)Glc branchesevery 8-12 residues

Up to 50,000

Energy storage:in bacteriaand animal cells

Cellulose

Homo-

(B1-+4)Glc

Up to 15,000

Chitin

Homo-

(B1-+4)GlcNAc

Very large

Structural: in plants, gives rigidity and strengthto cell walls Structural: in insects,spiders,crustaceans, givesrigidity and strength to exoskeletons

Dextran

Homo-

(a1-+6)GIc,with (a1-+3) branches

Wide range

Structural:in bacteria,extracellularadhesive

Peptidoglycan

Hptprn-'

4)Mur2Ac(F1-+4) GlcNAc(F1

Very large

Structural:in bacteria,givesrigidity and strength to cell enveloPe

Energystorage:in plants

peptides attached Agarose

Hetero-

3)o-Gal(F1+4)3,6anhydro-l-Gal(a1

1,000

Structural:in algae,cell wall material

Hyaluronan (a glycosaminoglycan)

Hetero-; acidic

a)GlcA(B1+3) GIcNAc(F1

Up to 100,000

Structural: in vertebrates,extracellular matrix of skin and corurectivetissue;viscosity and lubrication in joints

*Eachpolymer (hetero-). (homo-)or heteropolysaccharide is classified as a homopolysaccharide lTheabbreviated of thisdisaccharepeats contains thatthe polymer repeating unitsindicate namesforthe peptidoglycan, agarose, andhyaluronan unit. of thenextdisaccharide in peptidoglycan, theGlcNAc 0f onedisaccharide unitis (81+4)-linkedto thefirstresidue rideunit.Forexample,

PUr)

Carbohydrares andGtycobiotogy

SUMMAR 7Y .2 Polysaccharides r

Polysaccharides (glycans) serve as stored fuel and as structural components of cell walls and extracellular matrix.

r

The homopolysaccharides starch and glycogen are stored fuels in plant, animal, and bacterial cells. They consist of >glucose with (a1+4) linkages, and both contain some branches

r

The homopolysaccharidescellulose, chitin, and dextran serue structural roles. Cellulose, composed of (B1-+4)-Linked o-glucoseresidues,lends strength and rigidity to plant cell walls. Chitin, a pol}.'rner of (B 1-+4) -linked N-acetylglucosamine, strengthens the exoskeletons of arthropods. Dextran forms an adhesive coat around certain bacteria.

r

Homopolysaccharidesfold in three dimensions.The chair form of the pytanose ring is essentially rigid, so the conformation of the pol}rmers is determined by rotation about the bonds from the nngs to the oxygen atom in the gycosidic linkage. Starch and glycogen form helical structures with intrachain hydrogen bonding; cellulose and chitin form long, strarght strands that interact with neighboring strands.

r

Bacterial and algal cell walls are strengthened by heteropolysaccharides-peptidoglycan in bacteria, agar in red algae The repeating disaccharide in peptidoglycan is GlcNAc( Bl--+4)Mur2Ac; in sugar, it is n-Gal(B 1-+4)3,6-anhydro-l-Gal.

r

Glycosaminoglycans are extracellular heteropolysaccharidesin which one of the two monosaccharide units is a uronic acid (keratan sulfate is an exception) and the other an N-acetylated amino sugar. Sulfate esters on some of the hydroxyl groups and on the amrno group of some glucosamine residues in heparin and in heparan sulfate give these pol;rmers a high density of negative charge, forcing them to assume extended conformations. These polymers (hyaluronan, chondroitin sulfate, dermatan sulfate, and keratan sulfate) provide viscosity, adhesiveness,and tensile strength to the extracellular matrix.

7.3 Glycoconjugates: Proteoglycans,

Glycoproteins, andGlycolipids In addition to their important roles as storecl fuels (starch, glycogen, dextran) and as structural materials (cellulose, chitin, peptidoglycans), polysaccharides and oligosaccharidesare information carriers. Some provide communication between cells and their extracellular surroundings; others label proteins for transport to and localization in specific organelles, or for destruction when the protein is malformed or superfluous; and others serve as recognition sites for extracellular siAnal

molecules(growth factors,for example)or extracellular parasites(bacteriaor viruses). On almost every eukaryotic cell, specific oligosaccharidechains attached to componentsof the plasmamembraneform a carbohydrate layer (the glycocalyx), severalnanometersthick, that serves as an information-rich surface that a ceu shows to its surroundings.These oligosaccharidesare central playersin cell-cellrecognitionand adhesion,cell migration during development,blood clotting, the immune response,wound healing, and other cellular processes. In most of thesecases,the informationalcarbohydrate is covalentlyjoined to a protein or a lipid to form a glycoco4iugate, which is the biologicallyactive molecule. Proteoglycans are macromoleculesof the ceII surface or extracellular matrix in which one or more suHated$ycosaminoglycanchainsare joined covalently to a membraneprotein or a secretedprotein. The glycosaminoglycanchain can bind to extracellularproteins through electrostaticinteractionswith the negatively chargedgroups on the polysaccharide.Proteoglycans are major componentsof all extracellularmatrices. Glycoproteins have one or severaloligosaccharides of varying complexity joined covalently to a protein. They are usually found on the outer face of the plasmamembrane(as part of the glycocalyx),in the extracellular matrix, and in the blood. Inside cells they are found in specificorganellessuchas Golgicomplexes, secretorygranules,and lysosomes.The oligosaccharide portions of glycoproteinsare very heterogeneousand, like glycosaminoglycans,they are rich in information, forming highly specific sites for recognition and highaffinity binding by carbohydrate-bindingproteins called lectins. Some cytosolic and nuclear proteins can be glycosylatedas well. Glycolipids are membranesphingolipidsin which the hydrophilic head groups are oligosaccharides. As in glycoproteins,the oligosaccharides act as specificsites for recognition by lectins. The brain and neurons are rich in glycolipids,which help in nerve conduction and myelin formation. Glycolipidsalso play a role in signal transductionin cells.

Proteoglyca nsAre6lycosaminoglycan-(0ntainin g Macrornolecules ofthe(ell5urface and [xtracellular Matrix Mammalian cells can produce 40 types of proteoglycans. These molecules act as tissue organizers, and they influence various cellular activities, such as growth factor activation and adhesion. The basic proteoglycan unit consists of a "core protein" with covalently attached glycosaminoglycan(s). The point of attachment is a Ser residue, to which the glycosaminoglycan is joined through a tetrasaccharide bridge (Fig. 7-24). The Ser residue is generally in the sequence -Ser-Gly-X-Gly(where X is any amino acid residue), although not every protein with this sequence has an attached glycosamino$ycan.

Itr"]

a lnysc,o p r o t eainndsG, l y c o l i p i d s 7 . 3G l y c o c o n j u gPartoetse: o g l y cG

Carboxyl terminus

(a)

? ?

I

GIv

I

T (B1--3)

Glypican

Syndecan

(,Bt-+) "lt \Br+41(Br-'s)(pr+s)

Globular domain

Heparan sulfate Chondroitin sulfate

Co."p.ot"in- J

i

Amino terminus

FIGURE 7-24 Proteoglycanstructure, showing the tetrasaccharide bridge. A typicaltetrasaccharide linker(blue)connectsa glycosaminoglycan-in this casechondroitin4-sulfate(orange)-to a Ser residue (pink)in the core protein.The xyloseresidueat the reducingend of the linker is joined by its anomericcarbonto the hydroxylof the Serresidue.

Many proteoglycansare secretedinto the extracellular matrix, but some are integral membraneproteins (see Fig. 11-6) . For example,the sheet-likeextracellularmatrix (basal lamina) that separatesorganizedgroups of cellsfrom other groupscontainsa family of coreproteins (M,20,000 to 40,000),eachwith severalcovalentlyattachedheparansulfatechains.There are two major families of membrane heparan sulfate proteoglycans. Syndecans havea singletransmembranedomainand an extracellular domain bearing three to five chahs of heparan sulfate and in some caseschondroitin sulfate (Fig.7-25a). Glypicans areattachedto the membrane by a lipid anchor, a derivative of the membrane lipid phosphatidylinositol (Chapter11) Both s;mdecansand gllpicanscanbe shedinto the extracellularspace.A proteasein the ECM that cuts closeto the membranesurface releasessyndecanectodomains(those domains outside the plasmamembrane),and a phospholipase that breaks the connection to the membrane lipid releasesglypicans.Numerouschondroitin sulfate and dermatan sulfate proteoglycansalso exist, some as membrane-bound entities,others as secretedproducts in the ECM. The glycosaminoglycan chainscan bind to a variety of extracellularligandsand thereby modulatethe ligands' interaction with speci-flcreceptors of the cell surface. Detailed studies of heparansulfate demonstratea domain structure that is not random; some domains (typically 3 to 8 disaccharideunits long) differ from neighboringdomainsin sequenceand in ability to bind to specificproteins. Highly sulfated domains (called NS domains) alternate with domains having unmodified GlcNAc and GlcA residues (N-acetylated,or NA, domains) (Fig. 7-25b). The exactpattern of sulfationin the NS domarn depends on the particular proteoglycan; given the number of possible modifications of the GlcNAc-IdoA dimer, at least 32 different disaccharide units are possible.Furthermore,the samecore protein

Chondroitin sulfr Outside

Core protein

o-

Cleavage site

Membrane

Inside

coo-

(b) Heparan sulfate I GlcNAc

C Gtca I GIcNS

NS

@raoe

-NA

domain NS domain

2S 2-O-sulfate 6S 6-O-sulfate

7-25 Twofamiliesof membraneproteoglycans.(a)Schematic FIGURE Synand a glypicanin the plasmamembrane. of a syndecan diagrams bedecansare held in the membraneby hydrophobicinteractions tween a sequenceof nonpolaramino acid residuesand plasma cut near by a singleproteolytic lipids;theycan be released membrane aminothe extracellular In a typicalsyndecan, surface. the membrane (by linkers such tetrasaccharide attached terminaldomainiscovalently two chonchains and heparan sulfate 7-24\ to three in Fig as those droitin sulfatechains Clypicansare held in the membraneby a .l.l), lipid(CPlanchor;seeChapter and membrane attached covalently All bond is cleavedby a phospholipase. are shedif the lipid-protein whichformdisulfidebonds Cysresidues, have14 conserved glypicans to stabilizethe proteinmoiety,and eithertwo or threeglycosaminocloseto the memnearthe carboxylterminus, glycanchainsattached brane surface.(b) Along a heparansulfatechain, regionsrich in with regionswith alternate the NS domains(green), sulfatedsugars, ClcA, the NA domains of ClcNAc and chieflyunmodifiedresidues (gray).One of the NS domainsis shownin more detail,revealinga with a CIcNS(N-sulfoglucosamine), highdensityof modifiedresidues: sutfateesterat C-6; and both CIcA and ldoA, with a sulfateesterat C-2.The exactpatternof sulfationin the NS domaindiffersamong proteoglycans.

can display different heparan sulfate structures when in different cell types. sy'nthesized The NS domains bind speciflcally to extracellular proteins and signalingmoleculesto alter their activtties. The changein activity may result from a conformational change in the protein that is induced by the binding

[rt*_]C a r b o h y d ra nt edGs l y c o b i o l o g y (a) Conformational

(b) Enhanced protein-protein

activation Factor Xa

Thrombin

A conformational change induced in the protein antithrombin (AT) on binding a specifrc pentasaccharide NS domain allows its interaction with blood clotting factor Xa, preventing clotting.

(c) Coreceptor

for extracellular

interaction

ligands

Binding of AT and thrombin to two adjacent NS domains brings the two proteins into close proximity, favoring their interaction, which inhibits blood clotting

(d) Cell surface localization/concentration

NS domain FGF receptor dimer

LM.-b.un" NS domains interact with both the fibroblast erowth factor (FGF) and its receptor, bringing the oligomeric complex together and increasing the effectiveness of a low concentration ofFGF.

The high rlensity ofnegative charges in heparan sulfate attracts positively charged lipoprotein lipase molecules and holds them by electrostatic and sequence-specific interactions wiih NS domains.

tlGURt7-26 Fourtypesof proteininteractionswith NS domainsof heparansulfate.

(Fig. 7 -26a), or it may be due to the ability of adjacent domains of heparan sulfate to bind to two different proteins, bringing them into close proximity and enhancing protein-protein interactions (Fig. 7-26b). A third general mechanism of action is the binding of extracellular signal molecules (growth factors, for example) to heparan sulfate, which increases their local concentrations and enhances their interaction wrth growth factor receptors in the cell surface; in this case, the heparan sulfate acts as a coreceptor (Fig 7-26c). For example, flbroblast growth factor (FGF), an extracellular protein signal that stimulates cell division, flrst binds to heparan sulfate moieties of syndecan molecules in the target cell's plasma membrane. Syndecan presents FGF to the FGF plasma membrane receptor, and only then can FGF interact productively with its receptor to trigger cell dir.rsion. Finally, in another type of mechanism, the NS domains interact-electrostaticaily and otherwise-with a variety of soluble moiecules outside the cell, maintaining high local concentrations at the cell surface (Fig. 7-26d). The importance of correctly synthesizing sulfated domains in heparan sulfate is demonstrated in mutant ("knockout") mice lacking the en4rme that sulfates the C-2 hydroxyl of IdoA. These animals are born without kidneys and with very severe developmental abnormalities of the skeleton and eyes. Other studies demonstrate that membrane proteoglycans are important in lipoprotein clearance in the liver. There is growing evidence that the path taken by developing axons in the nervous system, and thus the wiring circuitry, is influenced by

proteoglycans containing heparan sulfates and chondroitrn sulfate, which provide directional cues for axon outgrowth. Some proteoglycans can form proteoglycan aggregates, enormous supramolecular assemblies of many core proteins all bound to a single molecule of hyaluronan. Aggrecan core protein Q'1, -250,000) has muitiple chains of chondroitin sulfate and keratan sulfate, joined to Ser residues in the core protein through trisaccharide linkers, to give an aggrecan monomer of M, -2 x 10t'. When a hundred or more of these "decorated" core proteins bind a single, extended molecule of hyaluronate (h-ig. 7-27), the resulting proteoglycan aggregate (M, > 2 x 10") and its associatedwater of hydration occupy a volume about equal to that of a bacterial cell! Aggrecan interacts strongly with collagen in the extracellular matrix of cartilage, contributing to the development, tensile strength, and resiliency of this connective tissue. Interwoven with these enormous extracellular proteoglycans are fibrous matrix proteins such as collagen, elastin, and flbronectin, forming a cross-linked meshwork that gives the whole extracellular matrix strength and resilience. Some of these proteins are multiadhesive, a single protein having binding sites for several different matrix molecules. Fibronectin, for example, has separate domains that bind flbrin, heparan sulfate, collagen, and a family of plasma membrane proteins called integrins that mediate signaling between the cell interior and the extracellular matrix (see Fig. 12-28). The overail picture of cell-matrix interactions that emerAes

l yncdo l i p i dI sZ S s I a lnysc,o p r o t e i n sG, a 7 . 3G l y c o c o n j u gPart o e tse: o g l y cG

Integrin isaccharides) Aggrecan core protein Proteoglycan

tl.!i:'i'"

_Fibronectin ,u+..

#/rd..,

Cross-linked frbers of collagen

ltJ

tIGURE7-27 Proteoglycanaggregateof the extracellularmatrix. with many aggrecanmolecules. Schematicdrawingof a proteoglycan with One verylongmoleculeof hyaluronan isassociated noncovalently .100 about moleculesof the core proteinaggrecanEachaggrecanmoleculecontainsmanycovalently boundchondroitin sulfateand keratan sulfatechains.Linkproteins at the junctionbetweeneachcoreprotein inandthe hyaluronan backbone mediatethe coreprotein-hyaluronan Themicrograph viewed teraction. showsa singlemoleculeof aggrecan, .l-'l with the atomicforcemicroscope(seeBox 1 ).

(FiS. 7-28)

shows an array of interactions between cel-

lular and extracellularmolecules.These interactions serve not merely to anchor cells to the extracellular matrix but alsoto provide pathsthat direct the mrgration of cells in developingtissue and to convey information in both directionsacrossthe olasmamembrane.

7-28 lnteractionsbetweencellsand the extracellularmatrix. FIGURE of the extracellubetweencellsand the proteoglycan The association and by an exprotein(integrin) lar matrixis mediatedby a membrane protein(fibronectin in this example)with bindingsitesfor tracellular of colNote the closeassociation both integrinand the proteoSlycan. and proteoglycan. lagenfiberswith the fibronectin

(ovalently 0ligosaccharides Attached Have 6lycoproteins Glycoproteinsare carbohydrate-proteinconjugatesin which the glycans are smaller, branched, and more structurally diverse than the glycosaminoglycansof proteoglycans.The carbohydrate is attached at its anomeric carbon through a glycosidiclink to the -OH of a Ser or Thr residue(O-linked),or through an l/-glycosyl link to the amide nitrogen of an Asn residue (Nlinked) (FiS. 7-29). Someglycoproteinshave a single

(a) O-linked

FIGURE 7-29 Oligosaccharide linkagesin glycoprohave a glycoteins. (a) O-linkedoligosaccharides sidicbondto the hydroxylgroupof SerorThrresidues (pink),illustrated herewith CalNAcasthesugarat the reducingend of the oligosaccharide. One simple chainandone complexchainareshown (b)N-linked have an N-glycosylbond to the oligosaccharides illustrated amidenitrogenof an Asn residue(green), herewith ClcNAcas the terminalsugarThreecomchainsthatareN-linked montypesof oligosaccharide of in glycoproteins areshownA completedescription structurerequiresspecification of the oligosaccharide (a or B) of each glycopositionand stereochemistry sidiclinkaee.

ft)N-linked

C:O

I

GalNAc

CH, GlcNAc Examples:

Asn

-_-l

andGlycobiology [256] [arbohydrates oligosaccharidechain, but many have more than one; the carbohydratemay constitute from 1% to 70o/oor more of the glycoproteinby mass.Mucins are secreted or membraneglycoproteinsthat can contain large numbers of Olinked oligosaccharidechains. Mucins are presentin most secretions;they give mucusits characteristic slipperiness.About half of all proteins of mammals are glycosylated,and about l% of all mammalian genes encode enzymesinvolved in the synthesisand attachmentof theseoligosaccharide chains.Sequences for the attachment of Olinked chainstend to be rich in Gly,Val,and Pro residues.In contrast the attachmentof N-linked chains dependson the consensussequence N-{P)-tSTl (see Box 3-3 for the conventionson representing consensussequences).As with proteoglycans, not all potentialsitesare used. One class of glycoproteinsfound in the cytoplasm and the nucleus is unique in that the glycosylatedpositions in the protein carry only single residues of l/-acetylglucosamine,in O-glycosidiclinkage to the hydroxyl group of Ser side chains.This modification is reversibleand often occurson the sameSer residues that are phosphorylatedat some stage in the protein's activity. The two modiflcationsare mutually exclusive, and this type of glycosylationmay prove to be important tn the regulation of protein activity. We discussit in the context of protein phosphorylationin Chapter12. As we shall seein Chapter11,the externalsurface of the plasmamembranehas many membraneglycoproteins with arraysof covalentlyattachedoligosaccharides of varyrngcomplexty. The first well-characterizedmembraneglycoproteinwasglycophorinA of the ery[hrocyte membrane(see Fig. I1-7). It contains60% carbohydrate by mass,in the form of 16 oligosaccharidechains (totaling60 to 70 monosaccharide residues)covalently attached to amino acid residuesnear the amino terminus of the polypeptidechain.Fifteen of the oligosaccharide chainsare O-linkedto Ser or Thr residues,and one is Nlinked to an Asn residue. Glycomics is the systematiccharacterizationof all of the carbohydratecomponentsof a givencell or tissue, includingthose attachedto proteins and to lipids. For glycoproteins,this also means determtning which proteins are glycosylatedand where in the amino acid sequence each oligosaccharideis attached. This is a challengingundertaking,but worthwhile becauseof the potential insightsit offers into normal patterns of glycosylation and the ways in which they are altered during developmentor in geneticdiseasesor cancer.Current methodsof characterizingthe whole carbohydratecomplement of cells depend heavily on sophisticatedapplicationof massspectroscopy(seeFig. Z-BZ). The structures of a large number of O- andrV-linked oligosaccharidesfrom a variety of glycoproteinsare known; Figure 7-29 shows a few typical examples.We considerthe mechanismsby which specificproteinsacquire speciflcoligosaccharide moietiesin Chapter22.

Many of the proteins secreted by eukaryotic cells are glycoproteins,including most of the proteins of blood. For example,immunoglobulins(antibodies) and certain hormones,such as follicle-stimulatinghormone, luteinizing hormone, and thyroid-stimulating hormone, are glycoproteirs. Many milk proteins, including lactalbumin, and some of the proteirLssecretedby the pancreas (such as ribonuclease)are glycosylated,as are most of the proteinscontainedin lysosomes. The biological advantagesof adding oligosaccharides to proteins are slowly being uncovered.The very hydrophilic clusters of carbohydratealter the polarity and solubility of the proteins with which they are conjugated.Oligosaccharidechainsthat are attachedto newly synthesizedproteins in the endoplasmicreticulum (ER) and elaboratedin the Golgicomplexserveas destination labels and also act in protein quality control, targeting misfolded proteins for degradation(see Fig. 27-39). When numerous negatively charged oligosaccharide chains are clustered in a single region of a protein, the chargerepulsionamongthem favorsthe formation of an extended,rodlike structure in that region. The bulkiness and negativechargeof oligosaccharidechainsalso protect some proteins from attack by proteolytic enzyrnes.Beyond these global physical effects on protein structure,there are alsomore specificbiologicaleffects of oligosaccharide chainsin glycoproteins(Section7.4). The importance of normal protern glycosylationis clear from the flnding ofat least 18 different genetic disorders of glycosylationin humans,all causing severelydefective physical or mental development;someof these disorders are fatal.

Glycolipids andLipopolysaccharides AreMembrane Components Glycoproteinsare not the only cellular componentsthat bear complexoligosaccharide chains;somelipids, too, have covalentlybound oligosaccharides.Gangliosides are membranelipids of eukaryoticcells in which the polar head group, the part of the lipid that forms the outer surface of the membrane,is a complex oligosaccharide containing a sialic acid (Fig. 7-9) and other monosaccharideresidues.Someof the oligosaccharide moieties of gangliosides,such as those that determine human blood groups (seeFig. 10-15), are identicalwith those found in certain glycoproteins,which therefore also contribute to blood group type. Like the oligosaccharide moieties of glycoproteins,those of membranelipids are generally,perhapsalways,found on the outer face of the plasmamembrane Lipopolysaccharides are the dominant surface ff feature of the outer membraneof gram-negative f bacteria such as Escheri,chi,acoli, and SaLmonetta typhzmuri,urzz.These molecules are prime targets of the antibodiesproduced by the vertebrate immune system in responseto bacterial infection and are therefore

3r1

n ao l e c u l e s :STuhgeaCr o d e 7 . 4C a r b o h y d raastIensf o r m a t i oM

O-Specific chain

Core

-o- o -o $ HO'

o ilo

P /oH

LipidA

7Y .3 Glycoconjugates: SUMMAR ns,Glycoproteins, Proteoglyca a n dG l y c o l i p i d s Proteoglycansare glycoconjugatesin which one or morelargeglycans,calledsulfated glycosaminoglycans (heparansulfate,chondroitin sulfate,dermatansulfate,or keratan sulfate) are covalentlyattachedto a coreprotein.Boundto the outside of the plasmamembraneby a transmembranepeptide or a covalentlyattached providepoints of adhesion, lipid, proteoglycans recognition,and information transfer betweencells, or betweenthe cell and the extracellularmatrix. Glycoproteinscontain oligosaccharidescovalently Iinked to Asp or Ser/Thr residues.The glycans are typically branchedand smallerthan glycosaminoglycans. Many cell surfaceor extracellularproteils are glycoproteins,as are most secretedproteins.The covalentlyattached influencethe folding and stability oligosaccharides of the proteins,provide critical information about the targeting of newly synthesizedproteins, and allowfor speciflcrecognitionby other proteins. Glycomicsis the determinationof the full complementof sugar-containingmoleculesin a cell or tissue,and the determinationof the function of eachsuchmolecule.

FIGURI7-30 Bacteriallipopolysaccharides. Schematicdiagramof the lipopolysaccharide of the outermembraneol Salmonella typhimurium. Kdo is 3-deoxy-D-manno-octulosonic acid (previouslycalled ketodeoxyoctonicacid);Hep is r-glycero-o-manno-heptose; AbeOAc is abequose(a 3,6-dideoxyhexose) acetylatedon one of its hydroxyls. Therearesix fattyacid residues in the lipidA portionof the molecule Differentbacterialspecieshavesubtlydifferentlipopolysaccharide structures/ but they havein commona lipid region(lipidA), a core oligosaccharide alsoknownas endotoxin, chain, and an "O-specific" which is the principaldeterminant of the serotype(immunological reactivity) of the bacterium.Theoutermembranes of the gram-negative bacteriaS.typhimuriumand E coll containso manylipopolysaccharide moleculesthat the cell surfaceis virtuallycoveredwith O-specific chains.

important determinantsof the serotype of bacterial strains (serotypesare strains that are distinguishedon the basisof antigenicproperties).The lipopolysaccharides of S typhi,murium contain six fatty acids bound to two glucosamine residues,oneof which is the point of (Fig. 7-30). attachmentfor a complexoligosaccharide E. coli, has similar but unique lipopolysaccharides.The Iipid A portion of the lipopolysaccharides of somebacteria is calledendotoxin;its toxicity to humansand other animals is responsiblefor the dangerouslylowered blood pressurethat occursin toxic shocksyndromeresulting from gram-negativebacterialinfections.I

Glycolipidsin plants and animalsand lipopolysaccharidesin bacteriaare componentsof the cell envelopewith covalentlyattachedoligosaccharide chainsexoosedon the cell'souter surface.

asInformational 7.4 Carbohydrates Code TheSugar Molecules: Glycobiology,the study of the structure and function of glycoconjugates,is one of the most active and exciting areas of biochemistry and cell biology. As is becoming increasinglyclear, cells use specific oligosaccharidesto encode important information about intracellular targeting of proteins, cell-cellinteractions,cell differentiation and tissue development,and extracellular signals. Our discussionusesjust a few examplesto illustratethe diversity of structure and the rangeof biologicalactivity In Chapter 20 we discussthe of the glycoconjugates. including peptidoglyof polysaccharides, biosymthesis of oligosaccharide assembly 27,the in Chapter and can; glycoproteins. on chains Improved methods for the analysisof oligosaccharide and polysaccharidestructure haverevealedremarkof able complexity and diversity in the oligosaccharides glycoproteinsand glycolipids.Considerthe oligosaccharide chains in Figure 7-29, typical of those found in many glycoproteins.The most complex of those shown

-

l

andGtycobiology !2581 Carbohydrates contains 14 monosaccharideresidues of four different kinds,variouslylinkedas (1-+2), (1-+3), (1-+4), (1-+6), (2-->3),and (2-+6), somewith the a and somewith the Branched structures, not found in nuB cor-Lflguration. cleic acids or proteins, are conrmonin oligosaccharides. With the reasonableassumptionthat 20 different monosaccharidesubunits are availablefor construction of oligosaccharides, we can calculatethat many billions of different hexameric oligosaccharidesare possible;this compareswith 6.4 x 107 (206) different hexapeptides possiblewith the 20 commonaminoacids,and4,096(4o) different hexanucleotideswith the four nucleotide subunits. If we also allow for variationsin oligosaccharides resulting from sulfation of one or more residues,the number of possibleoligosaccharides increasesby two orders of magnitude.In reality, only a subsetof possible combinationsis found, given the restrictionsimposedby the biosyntheticenz;.rnesand the availabilityof precursors.Nevertheless,the enormouslyrich structural information in glycansdoesnot merely rival but far surpasses that of nucleic acids in the density of information containedin a moleculeof modestsize.Eachof the oJigosaccharidesrepresentedin Figure 7-29 presentsa unique, three-dimensionalface-a word in the sugar codereadableby the proteins that interact with it.

(ode lectins AreProteins ThatRead the5ugar andMediate Many Biologi(al Processes Lectins, found in all organisms,are proteins that bind carbohydrateswith lugh specificityand with moderateto high affinity (Table 7-3). Lectins servein a wide variety of cell-cellrecognition,signaling,and adhesionprocesses and rn intracellular targeting of newly symthesizedproteins. Plant lectins, abundantin seeds,probablyserveas

Lectinsourceand leetin

deterrentsto insectsand other predators.In the laboratory puri-fledplant lectins are usefulreagentsfor detecting and separating glycans and glycoproteins with djfferent oligosaccharide moieties.Herewe discussjust a few examplesof the roles of lectins in animalcells. Somepeptide hormonesthat circulate in the blood have oligosaccharidemoieties that strongly influence their circulatory half-life. Luteinizing hormone and thyrotropin (polypeptidehormonesproducedin the adrenal cortex) have l/linked oligosaccharidesthat end with the disaccharideGalNAc4S(B 1--+4)GlcNAc,which is recognizedby a lectin (receptor) of hepatocytes. (GalNAc4Sis N-acetylgalactosamine sulfated on the -OH group at C-4.)Receptor-hormone interactionmediates the uptake and destruction of luteinizing hormone and thylotropin, reducing their concentrationin the blood.Thus the blood levelsof thesehormonesundergo a periodicrise (due to pulsatilesecretionby the adrenal cortex) and fall (due to continual destruction by hepatocltes). The residues of Neu5Ac (a sialic acid) situated at the ends of the oligosaccharidechains of many plasma glycoproteins (Fig. 7-29) protect those proteins from uptake and degradationin the liver. For example,ceruIoplasmin,a copper-containingserum glycoprotein,has several oligosaccharidechains ending in NeuSAc.The mechanismthat removessialicacid residuesfrom serun glycoproteinsis unclear.It may be due to the activity of the enz;rmesialidase(also called neuraminidase)produced by invadingorganismsor to a steady,slow release by extracellular enz;.'rnes. The plasmamembraneof hepatocyteshas lectin molecules(asialoglycoprotein receptors; "asialo-" indicating "without sialic acid") that specificallybind oligosaccharidechains with galactose residuesno longer "protected" by a terminal Neu5Ac

Abbreviation

tigand(s)

ConcanavalinA

ConA

Manal-OCH3

Gri,ffoni,a si,m,pli,ciJoli,alectin 4 Wheat germ agglutinin

GS4

Lewis b (Leb) tetrasaccharide

WGA

Neu5Ac(c2-+3)Gal(B1-+4)Glc

Plant

GlcNAc(B1--+4)GIcNAc Ricin

Gal(B1-+4)Glc

Animal Galectin-1 Marmose-bindingprotein A

Gal(B1--+4)GIc MBP-A

High-mannoseoctasaccharide

ViraI Influenzavirus hemagglutinin Polyomavirus protein 1

HA

NeubAc(a2--+6)Gaf(B 1--+4)Glc

VP1

Neu5Ac(a2--+3)Gal(B 1-+4)Glc

Enterotoxin

LT

Gal

Choleratoxin

CT

GMI pentasaccharide

Bacterial

Source: Weiss, W.l.& Drickamer, K.(1996)Structural basisof lectin-carbohydrate recognition.Annu. Rev.Biochen.65,44l-473.

P"l

7 . 4C a r b o h y d raastIensf o r m a t i oM n ao l e c u lTehs e :S u g aCr o d e

residue.Receptor-ceruloplasmin interactiontriggersendocytosisand destructionof the ceruloplasmin.

Glycoprotein ligand for integrin

Glycoprotein ligand for P-selectin

OH HOH2C\ | .*H H-C

IH

HO HN HeC-Q, \\

o

N-Acetylneuraminicacid(Neu5Ac) (a sialicacid)

A similar mechanism is apparently responsible for remonng "old" efihrocy'tes from the mammalian bloodstream. Newly sy'nthesized erythrocytes have several membrane glycoproteins with oJigosaccharidechains that end in Neu5Ac. When the sialic acid residues are removed by withdrawing a sample of blood from experimental arumals, treating it with sialidasein vitro, and reintroducing it into the circulation, the treated efihrocy'tes disappear from the bloodstream within a few hours; erythrocS,tes with intact oligosaccharides (withdrawn and reintroduced without sialidasetreatment) continue to circulate for days. Cell surface lectrns are important in the developffi ment of some human diseases-both human lectins E and the lectins of infectious agents. Selectins are a family of plasma membrane lecturs that mediate cell-cell recognition and adhesion in a wide range of cellular processes. One such process is the movement of immune cells (neutrophils) through the caprllary wall, from blood to tissues, at sites of infection or inflammation (Fig. 7-31). At an infection site, P-selectin on the surface of capillary endothelial cells interacts with a specific oligosaccharide of the glycoproteins of circulating neutrophils. This interaction slows the neutrophils as they adhere to and roll along the endothelial lining of the capillaries. A second interaction, between integrin molecules (p. 455) in the neutrophil plasma membrane and an adhesion protein on the endothelial cell surface, now stops the neutrophil and allows it to move through the capillary wall into the ilrfected tissues to initiate the rmmune attack. TWo other selectins participate in this "lymphocyte homing": Eselectin on the endothelial cell and L-selectin on the neutrophil bind their cognate oligosaccharides on the neutrophil and endothelial cell, respectively. Human selectins mediate the inflammatory responses in rheumatoid arthritis, asthma, psoriasis, multiple sclerosis,and the rejection of transplanted organs, and thus there is great interest in developing drugs that inhibit selectin-mediated cell adhesion. Many carcinomas express an antigen normally present only in fetal cells (sialyl Lewis x, or sialyl Le*) that, when shed into the circulation, facilitates tumor cell survival and metastasis. Carbohydrate derivatives that mimic the sialyl

FIGURE 7-31 Roleof lectin-ligandinteractionsin lymphocytemovement to the site of an infection or injury. A neutrophilcirculating betweenP-selectin througha capillaryis slowedby transientinteractions cells moleculesin the plasmamembraneof the capillaryendothelial As it ligandsfor P-selectin on the neutrophilsurface. and glycoprotein P-selectin molecules,the neutrophilrolls interactswith successive stronger interNeara siteof inflammation, alongthecapillarysurface. actionsbetweenintegrinin the capillarysurfaceand its ligandin the Theneutrophilstopsrolling neutrophilsurfaceleadto tightadhesion. sentout fromthe siteof inflammaand,underthe influenceof signals throughthe capillarywall-as it tion, beginsextravasation-escape movestowardthe siteof inflammarron.

Le" portion of sialoglycoproteins or that alter the biosynthesis of the oligosaccharide might prove effective as selectin-specific drugs for treating chronic hflammation or metastatic disease. Several animal viruses, includtng the influerza virus, attach to their host cells through interactions with oligosaccharides displayed on the host cell surface. The lectrn of the ffiuerza virus, known as the llA (hemagglutinin) protein, is essential for viral entry and hfection. After the virus has entered a host cell and has been replicated, the newly ryrrtheszed viral particles bud out of the cell, wrapped in a portion of its plasma membrane. A viral sialidase (neuraminidase) trims the terminal sialic acid residue from the host cell's oligosaccharides,releasing the viral particles from their interaction with the cell and preventing their aggregation with one another. Another round of in-fection can now begin. The antiviral drugs oseltamivir (Tamiflu) and zanamivir (Relerza) (next page) are used clinically in the treatment of hfluenza. These drugs are sugar analogs; they inhibit the viral sialidase by competing with the host cell's oligosaccharidesfor binding. This orevents the release ofviruses from the infected cell

Lruu-C a r b o h y d raant edGs l y c o b i o l o g y and also causesviral particles to aggregate,both ofwhich block another cycle ofinfectron

4

o

NH

\\

HN

o

,/..'.-NHz NH

HN

BarryJ Marshall Oseltamivir (Tamiflu)

Zanamivir (Relenza)

Lectins on the surface of the herpes simplex viruses HSV-I and HSV-2 (the causative agents of oral and genital herpes, respectively) bind specifically to heparan sulfate on the host cell surface as a flrst step in the infection cycle; infection requires precisely the right pattern of sulfation on this polymer. Analogs of heparan sulfate that mimic its interaction with the viruses are being investigated as possible antiural drugs, interfering with interactions between virus and cell. Some microbial pathogens have lectins that mediate bacterial adhesion to host cells or the entry of toxin into cells. For example, Helicobctcter py\orz-shown by Barry J. Marshall and J. Robin Warren in the 1980s to be responsible for most gastric ulcers-adheres to the inner surface of the stomach as bacterial membrane Iectins interact with speciflc oLigosaccharidesof membrane glycoproteins of the gastric epithelial cells (Fig. 7-32). Among the binding sites recognizedby H. pylori, is the ohgosaccharideLewis b (Leu), when it is part of the type O blood group determinant. This observation helps to explain the severalfold greater incidence of gastric ulcers in people of blood type O than in those of type A or B. Chemically syrrthesized analogs of the Leb oligosaccharide may prove useful in treating this t;,pe of ulcer. Administered orally, they could prevent bacterial adhesion (and thus infection) by competing with the gastric glycoproteins for binding to the bacterial iectin.

FIGURE7-32 An ulcer in the making. Helicobacter pylorl cells adheri n g t o t h e g a s t r i cs u r f a c e .T h i s b a c t e r i u m c a u s e su l c e r s b y i n t e r a c t i o n s b e t w e e na b a c t e r i a ls u r f a c el e c t i n a n d t h e L e bo l i g o s a c c h a r i d e( a b l o o d g r o u p a n t i g e n )o f t h e g a s t r i ce p i t h e l i u m

J RobinWarren

Some of the most devastating of the human parasitic diseases,wrdespread in much of the developing world, are caused by eukaryotic microorganisms that display unusual surface oligosaccharides,which in some cases are known to be protective for the parasites. These organisms include the trypanosomes, responsible for African sleeping sickness and Chagas disease;Plasmodi,um Jalci,paT"?)7n, the malaria parasite; and Entamoeba hi,sto|yt'ica, the causative agent of amoebic dysentery. The prospect of finding drugs that interfere with the syrrthesis of these unusual oligosaccharide chains, and therefore with the replication of the parasites, has inspired much recent work on the biosl'nthetic pathways of these oligosaccharides. The cholera toxin molecule (produced by the bacterium Vi,bri,o choLerae) triggers diarrhea after entering intestinal cells responsible for water absorption from the intestine. The toxin attaches to its target cell through the oligosaccharidemoiety of ganglioside GMl, a membrane phospholipid (for the structure of GMl see Box 10-2, Fig. 1), on the surface of intestinal epithelial cells. Similarly, the pertussis toxin produced by BordeteLla pertusses, the bacterium that causes whooping cough, enters target cells only after interacting with a host cell oligosaccharide (or perhaps several oligosaccharides) bearing a terminal sialic acid residue. Understanding the details of the oligosaccharide-binding sites of these toxins (lectins) may allow the development of genetically engineered toxin analogsfor use in vaccines. Toxin analogsengineered to lack the carbohydrate binding site would be harmless because they could not bind to and enter cells, but they might elicit an immune response that would protect against later exposure to the natural toxin. It is also possibie to imagine drugs that would act by mimicking cell surface oligosaccharides, binding to the bacterial lectins or toxins and preventing their productive binding to cell surfaces. Lectins also act intracellularly. An oligosaccharide containing mannose 6-phosphate marks newly sy'rthesized proteins in the Golgi complex for transfer to the lysosome (see Fig. 27-39). A common structural feature on the surface of these glycoproteins, the signal patch, is recognized by an enzyme that phosphorylates (in a two-step process) a mannose residue at the terminus of an oligosaccharide chain. The resulting mannose 6-phosphate residue is then recognized by the cationdependent mannose 6-phosphatereceptor, a membrane-

7 . 4C a r b o h y d raastIensf o r m a t i oM n ao l e c u l e s :5TuhgeaCr o d e[ t 6 t l

associatedlectin with its mannosephosphatebinding site on the lumenal side of the Golgi complex. When a section of the Golgi complex containingthis receptor buds off to form a transport vesicle,proteins containing mannosephosphateresiduesare draggedrnto the forming bud by interaction of their mannosephosphateswith the receptor;the vesiclethen movesto and fuseswith a lysosome,depositingits cargo therein. Many,perhaps all, ofthe degradativeenzyrnes(hydrolases)ofthe lysosome are targeted and deliveredby this mechanism. Someof the mannose6-phosphatereceptorscan capture enzymes containing the mannose 6-phosphate residueand direct them to the lysosome.This processis the basis for "enz;.,rnereplacementtherapy" to correct lysosomalstoragedisordersin humans.I Other lectins act in other kinds of protein sorting. Any newly synthesizedprotein in the endoplasmicreticulum already has a complex oligosaccharideattached, which can be bound by either of two ER lectins that are also chaperones:calnexin (membrane-bound)or calreticulin (soluble). These lectrns link the new protein with an enz).rnethat brings about rapid disulfide exchangeas the protein tries variousways to fold, leading eventuallyto the native conformation.At this point, enzJ,.rnes in the ER trim the oligosaccharide moietyto a form recognizedby anotherlectin, ERGIC53.which drawsthe folded protein (glycoprotein)into the Golgi complexfor further maturation.If a proteh hasnot folded effectively, the oLigosaccharide is tnmmed to another form, this one recogruzedby a lectin, EDEM,that initiatesmovementof the defectivelyfolded protein into the cytosol,where it will be degraded.Thus, protein glycosylationservesin the ER asa kind of quality control signal,allowingthe cell to eliminateimproperly folded proteins. (This processis describedin greaterdetail in Chapter27.)

(a) FIGURE 7-33 Detailsof a lectin-carbohydrate interaction.Structure of the bovine mannose6-phosphatereceptorcomplexedwith man(PDBlD 1M6P).Theproteinis represented nose6-phosphate asa surfacecontourimage,showingthe surfaceas predominantly negatively charged(red)or positivelycharged(blue).Mannose6-phosphate is shownasa stickstructure; a manganese ion is shownin violet (b)An view of the bindingsite.Mannose6-phosphate enlarged is hydrogen-

Lectin-Carbohydrate Interactions AreHighly Specifi c and0ftenPolyvalent In all the functionsof lectinsdescribedabove,andin many moreknownto involvelectin-oligosaccharide interactions, it is essentialthat the oligosaccharide havea uniquestructure, so that recognitionby the lectin is higtrly specific. proThe high densrtyof rnformationin oligosaccharides videsa sugarcodewith an essentiallyudimited numberof unique "words" small enoughto be read by a singleprotein. In ther carbohydrate-bindingsites, lectins have a subtle molecular complementarity that allows interaction only with their correct carbohydratecognates.The result is an extraordrnarilyhrgh specificityin theseinteractions. The affinity betweenan oligosaccharide and eachcarbohydrate binding domarn(CBD) of a lectin is sometimes modest (micromolarto millimolarK6 values),but the effectiveaffinity is in many casesgreatlyincreasedby lectin multivalency,in which a single lectin molecule has multiple CBDs. In a cluster of oligosaccharides-asis commonly found on a membranesurface,for example-each oligosaccharidecan engage one of the lectin's CBDs, strengtheningthe interaction.When cells expressmultiple receptors,the avidity of the interaction can be very high, enabling higirly cooperative events such as cell attachmentand rolling (seeFig. 7-3I). X-ray crystallographicstudies of the structures of several lectin-carbohydrate complexes have provided rich detailsof the lectin-sugarinteraction(Fig. 7-33). In humans,a family of 11lectinsthat bind to oligosaccharide chainsendingin sialicacid residuesplayssomeimportant biologicalroles.All of these lectins bind sialic acids at B sandwichdomainslike those found in immunoglobulins (Igs; seethis motif in the CDSprotein in F g 4-27), and the protehs are therefore called siglecs 1 to ll(sialic

(b) with the manganese ion (shown bondedto Arg111and coordinated smallerthan itsvanderWaalsradiusfor clarity).Eachhydroxylgroup to the protein.The Hisr05hydrogenof mannoseis hydrogen-bonded phosphate mannose6-phosphatemay be the to a oxygen of bonded residuethat, when protonatedat low pH, causesthe receptorto reinto the lysosome. leasemannose6-phosphate

?'1

C a r b o h y d raant edG s lycobiology

acid-recognizing1g-superfamilyle ctins), or sometimes sialoadhesins. The interactionof a siglecwith sialicacid (Neu5Ac)involveseachof the ring substituentsuniqueto Neu5Ac:the acetylgroupat C-5undergoesboth hydrogenbond and van der Waalsinteractions with the protein; the carboxylgroup makesa salt bridge with a conserved Arg residue; and the hydroxyls of the glycerol moiety hydrogen-bondwith the protein. Siglecsregulateactivities in the immune and nervous systemsand in blood cell development.Siglec-7,for example,by bindng to a specific ganglioside (GD3) containing two sialic acid residues,suppressesthe actinty of NK (natural killer) cells in the immune system,sparingcells targetedfor immune destruction from the NK killing activity. The elevated GD3 levels in tumors such as malignant melanomaand neuroblastomamay be a mechanismfor evadingthe protectiveaction of the immune system. The structure of the mannose6-phosphatereceptorAectin revealsdetails of its interaction with mannose 6-phosphatethat explainthe speciflcityof the binding and the role for a divalent cation in the lectin-sugar interaction (Fig. 7-33a). His105is hydrogen-bondedto one ofthe oxygenatomsofthe phosphate(Fig. 7-33b). When the protein tagged with mannose6-phosphate reachesthe Iysosome(which has a lower internal pH than the Golgi complex),the receptorlosesits affinity for mannose6-phosphate.Protonationof His105 may be responsiblefor this changein binding. In addition to thesevery specifi,cinteractions,there are more general interactionsthat contribute to the binding of many carbohydratesto their lectins. For ex-

{b

/H

Hydrophobic side

Indolyl moiety ofTrp . . . . . , . . , . . ...- . . . . . . . . . . . ::...:.t:,.-., ,:

FIGURE 7-34 Hydrophobicinteractionsof sugarresidues.Sugarunits havea morepolarside(thetop of thechairasshown suchasgalactose that is availableto here,with the ring oxygenand severalhydroxyls) with the lectin,and a lesspolarsidethatcan havehyhydrogen-bond with nonpolarsidechainsin the protein,such drophobicinteractions asthe indoleringofTrp residues

ample, many sugarshave a more polar and a less polar side (Fig. 74D; the more polar side hydrogen-bonds with the lectin, while the lesspolar undergoeshydrophobic rnteractionswith nonpolar amino acid residues.The sum of all theseinteractionsproduceshigh-affinitybinding and tugh specificityof lectins for their carbohydrates. This representsa kind of information transfer that is clearly central in many processeswithin and between cells.Figure 7-35 summarizessomeof the biologicalinteractionsmediatedby the sugarcode.

in recognition FIGURE 7-35 Rolesof oligosaccharides and adhesionat the cell surface.(a) Oligosaccharides as stringsof hexawith unique structures(represented gons),components of a varietyof glycoproteins or glycolipidson the outer surfaceof plasmamembranes, interactwith highspecificity and affinitywith lectinsin milieu.(b) Virusesthat infectanimal the extracellular cells,suchas the influenzavirus,bind to cell surface glycoproteinsas the first step in infection.(c) Bacterial toxins,bind to toxins,suchasthe choleraand pertussis glycolipidbeforeentering a cell.(d)Somebaca surface teria,suchas H pylori,adhereto and thencolonizeor (lectins)in the plasma infectanimalcells.(e) Selectins membrane of certaincellsmediatecell-cellinteractions, with theendothelial cellsof suchasthoseof neutroohils 6thecapillarywall at an infectionsite.(f) Themannose phosphatereceptor/lectin of the transColgi complex bindsto the oligosaccharide of lysosomal enzymes, targetingthem for transferinto the lysosome.

7 . 5W o r k i nwgi t hC a r b o h y d r a t e s

lD"]

SUMMAR 7Y . 4 C a r b o h y d r a st e s I n f o r m a t i oM n aol e c u l e s : I h eS u g aCr o d e r

Monosaccharides canbe assembledinto an almost limitless variety of oligosaccharides, which differ in the stereochemistry and positionof glycosidic bonds,the t;,pe and orientation of substituent groups,and the numberand type ofbranches. Glycansare far more information-densethan nucleicacidsor proteins.

r

Lectins, proteins with highly speciflccarbohydratebinding domains,are commonlyfound on the outer surfaceof cells,where they initiate interaction with other cells.In vertebrates,oligosaccharide tags "read"by lectinsgovernthe rate of degradationof certainpeptidehormones,circulatingproteins,and blood cells.

r

Bacterialand viral pathogensand someeukaryotic parasitesadhereto their animal-celltargets by the binding of lectins in the pathogensto oligosaccharides on the target cell surface,

r

Intracellular lectins mediateintracellular protein targeting to speciflcorganellesor to the secretory pathway.

r

X-ray crystallographyof lectin-sugarcomplexes showsthe detailed complementaritybetweenthe two molecules,which accountsfor the strength and speciicity of lectin interactionswith carbohydrates.

7.5 Working withCarbohydrates The growing appreciationof the importanceof oligosaccharide structure in biologicalrecognitionhas been the driving force behind the developmentof methodsfor analyzing the structure and stereochemistryof complex oligosaccharides.Oligosaccharideanalysisis complicatedby the fact that, unlike nucleic acidsand proteins, oligosaccharides canbe branchedand arejoinedby a variety of linkages. The high charge density of many oligosaccharides and polysaccharides, and the relative Iability of the sulfateestersin glycosaminoglycans, present further difficulties For simple,linearpolyrnerssuchas amylose,the positionsof the $ycosidicbondsaredeterminedby the classical method of exhaustivemethylation:treating the intact polysaccharide with methyliodidein a stronglybasicmediumto convertall free hydroxylsto acid-stablemethyl ethers,then hydrolyzingthe methylatedpolysaccharidein acid. The only free hydroxyls present in the monosaccharidederivatives soproducedare thosethat were involvedin $ycosidic bonds. To determine the sequenceof monosaccharide residues,includingarrybranchesthat are present,exoglycosidases of lcrownspecrficityare usedto removeresidues one at a time from the nonreducingend(s). The known speciflcityof theseexo$ycosidases often allowsdeduction of the positionand stereochemistry of the linkages.

For analysis of the oligosaccharidemoieties of glycoproteinsand glycolipids, the oligosaccharidesare releasedby purified enzymes-glycosidasesthat specifically cleave O- or l/-linked oligosaccharidesor lipases that remove lipid head groups. Alternatively, Olinked glycans can be releasedfrom glycoproteinsby treatment with hydrazine. The resulting mixtures of carbohydratesare resolved into their individual componentsby a variety of methods (Fig. 7-36), including the sametechniques used in protein and amino acid separation:fractional precipitationby solvents,and ion-exchangeand sizeexclusionchromatography(seeFig. 3-17). Highlypurifled lectins,attachedcovalentlyto an insolublesupport, are commonlyusedin affinity chromatographyof carbohydrates (see Fig. 3-l7c). Hydrolysis of oligosaccharidesand polysaccharides in strong acid yields a mixture of monosaccharides, which may be identifled and quantifled by chromatographic techniques to yield the overall composition of the polymer. Oligosaccharideanalysisrelies increasinglyon mass spectrometryand high-resolutionNMR spectroscopy. mass specMatrix-assistedlaser desorption/ionization MS) mass spectrometry and tandem trometry @ALDI (MSA4S),both describedin Box 3-2, are readily applicable to polar compoundssuch as oligosaccharides. MALDI MS is a very sensitivemethod for determining the mass of a molecular ion (in this case,the entire chain; FiS. 7-37). MSA4Srevealsthe oligosaccharide mass of the molecular ion and many of its fragments, which are usuallythe result of breakageof the glycosidic bonds.NMRanalysisalone(seeBox 4-5), especiallyfor oligosaccharidesof moderatesize,can yield much information about sequence,linkage position, and anomeric carbon conflguration.For example,the structure of the heparin segment shown as a space-fillingmodel in Figure 7-22was obtainedentirely by NMR spectroscopy. Automatedproceduresand commercialinstrumentsare used for the routine determination of oligosaccharide structure, but the sequencingof branched oligosaccharidesjoined by more than one type of bond remainsa far more formidable task than determining the linear sequencesof proteins and nucleic acids. Another important tool in working with carbohydrates is chemrcalslmthesis,which has proved to be a powerfr,rlapproachto understandingthe biologicalfimctions of glycosaminoglycansand oligosaccharides.The is diffict-tlt,but carchemistryinvolvedin such sSmtheses short segments bohydrate chemistscan now sSmthesize of almost any glycosaminoglycan,with correct stereochemistry chain length, and sulfation pattern, and oligosaccharidessigfficantly more complex than those shown in Figure 7-29. Solid-phaseoligosaccharidesynthesisis basedon the sameprinciples (and has the same advantages)as peptide synthesis(see Fig. 3-29), but requiresa set of tools unique to carbohydratechemistry: blockinggroupsand activatinggroupsthat allow the s1'nthesis of glycosidic linkageswith the correct hydroxyl

264

CarbohydratesandGlycobiology

Release oligosaccharides with endoglycosidase

I

I t r l o n - e x c h a n g ec h r o m a t o g r a p h y

I zr c"t frltratiir

,t| Purified polysaccharide

3) Lectin affinity chromatography

Separated oligosaccharides

Exhaustive methylation with CH3I, strong base

Hydrolysis with strong acid

Enzymatic hydrolysis with specifrc glycosidases

FuIIy methylated carbohydrate

Monosaccharides

High-performance liquid chromatography, or derivatization and gas-liquid chromatography

Composition of mixture Tlpes and amounts of monosaccharide units

NMR and mass spectrometry

Smaller oligosaccharides

Acid hydrolysis yields monosaccharides methylated at every -OH except those involved in glycosidic bonds

Sequence of monosaccharides; position and configuration of glycosidic bonds

Position(s) of glycosidic bonds

Sequence of monosaccharides; position and configuration of glycosidic bonds

FIGURE 7-35 Methodsof carbohydrateanalysis.A carbohydrate purifiedin the firststageof the analysis oftenrequires all fouranalvticalroutesfor itscomoletecharacterization

oo 80 2837 4

;

2192.2

oOU

.H+o

1579I 2285.2

20

2663

2908 4

2489 3

2540 m/z

FIGURE 7-37 Separation and quantificationof the oligosaccharides in a groupof glycoproteins.In thisexperiment, the mixtureof proteinsextractedfrom kidneytissuewastreatedto releaseoligosaccharides from glycoproteins, and the oligosaccharides were analyzedby matrixassisted laserdesorption/ionization (MALDIMS). massspectrometry

Eachdistinct produces oligosaccharide a peakat itsmolecular mass, and the areaunderthecurvereflects the quantityof thatol igosaccharide. The mostprominentoligosaccharide here(mass28374 u) is composed of 13 sugarresidues; in thissample,otheroligosaccharides containing as few as7 and as manyas 19 residues arealsoresolved by thismethod.

F'{

F u r t h eRre a d i n g

group. Highly purifled enzymes (glycosyltransferases) should greatly aid in the preparationof pure syr-rthetic compounds.Synthetic approacheslike this represent a current areaof great interest sinceit is difficult to purify in adequatequantity definedoligosaccharides from natural sources.Glycan microarraysof defined oJigosaccharides,analogousto the DNA arraysdescribedin Chapter9, can be probed with fluorescentlytaggedlectins to determine their binding speciflcity.

chondroitin sulfate 250 heparan sulfate 25I proteoglycan 252 glycoprotein 252 glycolipid 252 syndecan 253

SUMMAR 7Y . 5 W o r k i nwgi t h C ar b o h yr d ates

General

r

r

r

r

Establishingthe completestructureof oligosaccharides and polysaccharides requires determinationof linear sequence,branchingpositions,the conflgurationof eachmonosaccharide unit, and the positionsof the glycosidiclinkages-a more complexproblemthan protein and nucleic acid analysis. The structuresof oligosaccharides and poiysaccharidesare usually determtnedby a combinationof methods:speciflcenzynatic hydrolysisto determinestereochemistry at the glycosidicbond and producesmallerfragmentsfor further analysis;methylation to locate glycosidic bonds;and stepwisedegradationto determine sequenceand configurationof anomericcarbons. Massspectrometryand high-resolutionNMR spectroscopy, applicableto smallsamplesof carbohydrate,yield essentialinformation about sequence,configurationat anomericand other carbons,and positionsof glycosidicbonds. Solid-phase syntheticmethodsyeelddefined oligosaccharides that are ofgreat valuein exploring lectir-oligosaccharide interactionsand may prove clinically useful.

Further Reading Collins, P.M. & Ferriea R,J. (1995)Monosaccharides:The'ir Chemistry and Ther,rRolesi,n Na,tural Prodzcls, JolLnWiley & Sons,Chichester, UK A comprehensivetext at the graduatelevel. Lindhorst, T.K. (2003)Essenti,alsoJCarbohgdrate Chemi,strg and Br,ochemist?'a ,2nd edn, Wiley-VCH,Weinheim,Germany. Morrison, R.T. & Boyd, R.N. (1992)Organic Chem'[stty,6thedn, PrenticeHa1l,Upper SaddleRiver,NJ Chapters34 and 35 coverthe strucLure,sLereochemistry. nomenclature,and chemicalreactionsof carbohydrates P6rez, S. & Mulloy, B. (2005) Prospectsfor glycoinformalics Curr. Opin Struct Bi,ol 16,517-524 Brief introduction to somevery usefi:l matenalson carbohydrate structure, synthesis,chemistry,and biology on the Internet Varki, A., Cummings, 8., Esko, J., Freeze, H., Hart, G., & Marth, J. (eds) (2002)Essenti,alsoJGlgcobi.o\ogy,Cold Spring Harbor LaboratoryPress,Cold SpringHarbor,NY Structure,bioslrLthesis,metabolism,and function of glyproteoglycans,glycoproteins,and glycolipids,all cosaminoglycans, presentedat an intermediatelevel and very well illustrated. Glycosaminoglycans

and Proteoglycans

Bishop, J.R., Schuksz, M., & Esko, J.D. (2007) Heparansulfate proteoglycansfine-tunemammalianphysiology.Nature 446, 1030 1037 Biiloq H.E. & Hobert, O. (2006) The moleculardiversity of glycosaminoglycans shapesanimaldevelopmentAnnu Reu CeIl Bi,oL 22,375407 deAdvancedreview of recent studieson glycosaminoglycan fects in human diseaseand of the use of model organismsto study glycosamrnoglycan biology. Esko, J.D. & Selleck, S.B. (2002)Orderout of chaos:assemblyof Annu Reu Bi,ochem 71, ligand binding sitesin heparans;.;Jfate. 43547r

KeyTerms Tenns in bold are defined i,n the glossary glycoco4iugate 235 monosaccharide 235 oligosaccharide 235 disaccharide 235 polysaccharide 235 aldose 236 ketose 236 Fischer projection formulas 236 epimer 238 hemiacetal 238 hemiketal 238 pl.ranose 239 furanose 239 anomers 239

glgrican 253 glycomics 256 lectin 258 selectin 259 siglec 26I sialoadhesin 262

anomeric carbon 239 mutarotation 239 Haworth perspective formulas 239 reducing sugar 24I hemoglobinglycation 242 glycosidic bonds 243 reducing end 243 glycan 244 starch 245 glycogen 246 extracellularmatrix (ECM) 249 glycosaminoglycan 249 hyaluronan 250

Fears, C.Y. & Woods, A. (2006) The role of syndecansin disease and wound healing Matria BioI 25, 443-456 Intermedia te-levelreview. Gama, C.I. & Hsieh-Wilson, L.C. (2005) Chemicalapproachesto decipheringthe glycosaminoglycancode.Cut-r. Op'in Chem B'i,oI I, 609-619 Intemediate review of the use of chemicallysynthesizedglyin exploringthe functions of theseglycoconjugates. cosaminoglycans Hdker, U., Nybakken, K., & Perrimon, N. (2005) Heparansulphate proteoglycans:the sweetside of development. Nat Reu MoI CelIBiol 6, 530-541. Intermediatereviewof the rolesof proteoglycansin development. Holt, C.E. & Dickson, B,J. (2005) Sugarcodesfor axons?Neuron 46,169 1.72. Brief, intermediate-levelreview of the possiblerole of glycosaminoglycans in directing the outgrowth of axonsin the developingnelvous system lozzo,R.Y. (1998) Matrix proteoglycans:from moleculardesignto cellularfunction Annu Reu Bi,ochem 67. 609-652.

F..l

C a r b o h y d raant edG s lycobiology

A review focusing on genetic and molecular biological studies of the matrix proteoglycans. The structure-function relationships of some paradigmatic proteoglycans are discussed in depth, and novel aspects of their biology are examined

Dahms, N.M. & Hancock, M.K. (2002) P-t)-pelectins Biochim BiopLtgs Acta 1572, 317-340.

Roseman, S. (2001) Reflections on glycobiology J. Biol, Chem

Fukuda, M, (ed.). (2006) Glgcobi,ology,Methodsin Enzymology, Vol 415.AcademicPress.Inc NewYork

276,41,5274r,542. A masterfulrer,rewof the history of carbohydrateand glycosaminoglycanstudies,by one of the major contributorsto this field Sasisekharan, R., Raman, R., & Prabhakar, V. (2006) Glycomics approachto structure-functionrelationshipsof glycosaminoglycans. Annu Reu Bi,omed Eng 8,181-231 Advancedreview,including the methodologyof glycosaminoglycan analysis Glycoproteins Freeze, H.H. & Aebi, M. (2005)Alteredglycanstructures:the molecularbasisof congenitaldisordersof glycosylation.Curr. Opi,n Stntct Bi"ol 15, 490-498 Short, intermediatereview of the consequences of genetic defectsin glycoproternsynthesis. Gahrnberg, C.G. & Tolvanen, M. (1996) Why mammahancell surfaceproteins are glycoproteins Tlends Bzochem Sci, 21,308-311 Imberty, A., Mitchell, E.P., & Wimmerovd, M. (2005) Structural basisof high-affinityglycanrecognitionby bacterialand fungal \eclins.Cum Opzn Stntct Biol 15,525 534.Short, intermediatelevel review. Leroy, J.G. (2006) Congenitaldisordersof N-glycosylationincluding diseasesassociatedurth O- as well asN-glycosylationdefects Pediatr Res 60,643 656 Intermedialereviewof the medicalconsequences of defective protein glycosylatron Mendonca-Previato, J.O., Todeschini, A.R., Heise, N., & Previato, J.O. (2005) Protozoanparasite-specificstructures Cun Opi,n Stru,ct Bi.ol 16, 499-505. Short, intermediatelevel review of the structuresof parasite glycoproteinoligosaccharides Varki, A. (2007) Glycan-basedinteractionsinvohrng invertebrate proteins.Nature 446, 1023-1029 sialic-acid-recognizing Varki, A. & Angata, T. (2006) Siglecs the major subfamilyof I-typelectins Glgcobzologg 16, 1R-27R Intermediatelevel review,including both structure and blology. Tfleerapana,E. & Imperiali, B. (2006) AsparagineJinkedprotein glycosylation:from eukaryotlcto prokaryotic systems GLycobiologg 16, 91R-101R Intermediatelevel review of the biosyntheticprocessof protein glycosylation Glycobiology and the Sugar Code Angata, T. & Brinkman-Van der Linden, E. (2002) I-t1pe lectins Biochim Bi,ophys Acta L572, 294-316 Boraston, A.8., Bolam, D.N., Gilbert, H.J., & Davies, G,J. (2004) Carbohydrate-bindingmodules:fine-tuningpolysaccharide recognition Bt ochem J. 582, 769-781 Excellent reuew of the structural basisfor the spectflcityof sugar-bindingproteins Bor6n, T., Normark, S., & Falk, P. (1994)He\icobacterpg\ori,: molecularbasisfor host recognitionand bacterial adherenceTlends Microbr,oL2,221-228 How the oligosaccharides that determineblood type affect the adhesionof ulcer-causingH. pglori to the stomachlirung Brooks, S., Dwek, M.V., & Schumacher, U. (2002)Functi,onaL and MoLecularGlgcobiologE, GarlandScience,Oxford, UK Cornejo, C.J., lVinn, R.K., & Harlan, J,M. (1997) Anti-adhesion therapy Adu Pharmo,coL39,99-142 Analogsof recogmtionoligosaccharides are used to block adhesion of a pathogento its host-celltarget

Fukuda, M. (ed.). (2006)Functi,onal G|gcomzcs,Methodsin Enzl'rnology,Yol 477,AcademicPress,Inc , New York

Fukuda, M. (ed.). (2006) Glgcomics,Methodsin Enz5.'rnology, Vol 416,AcademicPress,Inc, NewYork. Gabius, H.-J. (2000) Biologicalinformationtransfer beyondthe geneticcode:the sugar code.Ncttut-wzssenschaJten 87, 108-121 Descriptionof the basisfor the high information density in with examplesof the importanceof the sugarcode. oligosaccharides, Gabius, H.-J., Andre, S., Kaltner, H., & Siebert, H.C. (2002) The sugarcode:functional lectinomics Bi,ochi,m B'i,ophysActa 1572,t65-t77 This reviewexaminesthe reasonsfor the relativelylate appreciaand polysaccharides. lion of the informationalrolesof oligosaccharides Ghosh, P., Dahms, N.M., & Kornfeld, S. (2003) Mannose 6-phosphatereceptors:new twists in the tale Nat Reu Mol CelI B'ioI 4,202-212 Handel, T.M., Johnson,2., Crown, S.E., Lau, E.K., Sweeney, M,, & Proudfoot, A.E. (2005) Regulationof protein function by glycosaminglycans-asexemplifledby chemoktnesAnnu Reu Biochem 74,385-410 Hebert, D.N., Garma.n,S.C., & Molinari, M. (2005)The $yca.ncode carbohydrates asproof the endoplasmicreticulum:asparagine-lirked tein maturationand qualrtycontrol tags Ttend,sCeLIBi.ol 15, 364-370 Intermediatelevel review. Helenius, A, & Aebi, M. (2004) Rolesof N-linked glycansin lhe endoplasmicreticulum Annu Reu Bi,ochem 73, 1019-1049 Hooper, L.A., Ma.nzella, S.M., & Baenziger, J.U. (1996) From legumesto leukocy'tes:biologicalroles for sulfatedcarbohydrates FASEBJ.10, 1137-1146 in peptide horEvidencefor roles of sulfatedoligosaccharides mone halflife, symbioticinteractionsin nitrogen-fixinglegumes,and I}.'rnphocyte homing intricatemolelozzo,R.Y. (2001)Heparansulfateproteoglycans: culeswith intriguing functions J. Clin Inuest 108, 165-167. Introduction to a seriesof paperson heparansulfatespublished in the sameissueof the journal; all are rewardinglreading Ito, Y., Hagihara, S., Matsuo, I., & Totani, K. (2005) Structural in glycoproteinquality approachesto the study of oligosacchandes control.Cun Opin Struct B'i,oL15,481-489. Kilpatrick, D,C. (2002) Animal lectrns:a historicalintroduction and overview.Biochi,rn,Bi,ophEsActa 1572, 187-197. Introduction to a seriesof excellentreviewson lectins and their biologicalroles,all publishedin the sameissueof the journal. Lederkremer, G.Z. & Glickman, M.H. (2005) A window of opportunity: timrng protein degradationby trimming of sugarsand ubiquitins Ttends Biochem. Scz 30,297-303 Intermediatelevel review of the role of protein glycosylationin quality control in the endoplasmicreticulum Liitteke, T., Bohne-La.ng,A., Loss, A., Goetz, T., Frank, M., & von der Lieth, C.-W.(2006)Glycosciences.de: an intemet poftal to support 16, 71R-81R $ycomicsandglycobiologyresearchGlycobi,o\ogy McEver, R.P.,Moore, K.L., & Cummings,R.D. (1995)Leukocyte interactions I Baol trafficking mediatedby selectin-carbohydrate Chem 27O.11.025-11.028 This short rer,rewfocuseson the interaction of selectinswith their carbohydrateligands Reuter, G. & Gabius, H.-J. (1999) Eukaryoticglycosylation:whim of nature or multipurposetool?CeLIMoI Li,JeSci 55,368-422 Excellent review of the chemicaldiversity of oligosaccharides and polysaccharidesand of biologicalprocessesdependenton protein-carbohydraterecognition

Problems fztzl L)

Sharon, N. & Lis, H. (2004) History of lectins:from hemagglutinins to biologicalrecognitionmoleculesGLycobiology 14, 53R-62R Excellent intermediate-levelintroduction to lectin structure and functton Tbylor, M.E. & Drickamer, K. (2006)Introducti,on to GLgcobi,o\og!),znd edn, Oxford UniversityPress,Oxford Weigel, P.H. & Yik, J.H. (2002) Glycansas endocy'tosissignals:the casesof the asialoglycoproteinand hyaluronan/chondroitinsulfate receptorsBzochim Bi,ophysActa 1572,341-363 W/

TTAGC

33 $t

ACCACGATT

trttl

s,#

AATCGTGGTGCTAA FIGURE 8-18 Palindromes and mirror repeats.palindromes are sequencesof double-stranded nucleicacidswith twofoldsymmetry. In orderto superimpose one repeat(shadedsequence) on the other,it mustbe rotated180' aboutthe horizontal axisthen1g0. aboutthevertical axis,as shownby the coloredarrows.A mirror repeat,on the otherhand,hasa symmetricsequence within eachstrand Superimposingone repeaton the other requiresonly a singlelg0" rotation aboutthe verticalaxis

Yr.'l---+3'

(a)

b't-TfT -

Hairpin TGCGAT

ATCGCA ]-Tl-t

ACGCTA

s,

TAGCGT

II + IA

AT GC

strands of DNA (F'ig. ti-18). Such sequences are selfcomplementary within each strand and therefore have the potential to form hairpin (crossor cruciform shaped) structures (Fig. ti-I9) When the inverted repeat occurs within each individual strand of the DNA, the

sequence is called a mirror repeat. Mirror repeats do not have complementary sequences wtthin the same strand and cannot form hairpin or cruciform structures Sequences of these types are found in virtualiy every large DNA molecule and can encompass a few base pairs or thousands. The extent to which palindromes occur as cruciforms in cells is not known, although some cruciform structures have been demonstrated in vivo in .Escheri,ch'in cole. Self-complementary sequences cause isolated single strands of DNA (or RNA) in solution to fold into complex structures contaimng multiple hairpins. Several unusual DNA structures involve three or even four DNA strands. Nucleotides participating in a Watson-Crick base pair (Fig. 8-11) can form additional hydrogen bonds, particularly with functional groups arrayed in the major groove. For example, a cytidine residue (if protonated) can pair with the guanosine residue of a G:C nucleotide pair (Fig. S-20); a thlmrdine can pair with the adenosine of an A:T pair. The N-7, 06, and l,/6 of purines, the atoms that participate in the hydrogen bonding of triplex DNA, are often referred to as Hoogsteen positions, and the non-Watson-Crick pairing is called Hoogsteen pairing, after Karst Hoogsteen,who in 1963first recognizedthe potential for these unusual pairings. Hoogsteen pairing allows the formation of triplex DNAs. The triplexes shornn in Figure 8-20 (a, b) are most stable at low pH because the C:G . C+ triplet requires a protonated cytosine. In the triplex, the pK. of this cltosine is >7.5, altered from its normal value of 4.2. The triplexes also form most readily within long sequences containing only pyrimidines or only purines in a given strand. Some triplex DNAs contain two pyrimi-

33 TA s'# 3'<

lll\

\111+8, /-ll->5'

AT

CG GC CG TA AT

(b)

Cruciform

FIGURE 8-19 Hairpinsand cruciforms.palindromicDNA (or RNA) sequences can form alternative structures with intrastrand basepairing (a)Whenonlya singleDNA (or RNA)strandis involved, thestructure is calleda hairpin.(b) When both strandsof a duplexDNA are involved,it is calleda cruciformBlueshadinghighlights asymmetric sequences that can pair with the complementary sequence eitherin the samestrandor in the complementary strand.

dine strands and one purine strand; others contain two pufine strandsand one pyrimidine strand. Four DNA strandscan alsopair to form a tetraplex (quadruplex),but this occursreadily only for DNA sequences with a very high proportion of guanosine residues(Fig. 8-20c, d). The guanosinetetraplex, or G tetraplex, is quite stableover a wide rangeof conditions. The orientation of strands in the tetraplex can vary as shownin Figure8-20e. In the DNA of living cells,sites recognizedby many sequence-speciflc DNA-bindingproteins (Chapter 28) are arrangedaspalindromes,and po\py'rimidine or poiypurine sequencesthat can form triple helicesare found within regions involved rn the regulation of expresslon of some eukaryotic genes.In principle, spthetic DNA strandsdesignedto pair with these sequencesto form

Frr]

8 . 2N u c l eAi cc i dS t r u c t u r e

c-1' T:A'T (a)

C.1'

H Guanosinetetraplex (c)

FIGURE 8-20 DNA structurescontainingthree or four DNA strands, (a) Base-pairing form of triplex patternsin one well-characterized pair in eachcaseis shownin red. (b) TripleDNA. The Hoogsteen helical DNA containingtwo pyrimidinestrands(poly(C))and one (derived Thedarkblueand purinestrand(poly(C)) fromPDBlD l BCE). areantiparallel and pairedby normalWatson-Crick lightblue strands patterns. strand(purple)is parThethird (all-pyrimidine) base-pairing hydroallelto the purinestrandandpairedthroughnon-Watson-Crick genbonds.Thetriplexisviewedend-on,with fivetriplesshown Only patternin the triplet closestto the viewer is colored.(c) Base-pairing tetrapletsfrom a the guanosinetetraplexstructure.(d) Two successive viewedend-onwith the one closestto the viewer C tetraplexstructure, in color. (e) Possiblevariantsin the orientationof strandsin a C tetraolex

triplex DNA could disrupt gene expression.This approach to controlling cellular metabolismis of commercial interest for its potential applicationin medicineand agriculture.

forPolypeptide Chains Messenger RNAs Code We now turn our attention to the expressionof the genetic information that DNA contains.RNA, the second major form of nucleic acid in cells, has many functions. In gene expression,RNA acts as an intermediary by using the information encoded in DNA to specify the amino acid sequenceof a functional protein. Given that the DNA of eukaryotes is largely conflned to the nucleuswhereasprotein slmthesisoccurs

Parallel (e)

on ribosomesin the cytoplasm,some molecule other than DNA must carry the genetic messagefrom the nucleusto the cytoplasm.As early as the 1950s,RNA was consideredthe logical candidate:RNA is found in both the nucleus and the cytoplasm,and an increasein protein synthesisis accompaniedby an increasein the amount of cytoplasmicRNA and an increasein its rate of turnover. These and other observationsled several researchersto suggestthat RNA carriesgeneticinformation from DNA to the protein biosynthetic machinery of the ribosome. In 1961 Frangois Jacob and JacquesMonod presented a unified (and essentially correct) picture of many aspectsof this process'They proposedthe name"messengerRNA" (mRNA) for that oortion of the total cellular RNA carrying the genetic

[ 2 8 4 _ ] N u c l e o t i daensdN u c l eAi cc i d s

Gene (a) Monocistronic

J

Gene 1 (b) Polycistronic

Gene 2

Gene 3

FIGURE 8-21 BacterialmRNA. Schematic diagrams show (a) monocistronicand (b) polycistronicmRNAsof bacteria.RedsegmentsrepresentRNA coding for a gene producu gray segmentsrepresent noncodingRNA.In thepolycistronic transcript, noncodingRNAseparatesthe threegenes.

information from DNA to the ribosomes,where the messengersproude the templatesthat specifi/amino acid sequencesin pollpeptide chains.AlthoughmRNAs from different genes can vary greatly in length, the mRNAsfrom a particular gene generallyhave a defined size.The processof formingmRNA on a DNA template is known as transcription. In bacteria and archaea,a single mRNA molecule may codefor one or severalpolypeptidechains.If it carries the code for only one polypeptide,the mRNA is monocistronic; if it codes for two or more different polypeptides,the mRNA is polycistronic. In eukaryotes,mostmRNAsaremonocistronic.(For the purposes ofthis discussion,"cistron"refersto a gene.The term itselfhashistoricalrootsin the scienceofgenetics,andits formal genetic definition is beyond the scope of this text.) The minimum length of an nRNA is set by the length of the polypeptide chain for which it codes.For example,a polypeptidechain of 100amino acid residues requires an RNA coding sequence of at least 800 nucleotides,becauseeach amino acid is coded by a nucleotide triplet (this and other details of protein synthesis are discussed in Chapter 2T). However, mRNAs transcribed from DNA are always somewhat longer than the length needed simply to code for a po\peptide sequence(or sequences).The additional, noncoding RNA includes sequencesthat regulate protein synthesis.Figure 8-21 summarizesthe general structure of bacterialmRNAs.

these RNAs reflect a diversity of structure much richer than that observedin DNA molecules. The product of transcription of DNA is always single-stranded RNA.The singlestrandtendsto assume a right-handedhelical conformationdominatedby basestackinginteractions(Fig. 8-22), which are stronger between two purines than between a purine and pyrimidine or betweentwo pyrimidines.The purine-purineinteraction is so strong that a pyrimidine separatingtwo purines is often displacedfrom the stackingpattern so that the purines can interact. Any seH-complementary sequencesin the molecule produce more complex structures. RNA can base-pairwith complementaryregions of either RNA or DNA. Basepainng matches the pattern for DNA: G pairs with C and A pairs with U (or with the occasionalT residue in someRNAs). One differenceis that basepairing betweenG and U residuesunusual in DNA-is fairly common in RNA (see Fig. 8-24). The paired strands in RNA or RNA-DNA duplexesare antiparallel,as in DNA. RNA has no simple, regular secondarystructure that servesas a referencepoint, as doesthe doublehelix for DNA. The three-dimensionalstructures of many RNAs,like those of proteins,are complexand unique. Weak interactions, especiallybase-stackinginteractions, help stabilizeRNA structures,just as they do rn DNA. Where complementarysequencesare present, the predominant double-strandedstructure is an Aform right-handeddouble helix. Z-form helices have been made in the laboratory (under very high-salt or

Many RNAs Have More Complex Three-Dimensional Structures MessengerRNA is only one of several classesof cellular RNA. Tlansfer RNAs are adapter molecules in protein synthesis; covalently linked to an amino acid at one end, they pair with the mRNA in such a way that amino acids are joined to a growing polypeptide in the correct sequence. Ribosomal RNAs are components of ribosomes. There is also a wide variety of special-function RNAs, including some (called ribozyrmes) that have enzymatic activity. All the RNAs are considered in detail in Chapter 26. The diverse and often complex functions of

FIGURE 8-22 Typicalright-handedstackingpattern of single-stranded RNA. Thebasesareshownin gray,the phosphate atomsin yellow,and the ribosesand phosphate oxygensin green.Creenis usedto represent RNAstrandsin succeeding justasblue is usedfor DNA. chapters,

Acid 5tructure 8.2Nucleic [zas--l

high-temperatureconditions).The B form of RNA has not been observed.Breaksin the regularA-form helix causedby mismatchedor unmatchedbasesin one or both strandsare commonand result in bulgesor internal loops (Fig. 8-23). Hairpin loops form between nearby self-complementarysequences.The potential for base-pairedhelical structuresin many RNAsis extensive(FiS. 8-24), and the resultinghairpinsare the most commontype of secondarystructurein RNA.Speciflc short base sequences(such as UUCG) are often found at the ends of RNA hairpins and are knov'rr to form particularly tight and stable loops. Such sequencesmay act as starting points for the folding of an strucRNA moleculeinto its precisethree-dimensional ture. Other contributionsare madeby hydrogenbonds that are not part of standardWatson-Crickbasepairs. For example,the 2'-hydroxyl group of ribose can hydrogen-bondwith other groups.Someof theseproperties are evident in the structure of the phenylalanine transferRNA of yeast-the tRNA responsiblefor inserting Phe residuesinto polypeptides-and in two RNA enzymes,or ribozymes,whose functions,like those of

Hairpin Single strands

Internal loop A

U

U "'r'" ' I

HairPin double helix ft) 8-23 Secondarystructureof RNAs. (a) Bulge,internalloop, FIGURE and hairpinloop. (b) The pairedregionsgenerallyhavean A-form helix,asshownfor a hairpin. right-handed cce

helicalstructuresin an RNA. Shownhere FIGURI8-24 Base-paired is the possiblesecondary structure of the M1 RNAcomponentof the enzymeRNaseP of E.coli,with manyhairpinsRNaseP,which also (notshown),functionsin the processing a proteincomponent contains of transferRNAs(seeFig.26-27\. The two bracketsindicateadditionalcomplementary sequences that may be paired in the threeC:U structure. Thebluedotsindicatenon-Watson-Crick dimensional basepairs(boxedinset).Notethat C:U basepairsareallowedonly whenpresynthesized strands of RNAfold up or annealwith eachother. (theenzymesthat synthesizeRNAson Thereare no RNA polymerases a DNA template)that inserta U oppositea templateC, or vice versa, duringRNAsynthesis

o

NH2 Guanine

c

cG2oo

lDtC

Nucleotides andNucteic Acids

o o:P-o-

Cytosine

H

,rNa

N2-Dimethylguanine

cH3 cH3

q*^*'"t"

NHz Adenine

FIGURE8-25 Three-dimensionalstructure in RNA. (a) Threedimensionalstructureof phenylalanine IRNA of yeast(pDB lD ITRA). Someunusualbase-pairing patternsfoundin thistRNAareshown.Note alsothe involvement of the oxygenof a ribosephosphodiester bond in one hydrogen-bonding arrangement, and a ribose2'-hydroxylgroupin another(bothin red).(b) A hammerhead ribozyme(sonamedbecause the secondarystructureat the activesite lookslike the headof a hammer),derivedfrom certainplantviruses(derivedfrom pDB lD IMME).

protein enz),rnes,depend on their three-dimensional structures(FiS. 8-25). The analysisof RNA structure and the relationship between structure and function is an emergingfleld of inquiry that has many of the same complexitiesas the analysisof protein structure. The importance of understanding RNA structure grows as we become increasingly aware of the large number of functional roles for RNA molecules.

Ribozymes,or RNA enzymes,catalyzea varietyof reactions,primarily in RNA metabolismand proteinsynthesis. The complexthreedimensionalstructuresof theseRNAsreflectthe complexityinherent in catalysis,as describedfor proteinenzymesin Chapter6. (c) A segment of mRNA known as an intron,from the ciliatedprotozoan Tetrahymena thermophila(derivedfrom PDB lD l CRZ).This intron(a ribozyme)catalyzesitsown excisionfrom betweenexonsin an mRNA strand(discussed in Chaoter26).

SUMMAR 8 .Y2 N u c l eAi cc i dS t r u c t u r e r

Many lines of evidenceshow that DNA bears genetic information. In particular, the Avery-Macleod-McCartyexperiment showedthat DNA isolatedfrom one bacterial strain can enter and transform the cells of another strain, endowing it with someof the inheritable characteristicsof the donor.The Hershey-Chaseexperiment showedthat

-2sl Acid themistrY 8.3Nucleic

the DNA of a bacterialvirus, but not its protein coat,carriesthe geneticmessagefor replicationof the virus in a host cell. Putting together the availabledata, Watsonand Crick postulatedthat native DNA consistsof two antiparallelchainsin a right-handeddouble-helical basepairs,A:T and arrangement.Complementary G:C, are formed by hydrogenbonding within the helix. The basepairsare stackedperpendicularto the long axis of the doublehelix, 3.4 A apart,with 10.5basepairsper turn. DNA can exist in severalstructural forms.TWo variationsof the Watson-Crickform, or B-DNA,are structural A- and Z-DNA.Somesequence-dependent molecule. DNA variationscausebendsin the DNA strandswith appropriatesequencescan form hairpin/ cruciform structuresor triplex or tetraplex DNA. MessengerRNA transfersgenetic information from DNA to ribosomesfor protein synthesis.T?ansfer RNA and ribosomalRNA are alsoinvolvedin protein synthesis.RNA canbe structurally complex;singleRNA strands can fold into hairpins, regions,or complexloops. double-stranded

AcidChemistry 8.3 Nucleic The role of DNA as a repositoryof geneticinformation dependsin part on its inherent stability. The chemical transformationsthat do occur are generallyvery slow in the absenceof an enz5.rnecatalyst.The long-term storageof information without alterationis so important to a cell, however,that even very slow reactions that alter DNA structure can be physiologically significant. and agingmay be intisuchas carcinogenesis Processes mately Iinked to slowly accumulating,irreversible alterations of DNA. Other, nondestructivealterationsalso occur and are essentialto function, such as the strand separationthat must precedeDNA replicationor transcription. In addition to providing insights into physiological processes,our understandingof nucleic acid chemistry has given us a powerful array of technologies that have applications in molecular biology, medicine, and forensic science.We now examine the chemical propertiesof DNA and someof thesetechnologies.

unwinding of the double helix to form two single strands,completelyseparatefrom each other along the entire length or part ofthe length (partial denaturation) of the molecule. No covalent bonds in the DNA are broken(Fig. 8-26). Renaturationof a DNA moleculeis a rapid one-step process,as long as a double-helicalsegmentof a dozen or more residuesstill unites the two strands.When the temperature or pH is returned to the range in which most organismslive, the unwound segmentsof the two strands spontaneouslyrewind, or anneal, to yield the intact duplex (Fig. 8-26). However,if the two strands are completelyseparated,renaturation occurs in two steps. In the first, relatively slow step, the two strands "find" each other by random collisionsand form a short segmentof complementarydouble helix. The second step is much faster: the remaining unpaired bases successivelycome into register as base pairs, and the two strands "zipper" themselvestogether to form the doublehelix. The close interaction between stackedbasesin a nucleic acid has the effect of decreasingits absorption of UV light relative to that of a solutionwith the same concentrationof free nucleotides,and the absorption is decreasedfurther when two complementarynucleic acid strandsare paired.This is calledthe hypochromic effect. Denaturationof a double-strandednucleic acid

Double-helical DNA Annealing

Denaturation

Separation of strands

(\

tl

{/

Association of strands by base parnng

Can BeDenatured DNA andRNA Double-Helical Solutions of carefully isolated, native DNA are highly viscousat pH 7.0 and room temperature(25'C). When sucha solutionis subjectedto extremesof pH or to temperaturesabove80 oC,its viscositydecreasessharply, indicating that the DNA has undergone a physical change.Just as heat and extremesof pH denatureglobular proteins, they also causedenaturation,or melting, of double-helicalDNA. Disruption of the hydrogen bondsbetweenpairedbasesand of basestackingcauses

SeParated strands of DNA in random coils of denaturationand annealing(renaturation) FIGURES-26Reversible DNA.

F"l

N u c l e o t i daensdN u c l eAi cc i d s

producesthe oppositeresult: an increasein absorption called the hyperchromiceffect. The transition from double-strandedDNA to the single-stranded,denatured form can thus be detected by monitoring UV absorptionat 260 nm. Viral or bacterial DNA moleculesin solution denature when they are heated slowly (Fig. 8-2?). Each speciesof DNA has a characteristicdenaturationtemperature,or meltingpoint (l^; formally,the temperature at which half the DNA is present as separated single strands): the higher its content of G:C base pairs, the higherthe meltingpoint of the DNA. This is because G:C base pairs, with three hydrogenbonds, require more heat energyto dissociatethan A:T base pairs Thus the meltingpoint of a DNA molecule,determined under fixed conditions of pH and ionic strength, can yield an estimateof its basecompositionIf denaturation conditionsare carefullycontrolled,regionsthat are rich

,--.'

tIGURE 8-28 Partially denatured DNA. ThisDNA waspartiallydenatured,then fixedto preventrenaturation duringsamplepreparation Theshadowing methodusedto visualize the DNA in thiselectronmicrographincreases itsdiameterapproximately fivefoldand obliterates mostdetailsof the helix.However,lengthmeasurements can be obtained,and single-stranded regionsare readilydistinguishable from double-stranded regions.The arrowspoint to somesingle-stranded bubbleswheredenaturation hasoccurred.The regionsthat denature arehighlyreproducible and arerich in A:T basepairs

in A:T basepairs will specrficallydenaturewhile most of the DNA remainsdouble-stranded. Such denatured regions (called bubbles) can be visualizedwith electron microscopy(FiS. 8-28). Note that in the strand separation of DNA that occursin vivo duringprocessessuch as DNA replicationand transcription,the sites where theseprocessesare initiatedare often rich in A:T base pairs,as we shallsee. Duplexes of two RNA strands or one RNA strand and one DNA strand (RNA-DNA hybrids) can also be denatured.Notably,RNA duplexesare more stablethan DNA duplexes.At neutralpH, denaturationof a doublehelicalRNA often requirestemperatures20'C or more higher than those required for denaturation of a DNA molecule with a comparablesequence.The stability of an RNA-DNAhybrid is generallyintermediate between that of RNA and that of DNA. The physical basis for these differencesin thermal stability is not known.

s f,so c)

(a)

100 @

o €80

Eoo ts+o

(anForm Nucleic Acids fromDifferent Species Hybrids

; +20

The ability of two complementaryDNA strands to pair with one another can be used to detect similar DNA sequencesin two different speciesor within the genome of a singlespecies.If duplex DNAs isolatedfrom human cells and from mouse cells are completelydenaturedby heating,then mixed and kept at about2b "C below their l* for many hours, much of the DNA will anneal.The rate of DNA annealingis affectedby temperature,the length and concentrationof the DNA fragments beingannealed,the concentrationof saltstn the reactionmixture, and propertiesof the sequenceitself

al

0 60 (b)

*l

70

80 90 ,_ ("C)

100

110

FIGURE 8-27 Heatdenaturation of DNA. (a)Thedenaturatron, or melting,curvesof two DNA specimens. Thetemperature at the midpointof the transition (t-) is the meltingpoint;it dependson pH and ionic strength and on the sizeand basecomposition of the DNA (b) Relationship betweent- and the C-t-Ccontentof a DNA.

2B9l ChemistrY 8 . 3N u c l eAi c i d

(e.g., complexrtyand G:C content). Temperatureis especiallyimportant. If the temperature is too low, short sequenceswith coincidentalsimilarity from distant, heterologousparts of the DNA moleculeswill anneal unproductively and i.nterfere with the more general alignment of complementary DNA strands Temperaturesthat are too high will favor denaturation. Most of the reannealingoccurs between complementary mouse DNA strandsto form mouse duplex DNA; similarly, most human DNA strands anneal with complementary human DNA strands. However, some strands of the mouse DNA will associatewith human DNA strandsto yield hybrid duplexes, in which segmentsof a mouseDNA strandform base-pairedregions wrth segmentsof a human DNA strand (Fig. 8-29). This reflectsa commonevolutionaryheritage;different organismsgenerallyhavemanyproteinsand RNAswith similarfunctionsand,often, similarstructures.In many cases,the DNAs encodingthese proteins and RNAs The closerthe evolutionaryrehavesimilarsequences. lationshipbetween two species,the more extensively their DNAs will hybridize. For example,human DNA hybridizes much more extensivelywith mouse DNA than with DNA from yeast. The hybridization of DNA strands from different sourcesforms the basisfor a powerfulset of techniques

Sample 1

Sample 2 to be compared Two DNA samples 8-29 DNA hybridization. tIGURE are completelydenaturedby heatingWhen the two solutionsare with of eachsampleassociate mixedand slowlycooled,DNA strands partnerand annealto form duplexeslf theirnormalcomplementary they alsotend to similarity, sequence the two DNAs havesignificant form partialduplexesor hybridswith each other:the Sreaterthe the numberof similaritybetweenthetwo DNAs,the greater sequence in severalways measured formation can be formed. Hybrid hybrids isotopeto simOne of the DNAsis usuallylabeledwith a radioactive olifvthe measurements.

essential to the practice of modern molecular genetics. A specific DNA sequence or gene can be detected in the presence of many other sequencesif one already has an appropriate complementary DNA strand (usually labeleclin some way) to hybridize with it (Chapter 9). The complementary DNA can be from a different species or from the same species, or it can be synthesized chemically in the laboratory using techniques described later in this chapter. Hybridization techniques can be varied to detect a speciflc RNA rather than DNA' The isolation and identiflcation of speciflc genes and RNAs rely on these hybridization techniques. Applications of this technology make possible the identification of an individual on the basis of a single hair left at the scene of a crime or the prediction of the onset of a diseasedecades before symptoms appear (see Box 9-1)'

[,!ndergo Acids andNucleic Nuclestides Transformations Nonenzymatic Purines and pyrimidines, along with the nucleotides of which they are a part, undergo spontaneousalterations in their covalent structure' The rate of these reactionsis generallyuery slow,but they are physiologicallysignificantbecauseof the cell's very low tolerancefor alterationsin its genetic information.Alterationsin DNA structurethat producepermanent changesin the genetic information encoded therein are calledmutations, and much evidencesuggests an intimate link between the accumulation of mutationsin an individual organismand the processes of agingand carcinogenesis. Several nucleotide bases undergo spontaneous loss of their exocyclic amino groups (deamination) (Fig. S-30a). For example, under typical cellular conditions, deaminationof cy'tosine(in DNA) to uracil occurs in about one of every 10' cy'tidineresiduesin 24 hours. This correspondsto about 100 spontaneous eventsper day,on average,in a mammaliancell' Deamination of adenineand guanineoccursat about 1/100th this rate. The slow cytosine deaminationreaction seemsinnocuousenough,but is almostcertainlythe reasonwhy DNA containsth;'rnine rather than uracil. The product of cytosinedeamination(uracil) is readily recognizedas foreign in DNA and is removed by a repair system (Chapter25).lt DNA normallycontaineduracil, recognition of uracils resulting from cytosine deamination would be more difflcult, and unrepaired uracils would Iead to permanent sequencechanges as they were parredwrth adeninesduring replication. Cy'tosinedeaminationwould graduallyleadto a decreasein G:C base pairs and an increasein A:U basepairs in the DNA of all cells. Over the millennia, c1'tosinedeaminationcould eliminateG:C basepairsand the geneticcodethat depends on them. Establishingthymine as one of the four basesin DNA may well have been one of the crucial

zooI

N u c l e o t i daensdN u c l eAi cc i d s

o

NHo lNZ\

o-

N

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OH

I nro-,,1

HN-\.-cHt

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ltl o4N/

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o4N/

I

5-Methylcytosine

Guanosine residue (in DNA)

,O

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ll ,uN-^-rN,, rt

Thymine

*'l\T'-\*,/ HzN ii

o lt

)

+

oi _O_p_O_ o H

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til) \N

I

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HN---ir-Nrt

Hypoxanthine

o

Xanthine (a) Deamination

turning points in evolution, making the long-term storage of genetic information possible. Another important reaction in deoryribonucleotides is the hydrolysis of the l/-B-glycosyl bond between the base and the pentose, to create a DNA lesion called an AP (apurinic, apyrimidinic) site or abasic site (Fig. 8-30b). This occurs at a higher rate for purines than for pyrimidines. As many as one in 105purines (10,000 per mammalian cell) are lost from DNA every 24 hours un_ der typical cellular conditions. Depurination of ribonucleotides and RNA is much slower and generally is not considered physiologically significant. In the test tube, loss of purines can be accelerated by dilute acid Incubation of DNA at pH 3 causes selective removal of the purine bases, resulting in a derivative called apurinic acid. Other reactions are promoted by radiation. W light induces the condensation of two ethylene groups to form a cyclobutane ring. In the cell, the same reactlon between adjacent pyrimicline bases in nucleic acids forms cyclobutane pyrimidine dimers. This happens most frequently between adjacent thymidine residues

Apurrn.tc resid (b) Depurination FIGURE 8-30 Some well-characterizednonenzymaticreactions (a) Deamination of nucleotides. reactionsOnly the baseis shown. (b) Depurination, in which a purineis lostby hydrolysis of the N-Bglycosylbond. Lossof pyrimidines via a similarreactionoccurs,bur much more slowly.The resultinglesion,in which the deoxyribose is presentbut the base is not, is called an abasicsite or an Ap site (apurinicsiteor, rarely,apyrimidinic site).Thedeoxyribose remaining afterdepurinationis readilyconvertedfrom theB-furanose to thealdehydeform (seeFig 8-3) Furthernonenzymatic reactionsare illustratedin Figures 8-31 and 8-32

on the same DNA strand (Fig. 8-31). A second type of pyrimidine dimer, called a 6-4 photoproduct, is also formed during UV irradiation. Ionizing radiation (x rays and gamma rays) can cause ring opening and fragmentation of bases as well as breaks in the covalent backbone of nucleic acids. Virtually all forms of life are exposed to energy-rich radiation capable of causing chemical changes in DNA. Near-LV radiation (wrth wavelengths of 200 to 400 nm), which makes up a signiflcant portion of the solar spectrum, is known to cause pyrimidine dimer formation and other chemical changes in the DNA of bacteria and of human skin cells. We are subject to a constant fleld of ionizing radiation in the form of cosmic rays, which can penetrate deep into the earth, as well as radiation emitted from radioactive elements, such as radium, pluto_ nium, uranium, radon, ]aC, and 3H. X rays used in medical and dental examinations and in radiation therapy of cancer and other diseases are another form of ionizing radiation. It is estimated that tIV and ionizing radiations are responsible for about 10% of all DNA damage caused by environmental agents.

8.3Nucleic Acid Chemistry Ittt l

cHa

Adjacent

thYmrnes

ox

.rH

-c-N--.. ,,,"

*\^-^j":o H

CHA

'I

CHe

H Cyclobutane thymine dimer

6-4 Photoproduct

ft)

(a) FIGURE 8-31 Formationof pyrimidinedimers inducedby UV light. (a) One type of reaction(on the left) resultsin the formationof a cy-

with a linkagebetweenC-6 of one pyrimidineandC-4 of itsneighbor. (b) Formation a bendor pyrimidinedimerintroduces of a cyclobutane kink intothe DNA.

clobutylring involvingC-5 and C-6 of adjacentpyrimidineresidues. An alternativereaction(on the right) resultsin a 6-4 photoproduct,

salts, is a potent acceleratorof the deaminationof bases.Bisulfite has similar effects. Both agents are used as preservativesin processedfoods to prevent the growth of toxic bacteria.They do not seemto increase cancer risks significantly when used in this way, perhapsbecausethey are used in small amounts and make only a minor contribution to the overall Ievels of DNA damage. (The potential health risk from food spoilageif these preservativeswere not used is much greater.)

DNA also may be damaged by reactive chemicals in-

troduced into the environmentas products of industrial activity. Such products may not be injurious per se but may be metabolizedby cells into forms that are. There are two prominentclassesof such agents(Fig. 8-32): (1) deaminatingagents,particularlynitrous acid (HNO2) or compoundsthat can be metabolizedto nitrous acid or nitrites, and (2) alkylatingagents Nitrous acid, formed from organic precursors such as nitrosaminesand from nitrite and nitrate FIGURE 8-32 Chemicalagentsthat cause DNA damage.(a) Precursors of nitrousacid, which promotes deamination reactions (b)Alkylatingagents.

*l

coo-

H3N-C-H l CHo methionine

NaNO3 Sodium nitrate

N

lCHO

NaNO2 Sodium nitrite

CH'

tI

N

o" ,o

CH,

,/"\

oo

N-N:O CHs Dimethylnitrosamine

CHs Dimethylsulfate

CHs R1 N-N:O R2 Nitrosamine

(a) Nitrous acid precursors

/cHz-cll2-cl

H3C-N\

cH2-cH2-cl Nitrogenmustard (b) Alkylating agents

a-l

292

N u c l e o t i daensdN u c l eAi cc i d s

Alkylating agents can alter certain bases of DNA. For example, the highly reactive chemical dimethylsulfate (Fig 8-32b) can methylate a guanine to yield Obmethylguanine, which cannot base-pair with cytosine.

Guanine tautomers

O6-Methylguanine

Many srmilar reactions are brought about by alkylating agents normally present in cells, such as ,S-adenosylmethionine. The most important source of mutagenic alterations in DNA is oxidative damage. Excited-oxygen species such as hydrogen peroxide, hydroxyl radicals, and superoxide radicals arise during irradiation or as a byproduct of aerobic metabolism. Of these species,the hydroxyl radicals are responsible for most oxidative DNA damage. Cells have an elaborate defense system to destroy reactive oxygen species, includirLg enzyrnes such as catalase and superoxide dismutase that convert reactive oxygen species to harmless products. A fraction of these oxidants inevitably escape cellular defenses, however, and damage to DNA occurs through any of a large, complex group of reactions ranging from oxidation of deoxyribose and base moieties to strand breaks. Accurate estimates for the extent of this damage are not yet available, but every day the DNA of each human cell is subjected to thousands of damaging oxidative reactions. This is merely a sampling of the best-r.mderstood reactions that damage DNA. Many carcinogenic compounds in food, water, or air exert their cancer-causing effects by modifying bases in DNA. Nevertheless, the integrity of DNA as a pollmer is better maintaured than that of either RNA or protein, because DNA is the only macromolecule that has the beneflt of biochemical repair systems. These repair processes(described in Chapter2S) greatly lessen the impact of damage to DNA. r

Some Bases ofDNA AreMethylated Certain nucleotide bases in DNA molecules are enzJ,rynaticallymethylated. Adenine and c;,'tosine are methylated more often than guanine and th;.rnine. Methylation is generally con_finedto certain sequences or regions of a DNA molecrile. In some cases the function of methylation is well understood; in others the ftrnction remains unclear. All known DNA methylases use S-adenosylmethionine as a methyl group donor (Fig. 8-32b).

E. co|i, has two prominent methylation systems.One servesas part of a defensemechanismthat helps the cell to distinguish its DNA from foreign DNA by marking its own DNA with methyl groups and destroying (foreign) DNA without the methyl groups (this is known asa restriction-modiflcation system;seep. 305). The other systemmethylatesadenos_ine residueswithin the sequence(5')GATC(3') to N"-methyladenosine (Fig. 8-5a). This is mediatedby the Dam (DNA adenine zzethylation)methylase,a componentof a systemthat repairs mismatched base pairs formed occasionally during DNA replication(seeFig.25-22). In eukaryoticcells, about 5% of cytidine residuesin DNA are methylatedto S-methylcytidine(Fig. 8-5a). producMethylationis most corrunonat CpGsequences, ing methyl-CpG s;,mmetrically on both strands of the DNA. The extent of methylationof CpGsequencesvaries by molecularregion in large eukaryoticDNA molecules.

(anBeDetermined TheSequences 0fLong DNA Strands In its capacityas a repositoryof information,a DNA molecule'smost important property is its nucleotide sequence.Until the late 1970s,determiningthe sequence of a nucleic acid containingeven flve or ten nucleotides was very laborious.The developmentof two new techniques in 1977,one by Alan Maxam and Walter Gilbert and the other by Frederick Sanger,made possiblethe sequencingof larger DNA molecules with an ease unimaginedjust a few yearsbefore.The techniquesdepend on an improved understanding of nucleotide chemistry and DNA metabolism,and on electrophoretic methodsfor separatingDNA strandsdiffering in sizeby only one nucleotide.Electrophoresisof DNA is similar to that of proteins (see Fig. 3-18). Polyacrylamideis often used as the gel matrix in work with short DNA molecules(up to a few hundrednucleotides);agaroseis generallyusedfor longerpiecesof DNA. In both Sangerand Maxam-Gilbertsequencing,the generalprincipleis to reducethe DNA to four setsof labeled fragments.The reaction producing each set is base-specific,so the lengths of the fragments correspondto positionsin the DNA sequencewherea certain base occurs.For example,for an oligonucleotidewith the sequencepAATCGACI labeledat the 5' end (the left end), a reactionthat breaksthe DNA after each C residue will generate two labeled fragments:a fournucleotideand a seven-nucleotide fragment;a reaction that breaksthe DNA after each G will produce only one labeled, five-nucleotidefragment. Becausethe fragmentsare radioactivelylabeledat their 5' ends,only the fragmentto the 5' side of the break is visualized.The fragmentsizescorrespondto the relativepositionsof C and G residuesin the sequence.When the sets of fragments correspondingto eachof the four basesare electrophoreticallyseparatedside by side, they produce a ladder of bandsfrom which the sequencecan be read directly (Fig. 8-33). We illustrate only the Sanger

8.3Nucleic Acid Chemistrv ' L l Izs:

dATP OH Primer strand

T 3'A

G C

G

G

Template strand (a)

o-o-o

-o-J-o-l-o-l- o-?"' il | oool-,.

(b)

ti

ddNTP analog

Primer

5',

OH CTAAGCTCGACT

3,

Template

FIGURE 8-33 DNA sequencingby the Sangermethod. Thismethod makesuseof the mechanismof DNA synthesis by DNA polymerases (Chapter 25). (a) DNA polymerases requireboth a primer(a short strand), oligonucleotide to which nucleotides areadded,and a templatestrandto guideselection of eachnewnucleotide. In cells,the3'hydroxylgroupof theprimerreactswith an incomingdeoxynucleoside triphosphate(dNTP)to form a new phosphodiester bond. (b) The procedureusesdideoxynucleoside Sangersequencing triphosphate (ddNTP) (TheSangermethodisalso analogs to interruptDNA synthesis. knownasthe dideoxymethod.)When a ddNTPis insertedin placeof a dNTP,strandelongationis haltedaftertheanalogisadded,because it lacksthe3'-hydroxylgroupneededfor the nextstep.(c)TheDNA to be is usedasthe templatestrand,and a shortprimer,radioacsequenced labeled,is annealed tivelyor fluorescently to it By additionof small amountsof a singleddNTP,for exampleddCTP,to an otherwisenormal reactionsystem,the synthesized strandswill be prematurely terminatedat some locationswhere dC normallyoccurs Civen the excessof dCTPoverddCTBthe chancethatthe analogwill be incorporatedwhenevera dC is to be added is small.However,ddCTPis presentin sufficientamountsto ensurethateachnew strandhasa high probability of acquiring at leastoneddCat somepointduringsynthesis Theresultis a solutioncontaining a mixtureof labeledfragments, EachC residuein the sequence genereachendingwith a C residue. atesa set of fragmentsof a particularlength,suchthat the differentsizedfragments, separated by electrophoresis, revealthe locationof C residues. This procedureis repeatedseparately for each of the four ddNTPs,and the sequencecan be read directlyfrom an autoradiogramof the gel. BecauseshorterDNA fragmentsmigratefaster,the fragments nearthe bottomof thegel represent the nucleotidepositions isread(inthe5' -+ closest to theprimer(the5' end),andthesequence direction) from bottom to top. Note that the sequence obtainedis 3' to the strandbeing analyzed. thatof the strandcomplementary

+ dCTP,dGTP,dATP,dTTP

I

+ ddATP

+ ddCTP

I-GATTCGAGCTGddA --GATTCGddA

I_GATTCGAGddC I_GATTddC

GT L2 11 10 9 8 7 6

+ ddGTP I_GATTCGAGCTddG }-GATTCGAddG

3'

T C A

A

T T

1

G

(c)

Autoradiogram of electrophoresis gel

Sequenceof complementary strand

+ ddTTP I-GATTCGAGCddT I--GATddT

294

Nucleotides andNucleic Acids

method,becauseit has proved to be technicallyeasier and is in morewidespreaduse.It requiresthe enzyrnatic synthesisof a DNA strandcomplementaryto the strand under analysis,using a radioactivelyIabeled"primer" and dideoxynucleotides. DNA sequencingis now automatedby a variation of Sanger'ssequencingmethod in which the dideoxynucleotidesused for eachreactionare labeledwith a differently coloredfluorescenttag (Fig. 8-34). With this technology,researcherscan sequenceDNA molecules containingthousandsof nucleotidesin a few hours.The entire genomesof more than a thousandorganismshave now been sequencedin this way (see Table 1-2), and many very large DNA-sequencingprojects have been completedor arein progress.For example,in the Human GenomeProject,researchers havesequencedall 3.2 billion basepairs of the DNA in a human cell (Chapter9).

Primer

Il',

, ,t,li, ,t'r r:1.,,

four dNTPs, four ddNTPs

-A

\*_

I

-T

-C -G

Q

of DNA N OideorySequencing

I

I o"r'"t,r""

The[hemical Synthesis 0ft]flA[-Nas Been Autamaterl Also important in nucleic acid chemistry is the rapid and accurate synthesis of short oligonucleotidesof knoum sequence.The methodswere pioneeredby H. Gobind Khorana and his colleaguesin the 1970s.Refinements by Robert Letsinger and Marvin Caruthers led to the chemistrynow in widestuse,calledthe phosphoramiditemethod(l-ie. 8-35). The synthesisis carried out with the growing strand attached to a solid support,using principlessimilarto those usedby Merrifleld for peptide synthesis(seeFig. 3-29), and is readily automated.The efflciencyof each addition step is very high, allowing the routine synthesis of pol;.rners containing70 or 80 nucleotidesand, in somelaboratories, much longer strands.The availabilityof relatively inexpensiveDNA poly.rners with predesignedsequences is having a powerful impact on all areasof biochemistry (Chapter9).

Temnlate of unKnown sequence

v /

* "---€ -".""-G

.*-,er

.,.--'-,&l

Dye-labeled segments of DNA, copied from template with unknown sequence

II

v Dye-labeled segments applied to a capillary gel and subjected to electrophoresis

*

*

FIGURt8-34 Strategyfor automatingDNA sequencingreactions. Eachdideoxynucleotide usedin the Sanger methodcan be linkedto a fluorescent moleculethat givesall the fragments terminating in that nucleotide a particular color.All four labeledddNTPsareaddedto a singletube.The resulting coloredDNA fragments arethen separated by sizein a singleelectrophoretic gel containedin a capillarytube(a refinementof gel electrophoresis that allowsfor fasterseparations). All fragments of a givenlengthmigratethroughthe capillarygel in a single peak,and the colorassociated with eachpeakis detectedusinga laserbeam.TheDNA sequence is readby determining thesequence of colorsin the peaksastheypassthe detector.This informationis fed directlyto a computer,which determines the sequence.

I Computer-generated result after bands migrate past detector

(hemistry Acid 8.3Nucleic [trtj DMT

I o

I s'CIJ2

Nucleoside protected at 5'hydroxyl

NC-(CHr), -O-Plruv>vu

r

faHrircu-N'--cHtc Diisopropylaminoactivating group

DMT

.t,

@

@

Protecting group removed

Repeatsteps @

"

@

Next nucleotide added (cH3)rcH-N-CH(CH3)2 H Diisopropylamine byproduct

o

I R

untilall residuesare added

Remove protecting groups from bases Remove cyanoethyl groups from phosphates

NC-(CH2)2 -O-

Oligonucleotide chain FIGURE 8-35 Chemicalsynthesisof DNA by the phosphoramidite method.Automated DNA synthesis is conceptually similarto the synthesisof polypeptides on a solidsupport. Theoligonucleotide is builtup on the solidsupport(silica), one nucleotide at a time,in a repeated seriesof chemicalreactions with suitablyprotectednucleotideprecursors. @ fn" first nucleoside(whichwill be the 3' end) is attachedto the silicasupportat the 3' hydroxyl(through a linkinggroup,R)and is protectedat the 5' hydroxylwith an acid-labiledimethoxytrityl group (DMT) The reactivegroupson all basesare alsochemicallyprotected. @ fn" protectingDMT groupis removedby washingthe columnwith acid (theDMT groupis colored,so this reactioncan be followedspectrophotometricallV). @ The next nucleotidehas a reactivephosphoramiditeat its 3' position:a trivalentphosphite(asopposedto the more oxidizedpentavalent phosphate normallypresent in nucleicacids)with one linkedoxygenreplaced by an aminogroupor substituted amine.In the commonvariantshown,one of the phosphoramidite oxygensis bondedto the deoxyribose, theotheris protectedby a cyanoethyl group,

and the third positionis occupiedby a readilydisplaceddiisopropylnucleotide formsa 5',3' with the immobilized aminogroup.Reaction ln step@, the groupis eliminated. andthe diisopropylamino linkage, phosphitelinkageis oxidizedwith iodineto producea phosphotriester are linkage.Reactions @ through@ "ru tepeateduntilall nucleotides added.At eachstep,excessnucleotideis removedbeforeadditionofthe nextnucleotide.In steps@ and@ the remainingprotecting Sroupson are removed,and in @ the oligonuthe basesand the phosphates from the solid supportand purified.Thechemical cleotideis separated of RNAis somewhatmorecomplicatedbecauseof the needto synthesis the reactivprotectthe 2' hydroxylof ribosewithoutadversely affectinB itv of the 3'hvdroxvl.

]96 )

N u c l e o t i daensdN u c l eAi cc i d s

S U M M A R8Y. 3 N u c l e A i cc i d( h e m i s t r y Native DNA undergoes reversible unwinding and separation of strands (melting) on heating or at extremes of pH. DNAs rich in G:C pairs have higher melting points than DNAs rich in A:T pairs.

tFd

o-o-o

-o-i-o-f-o-T-o-?H, o

o

o

Denatured single-stranded DNAs from two species can form a hybrid duplex, the degree of hybridization depending on the extent of sequence similarity. Hybridization is the basis for important techniques used to study and isolate specificgenesand RNAs. DNA is a relatively stable polymer. Spontaneous reactions such as deamination of certain bases. hydrolysis of base-sugarl/-glycosyl bonds, radiation-rnduced formation of pyrimidine dimers, and oxidative damage occur at very low rates, yet are important because of a cell's very low tolerance for changes in genetic material DNA sequencescan be determined and DNA poll.mers synthesized with simple, automated protocols invohrng chemical and enz;,matic methods.

8.4 Other Functions ofNucleotides In additionto their rolesasthe subunitsofnucleicacids, nucleotideshave a vari.etyof other functions in every cell: as energy carriers,componentsof enz1rnecofactors, and chemicalmessengers.

OH OH NMP NDP NTP Abbreviations of ribonucleoside 5'-phosphates

Base

Mono- Di-

Tri-

Adenine

AMP

ADP

ATP

Guanine GMP

GDP

GTP

C1'tosine CMP

CDP

CTP

Uracil

UDP

UTP

UMP

Abbreviations of deoxyribonucleoside 5'-phosphates Base

Mono-

Di-

Tri-

Adenine

dAMP

dADP

dATP

Guanine dGMP dGDP dGTP Cytosine dCMP dCDP dCTP

Itluclentides [arry(hemieal Energy in[ells The phosphate group covalently linked at the 5' hydroxyl of a ribonucieotide may have one or two additional phosphatesattached. The resulting molecules are referred to as nucleosidemono-, di-, and triphosphates( Fig. ft-36). Startingfrom the ribose,the three phosphatesare generally labeled a, B, and y. Hydrolysis of nucleosidetriphosphatesprovides the chemical energy to drive many cellular reactions. Adenosine 5'-triphosphate,ATP, is by far the most widelyusedfor this purpose,but UTP,GTP,and CTPare also used in somereactions.Nucleosidetriphosphates alsoserveas the activatedprecursorsof DNA and RNA synthesis,as describedin Chapters25 and26. The energyreleasedby hydrolysisof ATP and the other nucleosidetriphosphatesis accountedfor by the structureof the triphosphategroup.The bond between the ribose and the a phosphateis an ester linkage. The a, B and B,7 linkages are phosphoanhydrides (Fig. 8-37). Hydrolysis of the esterlinkageyields about 14 kJ/molunder standardconditions,whereashydrolysis of eachanhydridebond yields about 30 kJ/mol.AIP hydrolysisoften plays an important thermodynamicrole in biosyrrthesis.When coupledto a reactionwith a positive free-energychange,ATP hydrolysisshifts the equilibrium of the overallprocessto favor product formation

Thymine dTMP

dTDP

dTTP

tIGURE 8-36 Nucleoside phosphates. Ceneralstructure of the nucle(NMPs,NDPs,and NTPs)and oside5'-mono-,di-, and triphosphates In the deoxyribonucleoside phosphates their standardabbreviations. (dNMPs,dNDPs,and dNTPs), the pentoseis 2'-deoxy-o-ribose.

Anhydride Anhydride

ATP H3C-C-O-g-CH' il

ooo

il

Acetic anhydride, a carboxylic acid anhydride

OH OH

H3C-C-O-CH3 tl

Methyl acetate, a carboxylic acid ester

FIGURE 8-37 The phosphateester and phosphoanhydride bonds of ATP.Hydrolysis of an anhydridebond yieldsmore energythan hydrolysisof the ester.A carboxylicacidanhydride and carboxylicacid esterareshownfor comparison

?'1

r u n c t i oonfsN u c l e o t i d e s 8 . 4 0 t h eF

(recallthe relationshipbetweenequiJrbriumconstantand free-energychangedescribedby Eqn 6-3 on p. 188).

transferase(an enz;.'rneof lipid metabolism)by a factor of 106.Althoughthis requirementfor adenosinehasnot been investigatedin detail, it must involve the binding energy between enzyme and substrate (or cofactor) that is usedboth in catalysisand in stabilizingthe initial complex (Chapter6). In the caseof enzl'rne-substrate p-ketoacyl-CoAtransferase,the nucleotide moiety of coenz).rneA seemsto be a binding "handle"that helps to pull the substrate(acetoacetyl-CoA) into the activesite. Similar roles may be found for the nucleosideportion of other nucleotidecofactors. Why is adenosine, ratherthan someotherlargemolecule,used in these structures?The answerhere may involve a form of evolutionary economy.Adenosine is certainly not unique in the amount of potential binding energy it can contribute. The importance of adenosine probably lies not so much in some special chemical

Adenine Nucleotides Are[omponents ofMany (ofactors Enzyme A variety of enzl'rne cofactors serving a wide range of chemical functions include adenosine as part of their structure (Fig. 8-38). They are unrelated structurally except for the presence of adenosine. In none of these cofactors does the adenosine portion participate directly in the primary function, but removal of adenosine generally results in a drastic reduction of cofactor activities. For example, removal of the adenine nucleotide (3'-phosphoadenosine diphosphate) from acetoacetylCoA, the coenzyme A derivative of acetoacetate, reduces its reactivrty as a substrate for B-ketoacyl-CoA

NHo

H

lttl"ll

HS-CH2 -CH2 -N-C-CH2

H

HCH.

-CH2 -N-A-A-q-CH'

|tl

o p-Mercaptoethylamine

Pantothenic

OHCH3

O_

-tA* llr -^\Nl

O_

-O-P-O-PP-O-P

il oo

tl

acid

o

FIGURE 8-38 Somecoenzymescontainingadenosine.Theadenosine portion is shadedin light red. CoenzymeA (CoA)functionsin acyl group transferreactions;the acyl group (suchas the acetyl or acetoacetylgroup)is attachedto the CoA througha thioesterlinkageto the moiety.NAD+ functionsin hydridetransfers, B-mercaptoethylamine and FAD,theactiveformof vitaminB, (riboflavin), in electron transfers. Anothercoenzyme i ncorporati ngadenosine is 5'-deoxyadenosylcobalamin,theactiveformof vitamin8.,,(seeBox17-2),whichparticipates in intramolecular grouptransfers betweenadjacentcarbons.

I O:P-OI

o

3'-Phosphoadenosinediphosphate (3'-P-ADP) Coenzyme A

I

CHo

t-

Riboflavin

CHOH

I I

CHOH

o_

II o:P-oII

CHOH I CHo

to o-P:o o

OH OH

o

I

I

NH,

o:P-o-

-A* llt

l(.

-/\N'/?

OH OH

OH OH Nicotinamide

adenine dinucleotide

(NAD+)

Flavin adenine dinucleotide (FAD)

F"l

N u c l e o t i daensdN u c l eAi cc i d s

characteristicas in the evolutionaryadvantageof using one compoundfor multiple roles. OnceATP becamethe universalsourceof chemicalenergy,systemsdeveloped to s1'nthesizeATP in greater abundancethan the other nucleotides;becauseit is abundant,it becomesthe logical choice for incorporation into a wide variety of structures. The economy extends to protein structure. A singleprotein domain that binds adenosinecan be used in different enzymes.Such a domain, called a nucleotide-binding fold, is found in many enzyrnesthat bind ATP and nucleotidecofactors.

Some Nucleotides AreRegulatory Molecules Cells respondto their environmentby taking cues from hormonesor other external chemicalsignals.The interaction of theseextracellularchemicalsignals("first messengers")with receptorson the cell surfaceoften leadsto the production of second messengers inside the cell, which in turn leadsto adaptivechangesin the cell interior (Chapter l2). Often, the second messengeris a nucleotide (Fig. 8-39). One of the most common is

adenosine 3',5' -cyclic monophosphate (cyclic AMP, or cAMP), formed from ATP in a reaction catalyzedby adenylyl cyclase,an enzpe associatedwrth the inner face of the plasmamembrane.CyclicAMP servesregulatory functions in virtually every cell outside the plant kingdom.Guanosine3',5' -cycJicmonophosphate(cGMP) occursin many cellsand alsohas regulatoryfunctions. Another regulatorynucleotide,ppcpp (Fig. 8J9), is producedin bacteriain responseto a slowdownin protein synthesisduring amino acid starvation.This nucleotide inhibits the synthesisof the rRNA and IRNA molecules (seeFig. 28-24) neededfor protein sy'nthesis, preventing production of nucleicacids. the ururecessary

5UMM AR Y8 . 4 O t h eFr u n c t i oonfs Nu c l e o t i d e s r

ATP is the central carrier of chemicalenergyin cells.The presenceof an adenosinemoietyin a variety of enzlme cofactorsmay be related to binding-energyrequirements.

r

Cyclic AMP,formed from ATP in a reaction catalyzedby adenylylcyclase,is a commonsecond messengerproducedin responseto hormonesand other chemicalsignals.

KeyTerms Adenosine3',5'-cyclicmonophosphate (cyclic AMP; cAMP)

O-P:O

gene 271 ribosomal RNA (rRNA) 271 messenger RNA (mRNA) 271 transfer RNA (tRNA) 27\ nucleotide 271 nucleoside 272 pyrimidine 272 purine 272 deoxyribonucleotide 273 ribonucleotide 273 phosphodiester linkage 275 5'end 275 3'end 275 oligonucleotide 276 polynucleotide 276

O-P:O

Further Reading

l3

o:PI

o

Guanosine 3',5'-cyclic monophosphate (cyclic GMP; cGMP)

o

Terms i,n bold are defi,nedi,n the glossary.

o-

I I -o-P-o-P-o iltl oo

I I o I

I o

Guanosine5'-diphosphate,3'-diphosphate (guanosine tetraphosphate) (ppGpp) FIGURE 8-39 Threeregulatorynucleotides.

base pair 277 major groove 279 minor groove 279 B-form DNA 28I A-form DNA 28I Z-form DNA 28I palindrome 281 hairpin 282 282 cruciform triplex DNA 282 G tetraplex 282 284 transcription monocishonicmRllA 284 polycistronic nRNA 284 mutation 289 second messenger 298 adenosine 3'15'-cyclic monophosphate (cyclic AMP, cAMP) 298

General Chang, K.Y. & Varani, G. (1997) Nucleicacidsstructure and recognitionNat Stnrct Bzol 4 (Suppl),854-858. Describesthe applicationof NMR to determinationof nucleic acid structure

Problems["']

Friedberg, E.C., Walker, G.C., Siede, W.,Wood, R.D., Schultz, R.A., & Ellenberger, T. (2006)DNA Repai,r and Mutagenests,2nd edn,ASMPress,Washington, DC A good sourcefor more information on the chemistryof nucleotidesand nucleic acids. Hecht, S.M. (ed.), (1996)Bioorganic Chemzstrg:Nuclei,cAczds, Oxford UniversityPress,Oxford A very useful set of articles. Kornberg, A. & Baker, T.A. (1991) DNA Repli,cat'i,on, 2nd edn, W. H Freemanand Company,New York The best placeto start to learn more about DNA structure Historical Judson, H.F. (1996) The Ei,ghth Dag oJCreation: Ma,kersof ttte Reuolutzonin Bi,ology, expandededn, Cold SpringHarbor Laboratory Press,ColdSpringHarbor,NY Ofby, R.C. (1994) Ttrc Path to the Double Helzu: The DzscoueryoJ DNA, DoverPublications, Inc.,New York. Franklzn a,ndDNA, W W. Nofton & Co , Sayre, A. (1978)RosaL'i,nd Inc , New York. Watson, J.D. (1968) The Doubl"eHelin: A Persona| Account oJthe Di,scoueryoJthe Stn Lctureof DNA, Atheneum,New York [Paperbackedition,TouchstoneBooks,2001.1 Variations

in DNA Structure

Frank-Kamenetskii, M.D. & Mirkin, S.M. (1995) Triplex DNA structures Annu Reu B'lochem 64, 65 95 Herbert, A. & Rich, A. (1996) The biologyof left-handedZ-DNA I B'ioI Chem 271, 11,595-11,598 Keniry, M.A. (2000) Quadruplexstructuresin nucleic acids Bzopolymers 56, 123-146 Goodsummaryof the structural propertiesof quadruplexes Moore, P.B. (1999) Structural motifs in RNA Annu Reu Biochem 68, 287-300 Shafer, R.H. (1998) Stability and structure of model DNA triplexes and quadruplexesand their interactionswith small ligands Prog NucLeicAcidRes Mol BioL 59,55-94 Nucleic Acid Chemistry Bonetta, L. (2006) Genomesequencingin the fast lane. Nat Methods3, l4I-747 Fasterand more powerful DNA sequencingtechnologiesare under development Collins, A.R. (1999) OxidativeDNA damage,antioxidants,and cancer Bioessays 21, 238-246 Cooke, M.S., Evans, M.D., Dizdaroglu, M., & Lunt J. (2003)Oxidative DNA damage:mechanisms,mutatron,and disease.IHSEBI t7,1195 1214 Marnett, L.J. & Plastaras, J.P. (2001) EndogenousDNA damage and mutation Tlends Genet 17,214 221. ATP as Energy Carrier Jencks, W.P.(1987) Economicsof enzlanecatalysis Cold Spriw Harb Sgmp Quctnt Bi,oL 52,65-73 A relatively short article, full of insights.

Problems 1. Nucleotide Structure Which positionsin the purine ring of a purine nucleotidein DNA havethe potentialto form hydrogen bonds but are not involved in Watson-Crickbase pairing?

2. Base Sequence of Complementary DNA Strands One strand of a double-helicalDNA has the sequence(5')GCGTGCGC(3' ). Write the basesequence CAATILITTCTC&4IAATILI What specialtlpe of sequenceis strand. the complementary of Doesthe double-strandedDNA DNA segment? in this contained structures? potential form any alternative to have the 3. DNA of the Human Body Calculatethe weight in grams of a double-helicalDNA moleculestretchingfrom the Earth to the moon (-320,000km). The DNA doublehelix weighsabout 1 x 10-18g per 1,000nucleotidepairs;eachbasepair extends 3.4 A. For an interesting comparison,your body contains about0.5g of DNA! 4. DNA Bending Assumethat a poly(A) tract flve basepairs Iong producesa 20" bend in a DNA strand Calculatethe total (net) bend produced in a DNA if the center base pairs (the third of flve) of two successive(dA)5 tracts are located (a) 10 basepairs apart; (b) 15 basepairs apart Assume10 basepairs per turn in the DNA doublehelix. 5. Distinction between DNA Structure and RNA Structure Hairpins may form at palindromic sequencesin single strandsof either RNA or DNA. How is the helical structure of a long and fully base-paired(except at the end) hairpin in RNA different from that of a similar hairpin in DNA? 6. Nucleotide Chemistry The cells of many eukaryotic organismshave highly specializedsystemsthat specificallyrepair G-T mismatchesin DNA. The mismatch is repaired to form a G:C (not A:T) basepair. This G-T mismatchrepair mechanismoccurs in addition to a more general system that repairs virtually ail mismatches.Can you suggestwhy cells might require a specializedsystemto repair G-T mismatches? 7. Spontaneous DNA Damage Hydrolysisof theN-glycosyl bond betweendeoxyriboseand a purine in DNA createsan AP site. An AP site generatesa thermodynamic destabil\zation greater than that createdby any DNA mismatchedbasepair. This effect is not completelyunderstood.Examine the structure of an AP site (seeFig. 8-33b) and describesomechemical of baseloss. consequences 8. Nucleic Acid Structure Explain why the absorption of UV light by double-strandedDNA increases (the hyperchromic effect) when the DNA is denatured. 9. Determination of Protein Concentration in a Solution Containing Proteins and Nucleic Acids The concentration of protein or nucleic acid in a solution containingboth canbe estimatedby usingtheir different hght absorptionproperties: proteins absorb most strongly at 280 nm and nucleic acids at 260 nm. Estimatesof their respectiveconcentrations in a mixture can be madeby measuringthe absorbance(A) of the solutionat 280and 260nm and usingthe table on page300, at 280and 260nm; the ratio of absorbances which gives-R2ss7266, the percentageof total massthat is nucleicacid;and a factor,F , that correctstheA2gsreadhgand givesa more accurateprotein

L 3 0 0_ N u c l e o t i daensdN u c l eAi cc i d s estimate The protein concentration(in mg/ml) : F X A2ss (assumingthe cuvette is 1 cm wide). Calculatethe protein : 0.94. concentration in a solutionof A2so: 0.69and,4266

Rzeotzao

Proportion of nucleic acid,(o/o)

r.75 163 t.52 1.40 136 130 r25 1.16 109 1.03 0.979 0 939 0.874 0 846 0.822 0.804 0 784 0.767 0.753 0 730 0 705 0.671 0 644 0.615 0.595

0.00 025 0.50 0.75 100 r.25 r50 2.00 250 3.00 350 400 500 5.50 6.00 650 700 7.50 8.00 9.00 10.00 1200 1400 17.00 20.00

F 1.116 1 081 1.054 1.023 0 994 0.970 0 944 0.899 0.852 0 814 0 776 0.743 0 682 0.656 0 632 0 607 0.585 0 565 0.545 0 508 0.478 0.422 0.377 0 322 0.278

10. Solubility of the Components of DNA Draw the fotlowing structures and rate their relative solubilities in water (most soluble to least solubte): deoxyribose, guanine, phosphate. How are these solubilities consistent with the threedimensional structure of double-stranded DNA? ll. Sanger Sequencing Logic In the Sanger (dideoxy) method for DNA sequencing, a small amount of a dideox)'nucleotide triphosphate-say, ddCTP-is added to the sequencing reaction along with a larger amount of the corresponding dCTp What result would be obserued if the dCTp were omitted? 12. DNA Sequencing The following DNA fragment was sequenced by the Sanger method The red asterisk indicates a fluorescent label ':,5'-3'_OH 3i -

ATTACGCAAGGACATTAGAC---5,

A sample of the DNA was reacted with DNA pol;;rnerase and each of the nucleotide mixtures (in an appropriate buffer) Iisted below. Dideoxynucleotides (ddNTps) were added in relatively small amounts 1. dATP, dTTP, dCTP, dGTP, ddTTP 2

dAIP, dTTR dCTP, dGTq ddGTP

3. dATP,dCTP,dGTP,ddTTP 4

dATR dTTP,dCTP,dcTP

The resulting DNA was separatedby electrophoresison an agarosegel,andthe fluorescentbandson the gelwerelocated. The band pattern resultingfrom nucleotidemixture 1 is shown below. Assumingthat ali mixtures were run on the samegel, what did the remaininglanesof the gel look like?

t

* * * m

13. Snake Venom Phosphodiesterase An exonuclease is an enzlrne that sequentially cleaves nucleotides from the end of a polynucleotide strand Snake venom phosphodiesterase, which hydrolyzes nucleotides from the 3' end of any oligonucleotide with a free 3'-hydroxyl group, cieavesbetween the 3' hydroxyl of the ribose or deoxyribose and the phosphoryl group of the next nucleotide. It acts on single-stranded DNA or RNA and has no base speciflcity. This enz).'rnewas used in sequence determination experiments before the development of modern nucleic acid sequencing techniques What are the products of partial digestion by snake venom phosphodiesterase of an oligonucleotide with the following sequence? (5' )GCGCCAUUGC(3' )-OH 14. Preserving DNA in Bacterial Endospores Bacterial endospores form when the environment is no longer conducive to active cell metabolism. The soit bacterium Baci,llus subti,Lzs,for example, begins the process of sporulation when one or more nutrients are depleted. The end product is a small, metabolically dormant structure that can surwive aimost indeflnitely with no detectable metabolism. Spores have mechanisms to prevent accumulation of potentially lethal mutations in their DNA over periods of dormancy that can exceed 1,000 years -B subti,Li,sspores are much more resistant than are the organism's growing cells to heat, tIV radiation, and oxidizing agents, all of which promote mutations. (a) One factor that prevents potential DNA damage in spores is their greatly decreased water content. How would this affect some types of mutations? (b) Endospores have a category of proteins called small acid-soluble proteins (SASPs) that bind to their DNA, preventing formation of cyclobutane-type dimers. What causes cyclobutane dimers, and why do bacterial endospores need mechanisms to prevent their formation?

P r o b l e mLs3 0 !

15. Oligonucleotide Synthesis In the schemeof Figure 8-35, each new base to be added to the growing oligonucleotide is modified so that its 3'hydroxyl is activatedand the 5' hydroxyl has a dimethoxytrityl (DMT) group attached. What is the functlon of the DMT group on the incomingbase?

Biochemistry ontheInternet 16. The Structure of DNA Elucidation of the threedimensionalstructure of DNA helped researchersunderstand how this molecule conveysinformation that can be faithfully replicatedfrom onegenerationto the next. To seethe secondary structure of double-strandedDNA, go to the Protein Data Bank website (www.rcsborg). Use the PDB identiflers listed below to retrieve the structure summariesfor the two forms of DNA Open the structuresusing Jmol (linked under the Display Options), and use the controls in the Jmol menu (accessedwith a control-click or by clicking on the Jmol logo in the lower right corner of the imagescreen)to completethe followingexercises.Referto the Jmolhelp links as needed. (a) Obtainthe flie for 141D,a highlyconserved,repeated DNA sequencefrom the end of the HIV-1 (the virus that causesAIDS)genomeDisplaythe moleculeasa ball-and-stick structure(in the controlmenu,chooseSelect> All, then Render ) Scheme> Ball and Stick). Identify the sugar-phosphate backbonefor each strand of the DNA duplex. Locate and identifiz individual bases.Identify the 5' end of each strand Locate the major and minor grooves Is this a right- or lefthandedhelix? (b) Obtain the flle for 145D,a DNA with the Z conformation. Display the molecule as a ball-and-stickstructure. Identify the sugar-phosphatebackbonefor each strand of the DNA duplex. Is this a right- or left-handedhelix? (c) To fully appreciatethe secondarystructure of DNA, view the moleculesin stereo.On the control menu, Select) All, then Render > Stereographic> Cross-eyedor Walleyed Youwill seetwo imagesof the DNA molecule.Sit with your noseapproximately10 inchesfrom the monitor and focus on the tip of your nose (cross-eyed)or the opposite edgesof the screen (wall-eyed).In the backgroundyou should see three imagesof the DNA helix. Shift your focus to the middle image, which should appear three-dimensional.(Note that only one of the two authorscan make this work )

Problem DataAnalysis 17. Chargaff's Studies of DNA Structure The chapter section "DNA Is a Double Helix that Stores Genetic Information" includes a sununaryof the main findings of Erwin Chargaff and his coworkers,Iisted as four conclusions("Chargaff's rules"; p. 278).In this problem,you will examinethe data Chargaffcollectedin support of these conclusions In one paper, Chargaff (1950) describedhis analytical methods and some early results. Briefly, he treated DNA

sampleswith acid to removethe bases,separatedthe basesby paperchromatography,and measuredthe amountof eachbase with l-IVspectroscopy.His resultsare shownin the three tables below.The molar rati,o is the ratio of the number of moles of eachbasein the sampleto the number of molesof phosphatein the sample-this givesthe fraction of the total number of bases representedby eachparticular base.The recouerAis the sum of all four bases(the sum of the molar ratios); full recoveryof all basesin the DNA would give a recoveryof 1.0.

Molar ratios in ox DNA Liver

Spleen

Thymus

Prep.I Prep.2 Prep.3 Prep.1 Prep.2 Prep.1

Base

0.26 0.2I 0 16 0.25 0.88

Adenine Guanine Cytosine Thlrrune Recouery

0.30 0.22 0.r7 0.25 0 94

0.28 0.24 0 18 0.24 0.94

0.25 0.20 0.15 0.24 0.84

0.26 0.20

0.26 0.2r 0.17 0.24 0.88

Molar ratios in humanDNA

Base Adenine Guanine Cytosine Th5.rmne Recouerg

Sperm

Thymus

Frep.1 Prep.2

Prep.I

0.27 017 0.18 030 092

0.28 0.19 0.16 0.28 0.91

0 29 0.18 0.18 0 31 0 96

Liver NormalCarcinoma 0.27 0.19

0.27 018 015 0.27 087

Molar ratios in DNA of mieroorganisms Aviantuberclebacilli

Yeast Base Adenine Guanine Cy'tosine Th;.'rnine Recouery

Prep.1

Frep.2

0.24 0.14 013 0.25 0.76

0.30 018 0.15 0.29 0.92

Prep.I

0.r2 0.28 0.26 0.11 0.77

(a) Basedon these data, Chargaffconcludedthat "no differencesin compositionhave so far been found in DNA from different tissuesof the same species."This correspondsto conclusion2 in this chapter.However,a skeptic looking at the data abovemight say,"They certainly look different to me!" If you were Chargaff,how would you use the data to convince the skeptic to changeher mind? (b) The base compositionof DNA from normal and cancerous liver celts (hepatocarcinoma)was not distinguishabiy different.Wouldyou expect Chargaff'stechniqueto be capable of detectinga differencebetweenthe DNA of normal and cancerouscells?Explainyour reasorung. As you might expect,Chargaff'sdata were not completely convincing.He went on to improvehis techniques,as described

Ft{

N u c l e o t i da en sdN u c t eAi cc i d s

in a later paper (Chargaff,1951),in which he reported moiar ratios of basesin DNA from a variety of organisms: Source

A:G T:C A:T

Ox Human Hen Salmon Wheat Yeast Haemophi,Lus i,nfluenzae type c E. coliK-I2 Avian tubercle bacillus Serratia nnLrcesceyls Bacil|us schatz

t.29 L43 1.04 1.00 1.56 1.75 1.00 1.00 r 45 1..291.06 0.91 1.43 1.43 r.02 L02 t . 2 2 1 . 1 81.00 0.97 r 67 1..921.03 1.20

0.99 1,0

r.74 1..541.07 0.91 r.05 0.95 1.09 0.99

10 1.0

0.4 0.4

1.09 1.08

1.1

0.7 0.7 0.7 0.6

0.95 0.86 1.12 0.89

0.9 1.0

G:C Purine:pyrimidine 1.1 1.0 0.99

r.02

(c) Accordingto Chargaff,as statedin conclusion1 in this chapter,"The base compositionof DNA generallyvaries from one speciesto another." Provide an argument, based on the data presentedso far, that supportsthis conclusion. (d) Accordingto conclusion4, "In alt cellularDNAs,regardlessof the species. . . A + G : T + C." provide an argument, based on the data presented so far, that supports this conclusion. Part of Chargaffsintent wasto disprovethe "tetranucleotide hypothesis";this was the idea that DNA was a monotonous

tetranucleotidepolymer (AGCT)" and thereforenot capableof containirg sequenceinJormation.Although the data presented aboveshow that DNA cannot be simpty a tetranucleotide-if so, all sampleswould havemolar ratios of 0.25for eachbase-it was still possiblethat the DNAfrom djfferent organismswasa siightly more complex,but still monotonous,repeatingsequence. To address this issue, Chargaff took DNA from wheat germ and treated it with the enzyme deoxy'ribonuclease for different time intervals. At each time interval, some of the DNA was convertedto small fragments;the remaining,Iarger fragmentshe calledthe "core."In the table below,the "lg% core" correspondsto the Iarger fragments left behind when 8I0/oof the DNA was degraded;the "8%ocore" correspondsto the Iargerfragmentsleft after 92% degradation. Base Adenine Guanine Cytosine Th5'rnine Recouerg

Intact DNA

19%Core

8%Core

0.27 0.22 0.22 0.27 0.98

0.33 0.20 0.16 0.26 0.95

0.35 0.20 0.14 0.23 092

(e) How would you use these data to argue that wheat germ DNA is not a monotonousrepeatingsequence? References Chargaff, E. (1950) Chemicalspeciicity of nucleic acidsand mechanism of their enzymicdegradation.-Brperienti,a 6,201-209 Chargaff, E. (1951) Structure and function of nucleic acids as cell constituents Fed Proc. 10. 654-659.

O f a l l t h e n a t u r a sl y s t e m sl ,i v i n gm a t t e ri s t h e o n e w h i c h , i n t h e f a c e preservesinscribedin its organizationthe of great transformations, largestamountof its own pasthistory. -Emile Zuckerkandland LinusPauling, Biology,1965 articlein Journalof Theoretical

Information DNA-Based Technologies Y.l

DNA floning:The Basics304

9 . 7 Frorn Genes toGenomes315 From Genomes to Prsteomes314 Y.+

fienorne Alterations of andNewProduets Bioterhnology 330

e now turn to a technologythat is fundamental to the advanceof modern biologicalsciences, defining present and future biochemicalfrontiers and illustrating many important principles of biochemistry.Elucidation of the laws governingenzymatic catalysis,macromolecularstructure, cellular metaboIism, and information pathwaysaliowsresearchto be directed at increasinglycomplexbiochemicalprocesses. vision, taste, Cell division, immunity, embryogenesis, cognition-all are orchestratedin an elaboncogenesis, orate symphony of molecular and macromolecular interactionsthat we are now beginningto understand with increasingclarity.The real implicationsof the biochemicaljourney begun in the nineteenthcentury are powerto anafoundin the ever-increasing Iyze and alter living systems. To understand a complex biological process,a biochemistisolatesand studies the individual componentsin vitro, then piecestogetherthe parts to get a coherent picture of the overall process.A major source of molecularinsights is the cell's own information archive, its DNA. The however,presheersizeof chromosomes, sentsan enormouschallenge:how doesone PaulBerg find and study a particular geneamongthe

tens of thousandsof genesnestedin the billionsof base pairs of a mammaiian genome? Solutions began to emergein the 1970s. Decadesof advancesby thousandsof scientists working in genetics, biochemistry, cell biology, and physicalchemistrycametogetherin the laboratoriesof Paul Berg, Herbert Boyer,and StanleyCohento yield techniques for Iocating, isolating, preparing, and studying small segmentsof DNA derived from much larger chromosomes.Techniques for DNA cloning paved the way to the modern fields of genomics andl proteomics, the study of genes and proteins on the scaleof whole cellsand organisms.Thesenew methods are transformingbasicresearch,agriculture,medicine, ecology,forensics,and many other fields, while occasionally presenting society with difficult choicesand ethical dilemmas. We begin this chapter with an outline of the fundamental biochemicalprinciplesof the now-classicdiscipline of DNA cloning. Next, we illustrate the range of applicationsand the potentialof a rangeof newer technologies,with a broad emphasison modernadvancesin genomicsand proteomics.

HerbertBoyer

StanleyN. Cohen

i :ro:r

Iso+-D N A - B a sI nefdo r m a t iToenc h n o l o o i e s 9.1 DNA Cloning: TheBasics A clone is an identical copy. This term originally applied to cells of a single type, isoiated and allowed to reproduce to create a population of identical cells. DNA cloning involves separating a speciflc gene or DNA segment from a larger chromosome, attaching it to a small molecule of carrier DNA, and then replicating this modifled DNA thousands or millions of times through both an increase in host cell number and the creation of multiple copies of the cloned DNA in each cell. The result is selective ampliflcation of a particular gene or DNA segment. Cloning of DNA from any organism entails five general procedures:

Cutti,ngDNA at preci,seLocati,onsSequencespeciflcendonucleases (restrictionendonucleases) providethe necessarymolecularscissors. 2. Selectznga small moleculeoJDNA capabteoJ selJ-replicateonTheseDNAsare calledcloning vectors (a vector is a deliveryagent).They are typically plasmidsor viral DNAs.

Cloning vector (plasmid)

Eukaryotic chromosome @ ONe fragment of interest is obtained by cleaving chromosomewith a restriction endonuclease.

@ l;;,"#o'"'"' I g, o*o is introduced I V

into the host cell.

o \ --l

a

Joi,ning two DlttrAJragmentscoua\ently The enzymeDNA ligaselinks the cloningvector and DNA to be cloned CompositeDNA molecules comprisingcovalentlylinked segmentsfrom two or more sourcesare calledrecombinant DNAs. Mouing recombinant DNAJro.m the test tube to a host cell that will provide the en4"rnaticmachinery for DNA replication.

5 . Selectzngor identi,Jyinghost ce\Lsthat contar,n recomb'inantDNA. The methods used to accomplish these and related tasks are collectively referred to as reeombinant DNA technology or, more informaliy, genetie engineering. Much of our initial discussion will focus on DNA cloning in the bacterium Escheri,ch,i,a coli,, the flrst organism used for recombinant DNA work and still the most common host cell. E coLi,hasmany advantages:its DNA metabolism (like many other of its biochemical processes) is well understood; many naturally occurring cloning vectors associatedwith t. coli such as plasmicls and bacteriophages (bacterial viruses; also called phages), are well characterized; and techruques are available for moving DNA expeditiously from one bacterial cell to another. The principles discussed here are broadly applicable to DNA cloning in other organisms, a topic discussed more fully Iater in the chapter.

yield Restrictlon Indonucleases andnNALiqase Recombinant ilNA Particularly important to recombinant DNA technology is a set of enzy'rnes (Table 9-l) made available through decades of research on nucleic acid metabolism. TWo classesof enz5.'rnes lie at the heart of the classicapproach to generating and propagating a recombinant DNA molecule (Fig. 9-l). First, restriction endonucleases

tIGURE 9-1 Schematic illustration of DNA cloning.A cloningvector andeukaryotic chromosomes areseparately cleavedwith the samerestrictionendonuclease. Thefragments to be clonedarethen ligatedto the cloningvectorTheresulting recombinant DNA (onlyone recombinantvectoris shownhere)is introduced intoa hostcellwhereit can (cloned)Notethatthisdrawingis not to scale:the size be propagated of the f coli chromosome relativeto that of a typicalcloningvector (suchasa plasmid)is muchgreaterthandepictedhere.

(also called restriction enz}.'rnes) recognize and cleave DNA at specific sequences(recognition sequencesor restriction sites) to generate a set of smaller fragments. Second, the DNA fragment to be cloned is joined to a suitable cloning vector by using DNA ligases to link the DNA molecules together. The recombinant vector is then introduced into a host ceil, which ampli_f,esthe fragment in the course of many generations of cell division. Restriction endonucleasesare found in a wide range of bacterial species. Werner Arber discovered in the early 1960s that their biological function is to recognize and cleave foreign DNA (the DNA of an infecting virus,

Basics Cloning:The 9.1DNA Ft{

Enrpne(s)

Function

Tlpe II restriction endonucleases

CleaveDNAs at speciflcbasesequences Joinstwo DNA moleculesor fragments

DNA ligase

Fills gapsin duplexesby stepwiseaddition of nucleotidesto 3' ends Makesa DNA copy of an RNA molecule Adds a phosphateto the 5'-oH end of a polynucleotideto label it or permit ligation

DNA Pol5.tnerasel(E. coli,) Reversetranscriptase Poly'nucleotidekinase ExonucleaseIII

Adds homopolymertails to the 3'-OH ends of a linear duplex Removesnucleotideresiduesfrom the 3' ends of a DNA strand

Bacteriophage\ exonuclease Alkaline phosphatase

Removesnucleoticlesfrom the 5' endsof a duplex to exposesin$e-stranded3' ends Removesterminal phosphatesfrom either the 5' or 3' end (or both)

Terminaltransferase

for example);such DNA is saidto be restri,cted.In the host cell'sDNA, the sequencethat would be recognized is protectedfrom diby its own restrictionendonuclease gestionby methylationof the DNA, catalyzedbya specific DNA methylase.The restriction endonucleaseand the correspondingmethylaseare sometimesreferredto as a restriction-modification system. There are three types of restrictionendonucleases, designatedI, II, and III. \rpes I and III are generally large, multisubunit complexescontaining both the endonucleaseand methylaseactivities. \,pe I restriction endonucleasescleaveDNA at random sites that can be more than 1,000base pairs (bp) from the recognition cleavethe sequence.TlrpeIII restrictionendonucleases DNA about 25 bp from the recognitionsequence.Both

J

J*

BamHI

t),pesmovealongthe DNA in a reactionthat requiresthe energy of ATP. Tlpe II restriction endonucleases, first isolatedby Hamilton Smith in 1970,are simpler,require no ATP,and cleavethe DNA within the recognition sequenceitself, The extraordinaryutility of this group of restriction endonucleaseswas demonstratedby Daniel Nathans,who first used them to developnovel methods for mappingand analyzinggenesand genomes. havebeen Thousandsof restriction endonucleases discoveredin different bacterial species,and more than 100 different DNA sequencesare recognizedby one or more of these enzymes.The recognitionsequencesare usually4 to 6 bp long and palindromic(see Fig. 8-18). Table 9-2 lists sequencesrecognizedby a few type II restrictionendonucleases.

(s',)GGATCC(3',) CCTAGG

Hi.ndIII

*1

t

J

J*

Clal

(5',)ATCGAT(3',) TAGCTA

Notl

*t

EcoRl

J*

*v

(5',)GAATTC(3',) CTTAAG

J

HaeIll

(5',)GATATC(3',) CTATAG

t J*

(5',)GGCC(3',) CCGG

*t

(5',)GCGGCCGC(3') CGCCGGCG

t

PstI

*t

EcoRY

(5',)AAGCTT(3') TTCGAA

(s',)CTGCAG(3',) GACGTC

t*

v

Puull

(5')CAGCTG(3',) GTCGAC

t

v

TTUTI\I

(5')GACNNNGTC(3') CTGNNNCAG

bythe basesthataremethylated indicate Asterisks endonuclease bondscleaved byeachrestriction Arrows indicate the phosphodiester abbreviation of a three-letter consists (where anybase.Notethatthenameof eachenzyme known). N denotes methylase corresponding to distinguish andRomannumerals followed bya straindesignation sometimes (in italics)of ihe bacterial fromwhichit is derived, species characterized endonuclease EamHlis thefirst(l) restrlction species.Thus isolated fromthesamebacterial restriction endonucleases different strainH. fromBaci//usamy/o/iquefaciens,

1

Information Technologies | 306. DNA-Based Some restriction endonucleasesmake staggered cuts on the two DNA strands, leaving two to four nucleotidesof one strand unpairedat eachresultingend. Theseunpaired strands are referred to as sticky ends (Fig. 9-2a), becausethey can base-pairwith each other or with complementarysticky ends of other DNA fragments.Other restrictionendonucleases cleaveboth strandsof DNA at the opposingphosphodiesterbonds, leaving no unpaired bases on the ends, often called blunt ends (Fig. 9-2b). The averagesizeof the DNA fragmentsproducedby cleavinggenomic DNA with a restriction endonuclease depends on the frequency with which a particular restriction site occursin the DNA molecule;this in turn dependslargelyon the sizeof the recognitionsequence. Cleavage srte

In a DNA moleculewith a random sequencein which all four nucleotideswere equally abundant, a 6 bp sequencerecognizedby a restriction endonucleasesuch as BamHlwould occur on averageonceevery 46 (4,096) bp, assumingthe DNA had a 50% G:C content. Enzyrnesthat recognizea 4 bp sequencewould produce smaller DNA fragments from a random-sequenceDNA molecule;a recognitionsequenceof this sizewould be expectedto occuraboutonceevery4a (256) bp. In natural DNA molecules,particular recognitionsequences tend to occur less frequently than this becausenucleotidesequences in DNA are not randomand the four nucleotidesare not equally abundant.In laboratory experiments,the averagesizeof the fragmentsproduced by restriction endonucleasecleavageof a large DNA

Recognition _,/sequences

Cleavage site

- - - c c rEL4rr ch-romosomal r c c c Ar r AG. ^ J. r c r A Gc - - - - - c c a B r r e - e" l {, DNA q lcr c"rA A G CG r A A T cc r c c A C A r c c - - -

t

t

,,,,,." J,;i,

| "'ii,,*l,',,. --_G GT G

---c cAClTAri

AGCTTCGCATTAGCAG TCGAAGCGTAATCGTC

Sticky ends (a)

Plasmid cloning vector cleaved with EcoRI and PuulI

CTGTAGC__ GACATCG__ Blunt ends (b)

Smol

Pstl

A A T T C t g , r CC * GGAC cT: Synthetic polylinker

FIGURE 9-2 Cleavage of DNA moleculesby restrictionendonucleases. Restriction endonucleases recognizeand cleaveonly specificse_ quences,leavingeither(a)stickyends(withprotrudingsinglestrands) or (b) blunt ends.Fragments can be ligatedto other DNAs, suchas the cleavedcloningvector(a plasmid)shownhere.Thisreactionis facilitatedby the annealingof complementary stickyends Ligationis lessef_ ficient for DNA fragmentswith blunt ends than for those with complementary stickyends,and DNA fragmentswith different(non_ complementary) stickyendsgenerallyare not ligated.(c) A synthetic DNA fragmentwith recognitionsequences for severalrestrictionen_ donucleases can be insertedinto a plasmidthat hasbeencleavedby a restriction endonuclease The insertis calleda linker;an insertwith multiplerestrictionsitesis calleda polylinker. RestrictionEn$ donucleases

Plasmid cloning vector cleaved with,EcoRI

(c)

9 . 1D N A C l o n i n g : T h eLB 3a 0 s7i_c]s can be increasedby simplyterminatingthe reactionbefore completion;the result is called a partial digest. Fragmentsize can alsobe increasedby using a special class of endonucleasescalled homing endonucleases (see Fig. 26-38). Theserecognizeand cleavemuch longerDNA sequences(14 to 20 bp). Once a DNA moleculehas been cleavedinto fragments, a particular fragment of known size can be enriched by agaroseor acrylamidegel electrophoresisor by HPLC(pp. 89,88). For a typicalmammaliangenome, however,cleavageby a restriction endonucleaseusually yields too many different DNA fragmentsto permit convenient isolation of a particular fragment.A commonintermediatestepin the cloningof a speciic geneor DNA segmentis the constructionof a DNA library (described in Section9.2). After the target DNA fragmentis isolated,DNA ligase can be used to join it to a similarlydigestedcloningvector-that is, a vector digestedby the sam,erestrictionendonuclease;a fragmentgeneratedby EcoRI,for example, generallywill not link to a fragmentgeneratedby BamHI. As describedin more detail in Chapter 25 (see Fig. 25-17), DNA ligasecatalyzesthe formation of new phosphodiesterbondsin a reactionthat usesATP or a similar cofactor.The basepainng of complementarysticky ends greatly facilitatesthe Jigationreaction (Fig. 9-2a). Blunt endscanalsobe ligated,albeitlessefflciently.Researchers can create new DNA sequencesby inserting sy.nthetic DNA fragments(called linkers) between the ends that are beirrgligated.Inserted DNA fragmentswith multiple recognitionsequencesfor restriction endonucleases(often usefullater as points for insertulgadditionalDNA by cleavageand ligation) are calledpolylinkers (Fig. 9-2c) The effectivenessof sticky endsin selectivelyjoining two DNA fragments was apparent in the earliest recombinantDNA experiments.Before restriction endonucleaseswere widely available,someworkers found they cor.rldgeneratesticky endsby the combinedaction of the bacteriophagetr' exonucleaseand terminal transferase (Table 9-1). The fragmentsto be joined were given complementaryhomopoll.rnenctails. Peter Lobban and Dale Kaiserused this method in 1971in the first experiments to join naturally occurring DNA fragments. Similar methodswere used soon after in the laboratory of Paul Berg to join DNA segmentsfrom simianvirus 40 (SV40) to DNA derived from bacteriophage\, thereby creating the first recombinantDNA molecr.rlewith DNA segmentsfrom different species.

Plasmids Plasmidsare circular DNA molecules that replicate separatelyfrom the host chromosome.Naturally occurring bacterial plasmids range in size from 5,000to 400,000bp. They canbe introducedinto bacterial cells by a processcalled transformation. The cells (generallyE. coli,) and plasmid DNA are incubated together at 0 'C in a calcium chloride solution, then subjected to a shockby rapidly shifting the temperature to 37 to 43'C. For reasonsnot well understood,someof the cells treated in this way take up the plasmid DNA. Somespeciesof bacteria,suchasAcznetobacterbaylyi, are naturally competentfor DNA uptake and do not require the calcium chloride treatment. In an alternative method, cells incubatedwith the plasmid DNA are subjected to a high-voltagepulse. This approach,called electroporation, transiently renders the bacterial membranepermeableto largemolecules. Regardlessof the approach,few cells actually take up the plasmrdDNA, so a method is neededto select those that do. The usual strategyis to use a plasmidthat includes a gene that the host cell requires for growth under speciflcconditions,such as a gene that confers resistanceto an antibiotic. Only cellstransformedby the recombinant plasmid can grow in the presenceof that antibiotic, making any cell that contains the plasmid "selectable"underthosegrowth conditions.Sucha gene is calleda selectablemarker. Investigatorshave developedmany different plasmid vectors suitable for cloning by modifying naturally occurring plasmids.The now classicE. coli' plasmid pBR322offers a good exampleof the featuresuseful in a cloningvector (FiS. 9-3).

EcoRI

Tetracycline resistance (teF)

Ampicillin resistance (ampn)

Vectors Allow Amplifi cation Cloning DNA Segments ofInserted The principles that govern the delivery of recombinant DNA in clonableform to a host cell, and its subsequent ampliication in the host, are well illustrated by considering three popular cloning vectors commonly used in experimentswith E. cole-plasmids, bacteriophages, andbacterialartificialchromosomes-anda vectorused to clonelargeDNA segmentsin yeast.

\urrl

Puull

tfGURE9-3 The constructed E. coli plasmid pBR322. Note the locationof some importantrestrictionsites-for Pstl,fcoRl, BamHl, genes;and the 5al1,and Pvull;ampicillin-and tetracycline-resistance in '1977,thiswasone of the early origin(ori).Constructed replication for cloningin E coli expressly plasmids designed

ft

308

DNA-Basedlnformationlechnologies

Important pBR322 features include : 1. An origin of replication, ori, a sequence where replication is initiated by cellular enz).rnes (Chapter 25). This sequence is required to propagate the plasmid and maintain it at a level of 10 to 20 copiesper cell. 2. Two genes that confer resistance to different antibiotics (tetR, ampR), allo\Mingthe identiflcation of cells that contain the intact plasmid or a recombinant version of the plasmid (Fig. g-4). 3. Several unique recognition sequences (PslI, EcoRl, BamHI, SalI, PuulI) that are targets for different restriction endonucleases,providing sites where the plasmid can later be cut to insert foreign DNA.

Foreigrr DNA is ligated to cleaved pBR322. Where ligation is successful, the ampicillin-resistance element is disrupted. The tetracycline-resrstance element remains intact.

4. Small size (4,361 bp), which facilitates entry of the plasmid into cells and the biochemrcal manipulation of the DNA.

/a\ (,J ,,

Ttansformation of typical bacterial cells with purified DNA (never a very efficient process) becomes less successful as plasmid size increases, and it is difflcult to clone DNA segments longer than about 15,000 bp when plasmids are used as the vector.

E. coli ce\ls are transformed, then grown on agar plates containing tetracycline to select for those that have taken up plasmid.

Bacteriophages Bacteriophage \ has a very efflcient mechanism for delivering its 48,502 bp of DNA into a bacterium, and it can be used as a vector to clone somewhat larger DNA segments (Fig. 9-5). TWo key features contribute to its utilitv:

All colonies have plasmids

Agar containing tetracycline

(h Individual colonies are transferred to matching positions on additional

plates. One plate contains tetracycline, the other tetracycline and ampicillin.

coloniestransferred for testing

Colonies with recombinant plasmids

Agar containing tetracycline (control)

Agar containing ampicillin + tetracycline

/?\

\q/

Cells that grow on tetracycline but not on tetracycline + ampicillin contain recombinant plasmids with disrupted ampicillin resistance,hence the foreign DNA. Cells *itn pnnlZZ without foreign DNA retain ampicillin resistance and grow on both plates.

About one-thirdof the ), genomeis nonessential and can be replacedwith foreign DNA. 2 . DNA is packagedinto infectious phageparticles only if it is between40,000and 53,000bp long,a constraint that can be used to ensurepackagingof recombinantDNA only. Researchershave developedbacteriophage\ vectors that can be readily cleavedinto three pieces,two of which contain essentialgenes but which together are only about 30,000bp long. The third piece, "filler" DNA, is discardedwhen the vector is to be used for cloning, and additional DNA is inserted between the two essentialsegmentsto generateligatedDNA moleculeslong enoughto produceviablephageparticles.In effect, the packagingmechanismselectslor recombinant viral DNAs. Bacteriophage}" vectors permit the cloning of DNA fragmentsof up to 23,000bp. Oncethe bacteriophage ), fragmentsare ligated to foreign DNA fragmentsof suitablesize,the resultingrecombinantDNAs can be packaged into phage particles by adding them to crude bacterial cell extracts that contain all the proteins neededto assemblea completephage.This is calledin vitro packaging (Fig. 9-5). All viablephageparticles will contain a foreign DNA fragment. The subsequent transmissionof the recombinantDNA into E colacellsis highly efficient.

l-ronl

C l o n i nTgh: eB a s i c s 9 . 1D N A

Filler DNA (not needed for packaging)

Lack essential DNA and./orare too small to be packaged

Recombinant DNAs

Baeterial Artificial Chromosomes (BACs) Bacterial artificial chromosomesare simply plasmidsdesigned for the cloning of very long segments(typically 100,000 to 300,000bp) of DNA (FiS. 9-6). They generallyinclude selectablemarkerssuch as resistanceto the antibiotic cruoramphenicol(CmR),as well as a very stable origin of replication (ori) that maintainsthe plasmid at one or two copies per cell. DNA fragments of several hundred thousand base pairs are cloned into the BAC vector.The large circular DNAs are then introduced into host bacteriaby electroporation.Theseproceduresuse host bacteriawith mutationsthat compromisethe structure of their cell wall, permitting the uptake of the large DNA molecules.

Cloning sites (inchtde lacZ)

J F plasmid por genes

_-R m'

I in vitro I packaging

t

tIGURE 9-5 Bacteriophage DNA methcloningvectors,Recombinant ods are used to modify the bacteriophage \ genome,removingthe genesnot neededfor phageproductionand replacingthem with into "filler"DNA to makethe phageDNA largeenoughfor packaging with foreignDNA phageparticles. As shownhere,thefilleris replaced intoviablephage in cloningexperiments Recombinants arepackaged particlesin vitro only if they includean appropriately sizedforeign DNA fragmentas well as both of the essential}, DNA end fragments. 9-6 Bacterial artificial chromosomes(BACs) as cloning FIGURE vectors.Thevectoris a relativelysimpleplasmid,with a replicationoriBin (ori)that directsreplication.Thepar genes,derivedfrom a type of of plasmids plasmidcalledan F plasmid,assistin theevendistribution to daughter cellsat cell division. Thisincreases the likelihoodof each daughter cell carryingone copyof the plasmid,evenwhenfew copies are present.The low numberof copiesis usefulin cloning largesegfor unwantedrecommentsof DNA becauseit limitsthe opportunities alterlargeclonedDNAsover binationreactionsthatcan unpredictably time.The BAC includesselectablemarkers.A lacZ gene(requiredfor issituated in thecloning of theenzymep-galactosidase) theproduction regionsuchthat it is inactivated by clonedDNA insertsIntroductionof is promotedby the use recombinantBACsinto cellsby electroporation DNAsare cellwall.Recombinant of cellswith an altered(moreporous) (CmR). Plates for resistance to the antibioticchloramphenicol screened for B-galactosidase that yieldsa coloredprodalsocontaina substrate with activeB-galactosidase and henceno DNA insertin uct.Colonies activitythe BACvectorturn blue; colonieswithoutB-galactosidase andthuswith thedesiredDNA inserts-arewhite.

Recombinant BAC

electroporation

Agar containing chloramphenicol and substrate for galactosidase

Colonies with recombinant BACs are white.

3.t0

D N A - B a sI nefdo r m a t iToenc h n o l o o i e s

Yeast Artificial Chromosomes (YACs) E. coli, celts are by no meansthe only hostsfor geneticengineering. Yeastsare particularly convenienteukaryotic organisms for this work. As with E. col,i, yeast genetics is a welldevelopeddiscipline. The genome of the most commonly usedyeast,Saccharomycescereui,siae,contains only 14 x 106 bp (a simple genome by eukaryotic standards,iess than four times the size of the E. coti, chromosome),and its entire sequenceis knor.rm. Yeastis also very easyto maintain and grow on a large scalein the laboratory.Plasmidvectorshave been constructed for yeast,employingthe sameprinciplesthat governthe use of E. coli, vectors described above. Convenient methodsare now availablefor moving DNA into and out of yeast cells,facilitatingthe study of many aspectsof eukaryoticcell biochemistry.Some recombinantplasmids incorporate multiple replication origins and other elementsthat allow them to be used in more than one species(for example,yeast or E. co\i,).Plasmidsthat can be propagatedin cells of two or more different speciesare calledshuttle vectors. Researchwith large genomesand the associated need for high-capacitycloningvectorsied to the development of yeast artifrcial chromosomes (yACS; Fig. !f-7) YACvectorscontainail the elementsneeded to maintaina eukaryoticchromosomein the yeast nucleus:a yeastorigin of replication,two selectablemarkers, and specialized sequences (derived from the telomeresand centromere,regionsof the chromosome discussedin Chapter24) neededfor stabilityandproper segregationof the chromosomes at cell division.Before beingusedin cloning,the vector is propagatedas a circular bacterial plasmid. Cleavagewith a restriction endonuciease(BamHI in Fig. 9-7) removesa Iength of DNA betweentwo telomeresequences(TEL), leaving the telomeresat the endsof the linearizedDNA. Cleavage at another internal site (EcoRI in Fig. 9-7) divides the vectorinto two DNA segments,referredto asvector arms, eachwith a different selectablemarker. The genomic DNA is prepared by partial digestion with restriction endonucleases (EcoRIin Fig. 9-Z) to obtain a suitablefragmentsize.Genomicfragmentsare then separatedby pulsed field gel electrophoresis, a variation of gel electrophoresis (seeFig. 3-18) that allowsthe separation of very large DNA segments.The DNA fragmentsof appropriatesize (up to about 2 x 106bp) are mixed with the preparedvector armsand hgated.The ligation mixture is then used to transform treated yeast cells with very large DNA molecules. Culture on a medium that requiresthe presenceof both selectable markergenesensuresthe growth of only thoseyeastcells that contain an arti-flcialchromosomewith a large insert sandwichedbetweenthe two vector arms (Fig. 9-Z). The stability of YACclonesincreaseswith size (up to a point). Thosewith insertsof morethan 150,000bp are nearlyas stable as normal cellular chromosomes,whereasthose with insertsof lessthan 100,000bp aregraduallylostduring mitosis (so generallythere are no yeast cell clones

Selectable marker Y

Selectable markerX

B amHI digestion creates linear chromosomewith telomeric ends EcoRl

ii EcoRI digestion i creates two arms v TELXoTiCENYTEL Left arm has selectablemarker X

.rTrTilTI*r\ Right arm has selectablemarker Y

; I

^ r..a ; ' 1'J'^'/7/:' n< J*Fragments genomic of :{'DNA generated by light i , . i Lrgate dieestion with EcoRI al

,

YAC

Select for X andY

Yeast spheroplast

Yeast with YAC clone

FIGURE 9-7 Constructionof a yeastartificial chromosome(yAC).A YACvectorincludesan originof replication (ori),a centromere (CfN), (TEL), two telomeres and selectable markers(Xand Y).Digestion with BamHl and EcoRlgenerates two separateDNA arms,each with a telomericend and one selectable marker.A largesegmentof DNA (e g, up to 2 x 106bp fromthe humangenome)is ligatedto the two armsto createa yeastartificialchromosome. TheYACtransforms yeast cells(prepared by removalof the cell wall to form spheroplasts), and the cellsare selectedfor X and Y; the survivingcells propagate the DNA insert.

carryingonly the two vector endsligatedtogetheror with only short inserts). YACsthat lack a telomere at either end are rapidly degraded.

Speeific SNA Sequences Ar€Detectable byhiybridizatio*l DNA hybridization, a process outlined in Chapter 8 (see Fig. 8-29), is the most corrrmonsequence-basedprocess for detecting a particular gene or segment of nucleic acid. There are many variations of the basic method, most making use of a labeled (such as radioactive) DNA

Irr!

C l o n i n q : TBhaes i c s 9 . 1D N A

or RNA fragment,known as a probe, complementaryto the DNA beingsought.In one classicapproachto detect a particular DNA sequencewithin a DNA library (a colIection of DNA clones),nitrocellulosepaper is pressed onto an agar plate containingmany individual bacterial coloniesfrom the library each colony with a different recombinantDNA. Somecellsfrom eachcolonyadhere to the paper,forminga replicaof the plate.The paperis treated with alkali to disrupt the cells and denaturethe DNA within, which remains bound to the region of the paper around the colony from which it came.Added radioactiveDNA probe annealsonly to its complementary DNA. A-fterany unannealedprobe DNA is washedaway, the hybridizedDNA canbe detectedby autoradiography (Fis. e-8). A common limiting step in detecting and cloning a gene is the generationof a complementarystrand of nucleicacid to use as a probe.The origin of a probe dependson what is knov,nabout the geneunder investigation. Sometimes a homologous gene cloned from anotherspeciesmakesa suitableprobe. Or, if the protein product of a genehas beenpurified,probescan be designedand synthesizedby working backward from the amino acid sequence,deducingthe DNA sequence tlpthat would codefor it (Fig. 9-9). Now,researchers ically obtain the necessaryDNA sequenceinformation from sequencedatabasesthat detail the structure of millions of genesfrom a wide range of organisms.

Agar plate with transformed bacterial colonies

Press nitrocellulose paper onto the agar plate. Some cells from each colony stick to the paper.

q

t*

Nitrocellulose paper

d

Treat with alkali to disrupt cells and exposedenatured DNA. ,!:,

DNA bound to paper

.1i -. ..:r: ..t

L,-z .q

':i"-

\,9:

Radioiabeled DNA probe Incubate the paper with the radiolabeled probe, then wash.

Probe annealed to colonies ofinterest

FIGURE 9-8 Useof hybridizationto identifya clonewith a particular to complemenTheradioactive DNA probehybridizes DNA segment. Once the labeled tary DNA and is revealedby autoradiography colonieson theorigthe corresponding colonieshavebeenidentified, inalagarplatecanbe usedasa sourceof clonedDNA for furtherstudy.

Known arnino acid sequence

U3fr - - - GIy

Possible codons

(5') G GA GGC GGU GGG

Leu -

UUA UUG OUA CUO CUU CUG

9-9 Probeto detect the genefor a protein of known amino FIGURE can codefor morethan one DNA sequence Because acid sequence. the geneticcodeis saidto be "degenanygivenaminoacidsequence, erate" (Asdescribedin Chapter27, an aminoacid is codedfor by a set calleda codon Mostamino acidshavetwo or of threenucleotides for a more codons;see Fig.27-7)fhus the correctDNA sequence probe The in advance. known be cannot knownaminoacidsequence to a regionof the genewith minimal to be complementary is designed with the fewestpossiblecodonsfor the region is, a that degeneracy, amino acids-two codons at most in the example shown here. Oligonucleotidesare synthesizedwith selectivelyrandomized so thattheycontaineitherof the two possiblenucleotides sequences, (shaded in pink).Theoligonudegeneracy at eachpositionof potential a mixtureof eightdifferentsequences: cleotideshownhererepresents and all eightwill the geneperfectly, one of the eightwill complement positions. matchat least17 of the 20

Pro - Trp -

- Val GIu - Asp - Met - Trp-Phe

ArS---COO-

CCA lucc GAA GAQ AUG UGG UUC GUIE AC..q'2C 1 step in the liver 1 step in the kidney

HO

HO

Cholecalciferol (vitamin D3 r

1,25-Dihydroxycholecalciferol ( 1,25-dihydroxyvitaminDr)

FIGURE10-20 Vitamin D3 production and metabolism. (a)Cholecalciferol (vitaminD3)is producedin the skinby UV irradiation of 7-dehydrocholesterol, whichbreaksthe bondshadedpink ln the liver,a hydroxylgroupis addedat C-25;in the kidney,a second hydroxylationat C-1 produces the active hormone, 1,25dihydroxycholecalciferol This hormoneregulates the metabolism of Ca'* in kidney,intestine, and bone.(b) DietaryvitaminD prevents rickets,a diseaseonce commonin cold climateswhereheavyclothing blocksthe UV componentof sunlightnecessary for the productionof vitaminD3 in skin In thisdetailfroma IargemuralbyJohnSteuartCurry, (1943),the peopleand anTheSocialBenefitsof BiochemicalResearch imalson the leftshowthe effectsof poor nutrition,includingthe bowed legsof a boy with classicalrickets.On the rightarethe peopleand animalsmadehealthier withthe "socialbenefits of research," including the useof vitaminD to prevent andtreatrickets. Thismuralis in the Departmentof Biochemistry at the University of Wisconsin-Madison.

asSignals, Cofactors, andPigments 10.3 Lipids [ruf

CHt CHt

":qit':".

";i::;y

Retinoicacid ------+ Hormonal

(d)

CHt

sigrral to epithelial ceIIs

CHt

CHt visible light

\ --------------)

point of cleavage

Neuronal signal to brain

11'-c-i:g

1

11-cis-Retinal (visual pigment) (c)

(e)

CHt

B-Carotene (a) FIGURE 10-21 Vitamin 41 and its precursorand derivatives.(a) pCaroteneis the precursorof vitaminA1. lsoprenestructuralunitsareset off by dashedred lines(seep. 359) Cleavage of B-carotene yieldstwo (b).Oxidationat C-l 5 converts molecules of vitamin41 (retinol) retinol to the aldehyde,retinal(c),andfurtheroxidationproducesretinoicacid (d), a hormonethat regulates geneexpression. Retinalcombineswith the proteinopsin to form rhodopsin(not shown),a visualpigment

widespread in nature.In the dark,retinalof rhodopsinis in the 11-cis form (c).When a rhodopsinmoleculeis excitedby visiblelight,the a seriesof photochemicalreactionsthat con1.1-crs-retinal undergoes (e),forcinga changein the shapeof the entire vert it to all-trans-retinal in the rod cell of the verterhodopsinmolecule.This transformation brateretinasendsan electricalsignalto the brain that is the basisof a topicwe addressin moredetailin Chapter12. visualtransduction,

Vitamins EandKandtheLipid Are Quinones (ofactors 0xidation-Reduction

protein that holds blood clots together.Henrik Dam and EdwardA. Doisyindependentlydiscoveredthat vitaminK deflciencyslowsblood clotting, which can be fatal. Vitamin K deflciency is very unconunon in humans, aside from a smallpercentageof infants who suffer from hemorrhagic diseaseof the newborn, a potentially fatal disorder. In the United States, newborns are routinely given a I mg injection of vitamin K. Vitamin Kr (phylloquinone) is found in green plant leaves;a related form, vitamin K2 (menaquinone),is formed by bacteria living in the vertebrateintestine.

Vitamin E is the collective name for a group of closely related lipids called tocopherols, all of which contain a substituted aromatic ring and a long isoprenoid side chain (Fig. 10-22a). Because they are hydrophobic, tocopherols associate with cell membranes, lipid deposits, and Jipoproteins in the blood Tocopherols are biological antioxidants. The aromatic ring reacts with and destroys the most reactive forms of oxygen radicals and other free radicals, protecting unsaturated fatty acids from oxidation and preventing oxidative damage to membrane Lipids,which can cause cell fragility. Tocopherols are found in eggs and vegetable oils and are especially abundant in wheat germ. Laboratory animals fed diets depleted of vitamin E develop scaly skin, muscrilar weakness and wasting, and sterility. Vitamin E deflciency in humans is very rare; the principal syrnptom is fragile erythrocl'tes. The aromatic ring of vitamin K (Fig. 10-22b) undergoes a cycle of oxidation and reduction during the formation of active prothrombin, a blood plasma protein essential in blood clotting. Prothrombin is a proteolytic enzlnne that splits peptide bonds in the blood protein fibrinogen to convert it to flbrin, the insoluble flbrous

EdwardA Doisy, 1893-1986

HenrikDam, 1895-1976

-rt{

Lipids

(a) Vitamin

E: an antioxidant

CH.

(b) Vitamin Kr: a blood-clotting cofactor(phylloquinone)

(c) Warfarin: a blood anticoagulant

(d) Ubiquinone: a rnitochondrial electron carrier (coenzyme Q) (n:4to8)

o H3CO..,/r\...-/CH3

ll

ll

gH,

H3co'-YfcHr-cH-i-cHrr(cHr-cH:i-cn,)" o

gH,

gH, cHr-cH:C-cH3

(e) Plastoquinone: a chloroplast electron carrier (n : 4 to 8)

(f) Dolichol: a sugar carrier (n:9 to 22)

FIGURE 10-22 Someotherbiologically activeisoprenoid compounds or derivatives.Units derivedfrom isopreneare set off by dashedred lines.In mostmammalian (alsocalledcoenzyme tissues, ubiquinone Q)

has 10 isopreneunits.Dolicholsof animalshave17 to 21 isoprene 'l units(85 to 05 carbonatoms),bacterialdolicholshave11, and those of plantsandfungihave14 to 24.

Warfarin (Fig. 10-22c) is a synthetic compound that inhibits the formation of active prothrombin. It is particularly poisonous to rats, causing death by internal bleeding. Irorucally, this potent rodenticide is also an invaluable anticoagulant drug for treating humans at risk for excessive blood clotting, such as surgical patients and those with coronary thrombosis. r Ubiquinone (also called coenzyme Q) and plastoquinone (Fig. 10-22d, e) are isoprenoids that function as Iipophilic electron carriers in the oxidation-reduction reactions that drive ATP synthesis in mitochondria and chloroplasts, respectively. Both ubiquinone and plastoquinone can accept either one or two electrons and either one or two protons (see Fig. I9-2).

units to certain proteins (glycoproteins)and lipids (glycolipids)in eukaryotes,the sugarunits to be addedare chemically activated by attachment to isoprenoid alcohols calleddolichols (FiS. 10-220. Thesecompounds have strong hydrophobic interactions with membrane Iipids, anchoringthe attached sugarsto the membrane, where they participate in sugar-transferreactions.

Dolichols Artivate 5ugar Precurs0rs f0rBiosynthesis During assembly of the complex carbohydrates of bacterial cell walls, and during the addition of polysaccharide

(onjugated Many Natural Pigments AreLipidic Dienes Conjugated dienes have carbon chains with alternating single and double bonds. Because this structural arrangement allows the delocalization of electrons, the compounds can be excited by low-energy electromagnetic radiation (visible light), giving them colors visible to humans and other animals. Carotene (Fig. 10-21) is yelloworange; similar compounds give bird feathers their striking reds, oranges, and yellows (Fig. f0-23). Like sterols, steroids, dolichols, vitamins A, E, D, and K,

withLipids[so 10.4Working

Canthaxanthin (bright red)

HO Zeaxanthin (bright yellow)

FIGURE 10-23 Lipidsas pigmentsin plantsand bird feathers.Compoundswith longconjugated systems absorblightin the visibleregionof the spectrum.Subtledifferences in the chemistryof thesecompounds producepigmentsof strikinglydifferentcolors.Birdsacquirethe pig-

that redor yellowby eatingplantmaterials mentsthatcolortheirfeathers The andzeaxanthin. suchascanthaxanthin containcarotenoidpigments, betweenmaleandfemalebirdsarethe result in pigmentation differences of carotenoids. in intestinaluptakeand processing of differences

ubiquinone,and plastoquinone,these pigmentsare s)mthesized from flve-carbon isoprene derivatives; the biosyntheticpathwayis describedin detail in Chapter21.

r

Dolicholsactivate and anchor sugarsto cellular membranes;the sugargroups are then used in the symthesisof complex carbohydrates,glycolipids, and glycoproteins.

r

Lipidic conjugateddienesserveas pigmentsin flowers and fruits and give bird featherstheir striking colors.

1Y 0 . 3 [ i p i d sa sS i g n a l s , SUMMAR C o f a c t oar n s ,dP i g m e n t s r

Sometypes of lipids, althoughpresent in relatively small quantities,play critical roles as cofactorsor signals.

r

Phosphatidylinositolbisphosphateis hydrolyzedto yield two intracellular messengers,diacylglyceroland inositol 1,4,5-trisphosphate. Phosphatidylinositol 3,4,5-trisphosphate is a nucleationpoint for supramolecularprotein complexesinvolvedin biologicalsignaling.

r

Prostaglandins,thromboxanes,and leukotrienes (the eicosanoids), derivedfrom arachidonate, are extremely potent hormones.

r

Steroidhotmones,derivedfrom sterols,serveas powerfirl biologicalsignals,such as the sex hormones.

r

Vitamins D, A, E, and K are fat-solublecompounds made up of isopreneunits. AiI play essentialroles in the metabolismor physiologyof animals.Vitamin D is precursor to a hormonethat regulatescalcium metabolism.Vitamin A furnishesthe visual pigment ofthe vertebrateeye and is a regulatorofgene expressionduring epithelial cell growth. Vitamin E functions in the protection of membranelipids from oidative damage,and vitamin K is essentialin the blood-clottingprocess.

r

ubiquinonesand plastoquinones,alsoisoprenoid derivatives,are electron carriersin mrtochondria and chloroplasts,respectively.

withLipids 10.4Working Becauselipids are insoluble in water, their extraction and subsequentfractionation require the use of organic solvents and some techniques not commonly used in the purification of water-solublemolecules such as proteins and carbohydrates.In general, complex mixtures of Iipids are separatedby differences in polarity or solubility in nonpolar solvents. Lipids that contain ester- or amide-linkedfatty acids can be hydrolyzed by treatment with acid or alkali or with speciflchydrolytic glycosidases)to yield their enzymes(phospholipases, components for analysis. Some methods commonly used in lipid analysisare shown in Figure 10-24 and discussedbelow

5olvents 0rganic Requires Extraction Lipid Neutral lipids (triacylglycerols,waxes,pigments,and so forth) are readily extracted from tissues with ethyl ether, chloroform, or benzene,solventsthat do not permit lipid clustering driven by hydrophobicinteractions. Membranelipids are more effectivelyextracted by more polar organic solvents,such as ethanol or methanol, wtuch reduce the hydrophobicinteractions amonglipid moleculeswhile also weakeningthe hydrogen bonds and electrostaticinteractionsthat bind membranelipids to membraneproteins.A commonlyused extractant is a

!o-l

Lipids FIGURE 10-24 Commonproceduresin the extraction,separation, and identification of cellularlipids. (a) Tissueis homogenized in a chloroform/methanol/water mixture,which on additionof waterand removal of unextractable sedimentby centrifugationyields two phases.Differenttypesof extractedlipids in the chloroformphasemay be separated by (b) adsorption chromatography on a columnof silica gel, throughwhich solventsof increasingpolarityare passed,or (c) thin-layerchromatography (TLC),in which lipidsare carriedup a silicagel-coated plateby a risingsolventfront,lesspolarlipidstraveling fartherthan more polaror chargedlipids.TLC with appropriate solvents can alsobe usedto separate closelyrelatedlipid species; for example,the chargedlipidsphosphatidylserine, phosphatidylglycerol, and phosphatidylinositol areeasilyseparated by TLC. Forthe determination of fattyacid composition, a lipid fraction containing ester-linked fattyacidsis transesterified in a warmaqueous solutionof NaOH and methanol(d),producinga mixtureof fattyacyl methylesters.Thesemethylestersare then separatedon the basisof chain Iengthand degreeof saturation by (e) gas-liquidchromatography (CLC)or (f) high-performance (HPLC). liquidchromatography Precise determination of molecularmassby massspectrometry allows u n a m b i g u o ui dse n t i f i c a t i o fni n d i v i d u al ilp i d s

homogenized in chloroform,/methanoVwater

Water

MethanoVwater

Chloroform

hin-layer matography

Neutral Polar Charged lipids lipids lipids

mixture of chloroform, methanol, and water, initially in volume proportions (1:2:0.8) that are miscible, producing a single phase. After tissue is homogenized in this solvent to extract all lipids, more water is added to the resulting extract and the mixture separates into two phases, methanoi/water (top phase) and chloroform (bottom phase). The lipids remain in the chloroform layer, and the more polar molecules such as proteins and sugars partition into the methanol/water layer.

(hr0matography Adsorption Separates Lipids of Different Polarity Fatty acyl methyl esters

(e)

(f)

Highperformance liquid chromatography

Gas-liquid chromatography

18:0 1 6 : 1

14:0 k

16:0

Elution time

Complex mixtures of tissue lipids can be fractionated by chromatographic procedures based on the different polarities of each class of lipid. In adsorption chromatography (Fig. l0-24b), an insoluble, polar material such as silica gel (a form of silicic acid, Si(OH)n) is packed into a glass column, and the lipid mixture (in chloroform solution) is applied to the top of the column. (In highperformance liquid chromatography, the column is of smaller diameter and solvents are forced through the column under high pressure.) The polar lipids bind tightly to the polar silicic acid, but the neutral lipids pass directly through the column and emerge in the first chloroform wash The polar lipids are then eluted, in order of increasing polarity, by washing the column with solvents ofprogressively higher polarity. Uncharged but polar lipids (cerebrosides, for example) are eluted with acetone, and very polar or charged lipids (such as glycerophospholipids) are eluted with methanol. ThinJayer chromatography on silicic acid employs the same principle (Fig. 10-24c). A thin layer of silica gel is spread onto a glass plate, to which it adheres.A small sample of lipids dissolved in chloroform is applied near

withLipids SOi 10.4Working

one edge of the plate, which is dipped in a shallowcontainer of an organicsolventor solventmixture; the entire setupis enclosedin a chambersaturatedwith the solvent vapor.As the solvent rises on the plate by capillary action, it carries lipids with it. The less polar Iipids move farthest, as they haveIesstendencyto bind to the silicic acid. The separatedlipids can be detected by spraying the plate with a dye (rhodamine) that fluoresceswhen associatedwith lipids, or by exposingthe plate to iodine fumes.Iodine reactsreversiblywith the doublebonds in fatty acids,such that lipids containingunsaturatedfatty acids develop a yellow or brown color. Severalother sprayreagentsare alsousefulin detectingspecificlipids. For subsequentanalysis,regions containing separated lipids can be scraped from the plate and the lipids recoveredby extraction with an organicsolvent.

(hromatography Mixtures of Resolves Gas-Liquid Volatile LipidDerivatives Gas-liquidchromatographyseparatesvolatile components of a mixture accordingto their relative tendencies to dissolvein the inert material packed in the chromatography column or to volatilize and move through the column,carriedby a current of an inert gassuchas helium. Some lipids are naturally volatile, but most must first be derivatized to increase their volatility (that is, lower their boiling point). For an analysisof the fatty acidsin a sampleof phospholipids,the lipids are first transesterified:heated in a methanoVHOlor methanol/NaOHmixture to convert fatty acids esterified to glycerol into their methyl esters (Fig. l0-24d). Thesefatty acyl methyl estersare then loadedonto the gas-liquid chromatographycolumn, and the column is heated to volatilizethe compounds.Thosefatty acyl esters most soluble in the column material partition into (dissolvein) that material;thelesssolublelipidsare carried by the streamof inert gasand emergefirst from the column. The order of elution dependson the nature of the solid adsorbantin the column and on the boiling point of the componentsof the lipid mixture. Using these techniques,mixtures of fatty acids of various chain lengths and various degreesof unsaturation can be completelyresolved(Fig. 10-24e).

ofLipidStructure Hydrolysis Aidsin Determination Specific Certainclassesof lipids are susceptibleto degradationunder specificconditions.For example,all ester-lirkedfatty acidsin triacylglycerols,phospholipids,and sterol esters are releasedby mild acid or alkalinetreatment,and somewhat harsherhydrolysisconditionsreleaseamide-bound fatty acids from sphingolipids.Erzymes that speciflcally hydrolyzecertain lipids are alsouseful in the determination of lipid structure. PhospholipasesA, C, and D (ng. 10-16) eachspJitparticularbondsin phospholipids and yield products with characteristic solubilities and C,for example, behaviors.Phospholipase chromatographic

releasesa water-solublephosphorylalcohol(suchasphosphocholinefrom phosphatidylcholine)and a chloroformeachofwhich canbe characterized solublediacylgJycerol, the structure of the intact to determine separately phospholipid.The combinationof specifichydrolysiswith characteization of the products by thinJayer, gas-liquid, or high-perfolrnanceJiquid chromatographyoften allows determinationof a lipid structure.

Lipid Structure Complete Reveals Spectrometry Mass To establishunambiguouslythe length of a hydrocarbon chainor the positionof doublebonds,massspectrometric analysisof lipids or their volatile derivatives is invaluable.The chemicalproperties of similar lipids (for example,two fatty acidsof similar length unsaturatedat different positions,or two isoprenoidswith different numbers of isopreneunits) are very much alike, and their order of elution from the various chromatographic proceduresoften does not distinguishbetween them. When the eluate from a chromatographycolumn is sampledby mass spectrometry,however,the components of a lipid mixture canbe simultaneouslyseparated and identifled by their unique pattern of fragmentation

(Fis.r0-25). Functions AlltipidsandTheir toCatalog Seeks Lipidomics In exploring the biologicalrole of lipids in cells and tissues, it is important [o know which lipids are present and in what proportions,and to know how this lipid compositionchangeswith embryonicdevelopment,disease,or drug treatment. As lipid biochemistshave become aware of the thousands of different naturally occurringlipids, they have proposeda new nomenclature system,with the aim of making it easierto compile and searchdatabasesof lipid composition'The system placeseachlipid in one of eight chemicalgroups (Table 10-3) designatedby two letters. Within these groups, finer distinctionsare indicatedby numberedclassesand are For example,all glycerophosphocholines subclasses. with two glycerophosphocholines GP01;the subgroupof fatty acidsin ester linkageis designatedGP0101;with one fatty acid ether-linkedat position I and one in ester Iinkage at position 2, this becomesGP0102'Specific fatty acids are designatedby numbers that give every lipid its own unique identifler, so that each individual lipid, including lipid types not yet discovered,canbe unambiguouslydescribedin terms of a L2-characteridentifier. One factor used in this classfficationis the nature of the bioslmtheticprecursor.For example,prenol lipids (dolichols and vitamins E and K, for example) are formed from isoprenylprecursors.Polyketides,which we havenot discussedin this chapter,include somenatural products, many toxic, with biosynthetic pathways related to those for fatty acids.The eight chemicalcategories in Table 10-3 do not coincide perfectly with the divisions accordingto biological function that we have

['u"l L i p i d s

960 a Cd bU

30 20 10

60

80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 m/z

FIGURE10-25 Determination of fatty acid structure by mass spect r o m e t r y . T h e f a t t y a c i d i s f i r s tc o n v e r t e dt o a d e r i v a t i v et h a t m i n i m i z e s m i g r a t i o n o f t h e d o u b l e b o n d s w h e n t h e m o l e c u l e i s f r a g m e n t e db y e l e c t r o n b o m b a r d m e n t .T h e d e r i v a t i v es h o w n h e r e i s a p i c o l i n v l e s t e r o f l i n o l e i ca c i d - 1 B : 2 ( A e , r '()M , 3 7 1 ) - j n w h i c h t h e a l c o h o l i s p i c o l i _ n o l ( r e d ) W h e n b o m b a r d e d w i t h a s t r e a mo f e l e c t r o n s ,t h i s m o l e c u l e i s v o l a t i l i z e da n d c o n v e r t e dt o a p a r e n t i o n ( M + ; M , 3 7 1 ) , i n w h i c h t h e N a t o m b e a r s t h e p o s i t i v e c h a r g e , a n d a s e r i e so f s m a l l e r f r a g m e n t s p r o d u c e d b y b r e a k a g eo f C - C b o n d s i n t h e f a t t y a c i d T h e m a s ss p e c trometer separates these charged fragments according to their mass/charge"atio (m/z). (To review the principles of mass specrrometry, see Box 3 2 )

used in this chapter For example, the structural lipids of membranes include both glycerophospholipids and sphingolipids, separate categories in Tabie 10-3. Each method of categorization has its advantages. The application of mass spectrometric techniques with high throughput and high resolution can provide quantitative catalogs of all the lipids present in a specific cell type-the lipidome-under particular conditions, and of the ways in which the lipidome changes with differentiation, disease such as cancer, or drug treatment. An animal cell contains about a thousand different lipid species, each presumably havng a spe-

TheprominentionsaI m/z :92, 1OB,151,and 164 containthe pyridineringof thepicolinol andvariousfragments of thecarboxylgroup, showing thatthecompound isindeeda picolinyl ester. Themolecular ion, M- (m/z : 371), confirmsthe presence of a C1ofattyacidwrthtwo double bonds.Theuniformseriesof ions14 atomicmassunits(u)apartrepresentslossof eachsuccessive methylandmethylene groupfromthe methyl endof theacylchain(beginning at C-1B;therightendof themolecule as shownhere),untilthe ion at m/z: 300 is reached. Thisis followedby a gapof 26 u for thecarbonsof theterminaldoublebond,at m/z : 274;a furthergapol 14 u lor the C-l 1 methylene group,at m/z : 260; andso forth.Bythismeanstheentirestructure isdetermined, althoughthesedata alonedo not revealthe configuration (cisor trans)of the doublebonds cific function. These functions are known for a growing

number of lipids, but the stiil largely unexplored lipidome offers a rich source of new problems for the next generation of biochemists and cell biologists to solve.

SUMMAR 1Y 0 . 4 W o r k i nwgi t hL i p i d s r

In the determination of lipid composition, the lipids are flrst extracted from tissues with organic solvents and separated by thin-layer, gas-liquid, or high-performance Iiquid chromatography.

Category

Categorycode

Examples

Fatty acids

FA

Oleate, stearoyl-CoA, palmitoylcarnitine

Glycerolipids

GL

Di- and triacylglycerols

Glycerophospholipids

GP

Phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine

Sphingolipids

SP

Sphingomyelin, ganglioside GM2

Sterol lipids

ST

Prenol lipids

PR

Cholesterol, progesterone, bile acids Farnesol, geraniol, retinol, ubiquinone

Saccharolipids

SL

Lipopolysaccharide

Polyketides

PK

Tetracycline.aflatoxin B.

F u r t h eRre a d i n g[ r a l

Phospholipases speciflcfor one of the bondsin a phospholipidcanbe usedto generatesimpler compoundsfor subsequentanalysis. Individual lipids are identifled by their chromatographic behavior,their susceptibilityto hydrolysisby specificenzymes,or mass cnont

rnm

ofnr

Lipidomics combines powerful analy'ticai techniques to determine the full complement of lipids in a cell or tissue (the lipidome) and to assemble annotated databasesthat allow comparisons between lipids of different cell types and under different conditions.

Lipids as Nutrients pol''unsaturated Angerer,P.& von Schacky,C. (2000)Omega-3 fatty acids ancl the cardiovascular system Cz,rz Opi'n Li'pi'dol ll /

ll,

o.l

fattyacidsAm Fam Phgsict'an Covington,M.B. (2004)Omega-3 7 0 , 1 3 31 4 0 Succinctstatementof the findingsthat omega-3fatty acids reduce the risk of cardiovasculardisease de Logeril, M., Salen, P., Martin, J'L., Monjaud, I', Delaye, J., & Mamelle, N. (1999) Mediterraneandiet, tradltional rlsk factors, and the rate of cardiovascularcomplicationsafter myocardialinfarction: final report of the Lyon Diet Heart Study.CircuLati'on 99, 779-785. Mozaffarian, D., Katan, M.B., Ascherio, P.H., Starnpfe4 M.J.' & Willet, W.C. (2006) Transfatty acidsand cardiovasculardisease. N. Ensl J. Med 354,1601-1613 that dietary trans fatty acidspredisA summaryof the er,'rdence poseto coronaryheart disease.

KeyTerms Structural

Terms in bold are defined in the glossarg fatty acid 343 polyunsaturated fatty acids (PUFAs) 345 346 triacylglycerols lipases 346 phospholipid 349 glycolipids 349 glycerophospholipid 350 etherlipid 350 plasmalogens 350 galactolipids 352 sphingolipids 352 ceramicle 354 sphingomyelin 354 glycosphingolipids354 cerebrosides 354 globosides 354

neutralglycolipids 354 gangliosides 354 sterols 355 cholesterol 355 prostaglandins 358 thromboxanes 358 leukotrienes 359 vitamin 360 vitamin D3 360 cholecalciferol 360 vitamin A (retinol) 360 vitaminE 361 tocopherols 361 vitaminK 361 dolichols 362 lipidome 366

Lipids in Membranes

Bogdanov, M. & Dowhan, \ry.(1999) Lipid-assistedprotein folding J BioI Chem 274,36,827-36,830 A minirenew of the role of membranelipids in the folding of membraneproteins De Rosa, M. & Gambacorta, A. (1988) The lipids of archaebacteria Prog Li'pi'd Res 27,153-175 Dowhan, lV. (1997) Molecularbasisfor membranephospholipid diversity:why are there so rnanylipids?Annu Reu Bi'ochem 66, 199-232 Gravel, R.A., Kaback, M.M', Proia, R., Sandhoff, K., Suzuki, K', & Suzuki, K. (2001) The GM2gangliosidosesInThe Metabol'i'cand Molecular BasesoJInheri,ted'D'isease,Sth edn (Scriver,C R , Sly' W.S, Childs,B., Beaudet,A.L.,Valle,D , Kinzler,K W.,& Vogelstein, Inc , New York B , eds),pp. 3827 3876,McGraw-Hill, This article is one of many in a four-volumeset that containsdefinitive descriptionsof the clinical,biochemical,and geneticaspects of hundredsof humanmetabo[c diseases-an authoritativesource and fascinatingreading Hoekstra, D. (ed.). (1994) CeILLipids, Ctrtent Topicsin Membranes,Vol.4, AcademicPress,Inc , SanDiego. Lipids as Signals, Cofactors,

Reading Further General Fahy, E., Subramaniam, S., Brown, H.A., Glass, C.K., Merrill' A.H., Jr., Murphy, R.C., Raetz, C.R.H., Russell, D.W., Seyama, Y,, Shaw, W., Shimizu, T., Spener, F., van Meers, G., VanNieuwenhze, M.S., White, S.H., Witztum, J.L., & Dennis, E.A. (2005) A comprehensive classiflcation system for lipids J. Lip'id Res 46.839 862. A new system of nomenclature for biological lipids, separating thenr into eight maior categories The definitive reference on lipid classilication Gurr, M.I., Harwood, J,L., & Frayn, K.N. (2002) Liptd Biochemi.stry: Att, Introduction,Sth edn, Blackwell Science Ltd., Oxford A goocl general resource on lipid structure and metabolism, at the intermediate level Vance, D.E. & Vance, J.E. (eds). (2002) Bi'och,em:lstrg oJ Li,pids, L'|)oproteins, o,n.dMembranes, New Comprehensive Biochemistry, Vol 36, Elsevier ScrencePublishing Co , Inc , New York An excellent collection of reviews on vadous aspects of lipid structure, biosynthesis, and function

and Pig;ments

Bell, R.M., Exton, J'H.' & Prescott, S.M. (eds). (1996)Ltpi'd Second,Messengers,HandbookofLipid Research,Vol 8, Plenum Press,New York Binkley, N.C. & Suttie, J.W. (1995) Vitamin K nutrition and osteoporosisJ. Nutr I25,I812 1821 Brigelius-Floh6, R. & Thaber, M.G. (1999) Vitamin Fl:function FASEBJ. 13, 1145-1155. andrnetabolism. Chojnacki, T. & Dallner, G. (1988) The biologicalrole of dolichol Biochem J.251,1-g Clouse, S.D. (2002) Brassinosteroidsignaltransduction:clarifying the pathwayfrom liganclperceptionto geneexpressionMoL Cell LO, 973-982 Lemmon, M.A. & Ferguson, K.M. (2000) Signal-dependentmembrane targetingby pleckstrin homology(PH) domains.Bi'ochem J' 350, 1-18 Prescott, S.M., Zimmerman, G.A., Stafforini, D.M., & Mclntyre, T.M' (2000) Platelet-activatingfactor and related lipid mediators A'nnu Reu Biochem 69,419445 Schneiter R. (1999) Bravelittle yeast,pleaseguide us to Thebes: 21, 1004-1010' sphingolipidfunctionins cereutsiaeBi'oEssa'Es

["']

Lipids

Suttie, J.W. (1993) Synthesis of r,rtamin K-clependent proteins FASEB J. 7,445_452 Vermeer, C. (1990) 7-Carboxyglutamate-containing proteins and the !.ltamin K-dependent carboxylase Biochem J. 266, 625-636 Describes the biochemical basis for the requirement of vitamrn K in blood clotting and the importance of carboxylation in the svnthesis of the blood-clotting protein thrombin Viitala, J. & Jdrnefelt, J, (198b) The red cell surface revisited Tfe'nds Bzochem Sci 1O,392 395 Includes discussion of the human A, B, and O blood tlpe determinants

(a) What structural aspect of these l8-carbon fatty acids can be correlated with the melting point? (b) Draw ail the possibie triacylglycerols that can be constructed from glycerol, palmitic acid, and oleic acid. Rank them in order of increasing melting point. (c) Branched-chain fatty aclds are found in some bacterial membrane lipids. Would their presence increase or decrease the fluidity of the membranes (that is, give them a lower or higher melting point)? Why?

Zittermann, A. (2001) Effects of vitamin K on calcium and bone metabolism Curr Opin CLin Nutr Metab Co,re 4.495-4gT

3. Preparation of B6arnaise Sauce During the preparation of b6arnaise sauce,egg yolks are incorporated into melted butter to stabilize the sauce and avoid separation The stabilizing agent in the egg yolks is lecithin (phosphatidylcholine).

Working

Suggest why this works.

Weber, H. (2002) Fatty acid-derived signals in plants Trencls plant Sc'i 7,217-224.

with

Lipids

Christie, WW. (1998) Gas chromatography-mass spectrometry methods for structural analysis of fatty acids Lipid,s 3,J, 343_gb3 A detailed description of the methods used to obtain data such as those presented in Figure 10 2b Christie, fV.W. (2003) Lipid Analysis, 3rd ecln, The Oilv press, Bridgwater, England German, J.B., Gillies, L.A., Smilowitz, J.T., Zivkovic, IVatkins, S.M. (2007) Lrpidomics and lipid proflling in metabolomics Curr Opin Lipid,ot 18, 66 Z1 Short renew of the goals and methods of lipidomics

4. Isoprene

Units in Isoprenoids Geraniol, farnesol, and squalene are called isoprenoids, because they are synthesized from five-carbon isoprene units. In each compound, circle the f,ve-carbonunits representhg isoprene units (see Fig. 10-22)

A.M., &

Geraniol

Griffiths, W, Desiderio, D.M., & Nibbering, N.M. (2007) Lipid, Mass Spectrometry i,n Metabolomzcs and" Systems Bi,ctlogg, Wiley InterScience, New York. Hamilton, R.J. & Hamilton, S. (eds). (1992) Lipid, Analysis: A PracticaL Approach,lRL Press, New york Thrs text, now out ofprint, ts available as part ofthe iRL press PracticaL Approach Series on CD-ROM, from Oxford Universrty Press (www.oup-usa org/acadsci,/pasbooks.html)

Farnesol

Matsubara, T. & Hagashi, A. (1991) MB/mass spectrometry of lipids Prog LipidRes 30,301.-522 An advanced drscussion of the identification of lipicls by fast atom bombardment (FAB) mass spectrometry, a powerful technique for structure determination Watson, A.D. (2006) Lipidomrcs: a global approach to lipid analysis in biological systems J. Li,pirl Res 47,2101-2111 A short, rntermediatelevel review of the classes of lipids, the methods for extracting and separating them, and mass spectrometric means for identifuing and quantifying all lipids in a given cell, tissue, or organelle Wenk, M.R. (2005) The emerging field of lipidomics Na, Reu Dntq Discoa.4,594-6I0 Intermediate-level discussion of the methocls of lipidomics and the potential of this approach in biomedica.lresearch and drug development

Problems

Squalene 5. Naming Lipid Stereoisomers The two compounds beIow are stereoisomers of carvone with quite different properties; the one on the left smells like speamint, and that on the right, like caraway. Name the compounds using the RS system.

CH"

1. Operational Definition of Lipids How is the definition of "lipid" different from the types of definitions used for other biomolecules that we have considered, such as amino acids, nucleic acids, and proteins? 2. Melting Points of Lipids The melting points of a series of 18-carbon fatty acids are: stearic acid, 69.6 oC; oleic acid, 13 4 'C; linoleic acid, - 5 'C; and linolenic acid. - 11 .C

t" o--c. c --cH li H"C. -\

CH"

C cH"-Qt H tl CH, Spearmint

9H' ooa-Coa" ll

P r o b l e m[rrt] s

6. RS Designations for Alanine and Lactate Draw (using wedge-bondnotation) and iabel the (r?) and (S) isomersof 2-aminopropanoicacid (alanine) and 2-hydrorypropanoic acid Oacticacid). H

H

,, r r 2 r^'-Ca...-COOH \

I ^---c..-cooH 'cu, url

2-Aminopropanoic acid (alanine)

2-Hydroxypropanoic acid (Iactic acid)

I CH,

7. Hydrophobic and Hydrophilic Components of Membrane Lipids A common structural feature of membrane lipids is their amphipathicnature. For example,in phosphatidylcholine,the two fatty acid chainsare hydrophobicand the phosphocholineheadgroup is hydrophilic. For eachof the following membranelipids, name the componentsthat serve as the hydrophobic and hydrophilic units: (a) phosphatidylethanolamine;(b) sphingomyelin;(c) galactosylcere(e) cholesterol. broside;(d) ganglioside; 8. Structure of Omega-6 Fatty Acid Draw the structure of the omega-6fatty acid 16:1. 9. Catalytic Hydrogenation of Vegetable Oils Cataly'tichydrogenation,usedin the foodindustry convertsdoublebondsin How the fatty acidsof the oil triacylglycerolsto -CH2-CH2physical properties of the oils? doesthis affectthe 10. Alkali Lability of Ttiacylglycerols A common procedure for cleaningthe greasetrap in a sink is to add a product that containssodiumhydroxide Explain why this works 11. Deducing Lipid Structure from Composition Compositionalanalysisof a certain lipid shows that it has exactly one mole of fatty acid per mole of inorganicphosA A ganglioside? phate Couldthis be a glycerophospholipid? sphingomyelin? 12. Deducing Lipid Structure from Molar Ratio of Components Completehydrolysisof a glycerophospholipidyields glycerol,two fatty acids(16:1(As)and 16:0),phosphoricacid, and serinein the molar ratio 1:l:1:1:1.Namethis lipid and draw its structure. 13. Impermeability of Waxes What property of the waxy cuticles that cover plant leavesmakes the cuticies impermeableto water? 14. The Action of Phospholipases The venom of the Eastern diamondback rattler and the Indian cobra contains phospholipase A,2,which catalyzes the hydrolysis of fatty acids at the C-2 position of glycerophosThe phospholipid breakdown product of this reaction is lysolecithin (lecithin is phosphatidylcholine) At high concentrations, this and other lysophosphollpids act as detergents, dissoiving the membranes of erythrocytes and lysing the cells. Extensive hemolysis may be lifepholipids

threatening.

(a) AII detergents are amphipathic. What are the hydrophilic and hydrophobicportions of lysolecithin? (b) The pain and inflammationcausedby a snakebite can be treated with certain steroids.What is the basis of this treatment? (c) Though the high levels of phospholipase.A2in venom can be deadly,this enzltne is necessaryfor a variety ofnormal What are theseprocesses? metabolicprocesses. 15. Lipids in Blood Group Determination Wenote in Figure 10-15 that the structure ofgiycosphingolipidsdetermines the blood groupsA, B, and O in humans.It is alsotrue that giycoproteinsdetermineblood groups.How can both statements be true? 16. Intracellular Messengers from Phosphatidylinositols When the hormone vasopressin stimulates cleavage of phosphatidylinositol4,5-bisphosphateby hotmone-sensitive phospholipase C, two productsareformed.What arethey?Comin water,and predict pare thet propertiesand their solubi-lities whether either would diffuse readily through the cytosol 17. Storage of Fat-Soluble Vitamins In contrast to watersolublevitamins,which must be part of our daily diet, fat-soluble vitamins canbe stored in the body in amountssufflcient for many months Suggestan explanationfor this dj-fference. 18. Ilydrolysis of Lipids Namethe productsof mi-ldhvdrolysis with dilute NaOHof (a) 1-stearovl-2,3-dipalmitoylglycerol; (b) 1-palmitoyl-2-oleoylphosphatidylcholine. 19. Effect of Polarity on Solubility Rank the following in order of increasingsolubility in water: a triacylglycerol,a diacylglycerol, and a monoacylglycerol, all containing only palmitic acid 20. Chromatographic Separation of Lipids A mixture of lipids is applied to a silica gel column, and the column is then washed with increasingly polar solvents. The mixture consists of phosphatidylserine,phosphatidylethanolamine, phosphatidylcholine,cholesterylpalmitate (a sterol ester), sphingomyelin,palmitate,n-tetradecanol,triacylglycerol,and cholesterol.In what order will the lipids elute from the colurtrrr? Explain your reasomng. 21. Identification of Unknown Lipids JohannThudichum, who practicedmedicinein Londonabout 100yearsago,also dabbledin lipid chemistryin his sparetime. He isolateda variety of lipids from neural tissue, and characterizedand named many of them. His carefully seaiedand labeledvials of isolated lipids were rediscoveredmany yearslater (a) How would you conflrm, using techniquesnot availableto Thudichum,that the vials labeled"sphingomyelin"and "cerebroside"actually contain these compounds2 (b) How would you distinguishsphingomyelinfrom phosphatidylcholineby chemical,physical,or enzymatictests? 22. Ninhydrin to Detect Lipids on TLC Plates Ninhydrin reactsspeciflcallywith primary aminesto form a purplish-blue product. A thinJayer chromatogramof rat liver phospholipids

ft

L_370 1 Lipids is sprayed with ninhydrin, and the color is ailowed to develop Which phospholipids can be detected in this way?

GangliosideCeramide Glucose Galactose Galactosamine Normal Er-^^-^^+ r raSrrrgrrL

DataAnalysis Problem 23. Determining the Structure of the Abnormal Lipid in Tby-SachsDisease Box 10-2, Figure 1, showsthe pathway of breakdown of gangliosidesin healthy (normal) individuals and individualswith certain genetic diseasesSome of the data on which the figure is basedwere presentedin a paper by Lars Svennerholm(1962). Note that the sugar NeubAc, N-acetylneuraminicacid, representedin the Box 10-2 fleure as O, is a sialicacid Svennerholm reportedthat "about90%ofthe monosialiogangliosidesisolatedfrom normai human brain" consistedof a compoundwith ceramide,hexose,N-acetylgalactosamine, and N-acetylneuraminic acidin the molar ratio 1:3:1:1. (a) Which of the gangliosides(GMl through GM3and glo_ boside)in Box 10-2, Figure 1, flts this description?Explain your reasorung (b) Svennerholmreported Ihat g0o/oof the gangliosides from a patient with Tay-Sachshad a molar ratio (of the same four componentsgiven above) of 1:2:1:1.Is this consrsrenr with the Box 10-2 figure?Explain your reasoning To determine the structure in more cletail,Svennerholm treated the gangliosideswith neuraminidaseto removethe N_ acetylneuraminicacid. This resulted in an asialoganglioside that was much easier to analyze He hydrolyzed it with acid, collectedthe ceramide-containing products,and determined the molar ratio of the sugarsin each product. He did this for both the normal and the Tay-Sachsgangliosides His results are shownbelow.

I I

Fragment2 Fragment3 Fragment4 Tay-Sachs Fragment 1 Fragment2 Fragment3

1 I

1

0

1

l

I 1 2

1 I I I I

0 I 1

0 0 1 1 0 0 1

(c) Basedon thesedata,what canyou concludeaboutthe structure of the normal ganglioside?Is this consistentwith the structure in Box l0-2? Explain your reasoning. (d) What can you concludeabout the structure of the TaySachsganglioside?Is this consistentwith the structure in Box l0-2? Explainyour reasoning. Svemerholmalso reported the work of other researchers permethylation who "permethylated"the normalasialoganglioside is the sameas exhaustivemethylation:a methyl group is addedto every free hydroxyl group on a sugar.They found the followirg permethylated sugars: 2,3,6-trimethylglycop;,r.anose; 2,3,4,6tetramethylgalactopyranose; 2,4,6trimethylgalactopy'ranose; and 4,6-dimethyl-2-deoxy-2-aminogalactoplranose. (e) To which sugarof GM1doeseachof the permethylated sugarscorrespond?Explain your reasoning. (f) Basedon all the data presentedso far, what piecesof information about norma"lgangliosidestructure are missinq? Reference Svennerholm, L. (1962) The chemical structure of normal human brainandTay-Sachs gangliosides Bi,ochem Biophgs Res Comm g, 436 441

372 ofMembranes andArchitecture 11.1 TheComposition 381 Dynamics 11.2 Membrane 389 Membranes Transport across 11.3 5olute he first cell probably came into being when a membrane formed, enclosing a small volume of aqueous solution and separating it from the rest of the universe. Membranes define the external boundaries of cells and regulate the molecular traffic across that boundary (FiS. 11-l); in eukaryotic cells, they divide the internal space into discrete compartments to segregate processesand components. They organize complex reaction sequencesand are central to both biological energy conserr/ation and cell-to-cell communication. The biological activities of membranes flow from their remarkable physical properties. Membranes are flexible, self-sealing,and selectively permeable to polar solutes.

Membrane bilayer

Viewedin crosssection,all cell FIGURE 11-1 Biologicalmembranes. Thiserythrotrilaminarappearance. sharea characteristic membranes with an electron viewed cytewas stainedwith osmiumtetroxideand strucas a three-layer appears plasma membrane The microscope. ture, 5 to 8 nm (50 to 80 A) thick.The trilaminarimageconsistsof layers(theosmium,boundto the innerand outer two electron-dense surfacesof the membrane)separatedby a lessdensecentralregion.

Their flexibility permits the shapechangesthat accompany cell growth and movement (such as amoeboid movement). With their abitity to break and reseal,two membranescan fuse,as il exocytosis,or a singlemembrane-enclosedcompartment can undergo fission to yield two sealedcompartments,asin endocl'tosisor cell division, r,l'rthoutcreating gross leaks through cellular surfaces.Becausemembranesare selectivelypermeable, they retain certain compoundsand ions within cells and within speciflc cellular compartments,while excludingothers. Membranesare not merely passivebarriers.They include an array of proteins specializedfor promoting or catalyzingvariouscellular processesAt the cell surface, transportersmovespeci-ficorganicsolutesand inorganic ionsacrossthe membrane;receptorssenseextracellular signalsand trigger molecular changesin the cell; adhesion moleculeshold neighboring cells together. Within the cell,membranesorganizecellularprocessessuchas the synthesisof Iipids and certain proteins, and the energy transductionsin mitochondria and chloroplasts' Becausemembranesconsistof just two layersof molecules, they are very thin-essentially two-dimensional' Intermolecular collisionsare far more probable in this space, spacethan in three-dimensional two-climensional processes organen4''rne-catalyzed of efflciency the so ized within membranesis vastly increased. In this chapter we fi,rstdescribethe compositionof cellular membranesand their chemical architecturethe molecular structures that underlie their biological functions.Next, we considerthe remarkabledynamic features of membranes,in which lipids and proteins moverelativeto eachother.Cell adhesion,endocytosis, and the membranefusion accompanyingneurotransmitter secretionillustrate the dynamrcroles of membrane proteins.We then turn to the protein-mediatedpassage of solutesacrossmembranesvia transportersand ion channels.In later chapterswe discussthe rolesof membranes in signal transduction (Chapters 12 and 23), energy transduction (Chapter 19), lipid synthesis (Chapter21), and protein synthesis(Chapter27)'_ L371l

lEt{

Biotogicat Membranes andlransport

11.1The(omposition andArchitecture ofMernbranes One approach to understandingmembrane function is to study membrane composition-to determine, for example, which componentsare comlnon to all membranesand which are uniqueto membraneswith specific functions.Sobefore describingmembranestructure and ftmction we considerthe molecularcomponentsof membranes:proteinsandpolarlipids,which accountfor almost all the massof biologcal membranes,and carbohydrates, presentaspart of glycoproteinsand gycolipids.

cardiolipin(FiS. 11-2);this distributionis reversedin the inner mitochondrialmembrane,which hasvery low cholesteroland high cardiolipin.In all but a few cases, the functionalsignificanceof thesecombinationsis not yet known. Plasma o

3. o

Inner mitochondrial Outer mitochondrial

L

Lysosomal

0)

tachType ofMembrane Has Characteristic Lipids andProteins

Nuclear

The relative proportions of protein and lipid vary with the type of membrane(Table 11-l), reflecting the dlersity of biologicalroles.For example,certainneurons have a myelin sheath, an extended plasma membrane that wraps around the cell many times and acts as a passive electricalinsulator.The myelin sheathconsistsprimarily of lipids, whereas the plasma membranesof bacteriaand the membranesof mrtochondriaand chloroplasts,the sites of many enzynne-catalyzed processes, containmore protein than lipid (in massper total mass). For studiesof membranecomposition,the first task is to isolatea selectedmembrane.Wheneukaryoticcells are subjectedto mechanicalshear,their plasmamem_ branes are torn and fragmented,releasingcltoplasmic componentsand membrane-bounded organellessuchas mitochondria, chloroplasts, lysosomes, and nuclei. Plasmamembranefragments and intact organellescan be isolatedby techniquesdescribedin Chapter 1 (see Fig. 1-8) and in WorkedExample2-1, p. bB Cellsclearlyhavemechanismsto control the kinds and amountsof membranelipid they synthesizeand to target specific lipids to particular organelles.Each kingdom,eachspecies,eachtissueor cell type, and the organellesof eachcell type have a characteristicset of membranelipids. Plasmamembranes,for example,are enriched in cholesterol and contain no detectable

Rough ER SmoothER Golgi

0

20

40

60

80

100

Percent membrane lipid

I

Cholesterof

fCardiolipin [] Minor lipids

I

Sphingotipids

ffiPhosphatidylcholine ffi Phosphatidylethanolamine

FIGURE ll-2 Lipid compositionof the plasmamembraneand organellemembranesof a rat hepatocyte.Thefunctionalspecialization of eachmembranetype is reflectedin its uniquelipid composition. Cholesterol is prominentin plasmamembranes but barelydetectable in mitochondrial membranes. Cardiolipinis a majorcomponent of the inner mitochondrialmembranebut not of the plasmamembrane Phosphatidylserine, phosphatidylinositol, and phosphatidylglycerol arerelatively (yellow)of mostmembranes minorcomponents butserve criticalfunctions; phosphatidylinositol and itsderivatives, for example, are importantin signaltransductions triggeredby hormones.Sphin_ golipids,phosphatidylcholine, and phosphatidylethanolamine are presentin mostmembranes, but in varyingproportionsClycolipids, whicharemajorcomponents of the chloroplast membranes of plants, arevirtuallyabsentfromanimalcells.

Components(% by weight) Protein

Phospholipid

Sterol

Steroltlpe

Otherlipids

0

Cholesterol

Galactolipids, plasmalogens

Zt)

Cholesterol

Human myelin sheath

30

Mouseliver

45

30 27

Maizeleaf

47

zo

7

Sitosterol

GalactoJipids

Yeast

52

7

4

Ergosterol

Paramecium(ciliated protist) E. coli,

Ttiacylglycerols, steryl esters

56

40

4

Stigmasterol

75

'I

0

Note:valuesdo notaddupto 100%in everycase,because therearecomponents otherthanprotein, phospholipids, andsterol;plants, for example, havehighlevelsof glycolipids.

Ftr]

e embranes t inodnA r c h i t e c toufrM 1 1 . 1T h eC o m D 0 s i a

The protein composition of membranes from different sources varies even more widely than their lipid composition, reflecting functional specialization. In addition, some membrane proteins are covalently linked to oligosaccharides.For example, in glycophorin, a glycoprotein of the erythrocyte plasma membrane, 600/oof the mass consists of complex oligosaccharides covalently attached to specific amino acid residues. Ser, Thr, and Asn residues are the most common points of attachment (see Fig. 7-29). The sugar moieties of surface glycoproteins influence the folding of the proteins, as well as their stability and intracellular destination, and they play a signiflcant role in the speciic binding of ligands to glycoprotein surface receptors (see Fig. 7-35). Some membrane proteins are covalently attached to one or more lipids, which selve as hydrophobic anchors that hold the proteins to the membrane, as we shall see

Share Membranes Alltsiological Properties Fundamental Some Membranes are impermeable to most polar or charged solutes, but permeable to nonpolar compounds; they are 5 to 8 nm (50 to 80 A) thick and appear trilaminar when viewed in cross section with the electron microscope (Fig. 1l-1). The combined evidence from electron microscopy and studies of chemical composition, as well as physical studies of permeability and the mo-

tion of individual protein and lipid molecules within membranes, led to the development of the fluid mosaic model for the structure of biological membranes (Fig. lf -3). Phospholipids form a bilayer in which the nonpolar regions of the lipid molecules in each layer face the core ofthe bilayer and their polar head groups face outward, interacting with the aqueous phase on either side. Proteins are embedded in this bilayer sheet, held by hydrophobic interactions between the membrane lipids and hydrophobic domains in the proteins Some proteins protrude from only one side of the membrane; others have domains exposed on both sides. The orientation of proteins in the bilayer is asymmetric, glving the membrane "sidedness": the protein domains exposed on one side of the bilayer are different from those exposed on the other side, reflecting functional asymmetry The individual lipid and protein units in a membrane form a fluid mosaic with a pattern that, unlike a mosaic of ceramic tile and mortar, is free to change constantly. The membrane mosaic is fluid because most of the interactions among its components are noncovalent, leaving individual lipid and protein molecules free to move laterally in the plane of the membrane. We now look at some of these features of the fluid mosaic model in more detail and consider the experimental eviclencethat supports the basic model but has necessitated its reflnement in several ways.

Glycolipid Outside

Lipid bilayer

Phospholipid polar heads Peripheral protein covalently linked to lipid

Integral protein (singletransmembrane helix)

11-3 Fluid mosaicmodel for membranestructure.The fatty FIGURE forma fluid,hydrophobic acylchainsin the interiorof the membrane heldby hydrophobic lipid, in this sea of region.Integralproteinsfloat Bothproteins side chains. acid nonpolar amino with their interactions and lipidsare freeto move laterallyin the planeof the bilayer,but

Peripheral protern

movementof eitherfrom one leafletof the bllayerto the other is remoietiesattachedto some proteinsand stricted.The carbohydrate surare exposedon the extracellular lipidsof the plasmamembrane faceof the membrane

LrO)

Biological Membranes andTransporr

ALipidBilayer lstheBasic Structural Element ofMembranes Glycerophospholipids,sphingolipids, and sterols are virtually insoluble in water. When mixed with water, they spontaneously form microscopic Jipid aggregates,clus_ tering together, with their hydrophobic moieties in contact with each other and their hydrophilic groups interacting with the surrounding water. This clustering reduces the amount of hydrophobic surface exposed to water and thus minimizes the number of molecules in the shell of ordered water at the lipid-water interface (see Fig. 2-7), resulting in an increase in entropy. Hydrophobic interactions among lipid molecules pror,rde the thermodSmamrc driving force for the formation and maintenance of these clusters. Depending on the precise conditions and the nature of the lipids, three types of lipid aggregate can form when amphipathic lipids are mixed with water (Fig. f f-4). Micelles are spherical structures that contain anywhere from a few dozen to a few thousand amphi_ pathic molecules. These molecules are arranged with their hydrophobic regions aggregated in the interior, where water is excluded, and their hydrophilic head groups at the surface, in contact with water. Micelle for_ mation is favored when the cross-sectional area of the head group is greater than that ofthe acyl side chain(s), as in free fatty acids, Iysophospholipids (phospholipids lacking one fatty acid), and detergents such as sodium dodecyl sulfate (SDS; p. 89). A second type of lipid aggregate in water is the bi_ layer, in which two lipid monolayers (leaflets) form a two-dimensional sheet. Bilayer formation is favored if the cross-sectional areas of the head group and acyl side chain(s) are similar, as in glycerophospholipids and sphingolipids. The hydrophobic portions in each mono_ layer, excluded from water, interact with each other. The

Individual units are wedge-shaped (crosssectionofhead greater than that of side chain)

(a) Micelle

hydrophilic head groups interact with water at each surface of the bilayer. Because the hydrophobic regions at its edges (Fig. 11-4b) are in contact with water, the bilayer sheet is relatively unstable and spontaneously folds back on itselfto form a hollow sphere, a vesicle (Fig. ll-4c). The continuous surface of vesicles elimrnates exposed hydrophobic regions, allowing bilayers to achieve maximal stability in their aqueous environment. Vesicle forma, tion also creates a separate aqueous compartment. It is likely that the precursors to the first living cells resembled lipid vesicles, their aqueous contents segregated from their surroundings by a hydrophobic shell. The lipid bilayer is 3 nm (30 A) thick. The hydrocarbon core, made up of the -CHzand -CH3 of the fatty acyl groups, is about as nonpolar as decane, and vesicles formed in the laboratory from pure lipids (liposomes) are essentially impermeable to polar solutes, as is the lipid bilayer of biological membranes (although the latter, as we shall see, are permeable to solutes for which they have specific transporters). Plasma membrane l-ipids are as;,rnmetrically distributed between the two monolayers of the bilayer, although the as;rmmetry, unlike that of membrane proteins, is not absolute. In the plasma membrane of the erythroc;.,te, for example, choline-containing lipids (phosphatidylcholine and sphingomyelin) are typically found in the outer (extracellular, or exoplasmic) leaflet (Fig. ll-5), whereas phosphatidylserine, phosphatidylethanolamine, and the phosphatidylinositols are much more common in the inner (cyloplasmic) leaflet. Changes in the distribution of lipids between plasma membrane leaflets have biological consequences. For example, only when the phosphatidylserine in the plasma membrane moves into the outer leaflet is a platelet able to play its role in formation of a blood clot. For many other cell types, phosphatidylserine exposure on the outer surface marks a cell for destruction by programmed cell death.

Individual units are cylindrical (cross section ofhead equalsthat ofside chain)

G) Bilayer

FIGURE 1t-4 Amphipathiclipid aggregates that form in water.(a) In micelles,the hydrophobic chainsof the fattyacidsaresequesrered at the coreof the sphere. Thereis vrrtuallyno waterin the hydrophobic interior(b) In an openbilayer,all acylsidechainsexceptthoseat the

Aqueous cavity

(c) Vesicle edgesof the sheetareprotectedfrom interactionwith water.(c) When a two-dimensional bilayerfoldson itself,it formsa closedbilayer,a three-dimensional hollow vesicle(liposome)enclosingan aqueous cavity.

e e m b r a n e[ ,st t ] 1 1 . 1I h eC o m p o s i tai onndA r c h i t e c toufrM

Membrane phospholipid

Percent of total membrane phospholipid

Distribution in membrane

Outer Inner monolayer monolayer,OO O ,OO

change in pH; chelating agent; urea; carbonate

Phosphatidylethanolamine Phosphatidylcholine

27

Sphingomyelin

23

Phosphatidylserine

15

Phosphatidylinositol Phosphatidylinositol 4-phosphate Phosphatidylinositol 4,5-bisphosphate Phosphatidic acid

tIGURE11-5 Asymmetricdistributionof phospholipidsbetween the inner and outer monolayersof the erythrocyteplasma membrane.The distributionof a specificphospholipidis determinedby C, which cannotreach treatinBthe intactcell with phospholipase (leaflet) removes the headgroupsof but lipidsin the innermonolayer headSroupreof each The proportion monolayer lipidsin the outer leasedprovidesan estimateof the fractionof eachlipid in the outer monorayer

Differ Proteins Types ofMembrane Three withtheMembrane inTheir Association Integral membrane proteins are very firmly associated with the lipid bilayer, and are removable only by agents that interfere with hydrophobic interactions, such as detergents, organic solvents, or denaturants (FiS. 11-6). Peripheral membrane proteins associate with the membrane through electrostatic interactions and hydrogen bonding with the hydrophilic domains of integral proteins and with the polar head groups of membrane lipids. They can be released by relatively mild treatments that interfere with electrostatic interactions or break hydrogen bonds; a commonly used agent is carbonate at high pH. Amphitropic proteins are found both in the cytosol and in association with membranes. Their affinity for membranes results in some cases from the protein's noncovalent interaction with a membrane protein or lipid, and in other cases from the presence of one or more lipids covalently attached to the amphitropic protein (see Fig. 11-14) Generally, the reversible association of amphitropic proteins with the membrane is reguIated; for example, phosphorylation or ligand binding can force a conformational change in the protein, exposing a membrane-binding site that was previously inaccessible.

Integral protein (hydrophobic dornain coated with detergent)

11-6 Peripheral,integral,and amphitropicproteins' MemFIGURE reby theconditions distinguished canbe operationally braneproteins proteins Mostperipheral themfrom the membrane. quiredto release removalof Ca2*by a in pH or ionicstrength, by changes arereleased lntegralproteinsare a8ent,or additionof ureaor carbonate. chelating interachydrophobic which disruptthe with detergents, extractable indiaround clusters micelle-like tionswith the lipid bilayerandform to a attached covalently proteins lntegral vidual proteinmolecules. (CPl; see phosphatidylinositol a glycosyl as membranelipid, such AmC phospholipase with treatment by be released can Fig.11-14), with membranesand phitropicproteinsare sometimesassociated sometimesnot, dependingon sometype of regulatoryprocesssuchas palmitovlation reversible

Bilayer theLipid Span Proteins Membrane Many Membraneprotein topology(the localizationof protein domains relative to the lipid bilayer) can be determined with reagentsthat react rvtthprotein sidechains but cannotcrossmembranes-polar chemicalreagents that react with primary aminesof Lys residues,for example,or enzymessuchas trypsin that cleaveproteins but cannot cross the membrane.The human erythrocyte is convenientfor such studiesbecauseit has no organelles;the plasmamembrane membrane-bounded is the only membranepresent' If a membraneprotein in an intact erythrocyte reacts with a membrane-impermeantreagent,that protein must have at least one domain exposed on the outer (extracellular) face of the membrane.Trypsin cleavesextracellulardomains but does not affect domainsburied within the bilayer or exposedon the inner surfaceonly,unlessthe plasma membraneis broken to makethesedomainsaccessible to the enzYme.

-

l

L 3 7 6]

B i o l o g i cMael m b r a naensdT r a n s p o r t

Experiments with such topology-speciflc reagents show that the erythrocyte glycoprotein glycophorin spans the plasma membrane. Its amino-terminal domain (bearing the carbohydrate chatns) is on the outer surface and is cleaved by trypsin. The carboxfl termrnus protrudes on the inside of the cell, where it cannot react with impermeant reagents. Both the amino-terminal and carboxyl-terminal domains contain many polar or charged amino acid residues and are therefore hydrophilic. However, a segment in the center of the protein (residues Zb to 93) contains mainly hydrophobic amino acid residues, suggesting that glycophorin has a transmembrane segment arranged as shown in Figure l1-2. These noncrystallographic experiments also revealed that the orientation of glycophorin in the membrane is as5.'mmetric:its amrno-termrnal segment is always on the outside. Simr-larstudies of other membrane proteins show

FIGURE 11-7 Transbilayer dispositionof glycophorinin an erythro_ cyte.One hydrophilic domain,containing all the sugarresidues, is on the outersurface, and anotherhydrophilicdomainprotrudes from the inner faceof the membrane.Eachred hexagonrepresents a tetrasac_ charide(containing two Neu5Ac(sialicacid),Cal, and CalNAc)O_ linked to a Ser or Thr residue;the blue hexagonrepresents an oligosaccharide N-linkedto an Asn residue.The relativesizeof the oligosaccharide unitsis largerthan shownhere.A segment of 19 hy_ drophobicresidues (residues 75 to 93) formsan c helixtnattraverses the membranebilayer(seeFlg. 1l-1 1a).The segmentfrom residues 64 to 74 hassomehydrophobic residues and probablypenetrates the outerfaceof the lipid bilayer,assnown.

that each has a speciflc orientation in the bilayer, giving the membrane a distinct sidedness. I'or glycophorin, and for all other glycoproteins of the plasma membrane, the glycosylated domains are invariably found on the extracellular face of the bilayer. As we shall see, the as}..rnmetric arrangement of membrane proteins results in functional asymmetry. All the molecules of a given ion pump, for example, have the same orientation in the membrane and pump ions in the same directron.

Integral Proteins AreHeld intheMembrane by Hydrophobic Interactions withIipids The firm attachment of integral proteins to membranes is the result of hydrophobic interactions between membrane lipids and hydrophobic domains of the protein. Some proteins have a single hydrophobic sequence in the middle (as in gycophorin) or at the amino or carboxyl terminus. Others have multiple hydrophobic sequences, each of which, when in the a-helical conformation, is long enough to span the lipid bilayer (Fig. 1f-8). One of the best-studied membrane-spanning proteins, bacteriorhodopsin, has seven very hydrophobic internal sequences and crosses the lipid bilayer seven times. Bacteriorhodopsin is a light-driven proton pump densely packed in regular arrays in the purple membrane of the bacteriumHaLobacterium saL,inarum. X-ray crystallography reveals a structure with seven a-heJical segments, each traversing the lipid bitayer, connected by nonhelical loops at the inner and outer face of the membrane (Fig. 1l-g). In the amino acid sequenceof bacteriorhodopsin, seven segments of about 20 hydrophobic residues can be identified, each forming an a helix that spans the bilayer. The seven helices are clustered to_ gether and oriented not quite perpendicular to the bilayer plane, a pattern that (as we shall see in Chapter 12) is a corrmon motif in membrane proteins involved in signal reception. Hydrophobic interactions between the nonpo_ Iar amino acids and the fatty acyl groups of the membrane lipids flmly anchor the protein in the membrane. Crystallized membrane proteins solved (i.e., their molecular structure deduced) by crystallography often include molecules of phospholipids, which are pre_ sumed to be positioned in the crystals as they are in the native membranes. Many of these phospholipid mole_ cules lie on the protein surface, their head groups interacting with polar amino acid residues at the inner and outer membrane-water interfaces and their side chains associated with nonpolar residues. These annular lipids form a bilayer shell (annulus) around the protein, oriented roughly as expected for phospholipids in a bilayer (Fig. 1f-10). Other phospholipids are found at the interfaces between monomers of multisubunit membrane proteins, where they form a "greaseseal." yet others are embedded deep within a membrane protein, often with their head groups well below the plane of the biiayer. f'or example, succinate dehydrogenase (Complex II, found in mitochondria; see Fig. lg-10) has several deeply embedded phospholipid molecules.

ofMembranes andArchite(ture 11.1The(omposition 3r1

NHg

Outside

Type I

Type III

f,i protein. a membrane-spanning tIGURE11-9 Bacteriorhodopsin, (PDB lD 2AT9)The singlepolypeptidechain folds into sevenhythe lipid bilayerroughly drophobica helices,eachof whichtraverses The seventransmemto the planeof the membrane. perpendicular and betweenthem around and the space branehelicesareclustered, light-absorbing The lipids. membrane of is filledwith the acyl chains m e m b r a n ien t h e ( s e e i n d e e p b u r i e d F i g . 1 0 2 1 ) i s p i g m e nrt e t i n a l (not The helices shown). segments helical of the with several contact .l plot in Figure11-1 b. with the hydropathy arecoloredto correspond

h''"'Nn! $$

Type V

Type VI

Inside

Outside

11-8 Integralmembraneproteins. For known proteinsof FIGURE of proteindomainsto the spatialrelationships the plasmamembrane, TypesI and ll havea single the lipid bilayerfall into six categories helix;theamino-terminal domainis outsidethecell in transmembrane type lproteinsand insidein type ll Typelll proteinshavemultiple In type IV proteins, helicesin a singlepolypeptide. transmembrane to assemble differentpolypeptides domainsof several transmembrane areheldto the forma channelthroughthe membraneTypeVproteins linkedlipids(seeFig l1-1 4), andtype bilayerprimarilyby covalently helicesand Iipid(CPl)anchors Vl proteinshavebothtransmembrane the book,we represent figure, in figures throughout In this and as proteinsegments in theirmostlikelyconformations: transmembrane thesehelicesareshownsimc helicesof sixto seventurns.Sometimes have few membraneproteinstructures As relatively ply as cylinders. of the exour representation beendeducedby x-raycrystallography, to scale. and not necessarilV domainsis arbitrarv tramembrane

with two integralmembrane Lipid annuliassociated FIGURE'11-10 of sheepaquaporin(PDBlD 2B60), proteins.(a)Thecrystalstructure powaterchannel,includesa shellof phospholipids a transmembrane positions on the sitionedwith theirheadgroups(blue)atthe expected acylchains andtheirhydrophobic surfaces innerand outermembrane (gold)intimatelyassociated with the surfaceof the proteinexposedto seal"aroundtheprotein,whichis thebilayerThelipidformsa "grease

(b) The crystalstructureof depictedas a greensurfacerepresentation. from Entero' Na*-ATPase V-type the Fointegralproteincomplexof the with four each subunits, (PDB identical has 10 2BL2) lD coccushirae phoswith filled cavity a central helices, surrounding transmembrane havebeencut awayto (PC) Herefiveof the subunits phatidylglycerol with eachsubunitaroundthe inteassociated revealthe PC molecules riorof thisstructure.

F"l

B i o l o g i cMael m b r a naensdl r a n s p o r t

TheTopology ofanIntegral Membrane Protein Can 5ometimes BePredicted fromltsSequenee Determination of the three-dimensional structure of a membrane protein-that is, its topology-is generally much more difflcult than determining its amino acid sequence, either directly or by gene sequencing. The amino acid sequencesare known for thousands of membrane proteins, but relatively few three-dimensional structures have been established by crystallography or NMR spectroscopy.The presence ofunbroken sequences of more than 20 hydrophobic residues in a membrane protein is commonly taken as evidence that these sequences traverse the lipid bilayer, acting as hydrophobic anchors or forming transmembrane channels. Virtually all integral proteins have at least one such sequence. Application of this logic to entire genomic sequences leads to the conclusion that in many species, 20o/oto 300/oof all proteins are integral membrane proteins. What can we predict about the secondary structure of the membrane-spanning portions of integral proteins? An a-helical sequence of 20 to 25 residues is just long enough to span the ttuckness (30 A) of the lipid bilayer (recall that the length of an a helix is l.b A (0.15 nm) per amino acid residue). A pol;peptide chain surrounded by lipids, having no water molecules with which to hydrogen-bond, will tend to form a helices or p sheets, in which intrachain hydrogen bonding is maximized. If the side chains of all amino acids in a helix are nonpolar, hydrophobic interactions with the surrounding lipids further stabilize the helix Several simple methods of analyzing amino acid sequences yield reasonably accurate predictions of secondary structure for transmembrane proteins. The relative polarity of each amino acid has been determined experimentally by measuring the free-energy change accompanying the movement of that amino acid side chain from a hydrophobic solvent into water. This free energy oftransfer, which can be expressed as a hydropathy index (see Table S-1), ranges from very exergonic for charged or polar residues to very endergonic for amino acids with aromatic or aliphatic hydrocarbon side chains. The overall hydropathy rndex (hydrophobicity) of a sequence of amino acids is estimated by summing the free energies of transfer for the residues in the sequence. To scan a polypeptide se_ quence for potential membrane-spanning segments, an investigator calculates the hydropathy index for suc_ cessive segments (called windows) of a given size, fromT to 20 residues.For a window of seven residues, for example, the indices for residues I to 7,2 to g, 3 to 9, and so on, are plotted as in Figure 1f-11 (plotted for the middle residue in each window-residue 4 for residues I to 7, for example). A region with more than 20 residues of high hydropathy index is presumed to be a transmembrane segment. When the sequences of membrane proteins of known three-dimensional

100

130

t

Hydrophobic Hydrophilic

J 50

100

130

Residue number (a) Glycophorin

10

50

100

150

200

250

yB

4. I

x

Hydrophobic

dU

Hydrophilic tr

I

h

r

\y _J

10

50

100

150

200

250

Residue number (b) Bacteriorhodopsin FIGURE 11-11 Hydropathyplots.Hydropathy index(seeTable 3-i) is plottedagainstresiduenumberfor two integralmembraneproteins. The hydropathy indexfor eachaminoacid residuein a sequence of definedlength,or "window," is usedto calculatethe averagenydropathyfor thatwindow.Thehorizontalaxisshowsthe residuenumber in the middle of the window. (a) Clycophorinfrom human erythrocytes hasa singlehydrophobic sequence betweenresidues 75 and 93 (yellow);compare this with Figure 11-7. (b\ Bacteriorhodopsin, knownfrom independent physicalstudiesto haveseven transmembrane helices(seeFig. 1l-9), has sevenhydrophobicregions.Note,however, thatthe hydropathy plot is ambiguous in the region of segments 6 and 7. X+ay crystallography hasconfirmedthat thisregionhastwo transmembrane segments.

structure are scannedin this way,we find a reasonably good correspondencebetween predicted and known membrane-spanningsegments.Hydropathy analysis predicts a single hydrophobic helix for glycophorin (Fig. 11-1la) and seventransmembranesegmentsfor bacteriorhodopsin(Fig. 11-1lb)-in agreementwith experimentalstudies. On the basisof their aminoacid sequencesand hydropathy plots, many of the transport proteins described in this chapter are believedto have multiple membrane-spanning helical regions-that is, they are type III or g,pe IV integralproteins (Fig. 11-B). When predictionsare consistentwith chemicalstudiesof protein localization(suchas thosedescribedabovefor glycophorinand bacteriorhodopsin),the assumptionthat hydrophobicregionscorrespondto membrane-spanning domainsis much better justified.

ofMembranes andArchitecture 11.1 TheComposition Fttl

I

I

Charged residues

r'p

|o rv'

K* channel

Maltoporin

Outer membrane phospholipaseA

OmpX

Phosphoporin E

Tyr and Trp residuesof membraneproteinsclustering FIGURE'11-12 at the water-lipidinterface.The detailedstructuresof thesefive intestudies.The gral membraneproteinsare known from crystallographic lividans K* channel(PDBlD 1BL8)is from the bacteriumStreptomyces (seeFig 1.1-48); (PDBlD 1AF6), phosphomaltoporin outermembrane lipaseA (PDBlD 1QD5),OmpX (PDBlD 1QJ9),and phosphoporin

of E coli. of theoutermembrane E (PDBfD l PHO)areproteins where predominantly arefound andTrp(red) ofTyr(orange) Residues meets thepolarheadgroupregion. ofacylchains region thenonpolar almost (Lys, arefound inblue) Arg,Clu,Asp;shown residues Charged phases. intheaqueous exclusively

A further remarkablefeature of many transmembraneproteinsof knownstructureis the presenceof Tyr and T?presiduesat the interface between lipid and water (Fig. 1l-fZ). The sidechainsoftheseresiduesapparently sen/e as membraneinterface anchors,able to interact simultaneouslywith the central lipid phaseand the aqueousphaseson either sideof the membrane.Another generalizationabout amino acid location relative to the bilayeris describedby the positive-inside rule: the positively charged Lys, His, and Arg residues of membraneproteinsoccur more commonlyon the c;,toplasmicfaceof membranes. Not all integral membraneproteins are composedof transmembranea helices.Another structural motif common in bacterialmembraneproteinsis the B barrel (see Fig. 4-17b), in which 20 or more transmembranesegmentsform B sheetsthat line a cylinder(FiS. 11-13). The samefactors that favor a-helix formation in the hydrophobic interior of a Iipid bilayer also stabilize B

barrels: when no water molecules are available to hydrogen-bondwith the carbonyloxygenand nitrogen of the peptide bond, maximalintrachain hydrogenbonding gives the most stable conformation.Planar B sheetsdo not maximize these interactions and are generally not found in the membraneinterior; B barrelsallow all possible hydrogenbonds and are apparentlycommonamong membraneproteins.Porins, proteins that allow certain polar solutes to cross the outer membrane of gramnegativebacteriasuch asE. coli, havemany-strandedB barrelslining the polar transmembranepassage. A pobpeptide is more extendedin the B conformation than in an a helix; just sevento nine residuesof B conformationare neededto span a membrane.Recall that in the B conformation,alternatingsidechainsproject aboveand belowthe sheet (seeFig. 4-6). In B strandsof membraneproteins, every secondresidue in the membrane-spanningsegment is hydrophobic and interacts with the lipid bilayer;aromaticside chainsare commonly found at the lipid-protein interface. The other residues may or may not be hydrophilic. The hydropathy plot is not useful in predicting transmembranesegmentsfor proteins with B barrel motifs, but as the databaseof predicknown p-barrel motifs increases,sequence-based tions of transmembraneB conformationshave become feasible.For example,someouter membraneproteins of gram-negativebacteria (Fig. 11-13) have been correctly predicted,by sequenceanalysis,to containB barrels.

FepA

OmpLA

Maltoporin

tIGURE11-13 Membraneproteinswith B-barrelstructure.Threeproareshown,viewedin the planeof teinsof the f coll outermembrane FepA(PDBlD l FEP),involvedin iron uptake,has22 the membrane. from PDBlD 1QD5), membrane-spanning OmpLA(derived B strands. p barrelthatexistsasa dimerin the is a 12-stranded a phospholipase, from PDBlD l MAL),a maltosetransmembrane. Maltoporin(derived porter,is a trimer;eachmonomerconsists of 16 p strands

(ovalently Anchor Lipids Attached Proteins Membrane Some Some membrane proteins contain one or more covalently linked lipids, which may be of several types: longchain fatty acids, isoprenoids, sterols, or glycosylated

ft

!:001

B i o l o g i cMael m b r a naensdT r a n s p 0 r t

derivatives of phosphatidylznositol (GPIs; Fig. ll-14). The attached lipid provides a hydrophobic anchor that inserts into the lipid bilayer and holds the protein at the membrane surface. The strength of the hydrophobic interaction between a bilayer and a single hydrocarbon chain Iinked to a protein is barely enough to anchor the protein securely,but many proteins have more than one attached Iipid moiety. Other interactions, such as ionic attractions between positively charged Lys residues in the protein and negatively charged lipid head groups, probably contribute to the stability of the attachment. The association of these lipidJinked proteins with the membrane is certainly weaker than that for integral membrane proteins and is, in at least some cases, reversible But treatment with alkaline carbonate does not release GPl-linked proteins, which are therefore, by the working definition, integral proteins. Beyond merely anchoring a protein to the membrane, the attached lipid may have a more specific role. In the plasma membrane, proteins with GpI anchors are exclusively on the outer face and are clustered in certain regions, as we shall see (pp. 384-386), whereas other types of lipid-linked proteins (with farnesyl or geranylgeranyl groups attached; Fig. 11-14) are exclusively on the inner face. In polarized epithelial cells (such as intestinal epithelial cells, see F.ig. lI-44), in which apical and basal surfaces have

different roles, GPI-linked proteins are directed specificallyto the apicalsurface.Attachment of a specific lipid to a newly synthesizedmembraneprotein therefore has a targeting function, directing the protein to its correct membranelocation.

S U M M A R1Y1 . 1 T h eC o m p o s i t iaonnd A r c h i t e c t uorfeM e m b r a n e s r

Bioiogical membranes define celh-rlarboundaries, divide cells into discrete compartments, organize complex reaction sequences,and act in signal reception and energy transformations.

r

Membranes are composed of lipids and proteins in varf,'lng combinations particular to each species, cell type, and organelle. The lipid bilayer is the basic structural unit.

r

Peripheral membrane proteins are loosely associated with the membrane through electrostatic interactions and hydrogen bonds or by covalently attached lipid anchors. Integral proteins associatefirnrly with membranes by hydrophobic rnteractions between the lipid bilayer and their nonpolar amino acid side chains, which are oriented toward the outside of the protein molecule. Amphitropic proteins associate reversibly with membranes.

tIGURE 11-14Lipid-linked membrane proteins. Covalently attached Iipids anchor membrane proteins totheIipidbilayer. A palmitoyl groupisshownattached bythioester linkage to a Cysresidue; anN-myristoyl groupisgenerally attached to anamino-terminal Cly;thefarnesyl andgeranylgeranyl groups at_ .l tached to carboxyl{erminal Cysresidues areisoprenoids of 5 and20 carbons,respectively These threelipid-protein assemblies arefoundonlyon the innerfaceof theplasma membrane. Clycosyl (Cpl)anphosphatidylinositol chors arederivatives ofphosphatidylinositol inwhichtheinositol bears a short o l i g o s a c c h a r i d ec o v a l e n t l yj o i n e d t o t h e c a r b o x y l - t e r m i n a lr e s i d u eo f a p r o _ t e i n t h r o u g h p h o s p h o e t h a n o l a m i n eC p l - l i n k e d p r o t e i n sa r e a l w a y so n t h e e x t r a c e l l u l a rf a c e o f t h e p l a s m a m e m o r a n e .

Palmitoyl group on internal Cys (or Ser)

N-Myristoyl gr:oup on amino-terminal Gly

Farnesyl (or geranylgeranyl) group on carboxyt-teiminal Cys

H,N

o -O-P:O

I o I CHo t-

HQ -gH, I L O,r? ?,p

't( 'b( .

(' c

Outside

t? ?5

il tt

lti

Inside

coo

F"l

1 1 . 2M e m b r aDnyen a m i c s

Many membraneproteins spanthe lipid bilayer severaltimes,with hydrophobicsequencesof about 20 amino acid residuesforming transmembranea helices.MultistrandedB barrelsare alsocommonin integral proteins in bacterialmembranes.$zr and proteinsare Ttp residuesof transmembrane commonlyfound at the lipid-water interface.

(a) Paracrystalline

state (gel)

The lipids and proteins of membranesare insertedinto the bilayer with specificsidedness; thus membranesare structurally and functionally asymmetric.Plasmamembraneglycoproteinsare alwaysoriented with the oligosaccharide-bearing domain on the extracellularsurface.

1'1.2Membrane Dynamics One remarkablefeature of all biological membranesis their flexibility-their ability to change shape wrthout Iosing their integrity and becomingleaky.The basisfor this property is the noncovalentinteractions among lipids in the bilayer and the mobility allowedto individual lipids becausethey are not covalentlyanchoredto one another. We turn now to the dynamicsof membranes:the motionsthat occur and the transientstructures allowedby thesemotions.

AcylGroups intheBilayer lnterior Are Degrees Ordered toVarying Although the lipid bilayer structure is quite stable,its individual phospholipid and sterol moleculeshave much freedomof motion (Fig. 1l-fb). The structureand flexibility of the lipid bilayer depend on the kinds of lipidspresent,and changewith temperature.Belownormal physiologicaltemperatures,the lipids in a bilayer form a semisolidgel phase, in which all types of motion of individual lipid molecules are strongly constrained; the bilayeris paracrystalline(Fig. 11-15a).Abovephysiologicaltemperatures,individual hydrocarbonchainsof fatty acids are in constant motion produced by rotation about the carbon-carbonbonds of the long acyl side chains.In this liquid-disordered state, or fluid state (Fig. 11-15b), the interior of the bilayer is more fluid than solid and the bilayer is like a seaof constantlymoving lipid. At intermediate (physiological)temperatures, the lipids exist in a liquid-ordered state; there is less thermal motion in the acyl chainsof the lipid bilayer,but Iateral movement in the plane of the bilayer still takes place.Thesedifferencesin bilayer state are easilyobseruedin liposomescomposedof a singlelipid, but biological membranescontain many lipids with a variety of fatty acyl chains and thus do not show sharp phase changeswith temperature. At temperaturesin the physiologicalrange for a mammal (about 20 to 40'C), long-chainsaturatedfatty acids(suchas 16:0and 18:0)packinto a liquid-ordered array,but the kinks in unsaturatedfatty acids (see Fig.

11-15 Twoextremestatesof bilayerlipids.(a) In the paracrysFIGURE tallinestate,or gel phase,polarheadgroupsare uniformlyarrayedat andpackedwith andtheacylchainsarenearlymotionless thesurface, state,or fluid state,acyl regulargeometry(b) In the liquid-disordered chainsundergomuchthermalmotionand haveno regularorganizabetweentheseextremesis the liquid-orderedstate, tion. Intermediate can diffuselaterallybut molecules in which individualphospholipid lessordered. more or and extended remain groups the acyl

10-2) interfere with packing, favoring the liquid-disordered state. Shorter-chainfatty acyl groups have the sameeffect. The sterol content of a membrane (which varies greatly with organismand organelle;Table l1-1) is another important determinant of lipid state. The rigid planar structure of the steroid nucleus, inserted between fatty acyl side chains,reducesthe freedom of neighboringacyl chainsto move by rotation about their carbon-carbonbonds,forcing the chainsinto their fully extendedconformation.The presenceof sterolstherefore reducesthe fluidity in the core of the bilayer, thus favoring the liquid-orderedphase, and increasesthe thicknessof the lipid leaflet (as describedbelow)' Cells regulate their lipid composition to achieve a constantmembranefluidity under variousgrowth conditions. For example,bacteria synthesizemore unsaturated fatty acids and fewer saturated ones when cultured at low temperaturesthan when cultured at higher temperatures(Table l1-2). As a result of this adjustment in lipid composition,membranesof bacteria cultured at high or low temperatures have about the samedegreeof fluidity.

Catalysis Requires ofLipids Movement Transbilayer At physiologicaltemperatures,transbilayer-or "flipflop"-diffusion of a lipid molecule from one leaflet of

L 3 B 2]

B i o l o g i cMael m b r a naensdT r a n s p o r t

Percentage oftotal fatty acids*

l0 "c Mpistic acid (14:0) Palmiticacid (16:0) Palmitoleicacid (16:1) Oleicacid (18:1) HYdrox).'rnYristic acid Ratio of unsaturatedto saturatedj

20"c

30' c 4

4 ,tr,

4

18 26 38 13 29

40'c 8

to

48

ta

24 34 10 2.0

9

30 10 1.6

T2 8 0.38

Source:Datafroml\4arr, A G.& Ingraham, J.L.( 1962)Effectof temperature onthecomposition of fattyacidsin Escherichiacoti.J. Bacteriol. 84,7260. +Theexactfatty acidcomposition depends notonlyon growth temperature butongrowth stageandgrowthmediumcomposition. l R a t i o s c a l c u l a t e d a s t h ept oetracle n t a g e o f p l u s 1 8 : l d i v i d e d b y t h e t o t a l 16:1 p e r c e n t a g e1o4f: 0 p l u s 1 6 : 0H y d r o x y m y r i s t i c a c i d w a s omittedfromthiscalculation

the bilayer to the other (Fig. ll-16a) occurs very slowly if at all in most membranes, although lateral diffusion i,n the plane of the bilayer is very rapid (Fig. 11-16b). Ttansbilayer movement requires that a polar or charged head group leave its aqueous environment and move into the hydrophobic interior of the bilayer, a process with a large, positive free-energy change. There are, however, situations in which such movement is essential. For example, in the ER, membrane glycerophospholipids are synthesized on the cytosolic surface, whereas sphingoiipids are synthesized or modifled on the lumenal surface. To get from their site of synthesis to their eventual point of deposition, these lipids must undergo flip-flop diffusion. Several families of proteins, including the flippases,floppases,and scramblases(Fig. l1-16c), facilitate the transbilayer movement of lipids, providing a path that is energetically more favorable and much faster than the uncatalyzed movement. The combination of asymmetric biosynthesis of membrane lipids, very slow uncatalyzed flip-flop diffusion, and the presence of selective, energy-dependent lipid translocators is responsible for the transbilayer asymmetry in lipid composition shown in Figure ll-5. Besides contributing to this asymmetry of composition, the energydependent transport of lipids to one bilayer leaflet may, by creating a Iarger surface on one side of the bilayer, be important in generating the membrane curvature essential in the budding of vesicles. Flippases catalyze translocation of the ami,nophospholipids phosphatidylethanolamine and phosphatidylserine from the extracellular to the c1'tosolicleaflet ofthe plasma membrane, contributing to the as).,rnmetric distribution of phospholipids: phosphatidylethanolamine and phosphatidylserine primarily in the cytosolic leaflet, and the sphingolipids and phosphatidylcholine in the outer leaflet. Keeping phosphatidylserine out of the extracellular leaflet is important: its exposure on the outer

(a) Uncatalyzed transbilayer ("flip-flop") diffusion

very slow (ly, in days)

(b) Uncatalyzed

lateral

diffusion

very fast (1pmls)

(c) Catalyzed

transbilayer

translocations

Outside

Inside ATP

ADP+P1

Flippase (P-typeATPase) movesPE and PS from outer to cytosolicleaflet

ATP

ADP+Pi

Floppase Scramblase (ABC transporter) moveslipids in movesphospholipids either direction, from cytosolicto toward equilibrium outer leaflet

FIGURE 1l-16 Motion of singlephospholipids in a bilayer.(a) Uncatalyzedmovementfrom one leafletto the other is veryslow,but (b) lateral diffusionwithin the leafletis very rapid,requiringno catalysis. (c) Threetypesof phospholipidtranslocaters in the prasmamembrane. Flippasestranslocateprimarily aminophospholipids (phos(PE),phosphatidylserine phatidylethanolamine (PS)) fromthe outer(exoplasmic)leafletto the inner(cytosolic) leaflet;they requireATp and are membersof the P-typeATPase family.Floppases move phospholipids from the cytosolicto the outerleaflet,requireATB and are membersof the ABC transporter family.Scramblases equilibratephospholipids acrossboth leaflets;theydo not requireATp but areactivatedby Ca2+.

F*'l

1 1 . 2M e m b r aDnyen a m i c s

surface triggers apoptosis (programmed cell death; see Chapter 12) and engulfment by macrophagesthat carry phosphatidylserine receptors. Flippases also act in the ER, where they move newly synthesized phospholipids from their site of synthesis in the cytosolic leaflet to the Iumenal leaflet. Flippases consume about one ATP per molecule of phospholipid translocated, and they are structurally and functionally related to the P-type ATPases (active transporters) described on page 396. Floppases move plasma membrane phospholipids from the cytosolic to the extracellular leaflet, and like flippases are MP-dependent. Floppasesare members of the ABC transporter family described on page 400, all of which actively transport hydrophobic substrates outward across the plasma membrane. Scramblases are proteins that move any membrane phospholipid across the bilayer down its concentration gradient (from the leaflet where it has a higher concentration to the ieaflet where it has a lower concentration); their activity is not dependent on ATP Scramblase activity ieads to controlled randomization of the head-group composition on the two faces of the bilayer. The activity rises sharply with an increase in cytosolic Ca'* concentration, which may result from cell activation, cell injury, or apoptosis; as noted above, exposure of phosphatidylserine on the outer surface marks a cell for apoptosis and engulfment by macrophages.Finally, a group of proteins that act primarily to move phosphatidylinositol lipids across lipid bilayers, the phosphatidylinositol transfer proteins, are believed to have important roles in Iipld signaling and membrane trafflcking.

Fluorescent probe on lipids

Intense laser beam bleaches small area

intheBilayer andProteins Diffuse Laterally Lipids Individual lipid molecules can move laterally in the plane of the membrane by changing places with neighboring lipid molecules; that is, they undergo Brornmian movement within the bilayer (Fig. 11-16b), which can be quite rapid. A molecule in the outer leaflet of the erythrocyte plasma membrane, for example, can diffuse laterally so fast that it circumnavigates the erythrocyte in seconds This rapid lateral diffusion in the plane of the brlayer tends to randomize the positions of individual molecules in a few seconds. Lateral diffusion can be shown experimentally by attaching fluorescent probes to the head groups oflipids and using fluorescence microscopy to follow the probes over time (Fig. 11-17) In one technique, a small region (5 pm21 of a cell surface with fluorescence-tagged lipids is bleached by intense laser radiation so that the irradiated patch no longer fluoresces when viewed with Iess-intense(nonbleaching) light in the fluorescencemicroscope. However, within milliseconds, the region recovers its fluorescence as unbleached lipid molecules diffuse into the bleached patch and bleached lipid molecules diffuse away from it The rate of/uorescence recovery after photobleaching, or FRAP, is a measure of the rate of Iateral diffusion of the lipids. Using the FRAP

With time, unbleached phospholipids diffuse into bleached area

Measure rate of fluorescence return

of lateraldiffusionratesof lipids by 11-17 Measurement FIGURE (FRAP).Lipids in the photobleaching after recovery fluorescence outerleafletof the plasmamembraneare labeledby reactionwith a probe(red),so the surfaceis unifluorescent membrane-impermeant A small microscope. formlylabeledwhenviewedwith a fluorescence with an intenselaserbeamand bearea is bleachedby irradiation of time, labeledlipid moleWith the passage comesnonfluorescent cules diffuse into the bleachedregion, and it again becomes recan trackthe time courseof fluorescence Researchers fluorescent. The diflipid labeled for the coefficient a diffusion turnanddetermine fusion ratesare typicallyhigh; a lipid movingat this speedcould an E.coli cell in one second.(TheFRAPmethodcan circumnavigate proteins') lateraldiffusionof membrane alsobe usedto measure

L 3 B 4l

B i o l o g i cMael m b r a naensdT r a n s p o r t

technique, researchers have shown that some membrane lipids diffuse laterally at rates of up to 1 s,m/s. Another technique, single particle tracking, allows one to follow the movement of a stngle tipid molecule in the plasma membrane on a much shorter time scale.Results from these studies conflrm rapid lateral diffusion within small, discrete regions of the cell surface and show that movement from one such region to a nearby region ("hop diffusion") is inhibited; membrane lipids behave as though corralled by fences that they can occasionally cross by hop diffusion (FiS. 11-1S). Many membrane proteins seem to be afloat in a sea of lipids Like membrane lipids, these proteins are free to diffuse laterally in the plane of the brlayer and are in constant motion, as shov,n by the FRAP technique with fluorescence-tagged surface proteins. Some membrane proteins associate to form large aggregates ("patches") on the surface of a cell or organelle in which individual protein molecules do not move relative to one another; for example, acetylcholine receptors form dense, nearcrystalline patches on neuronal plasma rnembranes at syrapses. Other membrane proteins are anchored to internal structures that prevent their free drffusion. In the erythrocyte membrane, both glycophorin and the cNoride-bicarbonateexchanger (p. 395) are tethered to spectrin, a fllamentous cytoskeletal protein (Fig. ll-f g). One possible explanation for the pattern of lateral diffusion of lipid molecules shov,n in Figure 1l-18 is that membrane proteins immobilized by their association with spectrin form the "fences" that define the regions ofrelatively turrestricted Jipid motion.

F I G U R E1 1 - 1 8 H o p d i f f u s i o n o f i n d i v i d u a l l i p i d m o l e c u l e s .T h e m o t i o n o f a s i n g l ef l u o r e s c e n t l yl a b e l e d l i p i d m o l e c u l e i n a c e l l s u r f a c ei s r e c o r d e do n v i d e o b y f l u o r e s c e n c em i c r o s c o p y ,w i t h a t i m e r e s o l u t i o n of 25 p"s (equivalent to 40,000 frames/s).The track shown nere reores e n t sa m o l e c u l e f o l l o w e d f o r 5 5 m s ( 2 , 2 5 0 f r a m e s ) ;t h e t r a c e b e g i n si n t h e p u r p l e a r e a a n d c o n t i n u e st h r o u g h b l u e , g r e e n , a n d o r a n g e T h e p a t t e r no f m o v e m e n t i n d i c a t e sr a p i d d i f f u s i o nw i t h i n a c o n f i n e c lr e g i o n ( a b o u t 2 5 0 n m i n d i a m e t e r ,s h o w n b y a s i n g l e c o l o r ) , w i t h o c c a s i o n a l h o p s i n t o a n a d j o i n i n g r e g i o n .T h i s f i n d i n g s u g g e s t st h a t t h e l i p i d s a r e c o r r a l l e d b y m o l e c u l a r f e n c e st h a t t h e y o c c a s i o n a l l yj u m p

Chloride-bicarbonate exchangeproteins

Glycophorin Outside

I Plasma membrane Ankyrin Spectrin Path of single lipid molecule Junctional complex (actin) Inside

FIGURE l1-19 Restrictedmotion of the erythrocytechloridebicarbonateexchangerand glycophorin.The proteinsspanthe membraneand are tetheredto spectrin,a cytoskeletal protein,by another protein,ankyrin,limitingtheirlateralmobility.Ankyrinis anchoredin the membraneby a covalentlybound palmitoylsidechain (seeFig. 'l 1-.14).Spectrin, a long,filamentous protein,is cross-linked at iunctionalcomplexes containingactin.A networkof cross-linked spectrin molecules attached to the cytoplasmic faceof the plasmamembrane stabilizes the membrane, makingit resistant to deformation. Thisnetwork of anchoredmembraneproteinsmayform the "corral',suggested .l'l-1 by the experiment shownin Figure B; the lipidtracksshownhere are confinedto regionsdefinedby the tetheredmembraneproteins.

Sphingolipids andCholester0l Cluster Together inMembrane Rafts We have seen that diffusion of membrane lipids from one brlayer leaflet to the other is very slow unless catalyzed,, and that the different Jipid species of the plasma membrane are asymmetrically distributed in the two leaflets of the bilayer (Fig. 11-5). Even within a single leaflet, the lipid distribution is not random. Glycosphingolipids (cerebrosides and gangliosides), which typically contain longchain saturated fatty acids, form transient clusters in the outer leaflet that largely exclude glycerophospholipids, which typically contain one unsaturated fatty acyl group and a shorter saturated acyl group. The long, saturated acyl groups of sphingoJipidscan form more compact, more stable associationswith the long ring system of cholesterol than can the shorter, often unsaturated, chains of phospholipids. The cholesterol-sphingolipid microdomains in the outer monolayer of the plasma membrane, visible with atomic force microscopy (Box 11-1), are slightly thicker and more ordered (less fluid) than neighboring microdomains rich in phospholipids and are more difflcult to dissolve with nonionic detergents; they behave like liquidordered sphingoJipid rafts adri_fton an ocean of liquiddisordered phospholipids (Fig. ll-20, p. 386). These lipid rafts are remarkably enriched in two classes of integral membrane proteins: those anchored to the membrane by two covalently attached long-chain saturated fatty acids (two palmitoyl groups or a palmitoyl and a myristoyl group) and GPl-anchored proteins

Dynamics 11.2Membrane [t*t]

In atomic force microscopy(AFM), the sharptip of a microscopic probe attached to a flexible cantilever is drawn across an uneven surface such as a membrane (Flg i). Electrostaticand van der Waalsinteractions between the tip and the sample produce a force that movesthe probe up and down (in the e dimension)as it encountershills and valleysin the sample.A laserbeam reflected from the cantileverdetectsmotions of as little as 1 A. In one type of atomic force microscope,the force on the probe is held constant (relative to a standard force, on the order of piconewtons) by a feedbackcircuit that causesthe platform holding the sampleto rise or fall to keep the force constant.A seriesof scansin the r and y dimensions(the plane of the membrane)yields a three-dimensional contour map of the surface with resolution near the atomic scale-0.1 nm in the vertical dimension,0.5 to 1.0nm in the lateral dimensions.The membranerafts shownin Figure 11-20bwere visualized by this tectLrLique. In favorablecases,AFM can be used to study single membraneprotein molecules.Sin$e moleculesof bacteriorhodopsin(seeFig. 11-9) in the purplemembranes of the bacterium Halobacteri,um salinantTTzare seen as highly regular structures (Fig. 2a). When severalimagesof individual units are superimposedwith the help of a computer,the real parts of the imagereinforce each other and the noisein individual imagesis averagedout, yielding a high-resolutionimage of the protein (inset in Fig. 2a). AFM of purifiedE. coli,aquaporin,reconstituted

(a)

Platform moves to maintain constant pressure on cantilever tip. Excursions in the z dimension are plotted as a function of.*,y.

I FIGURE into Iipid bilayers and viewed as if from the outside of a cell, shows the fine details of the protein's periplasmic domains(Fig. 2b). And AFM revealsthat Fo,the protondriven rotor ofthe chloroplastATP synthase(p. 760), is composedof many subunits (14 in Fig. 2c) arrangedin a circle.

1o nm

2 FIGURE

(Fig. 11-14). Presumablythese lipid anchors,like the acyl chains of sphingolipids,form more stable associations with the cholesteroland long acyl groups in rafts than with the surroundingphospholipids.(It is notable that other lipid-Iinked proteins, those with covalently attachedisoprenylgroupssuch asfarnesyl, arenot preferentially associatedwith the outer leaflet of sphingolipid/cholesterolrafts (Fig. 11-20a).) The "raft" and "sea" domains of the plasmamembraneare not rigidly separated;membraneproteins can move into and out of

Iipid rafts on a time scaleof seconds.But in the shorter time scale(microseconds)more relevantto many membrane-mediatedbiochemicalprocesses,many of these proteins reside primarily in a ra"ft. We can estimatethe fraction of the cell surfaceoccupied by rafts from the fraction of the plasma membrane that resistsdetergentsolubilization,which can be as high as 50% in somecases:the rafts cover half of the ocean(Fig. 11-20b). Indirect measutementsin cr-rltured flbroblasts suggesta diameter of roughly 50 nm for an

L 3 B 6 _ _B] i o l o g i cMael m b r a naensdT r a n s p o r t

Raft, enriched in sphingolipids, cholesterol

Acyl groups (palmitoyl,

Caveolin is an integral membraneprotem with two globular domains connected by a hairpin-shapedhydrophobicdomain,whichbindsthe proteinto the cytoplasmic leaflet of the plasma membrane. Three palmitoyl groups attachedto the carbo4Fl-terminalglobular domain further anchor it to the membrane.Caveolin (actually, a family of related caveolins)binds cholesterolin the membrane,and the presenceof caveolinforcesthe associated lipid bilayer to curve inward, forming caveolae ("little caves")in the surfaceof the cell (Fig. 11-21). Caveolae are nnusualrafts: they involve bothleafletsofthe bilayerthe cytoplasmicleaflet, from which the caveolin$obular domainsproject, and the extracellularleaflet, a flpical sphingolipid/cholesterol raft with associatedGPl-anchored proteins. Caveolaeare implicatedin a variety of cellular functions, includr4gmembranetrafflcking within cells and the transductionofexternalsignalsinto cellularresponses. The receptorsfor ursulinand other growth factors,aswell as ceftain GTP-bindingproteinsand protein kinasesassociatedwith transmembranes€naling,seemto be localized

FIGURE l1-20 Membranemicrodomains(rafts).(a)Stableassociations of sphingolipids and cholesterolin the outer leafletproducea microdomain,slightlythickerthan othermembraneregions,that is enrichedwith specifictypesof membraneproteinsCpl-linkedproteins arecommonin the outer leafletof theserafts,and proteinswith one or several covalently attached long-chain acylgroupsarecommonin the innerleaflet.Caveolinis especially commonin inwardlycurvedrafts (seeFig.11-21) Proteins calledcaveolae with attached prenylgroups (suchasRas;seeBox 12-2) tendto be excludedfrom rafts.(b) ln thisartificial membrane-reconstituted (on a mica surface)from cholesterol, syntheticphospholipid(dioleoylphosphatidylcholine), and the Cptlinkedproteinplacental alkalinephosphatase--the greater thickness of raft regionsis visualizedby atomicforce microscopy(seeBox .l'l_1). Theraftsprotrudefrom a lipid bilayerocean(theblacksurfaceis thetop of the upper monolayer);sharppeaksrepresentCpl-linkedproteins. Notethatthesepeaksarefoundalmostexclusivelyin the rafts

Plasma membrane

Outside Inside

%

>'s -F

a

Caveola

9rk

r-

f

"s

individual raft, which corresponds to a patch containing a few thousand sphingolipids and perhaps 10 to 50 membrane proteins. Because most cells express more than 50 different kinds of plasma membrane proteins, it is likely that a single raft contains only a subset of mem-

brane proteins and that this segregation of membrane proteins is functionally signiflcant. For a process that involves interaction of two membrane proteins, their presence in a single raft would hugely increase the likelihood of their collision. Certain membrane receptors and signaling proteins, for example, seem to be segregated together in membrane rafts. Experiments show that signaling through these proteins can be disrupted by manipulations that deplete the plasma membrane of cholesterol and destroy lipid rafts.

ft) FIGURE 11-21 Caveolinforces inward curvature of a membrane. C a v e o l a ea r e s m a l l i n v a g i n a t i o n si n t h e p l a s m a m e m b r a n e ,a s s e e n i n (a) an electron micrograph of an adipocyte surface-labeled with an electron-dense marker (b) Each caveolin monomer has a central hyd r o p h o b i c d o m a i n a n d t h r e e l o n g - c h a i na c y l g r o u p s ( r e d ) ,w h i c h h o l d the molecule to the inside of the plasma membrane When several caveolin dimers are concentrated in a small region (a raft), they force a c u r v a t u r ei n t h e l i p i d b i l a y e r ,f o r m i n g a c a v e o l a .C h o l e s t e r o m l olecules i n t h e b i l a y e ra r e s h o w n i n o r a n g e .

-387 11.2Membrane Dynamics in ra^ftsand perhapsin caveolae. We discusssomepossible rolesof rafts in signahngin Chapter12.

Mernbrane Curvature andFusion AreCentral toMany Biological Processes Caveolinis not urLiquein its ability to induce curvaturem membranes.Changesof curvature are central to one of the most remarkablefeatures of biologicalmembranes: their ability to undergo fusion with other membranes without losingtheir contimlty. Although membranesare stable,they are by no meansstatic.Within the eukaryotic endomembrane system(which includesthe nuclearmembrane, endoplasmicreticulum, Golgi, and various small vesicles),the membranouscompartmentsconstantlyreorganize.Vesiclesbud from the ER to carry newly slmthesized lipids and proteins to other organellesand to the plasmamembrane.Exocytosis,endocy[osis,cell division, fusion of egg and sperrncells,and entry of a membraneenvelopedvirus into its host cell all involvemembranereorganizationin wtuch the fundamentaloperationis fusion of two membrane segmentswithout Ioss of continuity (FiS. 11-22). Mostof theseprocesses beginwith a local increasein membranecuvature. Three mechanismsfor inducing membrane curvature are shown in Figure ll-23 A protein that is intrinsically curved may force curvaturein a bilayerby bindingto it; the binding energy providesthe drivingforcefor the increasein bilayercurvature. Alternatively,many suburutsof a scaffold protein may assembleinto curuedsupramolecularcomplexesand

stabilizecurvesthat spontaneously form in the bilayer.Or, a proteinmay insert oneor more hydrophobichelicesrnto one face of the bilayer,expandingits arearelativeto the other faceand therebyforcing curvature. Specificfusion of two membranesrequires that (1) they recognizeeach other; (2) their surfacesbecome closelyapposed,which requiresthe removal of water molecules normally associatedwith the polar head groups of Jipids;(3) their bilayer structuresbecomelocally disrupted,resultingin fusion of the outer leaflet of eachmembrane(hemifusion);and (4) their bilayersfuse to form a singlecontinuousbilayer.The fusion occurring in receptor-mediatedendocltosis,or regulatedsecretion, also requiresthat (5) the processis triggered at the appropriatetime or in responseto a speci-flcsignal.Integral proteins called fusion proteins mediate these events, bringing about speciic recognitionand a transient local distortion of the bilayer structure that favorsmembrane fusion. (Note that these fusion proteins are unrelatedto the productsencodedby two fusedgenes,alsocalledfuin Chapter9.) sionproteins,discussed

A protein with intrinsic cuwature on its surface interacts strongly with a curved membrane surface, allowing both membrane and protein to achieve their lowest energy.

Budding ofvesicles from Golgi complex Exocytosis Endocytosis Fusion ofendosome and lysosome

Virai infection

If a membrane region spontaneously curves, monomeric subunits of certain proteins can polymerize into a superstructure that favors and maintains the curvature

ofsperm and egg Fusion of small vacuoles (plants) Separation of two plasma mernbranes at cell division

FIGURE 11-22 Membranefusion.The fusionof rwo membranes is centralto a varietyof cellularprocesses involvingorganelles and the plasmamembrane.

A protein with one or more amphipathic helices inserted into one leaflet ofthe bilayer crowds the lipids in that leaflet, forcing the membrane to bend

FIGURE 11-23rhree models for protein-induced curvature of membranes.

F"-lB i o l o g i cMael m b r a naensdT r a n s p o r t Cytosol Secretory vesicle

Neurotransmitter-frlled vesicle

v-SNARE

Plasma membrane

v-SNARE and I-SNARE bind to each other, zipping up from the amino termini and drawing the two membranes together.

II I

Y

Zipping causescurvature and lateral tension on bilayers, favoring hemifu sion between outer leaflets.

A well-studied exampleof membranefusion is that occurring at synapses,when intracellular vesicles loaded with neurotransmitter fuse with the plasma membrane.This processinvolvesa family of proteins called SNARES(FiS. ll-24). SNAREsin the cytoplasmic face of the intracellular vesicle are called vSNAREs;thosein the target membranewith which the vesiclefuses(the plasmamembraneduring exocltosis) are t-SNAREs. TWoother proteins,SNAP25and NSF, are also invoived. During fusion, a v-SNARE and tSNARE bind to each other and undergo a structural changethat producesa bundle of long thin rods made up of helicesfrom both SNARESand two helicesfrom SNAP25(Fig. 11-24).The two SNAREsinitiallyinteract at their ends,then zip up into the bundleof helices.This structural changepulls the two membranesinto contact and initiates the fusion of their lipid bilayers. The complexof SNAREsand SNAP25is the target of the powerful Clostridtum botulznum toxin, a proteasethat cleavesspecificbondsin theseproteins,preventing neurotransmissionand thereby causing the death of the organism.Becauseof its very high speciflcity for these proteins,purifled botulinum toxin has servedas a powerful tool for dissectingthe mechanism of neurotransmitterreleasein vivo and in vitro.

Arelnvolved in Integral Proteins ofthePlasma Membrane Pr0cesses and0ther(ellular Surface Adhesion, Signaling, Hemifusion: inner leaflets of both membranes come into contact.

Completefusion createsa lusron pore.

I

t J

Pore widens; vesicle contents are released outside cell.

tIGURE11-24 Membranefusionduringneurotransmitter releaseat a synapse.The secretoryvesiclemembranecontainsthe v-SNARE (red).The target (plasma)membranecontainsthe synaptobrevin (blue)andSNAP25 (violet). t-SNAREs syntaxin Whena localincrease in of neurotransmitter, the v-SNARE, SNAP25, and [Ca2*]signalsrelease I-SNARE interact, forminga coiledbundleof foura helices, pullingthe two membranes togetherand disrupting the bilayerlocallyThisleads joiningthe innerleafletsof the two memDranes, firstto hemifusion, thento completemembranefusionand neurotransmitter release

Severalfamilies of integral proteins in the plasmamembrane provide specific points of attachmentbetween cells, or between a cell and extracellularmatrix proteins.Integrins are surfaceadhesionproteinsthat mediate a cell's interaction with the extracellular matrix and with other cells, including some pathogens.Integrins also carry signalsin both directions acrossthe plasma membrane,integrating information about the extracellularand intracellular environments.All integrins are heterodimericproteins composedof two unIike subunits,d and B, each anchoredto the piasma membraneby a single transmembranehelix. The large extracellular domainsof the a and B subunits combine to form a speciflcbinding site for extracellularproteins such as collagenand flbronectin, which contain a common determinant of integrin binding, the sequence Arg-Gly-Asp (RGD). We discussthe signalingfunctions of integrinsin more detailin Chapter12 @. a5Q. Other plasmamembraneproteins involvedin surface adhesionare the cadherins, which undergo homophilic ("with samekind") interactions with identical cadherinsin an adjacentcell. Selectins have extracellular domainsthat, in the presenceof Caz*, bind speciflc polysaccharides on the surfaceof an adjacentcell. present primarily in the varioustypes of Selectinsare blood cells and in the endothelialcells that line blood vessels(seeFig. 7-31). They are an essentiaipart of the process. blood-clotting Integralmembraneproteinsplay rolesin many other cellularprocesses.They serveas transportersand ion

11.3Solute Transport across Membranes lttrl charurels(discussed in Section11.3)and as receptorsfor hormones,neurotransmitters,and growth factors (Chapter 12).They are centralto oxidativephosphorylationand photophosphorylation(Chapter 19) and to cell-cell and cell-antigenrecognitionin the immune system (Chapter 5) Integralproteinsare alsoimportant playersin the membranefusionthat accompanies exocl'tosis,endocytosis,and the entry of manytypes of virusesinto host cells.

S U M M A R1Y1 . 2 M e m b r a nDey n a m i c s r

Lipids in a biological membrane can exist in liquid-ordered or liquid-disordered states; in the latter state, thermal motion of acyl chains makes the interior of the bilayer fluid. F luidity is affected by temperature, fatty acid composition, and sterol content.

r

Flip-flop diffusion of lipids between the inner and outer leaflets of a membrane is very slow except when speciflcally catalyzed by flippases, floppases, or scramblases.

r

Lipids and proteins can diffuse laterally within the plane of the membrane, but this mobility is limited by interactions of membrane proteins with internal cytoskeletal structures and interactions of lipids with lipid rafts. One class of lipid rafts consists of sphingolipids and cholesterol with a subset of membrane proteins that are GPl-linked or attached to several long-chain fatty acyl moieties.

Simple diffusion (nonpolar compounds only, down concentration gradient)

r

Caveolinis an integral membraneprotein that associateswith the inner leaflet of the plasma membrane,forcing it to curve inward to form caveolae,probablyinvolvedin membranetransport and signaling.

r

Speci-ficproteins causelocal membranecurvature and mediatethe fusion of two membranes,which processessuchas endocytosis, accompanies exocy'tosis,and viral invasion.

r

Integrinsare transmembraneproteinsof the plasma membranethat act both to attach cellsto eachother and to carry messagesbetweenthe extracellular matrix and the cytoplasm.

11.3Solute Transport across Membranes Every living cell must acquire from its surroundings the raw materials for bioslmthesis and for energy production, and must release to its environment the byproducts of metabolism. A few nonpolar compounds can dissolve in the Jipid bilayer and cross the membrane unassisted, but for transmembrane movement of any polar compound or ion, a membrane protein is essential In some cases a membrane protein simply facilitates the diffusion of a solute down its concentration gradient, but transport can also occur against a gradient of concentration, electrical charge, or both, in which case the process requires energy (l'ig. l1-"25). The energy may come directly

Facilitated diffusion (down electrochemical gradient)

Primary active transport (against electrochemical . sradient) Sort

Ionophoremediated ron transport (down electrochernical gradient)

Ion channel (down electrochemical gradient; may be gated by a ligand or ion)

o

from ATP hydrolysis or may be suppliedin the form of one solute moving down its electrochemical gradient, with the release of enough energy to drive another solute up its gradient. Ions may also move acrossmembranesvia ion channelsformed by proteins, or they may be carried acrossby ionophores,small moleculesthat mask the chargeof ions and allow them to diffuse through the lipid bilayer.With very few exceptions, the traffic of small molecules across the plasma membraneis mediated by proteins such as transmembranechamels. carriers. or pumps. Within the eukaryotic cell, dj-fferentcompartmentshave different concentrations of ions and of metabolic intermediates andproducts,and these,too, must move across intracellular membranes in tightly regulated, proprocesses. tein-mediated

Ion

w

Ion

Secondary active transport (against electrochemical gradient, driven by ion moving down its gradient)

FIGURI 11-25Summarv of transport types.

lt

390

B i o l o g i cMael m b r a naensdT r a n s p o r t

FIGURE11-26 Movement of solutes across a permeable membrane, (a) Net movement of an electrically neutralsolute is toward the side of lower s o l u t e c o n c e n t r a t i o nu n t i l e q u i l i b r i u m i s a c h i e v e d . The soiute concentrationson the left and right s i d e s o f t h e m e m b r a n e a r e d e s i g n a t e dC 1 a n d C 2 T h e r a t e o f t r a n s m e m b r a n em o v e m e n t ( i n d i c a t e d b y t h e a r r o w s )i s p r o p o r t i o n a lt o t h e c o n c e n t r a t i o n

Ct>

Cz

Before equilibrium Net flux

gradient, C2/C1 (b) Net movement of an electri-

Ct= Cz

V*t0

V^=0

At equilibrium No net flux

Before equilibrium

At equilibrium

c a l l y c h a r g e ds o l u t e i s d i c t a t e d b y a c o m b i n a t i o n of the electrical potential (V-) and the chemical c o n c e n t r a t i o nd i f f e r e n c e( C : / C r ) a c r o s st h e m e m b r a n e ; n e t i o n m o v e m e n t c o n t i n u e su n t i l t h i s e l e c t r o c h e m i c a lp o t e n t i a l r e a c h e sz e r o

Fassive Transport lsFacilitated byMembrane Proteins When two aqueous compartments containing unequal concentrations of a soluble compound or ion are separated by a permeable divider (membrane), the solute moves by simple diftrsion from the region of higher concentration, through the membrane, to the region of lower concentration, until the two compartments have equal solute concentrutions (Fig. l1-26a) When ions of opposite charge are separated by a permeable membrane, there is a transmembrane electrical gradient, a membrane potential, Iz^ (expressed in millivolts) This membrane potentiaLproduces a force opposingion movements that increase 7,. and drivirlg ion movements that reduce 7* (Fig 11-26b). Thus the direction rn wtuch a charged solute tends to move spontaneously across a membrane depends on both the chemical gradient (the difference in solute concentration) and the electrical gradient (Z*) across the membrane Together, these two factors are referred to as the electrochemical gradient or electrochemical potential. This behavior of solutes is in accord with the second law of thermodyeramics:molecules tend to spontaneously assume the distribution of greatest randomness and lowest energy. To pass through a Iipid bilayer, a polar or charged solute must first give up its interactions with the water molecules in its hydration shell, then diffuse about 3 nm (30 A) through a substance (lipid) in which it is poorly soluble (Fig. I 1-2 7 ). The energy used to strip away the hydration shell and to move the polar compound from water into Lipid, then through the lipid bilayer, is regained as the compound leaves the membrane on the other side and is rehydrated. However, the intermediate stage of transmembrane passage is a high-energy state comparable to the transition state in an enzyme-catalyzed chemical reaction. In both cases, an activation barrier must be overcome to reach the intermediate stage (Fig. 11-27; compare with Fig. 6-3). The energy of activation (AG+) for translocation of a polar solute across the bilayer is so large that pure lipid bilayers are virtually impermeable to polar and charged species over periods of time relevant to cell growth and diusion.

Membrane proteins lower the activation energy for transport of polar compotrnds and ions by providtng an alternative path through the bilayer for specifi.csolutes. Proteins diftrsion, or passive that bring about this facilitated transport, are not enz).Tnesin the usual sense; their "substrates" are moved from one compartment to another but are not chemically altered Membrane proteins that speed

(a)

T

*

AGI. slmpte dlffusion

!

F

Auiransport

(b)

Transporter 11-27 Energychangesaccompanying passage FIGURE of a hydrophilic solutethroughthe lipid bilayerof a biologicalmembrane.(a) In simple diffusion,removalof the hydration shellis highlyendergonic, and (AC+)for diffusionthroughthe bilayeris very the energyof activation proteinreduces high.(b)A transporter the AC+for transmembrane difinteractions fusionof the solute lt doesthis by formingnoncovalent with the dehydrated soluteto replacethe hydrogen bondingwith wapathway ter and by providinga hydrophilic transmembrane

Transport across Membranes 11.35olute lrrtl the movementof a soluteacrossa membraneby faciJitaturg diffrrsionare calledhansporters or perrneases. Like enzymes,transportersbind their substrateswith stereochemical speciflcitythrough multiple weak,noncovalentinteractions.The negativefree-energychange associatedwith these weak interactions, AGui,,.ri.s, counterbalancesthe positive free-energychangethat accompaniesloss of the water of hydration from the substrate,AGdehvdration, therebyloweringAG$for transmembranepassage(Fig. 11-27).Ttansportersspanthe lipid bilayer severaltimes, forming a transmembrane charurellined with hydrophilic amino acid side chains. The channelprovides an alternative path for a specifi.c substrateto move acrossthe Iipid bilayer without its having to dissolvein the bilayer, further lowering AG+ for transmembranediffusion.The result is an increase of several to many orders of magnitude in the rate of passageof the substrate. fransmembrane

Transporters CanBeGrouped into5uperfamilies Based onTheir Structures We know from genomicstudiesthat transportersconstitute a signiflcantfraction of all proteins encodedin the genomesof both simple and complex organisms There are probablya thousandor more different transporters in the human genome.Ttansportersfall within two very broad categories:carriersand channels(Fig. 11-28). Carriers bind their substrateswith high stereospecificity,catalyzetransport at rates well below the limits of free diffusion, and are saturable in the same senseas are enz).rnes: there is somesubstrateconcentration abovewhich further increaseswill not producea greater rate of transport. Channels generally allow transmembrane movementat ratesordersof magnitude greaterthan thosetypical of carriers,ratesapproaching the limit of unhindered diffusion. Channelstypically showlessstereospecificity than carriersand are usually not saturable.Most channelsare oligomericcomplexes of several,often identical,subunits,whereasmany carriers function asmonomericproteins.The classification as carrier or channelis the broadestdistinctionamong transporters.Within eachofthese categoriesare superfamiliesof varioustypes, definednot only by their pri-

mary sequencesbut by their secondary structures. Some channels are constructed primarily of helical transmembranesegments,others have B-barrel structures Among the carriers, some simply facilitate diffusion down a concentration gradient; they are the passive transporter superfamily. Active transporters can drive substratesacross the membrane against a concentrationgradient, some using energy provided directly by a chemicalreaction (primary active transporters)and somecouplinguphill transportof one substratewith downhill transport of another (secondary active transporters). We now consider some well-studied representativesof the main transporter superfamilies.Youwill encountersomeof thesetransporters again in later chapters in the context of the metabolicpathwaysin which they participate.

Transporter ofErythrocytes TheGlucose Passive Transport Mediates Energy-yieldingmetabolismin erythrocytesdependson a constant supply of glucosefrom the blood plasma, where the glucoseconcentrationis maintainedat about 5 mv. Glucoseenters the erythrocfie by facilitated diffusionvia a speciflcglucosetransporter,at a rate about 50,000times greaterthan uncatalyzedtransmembrane diffusion. The glucose transporter of erythrocytes (called GLUT1 to distinguishit from related glucose transportersin other tissues)is a tlpe III integralproIein (M, -45,000) with 12 hydrophobicsegments,each helix. of which is believedto form a membrane-spanning yet known,but The detailedstructure of GLUT1is not one plausiblemodel suggeststhat the side-by-sideassembly of severalhelices producesa transmembrane channelIined with hydrophilic residuesthat can hydrogen-bondwith glucoseas it movesthrough the channel (Fis. 1r-29). The processof glucosetransport can be described by analogy with an enz;.'rnaticreaction in which the "substrate"is glucoseoutsidethe cell (So,t),the "product" is glucoseinside (S1), and the "enzyme"is the transporter,T. Whenthe initial rate of glucoseuptakeis measuredas a function of externalglucoseconcentration (Fig. 1l-30), the resulting plot is hyperbolic;at high externalglucoseconcentrationsthe rate of uptake approachesZ-u*. Formally, such a transport process canbe describedby the equations

+Tlfr ,"",. sout ",

u-^llu^ u-,]lu,

Secondary actrve transporters

Passive transporters

Primary actrve transporters

FIGURE 1l-28 Classification of transporters.

si. + T2 --A 5rr. T, R-g

in which kr k-t, and so forth, are the forward and reverse rate constants for each step; Tr is the transporter conformation in which the glucose-binding site faces

392

B i o l o g i cMael m b r a naensdT r a n s p o r t

@

b!F

o>

x.+

Outside

5> oX >h Inside ,

*NHs

coo-

(a) -Ser-Leu-Val

Extracellular glucose concentration, [Slor1(mu)

(a)

-Thr -Asn-Phe -IIe2

t

(b)

1 Kt

(*) "*

(c) IIGURE 1I *29 Proposed structureof GtUTl . (a)Transmembrane helicesare represented hereas oblique(angledlrowsof threeor four aminoacidresidues, eachrow depictingoneturnof thea helix.Nine .l (blue of the 2 helicescontainthreeor morepolaror chargedresidues or red),oftenseparated by severalhydrophobicresidues(yellow).This representation of topologyis not intendedto represent three-dimensionalstructure(b) A helicalwheeldiagramshowsthedistribution of polarand nonpolarresidues on the surfaceof a helicalsegment. The helixis diagrammed asthoughobserved alongitsaxisfromthe amino terminus. Ad.jacent residues in the linearsequence areconnected, and eachresidueis placedaroundthe wheelin the positionit occupiesin the helix;recallthat3.6 residues are requiredto makeone complete (blue)areon one turnof thea helix.In thisexample, thepolarresidues (yellow)on the other. sideof the helixand the hydrophobic residues Thisis, by definition,an amphipathic helix.(c) Side-by-side association of four amphipathichelices,each with its polar face oriented towardthecentralcavity,can producea transmembrane channellined with polar(andcharged) residuesThischannelprovidesmanyoppor tunitiesfor hydrogen bondingwith glucoseas it movesthrough.

out, and T2 the conformation in which it faces in. The steps are surnrnarized in tsigrrre I 1*3 l. Given that every step in this sequence is reversible, the transporter is, in principle, equally able to move glucose into or out of the cell. However, glucose always moves down its concentration gradient, which normally means ,into the cell. Glucose that enters a cell is generally metabolized immediately, and the intracellular glucose concentration is thereby kept low relative to its concentration in the bloOd.

(a) The f IGURE 11-30 Kineticsof glucosetransportinto erythrocytes. initial rate of glucoseentry into an erythrocyte,Ve,dependson the initial concentration of glucoseon the outside,[S]",,.(b) Doubleplot of the datain (a).Thekineticsof facilitated reciprocal diffusionis to the kineticsof an enzyme-catalyzed reaction.Compare analogous theseplotswith Figure6-11, and with Figure1 in Box 6-1. Notethat K, is analogous to K-, the Michaelis constant.

o-Glucose

-

FlGUnt1l-31 Model of glucosetransportinto erythrocytesby CLUT1, The transporter existsin two conformations: T1, with the glucose-binding siteexposed on the outersurface of the plasmamembrane,andT2,with the bindingsiteexposed on the innersurfaceClucosetransport occursin four steps. @ Clucosein bloodplasmabinds to a stereospecific site on T1; this lowers the activationenergy l o r @ a c o n f o r m a t i o ncahl a n g ef r o mg l u c o s e o , . . Tt 1 o glucose;n.T2, effectingthe transmembrane passage of the glucose @ Glucoseis releasedfromT2into the cytoplasm,and @ the transporter returnsto the T1conformation,readyto transportanotherglucosemolecule.

Fr{

I 1 . 3S o l u tTer a n s p o a rctr o sMse m b r a n e s

Tlansporter fissue(s) whereexpressed

Gene

Rolex

GLUTl

SLC2Al

GLUT2

tlbiquitous Liver, pancreaticisiets, intestine

GLUT3

Brain (neuronal)

SLC2A3

Basal$ucose uptake In )iver,removalof excessglucosefrom blood; in pancreas,regulationofinsulin release Basalglucoseuptake

GLUT4

Muscle,fat, heart

SLC2A4

Activity increasedby insulin

SLC2A2

GLUTs

Intestine,testis, kidney,sperm

SLC2A5

Primarily fructose transport

GLUT6

Spleen,leukocytes,brain

SLC2A6

Possiblyno transporter function

GLUTT

Liver microsomes

SLC2A7

GLUT8

SLC2A8

GLUT9

Testis,blastocyst,brain Liver, kidney

GLUTlO

Liver, pancreas

SLCzAlO

GLUTl1

Heart, skeletalmuscle

SLCzAl1

GLUT12

Skeletalmuscle,adipose,smallintestine

SLC2A12

SLC2A9

*Dashindicates roleuncertain.

The rate equationsfor glucose transport can be derived exactly as for enzyme-catalyzedreactions (Chapter 6), yielding an expressionanalogousto the Michaelis-Menten eouation: (11-1)

in which Ze is the initial veiocity of accumulationof glucoseinside the cell when its concentrationin the surroundingmedium is [S]or1,and K, (Kt.u."port)is a constantanalogousto the Michaelisconstant,a combination of rate constantsthat is characteristicof each transport system.This equationdescribesthe i,ni,ti,al velocity, the rate observedwhen [5]6 : 0. As is the case for enzyme-catalyzed reactions,the slope-intercept form of the equationdescribesa linear plot of 1/76 againstl/[S]o,t, from which we can obtain valuesof K, and 7-u" (Fig. 11-30b). When [S]o"t: K,, the rate of uptake is \/zV^ ,; the transport processis half-saturated.The concentrationof glucosein bloodis 4.5 to 5 mM,aboutthree timesKr, which ensuresthat GLUT1is nearly saturated with substrate and operates near 7^u"' Becauseno chemicalbonds are made or broken in the conversionof Sor, to 56, neither "substrate"nor "product" is intrinsicallymore stable,and the process of entry is thereforefully reversible.As [S]'. approaches [S]o,5,the rates of entry and exit becomeequal.Sucha systemis thereforeincapableof accumulatingglucose within a cell at concentrationsabovethat in the surrounding medium; it simply equilibratesglucoseon the two sidesof the membranemuch fasterthan would occur in the absenceof a specifictransporter.GLUT1 is specificfor o-glucose,with a measuredK, of 1.5 mrr.l. For the close analogs D-mannoseand o-galactose,

which differ only in the position of one hydroxyl group, the valuesof K, are 20 and 30 mlt, respectively;and for K, exceeds3,000mnt.Thus GLUTl showsthe L-glucose, three hallmarksof passivetransport: high rates of diffusion dolnrra concentrationgradient, saturability,and speciflcity. Threlveglucose transporters are encoded in the human genome,each with its unique kinetic properties, patterns of tissue distribution, and function (Table11-3). In liver, GLUT2transportsglucoseout of hepatocyteswhen liver glycogen is broken down to replenishblood glucose.GLUT2 has a Kl of about 66 mnaand canthereforerespondto increasedlevelsof intracellularglucose(producedby glycogenbreakdown) by increasing outward transport. Skeletal and heart muscle and adiposetissue have yet another glucose transporter, GLUT4 (Kt : 5 mlt), which is distinguishedby its responseto insulin:its activity increases when insulin signalsa high blood glucoseconcentration, thus increasingthe rate of glucoseuptake into muscle and adiposetissue (Box 11-2 describessome malfunctionsof this transporter).

The(hloride-Bicarbonate fttalyzes Exchanger (otransport Hectroneutral ofAnions across Membrane thePlasma The erythrocyte containsanother facilitated dlffusion system, an anionexchangerthat is essentialin COztransport to the lungsfrom tissuessuchas skeletalmuscleand liver. WasteCO2releasedfrom respiringtissuesinto the blood plasmaentersthe erythrocy'te,whereit is convertedto bicarbonic anhydrase. carbonate(HCOt) by the enzJ,Tne (Recallthat HCO3 is the primary buffer of blood pH; see Fig. 2-20.) The HCO| reenters the blood plasmafor

391

B i o l o g i cMael m b r a naensdT r a n s p o r t

When ingestion of a carbohydrate-rich meal causes blood glucose to exceed the usual concentration between meals (about 5 mu), excess glucose is taken up by the myocytes of cardiac and skeletal muscle (which store it as glycogen) and by adipocytes (which convert it to triacylglycerols). Glucose uptake into myocytes and adipocytes is mediated by the glucose transporter GLUT4. Between meals, some GLUT4 is present in the plasma membrane, but most is sequestered in the membranes of small intracellular vesicles (Fig. 1) Insulin released from the pancreas in response to high blood glucose triggers the movement of these intracellular vesicles to the plasma membrane, where they fuse, thus exposing GLUT4 molecules on the outer surface of the cell (see Fig. 12-16) . Wth more GLUT4 molecules in action, the rate of glucose uptake increases l5-fold or more. When blood glucose levels return to normal, insulin release slows and most GLUT4 molecules are removed from the olasma membrane and stored in vesicles. In type 1 fiuvenile-onset) diabetes mellitus, the inability to release insulin (and thus to mobilize glucose

transporters) results in low rates of glucose uptake into muscle and adipose tissue. One consequence is a proIonged period ofhigh blood glucose after a carbohydraterich meal. This condition is the basis for the glucose tolerance test used to diagnose diabetes (Chapter 23). The water permeabrlity of epithelial cells lining the renal collecting duct in the kidney is due to the presence of an aquaporin (AQP-2) in their apical plasma membranes (facing the lumen of the duct). Vasopressin (antidiuretic hormone, ADH) regulates the retention of water by mobilizing AQP-2 molecules stored in vesicle membranes within the epithelial cells, much as insulin mobilizes GLUT4 in muscle and adipose tissue. When the vesicles fuse with the epithelial cell plasma membrane, water permeability greatly increases and more water is reabsorbed from the collecting duct and returned to the blood. When the vasopressin level drops, AQP-2 is resequestered within vesicles, reducing water retention In the relatively rare human disease diabetes insipidus, a genetic defect in AQP-2 leads to impaired water reabsorption by the kidney. The result is excretion of copious volumes of very dilute urine.

"stored' wrlnrn cel.ttn

small veslcles, ready to return to the surface when insulin levels rise again.

larger endosome'

FIGURE I Transport of glucoseintoa myocyteby CLUTais regulated by insulin.

Frrl

a rctr o sMse m b r a n e s 11 . 3S o l u tTer a n s p o

Carbon dioxide produced by catabolism enters erythrocyte.

Bicarbonate dissolves in blood plasma

Chloride-bicarbonate exchange protein In respiring

tissues

cuhrnic

anhydrasc

+ H2O

HCO; + H"

C]-

CO2 + H2O

HCO5 + H"

Cl

Coz

HCO;

Carbon dioxide leaves er1'throcl'te and is exhaled

cl

Bicarbonate enters erythroclte from blood plasma.

FIGURE 11-32 Chloride-bicarbonate exchangerof the erythrocyte membrane. Thiscotransport systemallowsthe entryandexitof HCO3 withoutchangingthe membranepotential.lts role is to increase the capacityof the blood CO2-carrying

transportto the lugs (Fig. 1l-32). BecauseHCO| is much more solublein blood plasmathan is CO2,this roundaboutroute increasesthe capacityof the blood to carry carbondioxidefrom the tissuesto the lungs.In the lungs,HCO| reentersthe erythrocyteand is conver[edto CO2,which is eventuallyreleasedinto the lung spaceand exhaled.To be effective,this shuttle requiresvery rapid movementof HCO| acrossthe erythroc;,'temembrane. The chloride-bicarbonate exchanger, also called the anion exchange (AE) protein, increasesthe rate of HCOt transport acrossthe erythrocyte membrane more than a millionfold.Like the glucosetransporter,it is an integral protein that probablyspansthe membraneat least 12 times. This protein mediatesthe simultaneous

movementof two anions:for eachHCOt ion that moves in onedirection,oneCl- ion movesrn the oppositedirection (Fig. 11-33), wrth no net transferof charge;the exchange is electroneutral. The coupling of CI- and HCOt movementsis obligatory;in the absenceof chloride,bicarbonatetransportstops.In this respect,the anion exchanger is typical of those systems, called cotransport systems, that simultaneouslycarry two solutesacrossa membrane.When, as in this case,the two substratesmovein oppositedirections,the process is antiport. In symport, two substratesare moved simultaneouslyin the same direction. T?ansportersthat carry only one substrate,suchasthe erythrocyteglucose transporter,are known asuniport systems(F'ig.11-33). The humangenomehas genesfor three closelyrelated chloride-bicarbonateexchangers,all with the samepredictedtransmembranetopology.Effihrocltes containthe AEI transporter,AE2 is prominentin liver, and AES is presentin plasmamembranesof the brain, heart, and retina. Similar anion exchangersare also found in plantsand microorganisms.

Movennent agalnst a inSolute Results Transpsrt Active Gradient 0r Electrochemical enncentration In passivetransport,the transportedspeciesalwaysmoves gradientand is not accumulated dovrmits electrochemical above the equilibrium concentration.Active transport, by contrast,resultsin the accumulationof a soluteabove the equilibriumpoint. Active transport is thermodlmamically unfavorable(endergonic)and takes place only when coupled (directly or indirectly) to an exergonic processsuch as the absorptionof sunlight,an oxidation reaction,the breakdovrnof ATP,or the concomitantflow of someother chemicalspeciesdown its electrochemical gradient.In primary active transport, soluteaccumulation is coupleddirectly to an exergonicchemicalreaction, such as conversionof ATP to ADP + P' (Fig. 1f€4).

5

k

Uniport

Symport

Artiport

Cotrairsport FIGURE 11-33 Threegeneralclasses Transporters of transportsystems. transported and the direcdifferin the numberof solutes(substrates) of all threetypesof transtion in which eachsolutemoves.Examples tellsus are discussed in the text. Notethat this classification Dorters (activetransport) or nothingaboutwhethertheseareenergy-requiring (passive energy-independent transport)processes.

(a) Primary active transport

(b) Secondary active transPort

11-34 Two types of active transport.(a) In primary active FIGURE transport,the energyreleasedby ATP hydrolysisdrivessolutemovegradient'(b) In secondaryactive ment againstan electrochemical by pritransport,a gradientof ion X (oftenNa*) hasbeenestablished gradiits electrochemical maryactivetransportMovementof X down (S) solute a second of cotransport ent now providesthe energyto drive gradient. againstitselectrochemical

f-

Membranes andTransport !396] Biological Secondary active tra,nsport occurs when endergonic (uphill) transportof onesoluteis coupledto the exergonic (downhill) flow of a djfferent solute that was originally pumped uphill by primary active transport. The amount of energyneededfor the transport of a solute againsta gradient can be calculatedfrom the initial concentrationgradient.The generalequationfor the free-energychangein the chemicalprocessthat convertsStoPis AG : AG'"+,R?ln(tPl/tsl)

(7r-2)

the insidenegativerelativeto the outside),so the second term of Equation 11-4 canmakea signi-flcantcontribution to the total free-energychangefor transporting an ion. Most cells maintain more than a l0-fold difference in ion concentrationsacrosstheir plasmaor intracellular membranes,and for many cells and tissues active transport is therefore a major energy-consuming process.

I

W0RKED EXAMPTE 11-2 EnergyCostof Pumping a(harged Solute

where AG'ois the standardfree-energychange,R is the gasconstant,8.315J/mol . K, and 7 is the absolutetemperature.When the "reaction"is simply transport of a solutefrom a regionwhere its concentrationis Cl to a regionwhereits concentrationis C2,rrobondsare made or broken and AG'ois zero.The free-energychangefor transport,AG,,is then

Calculate the energy cost (free-energy change) of pumping Ca2+from the cytosol,where its concentration is about 1.0 x 10-zu, to the extracellularfluid, whereits concentrationis about 1.0mu. Assumea temperatureof 37'C (body temperaturein a mammal)and a standard transmembranepotential of 50 mV (inside negative)for the plasmamembrane.

AG': PTIn (Cz/C)

Solution:In this calculation,both the concentrationgradient and the electricalpotential must be taken into account.In Equation11-4, substitute8.315J/mol. K for.R, 310K for f, 1.0x 10-3for C2,1.0x 10-7for C1,96,b00 J/V . mol for 'J, +2 (the chargeon a Ca2*ion) for Z, and 0.050V for Arl. Note that the transmembranepotential is 50 mV (insidenegative),so the changein potential when an ion movesfrom inside to outsideis 50 mV.

( 11-3)

If there is a l0-fold differencein concentrationbetween two compartments,the cost of moving I mol of an unchargedsoluteat 25 'C uphill acrossa membraneseparating the compartmentsis AG = (8.315 J/mol.K)(298K) tn (10/1): 5.700J/mol : 5.7kJ/mol Equation11-3 holdsfor all unchareedsolutes.

I

pumping W0RKED EXAMPIE 11-1 EnergyCostof anUncharged Solute

Calculate the energy cost (free-energy change) of pumpingan unchargedsoluteagainsta 1.0 x 104-fold concentrationgradientat 25 "C. Solution:Beginwith Equation11-3.Substitute1.0 x 104 for (C2/C),8.315J/mol. K forR, and 298K for ?: A,G1: RTln (C2/Cr) : ( 8 . 3 1 5J / m o l . K ) ( 2 9 8K ) l n ( 1 . 0x 1 0 4 ) : 23 kJ/mol

Whenthe soluteis an,ion,its movementwithout an accompanying counterionresultsin the endergonicseparation of positive and negative charges,producing an electricalpotential;such a transport processis said to be electrogenic. The energeticcost of moving an ion dependson the electrochemical potential (p. 390), the sum of the chemicaland electricalgradients: AG,:471" (C2/C)+ Zd Lr!

Srrr-+t

where Z is the chargeon the ion, J is the Faradayconstant (96,480J/V . mol), and Ary'is the transmembrane electricalpotential (in volts). Eukaryoticcellstypically haveplasmamembranepotentials of about 0.05V (with

AGr:47hr (C2/C)+ 2,7L{r 1'0x 10-: : (8.315 J/mol.K)(310 K) --h 1.0x 10-7 + 2 (96,500 J/V.mol)(0.050 V) : 33kJ/mol

The mechanismof active transport is of fundamental importanceur biology.As we shall seein Chapter 19, ATP is formed in mitochondria and chloroplasts by a mechanismthat is essentiallyATP-drivenion transport operating in reverse.The energy made availableby the spontaneousflow of protonsacrossa membraneis calculablefrom Equation11-4; rememberthat AG for flow down an electrochemicalgradient has a negativevalue, and AG for transport of ions agai,nstan electrochemical gradient has a positive value.

P-Type ATPases Undergo Phosphorylation (atalytic (ycles during Their The family of active transporters called P-type ATPases are cationtransportersthat are reversiblyphosphorylated by ATP (thus the name P-type) as part of the transport cycle.Phosphorylationforcesa conformational changethat is central to movementof the cation across the membrane.The humangenomeencodesat leastZ0 P-type MPases that sharesimilarities in amino acid sequenceand topology,especiallynear the Asp residue

'I 5olute across Membranes Transport 1.3 [rtf that undergoesphosphorylation.All are integral proteins with B or 10 predicted membrane-spanning regionsin a singlepolypeptide(type III in Fig. 11-8), and all are sensitive to inhibition by the phosphateanalogvanadate

o-o tl o:P-o tl

OH Phosphate

Transmembrane(M) ilomain

Lumon

o:v-o OH Vanadate

The P-type ATPasesare widespreadin eukaryotes and bacteria.In animal tissues,the Caz+ATPase(a uniporter for Caz* ions) and the Na*K* ATPase(an antiporter for Na+ and K* ions) are P-type MPases that maintaindifferencesin ionic compositionbetweenthe cytosol and the extracellularmedium. Parietal cells in the IrurLgof the mammalianstomachhave a P-type ATPase that pumps H* and K* acrossthe plasmamembrane, thereby aciffiing the stomachcontents.Lipid flrppases, as we noted above,are structurally and functionally related to P-type transporters.In vascularplants,a P-type ATPasepumps protons out of cells,estabJishing an electrochemicaldifferenceof as much as 2 pH units and 250 mV acrossthe plasmamembrane.A similar P-type ATPase in the bread mold Neurospora pumps protons out of cells to establishan inside-negativemembranepotential, which is used to drive the uptake of substratesand ions from the surroundingmedium by secondaryactive transport.BacteriauseP-typeATPasesto pump out toxic hear,ymetalionssuchasCdz' andCuzt. The best-understoodP-type pumps are the Caz* pumps that maintain a low concentrationof Ca2* in the c1'tosolof virtually all cells. The plasma membrane Ca2* pump movescalciumions out of the cell,and another P-typepump in the endoplasmicreticulummoves ^ t+. ^ rl Ca'- into the ER lumen.(ln musclecells,Ca"' is normally sequestered in a specialized form of ER calledthe reticulum; release sarcoplasmic of this Ca2* triggers musclecontraction.) The sarcoplasmic and endoplasmic reticulum calcium (SERCA) pnmps are P-typeMPases closely relatedin structureand mechanism.The SERCApump reticulum,which comprisesB0%of of the sarcoplasmic the protein in that membrane,is a singlepolypeptide (M. -110,000) that spans the membrane 10 times (Fig. I l-35). Three cytosolicdomainsformed by long loops connect the transmembranehelices:the N domain, where the nucleotideATP and Mg2* bind; the P domain,which containsthephosphorylatedAsp residue characteristicof all P-type ATPases;and the A (octuator) domain,which communicatesmovementsof the N and P domainsto the two Ca2*-bindingsites.The M dohelicesand the Caz*main containsthe transmembrane binding sites, which are located near the middle of the membranebilayer,40 to 50 A from the phosphorylated Asp residue-thus Asp phosphorylation-dephosphoryIationdoesnot directlyaffectCaz*bincling.

Qytocol Actuator (A) domain

ca9*

(F) Phosphorylation domain Nucleotidebinding (N) domain

Phosphorylation site ATP-binding site reticulum:a SERCA 11-35 The Ca2+pump of sarcoplasmic FIGURE helices(the M domain, pump. (PDB lD lEUL) Ten transmembrane framedin pink) surroundthe path for Ca2* movementthroughthe membraneTwo of the helicesare interruptednearthe middle of the bilayer;their nonhelicalregionsform the bindingsitesfor two Ca2+ groupsof an Asp residuein one helix ions(purple)The carboxylate sites and a Clu residuein anothercontributeto the Ca2+-binding Three globular domains extend from the cytosolicside: the N (nucleotide-binding) domain(framedin green)containsthe binding (phosphorylation) domain(orangecircle)contains sitefor ATP;the P andthe phosphorylation, reversible that undergoes residue theAsp351 changes that mediates thestructural A (actuato|domain(bluetriangle) to cytosol siteand its exposure alterthe a{finityof the Ca2*-binding site or lumen.Note the long distancebetweenthe phosphorylation is the prototype pumpstructure site.ThisSERCA andtheCa2*-binding and it containssome residues(shownin red) for all P-typeATPa.ses, family. that are conservedin all membersof the Ptype ATPase

The mechanism postulated for SERCA pumps (F-iS. f 1-3ti) takes into account the large conformational changesand the phosphorylation-dephosphorylation of the critical Asp residue in the P domain that is known to occur during a cataly'ticcycle. Each catalytic cycle movestwo Ca2* ions acrossthe membraneand convertsan ATP to ADP and P1.The role of ATP binding and hydrolysisis to bring about the interconversionof two conformations(El and E2) of the transporter'In the E1 conformation,the two Ca"--bindingsitesare exposed on the cytosolicside of the ER or sarcoplasmic reticulum and bind Ca2+with high affinity. ATP binding and Asp phosphorylationdrive a -conformational changefrom El to E2 in which the Ca2+-bindingsites are now exposedon the lumenalside of the membrane and their affinity for Ca2* is greatly reduced,causing Caz+releaseinto the lumen. By this mechanism,the energy released by hydrolysis of ATP during one

[trt]

Biological Membranes andTransport El-Pi

e ^

9+

Ua-

Transporter binds 3 Na+ from the inside ofthe cell.

and

ATP bind; N domain moves.

Phosphorylation favors P-Enz11.

Transporter releases 3 Na+ to the outside and binds 2 K+ from the outside ofthe cell.

ca2*

cc

--

o

Na+

2K*

Dephosphorylation favors Enz1.

Me*

Transporter releases 2 K+ to the inside. FIGURE 1l-36 Postulatedmechanismof the SERCApump. Thetransport cycle beginswith the proteinin the El conformation,with the Ca2+bindingsitesfacingthe cytosol.Two Ca2+ionsbind,thenATpbindsto the transporterand phosphorylates Asp3sl,forming E1-p.phosphorylationfavorsthe secondconformation, E2-p,in which the Ca2+-binding sites,now with a reducedaffinityfor Ca2*,are accessible on the other side of the membrane(the lumen or extracellular space),and the releasedCa2+diffusesaway.Finally,E2-Pis dephosphorylated, returning the proteinto the El conformation for anotherroundof transpon.

phosphorylation-dephosphorylation cycle drives Ca2+ acrossthe membraneagainsta large electrochemical gradient. A variation on this basic mechanism is seen in the Na+K+ AfPase of the plasmamembrane,discovered by Jens Skou in 1957.This cotranspofter couples phosphorylation-dephosphorylationof the critical Asp residueto the simultaneousmovementof both Na+ and K+ againsttheir electrochemicalgradients The Na*K' ATPaseis responsiblefor m Na+ and high K+ concentrationsin the cell extracellular fluid (Fig. 1f€8). For each molecule of FIGURE I I -38 Roleof the Na+K+ATpasein animalcells.In animalcells, thisactivetransport systemis primarilyresponsible for seftingand maintainingthe intracellular concentrations of Na+ and K+ andfor generating the membranepotential.lt doesthisby movingthreeNa+ out of the cell for everytwo K+ it movesin. The electricalpotentialacrossthe plasma membrane is centralto electricalsignalingin neurons, andthegradientof Na+ is usedto drivethe uphillcotransport of solutesin manycell types.

Inside

Outside

FIGURE 11-37 Postulatedmechanismof the Na+K+ATpase.

cytoeol rN;filil;,

+ Extracellular fluid or blood plasma

[K+1 = 4 -rrr [Na+] = 146 mu

39 across Membranes Transport 11.3Solute

JensSkou

ATP convertedto ADP and P1, the lransportermovestwo K* ionsinward and three Na* ions outward across the plasma membrane Cotransport is therefore electrogenic-it createsa net separationof charge acrossthe membrane;in animals, this producesthe membranepotentialof -50 to -70 mV (insidenegativerelativeto outside) that is characteristic

of most cells and is essential to the conduction of action potentials in neurons. The central role of the Na-K- ATPase is reflected in the energy invested in this single reaction: about 25o/oof the total energy consumption of a human at rest!

ProtonPumps F-Type ATPases AreReversible, ATP-Driven F-type ATPase active transporters catalyzethe uphill transmembranepassageof protons driven by ATP hydrolysis. The "F-type" designationderives from the identification of these ATPasesas energy-coupling /actors. The Fo integral membrane protein complex (FiS. 11-39; subscripto denotesits inhibition by the drug oligomycin) provides a transmembranepathway for protons,and the peripheralprotein F1 (subscript1 indicating this was the flrst of several factors isolated from mitochondria)usesthe energyof ATPto driveprotons uphill (into a region of higher H* concentration).

P1

The FoFl organizationof proton-pumping transporters must have developedvery early in evolution. Bacteria such as E coli, use an FoFr MPase complexin their plasmamembraneto pump protons outward, and archaeahavea closelyhomologousprotonPumP,the AoAt ATPase. The reaction catalyzedby F-type ATPasesis reversible,so a proton gradientcan supplythe energyto drive the reversereaction,ATP slmthesis(Fig. 1f-40). When functioning in this direction, the F-type ATPases are more appropriatelynamed ATP synthases. ATP synthasesare central to ATP production in mitochondria during oxidative phosphorylationand in chloroplasts during photophosphorylation,as well as in bacteria and archaea.The proton gradient neededto drive ATP synthesisis producedby other types of proton pumps powered by substrateoxidation or sunlight. We providea detaileddescriptionof theseprocessesin Chapter19. V-type ATPases, a class of proton-transporting ATPasesstructurally (and possiblymechanistically)reIatedto the F-typeATPases,are responsiblefor acidifying intracellular compartments in many organisms (thus 7 for uacuolar).Proton pumps of this type maintain the vacuolesof fungi and higher plants at a pH between 3 and 6, well below that of the surrounding cytosol (pH 7.5). V-type MPases are also responsible the Golgi for the acidiflcationof lysosomes,endosomes, All Vcells. complex,and secretoryvesiclesin animal with an type ATPaseshave a similar complex structure, (Vo) as that serves a integral (transmembrane)domain proton channeland a peripheraldomain (V1) that contains the ATP-bindingsite and the ATPaseactivity. The mechanismby which V-type ATPasescouple ATP hydrolysisto the uphill transport of protonsis not understoodin detail.

Fr ADP rPi

Fo

havea periphF-type.AThses tlGURt11-19 FoFtATPase/ATP synthase. one6subthreeBsubunits, eraldomain,F1,consistingof threeasubunits, anda centralshaft(the7 subunit,green)joinedto the integral unit(purple), hasmultiplecopiesof domainthroughe.Theintegraldomain,Fo(orange), Foprothec subunit(.12shownhere),one a subunit,andtwo b subunits. videsa transmembrane channelthroughwhich protonsarepumped(red of F1. Thereon theB subunits aboutfourfor eachATPhydrolyzed arrows), markablemechanism by whichthesetwo eventsarecoupledis described '19 to Fr (blackanow). rotationof Forelative in detailin Chapter lt involves similarto that areessentially Thestructures of theVoVrandAoA1ATPases areprobablysimilar,too. of FoF1, andthe mechanisms

An ATP-drivenproton 11-40 Reversibilityof F-typeATPases. FIGURE (redarrows)asprotonsflow alsocan catalyzeATPsynthesis transporter gradientThis is the centralreactionin the down Iheirelectrochemical and photophosphorylation, of oxidativephosphorylation processes 19. in in detail Chapter bothdescribed

[oo'_lB i o l o g i cMael m b r a naensdT r a n s p o r t NBDs

tlGURt1l-41 AnABCtransporter of E coli.Thevitamin8,, importer BtuCD(PDBlD l LZV)is a homodimer with 10 rransmembrane helical domains(blue)in eachmonomerandtwo nucleotide-binding domains (NBDs;red)that extendinto the cytoplasmThe residues involvedin ATPbindingand hydrolysis areshownasball-and-stick structures.

ABC Transporters UseATP to DrivetheActiveTransport ofaWide Variety of5ubstrates ABC transporters (l'ig. I l-4f ) constitute a large family of ATP-dependenttransporters that pump amino acids,peptides,proteins,metal ions,variouslipids,bile salts,andmanyhydrophobiccompounds, including drugs,out of cells againsta concentrationgradient. OneABC transporterin humans,the multi-drug transporter (MDRI), is responsiblefor the striking resistanceof certain tumors to somegenerallyeffective antitumordrugs.MDR1hasa broadsubstratespecificity for hydrophobiccompounds,including,for example,the chemotherapeuticdrugs adriamycin, doxorubicin, and vinblastine.By pumpingthesedrugsout of the cell,the transporter prevents their accumulationwithin a tumor and thus blockstheir therapeuticeffects.MDR1is an integral membraneprotein (M. 170,000)with 12 transmembrane segments and two ATp-binding domains ("cassettes"), which givethe familyits name:,ATp-binding cassettetransporters.I AXABC transportershavetwo nucleotide-bindingdomains (NBDs) and two transmembranedomains.In some cases,all these domainsare in a sin$e long polypeptide; other ABC transporters have two subunits, each contributing an NBD and a domainwith six (or in somecases 10;Fig. 11-41)transmembrane helices.ManyABC transporters are in the plasmamembrane,but sometypes are alsofound in the endoplasmicreticulum and in the membranesof mitochondnaand lysosomes.Most ABC transporters act as pumps,but at Ieast somemembersof the superfamily act as ion channels that are opened and closedbyATP hydrolysis.The CFTRtransporter(seeBox 11-3) is a Cl- channeloperatedby ATp hydrolysis. The NBDs of all ABC proteins are similar in sequenceand presumablyin three-dimensionalstructure; they are the conservedmolecularmotor that can be

coupledto a wide variety of pumps and channels.When coupled with a pump, the ATP-driven motor moves solutesagainsta concentrationgradient; when coupled with an ion channel,the motor opens and closesthe channel,usingATP as energysource.The stoichiometry of ABC pumpsis aboutoneATP hydrolyzedper molecule of substratetransported,but neither the mechanismof coupLingnor the site of substratebinding is known. SomeABC transporters have very high specificrty ffi for a srrglesubstrate;othersaremorepromiscuous. S The humangenomecontainsat least48 genesthat encode ABC transporters,many of which are involvedin maintaining the lipid bilayer and in transporting sterols, sterol derivatives,and fatty acids throughout the body. The flippasesthat movemembranelipidsfrom oneleafletof the bilayer to the other are ABC transporters,and the cellular machineryfor exporting excesscholesterolincludes an ABC transporter. Mutations in the genes that encode someof theseproterrs contributeto severalgeneticdiseases,includingcysticf,brosis(Box 11-3), Tangierdisease anemia,andliver failure. Cr.843),retinal degeneration, ABC transportersare alsopresentin simpleranimals and in plants and microorganisms.Yeasthas 31 genes that encodeABC transporters,Drosopthi,lahas 56, and E coli, has 80, representing2o/oof its entire genome. The presenceofABC transportersthat conferantibiotic resistance in pathogenic microbes (Pseudomonas aerug i,nosa, Staphy lococcus aureus, Candi,da albi,ca:ns,Ne'isseri,agonorrhoeae, andPlasmodi,umJalcipantm) is a serious public health concern and makes these transporters attractive targets for drug design.r

lonGradients Provide theEnergy forSecondary Active Transport The ion gradientsformed by primary transport of Na+ or H* can in turn provide the driving force for cotransport of other solutes.Many cell types containtransportsystems that couple the spontaneous,downhill flow of these ions to the simultaneousuphill pumping of another ion, sugar,or amino acid (Table lI-4).

0rganism/ tissue/celltype

Tfansported Cotransported solute (moving solute (moving againstits downits llpeof gfadient) gradient) transport

E coli,

Lactose

TT+ n

S;'rnport

Proline

Lr+

S;rmport

rr+ 11

S}.'rnport

NaNa*

Syrnport S;'rnport

Na*

Antiport

rr+ n

Antiport

H-

Antiport

Dicarboxylicacids Intestine,kidney Glucose (vertebrates) Amino acids Vertebratecells Ca2* (many tlpes) Higher plants K+ Fungl (Neurospora)

r.+ I

s a rctr 0 sMse m b r a n e[-"] 11 . 3S o l u tTer a n s p o

Cystic flbrosis (CF) is a seriousand relatively common hereditarydiseaseof humans.About 5% of white Americans are carriers,having one defectiveand one normal copy of the gene. Only individuals with two defective copies show the severesyrnptomsof the disease:obstruction of the gastrointestinaland respiratorytracts, commonly Ieading to bacterial infection of the airways and death due to respiratoryinsufficiencybefore the age of 30. In CF, the thin layer of mucus that normally coats the internal surfaces of the lungs is abnormally thick, obstructing air flow and providing a haven for pathogenicbacteria,particularly Staphglococcusaureus andPseudomonasaerugInosa. The defectivegenein CF patientswas discoveredin 1989.It encodesa membraneprotein calledcysticy'brosis fransmembraneconductanceregulator,or CFTR. This protein hastwo segments,eachcontainingsix transmembrane helices,two nucleotide-bindingdomains (NBDs), and a regulatoryregion (Fig 1) CFTRis thereforevery similar to other ABC transporter proteins. The normal CFTRprotein provedto be an ion channelspeci-flcfor Clions.The channelconductsCl- acrossthe plasmamembrane when both NBDs have bound ATP. and it closes FIGURE I Threestatesof the cysticfibrosistransmembrane conductanceregulator, CFTR.The proteinhastwo segments, eachwith six transmembrane helices,and threefunctionallysignificantdomains extendfrom the cytoplasmic surface:NBDI and NBD2 (green)are nucleotide-binding domainsthat bind ATB and a regulatory domain (blue)is the site of phosphorylation protein by cAMP-dependent kinase. WhenthisR domainis phosphorylated but no ATPis boundto the NBDs(left),the channelis closed.The bindingof ATPopensthe channel(middle)untilthe boundATPis hydrolyzed. When the regu(right),it bindsthe NBD domains latorydomainis unphosphorylated and prevents ATPbindingand channelopening.Themostcommonly occurringmutationleadingto CF is the deletionof Phesosin the in all but two NBDl domain(left) CFTRis a typicalABC transporter respects: mostABCtransporters lackthe regulatory domain,andCFTR actsasan ion channel(forCl ), not asa typicaltransporter

2 Mucus lining the surfaceof the lungstrapsbacteria.In FIGURE healthyIungs(shownhere),thesebacteriaare killedand sweptaway resulting in reis impaired, by theactionof cilia In CF,thismechanism lungs. to the damage and progressive curringinfections

when the ATP on one of the NBDsis broken doumto ADP and P1.The Cl- channelis further regulatedby phosphorylation of severalSer residuesin the regulatorydomain, catalyzedby cAMP-dependentprotein kinase (Chapter 12). When the regulatorydomainis not phosphorylated, the Cl- channelis closed.The mutation responsiblefor CF in 70%of casesresultsur deletionof a Phe residueat position508.The mutant protein folds incorrectly,which interfereswith its insertionin the plasmamembrane,resulting in reduced Cl movement across the plasma membranesof epithelial cells that line the airways (Fig. 2), the digestivetract, and exocrine glands (pancreas, sweatglands,bile ducts,andvasdeferens), Diminishedexport of Cl- is accompaniedby diminished export of water from cells, causingthe mucus on their surfacesto becomedehydrated,thick, and excessivelysticky.In normalcircumstances,cilia on the epithelial cellsthat line the inner surfaceof the lungsconstantly sweep away bacteria that settle in this mucus, but the thick mucus in individualswith CF hinders this process. Frequenthfections by bacteriasuchasS. aureus andP. aentg'inosa result, causingprogressivedamageto the Iungsand reducedrespiratoryefficiency.Respiratoryfailure is commonlythe causeof deathin peoplewith CF.

Channel open R domain phosphorylated AIP bound to NBDs

Channel closed R domain phosphorylated No ATP bound to NBDs Outside

R domain

(@ser)

Inside

ooc

Channel closed R domain unphosphorylated No ATP bound to NBDs

Biological Membranes andTransport

Ftt (a)

(b) Il-r

Lactose rransporter

H+ H+ g+ '

r*

^H^- .

H-

Proton purnp (inhibitedby CN-)

+ T" +

Fl

[LactoseJ-"6;;

H+ (inside)

+ CN-, or mutation at Glu325orArgJo2

H+

Time

FfGURE 11-42 lactose uptakein E. coli. (a) The primarytransporrof H+ out of the cell, drivenby the oxidationof a varietyof fuels,establishesboth a protongradientand an electrical potential(insidenegative) acrossthe membrane.Secondaryactivetransportof lactoseinto the cell involvessymportof H+ and lactoseby the lactosetransporter. The uptakeof lactoseagainstits concentration gradientis entirely

dependenton this inflowof protonsdrivenby the electrochemical gradient.(b)Whenthe energy-yielding oxrdation reactions of metabolism areblockedby cyanide(CN-),the lactose transporter allowsequilibration of lactoseacrossthe membranevia passivetransportMutations thataffectClu32sor Arg3o2 havethe sameeffectascyanide.Thedashed linerepresents theconcentration of lactosein thesurrounding medium.

The lactose transporter (lactose permease) of E. coli, is the well-studied prototlpe for proton-driven cotransporters. This protein consists of a single polj.pep-

superfamily have 12 transmembrane domains (the few exceptions have 14). The proteins share relatively little

tide chain(417residues)that functionsasa monomerto transportone proton and one lactosemoleculeinto the cell,with the net accumulationof lactose(Fig. 11-42). E coli, normally produces a gradient of protons and charge across its plasma membraneby oxidizing fuels and using the energyof oxidation to pump protons outward. (This mechanismis discussedin detail i_nChapter 19.) The lipid bilayeris impermeableto protons,but the lactosetransporterprovidesa route for proton reentry, and lactoseis simultaneouslycarrieclinto the cell by s)./mport.The endergonicaccumulationof lactose is therebycoupledto the exergonicflow ofprotonsinto the cell, with a negativeoverallfree-energychange. The lactosetransporteris one memberof the major facilitator superfamily (MFS) of transporters, which comprises28 families Almost all proteinsin this

sequencehomology,but the similarity of their secondary structures and topology suggestsa common tertiary structure.The crystallographicsolutionof the E co\i,lactose transporter may proude a ghmpseof this general structure(Fig. f 1-43a). The proteinhas 12 transmembranehelices,and connectlngloopsprotrude into the cytoplasm or the periplasmicspace (between the plasma membraneand outer membraneor cell wall). The six amino-terminaland six carboxyl-termrnalhelices form very similardomains,to producea structurewith a rough twofold symmetry.In the crystallizedform of the protein, a largeaqueouscavity is exposedon the cy[oplasmicside of the membrane.The substrate-bindingsite is in this cavity,more or lessin the middle of the membrane.The side of the transporter facing outward (the periplasmic face) is closedtightly, with no channelbig enough for lactose to enter. The proposed mechanismfor

Cy'toplasm

111

'ii: itl:*tt l') i-

,.i

(a)

Periplasmic space

FfGURE l1-43 The lactosetransporter(lactosepermease)ol E. coli. (a) Ribbonrepresentation viewedparallelto the planeof the membrane showsthe 12 transmembrane helicesarrangedin two nearlysymmetric domains,shownin differentshadesof purple In the form of the protein for which the crystalstructurewasdetermined, the substrate sugar(red) isboundnearthe middleof the membranewhereit isexposedto the cytoplasm(derivedfrom PDBlD -lPV7).(b)Thepostulated secondconfor-

(b)

mationof thetransporter, relatedto the firstby a large,reversible conformationalchangein which the substrate-binding site is exposedfirstto the periplasm(conformation on the right),wherelactoseis pickedup, thento the cytoplasm(conformation on the left),wherethe lactoseis released. Theinterconversion of the two formsis drivenby changesin the (protonatable) pairingof charged sidechainssuchasthoseof Clu325 and Arg3o2 which is affectedby the transmembrane protongradient. lgreen),

s a rctr o sMse m b r a n e[-rr] 11 . 35 o l u tTer a n s p o

transmembranepassageof the substrate(Fig. 11-43b) involvesa rocking motion between the two domains, driven by substratebinding and proton movement,alterdomainto the cynately exposingthe substrate-bindtrLg toplasm and to the periplasm.This "rocking banana" modelis similarto that shownrn Figure 11-31 for GLUTl. How is proton movementinto the cell coupledwith Iactoseuptake?Extensivegenetic studiesof the lactose transporter have establishedthat of the 417 residuesin the protein,only 6 are absolutelyessentialfor cotransport of H+ and lactose-some for lactosebinding, others for proton transport. Mutation in either of two residues (Glu325and Arg302;Frg. 11-43) results ur a protem still able to catalyzefacilitated djffusion of lactosebut incapable of coupling H* flow to uphill lactosetransport A simrlareffectis seenin wild-type (unmutated)cellswhen their ability to generatea proton gradientis blockedwith CN : the transportercarriesout facilitateddi-ffusionnormally,but it carLnotpump lactoseagainsta concentration gradient (Fig. 11-42b). The balancebetweenthe two conformationsof the lactose transporter is affected by changesin chargepairingbetweenside chatns. In intestinal epithelial cells, glucose and certain amino acids are accumulatedby symport with Na*, down the Na- gradientestablishedby the Na-K- ATPase of the plasmamembrane(Fig. 11-44). The apical surface of the intestinal epithelial cell is covered with microvilli, Iongthin projectionsof the plasmamembrane that greatly increasethe surfacearea exposedto the intestinal contents.Na*-glucose symporters in the apicalplasmamembranetake up glucosefrom the intestine in a processdriven by the dov'nhill flow of Na-: 2Naj.1 * glucoseo,, ------+2Nai' + glucosei,

Microvilli Epithelial cell

a. 2 Na+ 1;

ar

Pumping 11-3 Energeticsof EXAMPTE W0RKED bySymport

Calculate the maximu^

2K+

Solution:Using Equation 11-4 (p. 396), we can calculate the energy inherent in an electrochemicalNagradient-that is, the cost of moving one Na+ ion up this gradient: LG,: P71n*F

Na+-glucose symporter (driven by extracellul

We then substitute standard values for R , T , and J, and the given values for [Na+] (expressed as molar concentrations), *1 for Z (because Na- has a positive charge), and 0.050 V for Arl. Note that the membrane potential is - 50 mV (inside negative) , so the change in potential when an ion moves from inside to outside is 50 mV. ' '--13!119 2 J/mol'K)(3101111" AG, = 13.315 1.2 x to V) + 1(96,500 J/V'mol)(0.050

1SNa-

Glucose umporter (facilitates efflux)

11-44 Glucosetransportin intestinalepithelialcells.CluFIGURE with Na* acrossthe apicalplasmamembrane coseis cotransported into the epithelialcell. lt movesthroughthe cell to the basalsurface, glucoseuniporter. intothebloodvia CLUT2,a passive whereit passes continues to pump Na* outwardto maintainthe The Na+K" ATPase Na* gradientthatdrivesglucoseuptake.

+ zJ ^ir

lNali.

= p7t".qEqts LG. Iglucoselout

,a

ATPase

()

ratio that can be

achievedby the plasmamembraneNa*-glucoses;rrnporter of an epithelial cell, when [Na+]6 is 12 mnt, -50 mV [Na+]o,tis 145 mvt,the membranepotentialis (insidenegative),and the temperatureis 37'C.

A

Glucose

[4ucose-i" lglucoselout

This AG1is the potential energy per mole of Na+ in the Na+ gradient that is available to pump glucose' Given that two Na* ions pass down their electrochemical gradient and into the cell for each glucose carried in by slrnport, the energy available to pump I mol of glucose is 2 x ll.2 kJ/mol = 22.4 kJ/mol' We can now calculate the concentration ratio of glucose that can be achieved by this pump (from Equation 11-3, p. 396):

\ru

Intestinal lumen

I

: 11.2kJ/mol

Basal surface

Apical surface

The energyrequired for this processcomesfrom two sources:the greaterconcentrationof Na* outsidethan inside (the chemical potential) and the membrane (electrical) potential, which is inside negative and therefore draws Na+ inward.

Rearranging,then substitutingthe valuesof AGr,-R,and 7, gives 22.4kJlmoI lglucoselin : AG, : _ ,.U, ,(8.315J/mol'K)(310 K) "' [glucoselo,, R,T [glucose]i' - ,r..n [glucose]o', : 5.94 x 103

Thus the cotransporter can pump glucoseinward until its concentrationinside the epitheliatcell is about 6,000 times that outside (in the intestine).

Membranes andTransport !404] Eiological As glucoseis pumpedfrom the intestineinto the epithelial cell at the apical surface,it is simultaneously movedfrom the cell into the bloodby passivetransport througha glucosetransporter(GLUT2)in the basalsurface (Fig. Il-44). The crucial role of Na* in s1rnport and antiportsystemssuchasthis requiresthe continued outward pumping of Na+ to maintain the transmembraneNa* gradient. Begauseof the essentialrole of ion gradientsin ffi actlve transport and energy conservation,com_ E pounds that collapse ion gradients across cellular membranesare effective poisons,and those that are specificfor infectiousmicroorganrsms can serveas antibiotics. One such substanceis valinomycin,a small cyclic peptide that neutralizesthe K+ chargeby surrounding it with six carbonyl oxygens(FiS. lf_4b). The hydrophobicpeptide then acts as a shuttle, carrying K- acrossmembranesdown its concentrationgradient and deflating that gradient. Compounds that shuttle ions acrossmembranesin this way are called ionophores ("ion bearers").Both valinomycinand monensin (a Na--carrying ionophore) are antibiotics; they kill microbialcells by disrupting secondarytransport processesand energy-conserving reactions.Mon_ ensin is widely used as an antifungaland antiparasitic agent.r

FIGURE 11-45 Valinomycin, a peptideionophore thatbindsK+. In this image,thesurfacecontoursareshownasa transparent mesh,through whicha stickstructure of thepeptideanda K+ atom(green) arevisible. Theoxygenatoms(red)thatbind K+ are partof a centralhydrophilic cavity.Hydrophobic aminoacid sidechains(yellow)coatthe outside of the molecule.Because the exteriorof the K+-valinomycin comolex is hydrophobic, the complexreadilydiffuses throughmemoranesi car_ ryingK- down its concentration gradientTheresulting dissipation of the transmembrane ion gradientkills microbialcells,makingvalinomycina potentantibiotic

Aquaporins Form Hydrophilic Transmembrane (hannels forthePassaqe ofWater A family of integral membrane proteins discovered by Peter Agre, the aquaporins (AQPs), provide channels for rapid movement of water molecules across all plasma membranes. Eleven aquaporinsare known in mammals,each with a specific localizationand role (Table11-5, p. 406). Erythrocl'tes, which swell or shrink rapidly ir rePeterAgre sponseto abrupt changesin extracellular osmolarityas blood travels through the renal medulla,havea hrgh densityof aquaporinin their plasma membrane(2 X 105copiesof AQP-1per cell). Watersecretionby the exocrineglandsthat producesweat,saliva, and tears occurs through aquaporins.Seven different aquaporinsplayrolesin urine productionandwater retention in the nephron (the functional urLitof the kidney). EachrenalAQPhasa specificlocalizationin the nephron, and eachhas specificpropertiesand regulatoryfeatures. For example,AQP-2in the epithelialcellsof the renal collecting duct is regulatedby vasopressin(also calledantidiuretichormone):morewateris reabsorbedin the kidney when the vasopressinlevel is high. Mutant mice with no AQP-I gene have increasedurine output (polyuria) and decreasedurine-concentratingabitity, the resr.rltof decreasedwater permeabilityof the proximal tubule.In humans, genetically defective AQPs are known to be responsiblefor a variety of diseases,includinga relativeiy rare form of diabetesthat is accompaniedby polyuria (Box 11-2). Aquaporins are found in all organisms.The plant Arabidopsi,s thali,ana has 38 AQP genes,reflecting the critical roles of water movementin plant physiology. Changesin turgor pressure,for example,require rapid movementof water acrossa membrane(seep. b2). Watermoleculesflow through an AQp-l channelat a rate of about 10es-1. For comparison,the highest known turnover number for an enzl'rneis that for catalase, 4 x 107 s-1, and many enzymeshave turnover numbersbetween 1 s-r and 104 s-1 (see Table 6-Z). The low activation energy for passageof water through aquaporinchannels(AG* . 15kJ/mol)suggeststhat water movesthrough the channelsin a continuousstream, in the directiondictatedby the osmoticgradient.(For a discussionof osmosis,seep 52.) Aquaporinsdo not alIow passageof protons (hydroniumions,H3O+),which would collapsemembraneelectrochemicalgradients. What is the basisfor this extraordinaryselectivity? We find an answerin the structure of Aep-1, as detennined by x-ray crystallography. Aep-1 (Fig. f l-46a) consistsof four identicalmonomers(eachM,28,000), each of which forms a transmembrane pore with a

r a n s p oar ct r 0 sMse m b r a n e| s4 0 5 | I 1 . 3S o l u tl e

Electrostatic repulsion

FIGURE 11-46 Aquaporin.The proteinis a tetramerof identicalsubunits, each with a transmembranepore. (a) A monomer of (derivedfrom PDB lD 285F),viewedin spinachaquaporinSoPlP2;1 The helicesform a centralpore,and two the planeof the membrane. (green) containtheAsn-Pro-Ala(NPA)sequences, shorthelicalsegments found in all aquaporins, thatform partof the waterchannel.(b) This from PDBlD 1J4N)showsthat cartoonof bovineaquaporin1 (derived shownin redand white) the pore(brown;filledwith watermolecules

of 2.8 A (aboutthesizeof a watermolnarrowsat Hisl80to a diameter of moleculeslargerthan H2O.The positive ecule),limitingpassage theirpaschargeof Arglesrepelscations,includingH3O', preventing are oriin green shown helices sagethroughthe pore.The two short pore in such at the pointed positively dipoles charged entedwith their this passes through; as it reorient to molecule a water a way asto force preventing molecules, water of chains hydrogen-bonded up breaks by "protonhopping"(seeFig.2-1 3). protonpassage

diameter sufflcient to allow passage of water molecules in single file. Each monomer has six transmembrane helical segments and two shorter helices, both of which contain

ter molecules close enough to allow proton hopping (see Fig. 2-13), which would effectively move protons across

the sequenceAsn-Pro-Ala (NPA). The six transmembrane helicesform the pore through the monomer,and the two short loopscontainingthe NPAsequencesextend toward the middle of the bilayer from oppositesides. Their NPAregionsoverlapin the middle of the membrane to form part of the specificityfllter-the structurethat alIowsonly water to pass(Fig. 11-46b). The water channelnarrowsto a diameterof 2.8 A near the center of the membrane,severelyrestricting the size of moleculesthat can travel through. The positive chargeof a highly conservedArg residueat this bottleneck discouragesthe passageof cations such as H3O* The residuesthat line the channelof eachAQP-I monomer are generallynonpolar,but carbonyl oxygens in the peptidebackbone,projectinginto the narrowpart of the channelat intervals, can hydrogen-bondwith individual water moleculesas they passthrough; the two Asn residues(Asn76and Asnle2)in the NPA loops also form hydrogen bonds with the water. The structure of the channeldoesnot permit formation of a chain of wa-

the membrane.Critical Arg and His residues and electric dipolesformed by the short helicesof the NPAloops provide positive chargesin positionsthat repel any protons that might leak through the pore, and prevent hydrogenbondingbetweenadjacentwater molecules. An aquaporinisolated from spinachis known to be "gated"-open when two critical Ser residuesnear the intracellular end of the channelare phosphorylated,and Both the open closedwhen they are dephosphorylated. by crystaldetermlned and closedstructures havebeen that a conformation lography. Phosphorylationfavors into residue His a pressestwo nearbyLeu residuesand past that water of the channel,blocking the movement point and effectively closing the channel. Other aquaporins are regulated in other ways, allowing rapid changesin membranepermeabilityto water. Although generally highly speciflc for water, some AQPs also allow glycerol or urea to pass at high rates (Table 11-5); theseAQPsare believedto be important in the metabolism of glycerol. AQP-7, for example, found in the plasma membranesof adipocytes (fat

[-td

Biotogicat Membranes andlransport

Aquaporin

Permeant(permeability)

fissue distribution

Subcellulardistribution*

AQP-O

Water (low)

Lens

Plasmamembrane

AQP-1

Water (high)

Erythrocyte, kidney,Iung, vascular endothelium,brain, eye

Plasmamembrane

AQP-2

Water (high)

Kidney,vas deferens

Apical plasmamembrane, intracellular vesicles

AQP-3

Water (high), glycerol (high), urea (moderate)

Kidney,skin, lung, eye, colon

Basolateralplasmamembrane

AQP-4

Water (high)

Basolateralplasmamembrane

AQP-5

Water (high)

Apical piasmamembrane

AQP-6

Water (low), anions (NOt > Cl-)

Brain, muscie,kidney,lung, stomach, small htestine Salivarygland,lacrimal gland,sweat gland,lung, comea Kidney

AQP-7

Water (high), gycerol (high), urea (high), arsenite

Adiposetissue,kidney,testis

Plasmamembrane

AQP-8r

Water (high)

Testis,kidney,liver, pancreas,small intestine, colon

Plasmamembrane,intracellular vesicles

AQP-9

Water (low), glycerol (high), urea (high), arsenite

Liver, Ieukocy'te,brain, testis

Plasmamembrane

AQP-10

Water (low), glycerol (h€h), urea (high)

Smallintestine

Intracellularvesicles

Intracellularvesicles

source:DatafromKing,1.S.,Kozono, D.,& Agre,P (2004)Fromstructure to disease: theevolving taleof aquaporin biology. Nat Rev.5, 6gg. *Aquaporins thatarepresent primarily in theapicalor in thebasolateral membrane arenotedas localized in oneof thesemembranes; those present in bothmembranes aredescribed as localized in theplasmamemDrane. rAgP-8mightalsobe permeated by urea.

cells), transportsglycerol efficiently.Mice with defective AQP-7 develop obesity and adult-onsetdiabetes, presumablyas a result of their inability to moveglycerol into or out of adipocytesas triacylglycerolsare converted to free fatty acidsand glycerol,and \,'lceversa.

lon-5elective Channels AllowRapid Movement oflonsacross Membranes Ion-selective channels-first recognized in neurons and now known to be present in the plasma membranes of all cells, as well as in the iltracellular membranes of eukaryotes-provide another mechanism for moving rnorganic ions across membranes. Ion channels, together with ion pumps such as the Na+K+ ATpase, determine a plasma membrane's permeability to specific ions and regulate the cytosolic concentration of ions and the membrane potential. In neurons, very rapid changes in the activity of ion channels cause the changes in membrane potential (action potentials) that carry signals from one end of a neuron to the other. In myocy,tes, rapid opening of Caz+ chamels in the sarcoplasmic reticulum releases the Ca2+ that triggers muscle contraction. We discuss the signaling functions of ion channels in Chapter 12. Here we describe the structural basis for ion-channel

function, using as examples a voltage-gated K+ channel, the neuronal Na+ channel, and the acetylcholine receptor ion channel Ion channels are distinct from ion transporters in at least three ways. First, the rate offlux through channels can be several orders of magnitude greater than the turnover number for a transporter-lO7 to 108 ions/s for an ion channel, approaching the theoretical maximum for unrestricted diffusion. By contrast, the turnover rate of the Na*K* ATPase is about 100 s-1! Second, ion channels are not saturable: rates do not approach a maximum at high substrate concentration. Third, they are gated in response to some cellular event. In ligand-gated channels (which are generally oligomeric), binding of an extracellular or intracellular small molecule forces an allosteric transition in the protein, which opens or closes the channel. In voltage-gated ion channels, a change in transmembrane electrical potential (Z-) causes a charged protein domain to move relative to the membrane, opening or closing the channel. Both types of gating can be very fast. A channel typically opens in a fraction of a millisecond and may remain open for only milliseconds, making these molecular devices effective for very fast signal transmission in the nervous system.

a rctr o sMse m b r a n e s{ 0 11 . 3S o l u tTer a n s p o

lon-(hannel Funrtion lsMeasured ilectrieally Because a single ion channel typicaily remains open for only a few milliseconds, monitoring this process is beyond the limit of most biochemical measurements. Ion fluxes must therefore be measured electrically, either as changes in 7- (in the millivolt range) or as eiectric currents 1 (in the microampere or picoampere range), using microelectrodes and appropriate amplifiers. In patchclamping, a technique developed by Erwin Neher and Bert Sakmann in 1976, very small currents are measured through a tiny region of the membrane surface containing only one or a few ion-channel molecules (Fig. 11-47). The researcher can measure the size and duration of the current that flows during one opening of an ion channel and can determine how often a channel

I

opens and how that frequency is affected by membrane potential, regulatory ligands, toxins, and other agents Patch-clamp studies have revealed that as many as 10* ions can move through a single ion channel in 1 ms. Such an ion flux represents a huge amplification of the initial signal; for example, only two acetylcholine molecules are needed to open an acetylcholine receptor channel (as described below).

Channel

ErwinNeher

BertSakmann

Reveals ofaK* (hannel TheStrueture forltsSpecifrcity tlrefiasis Micropipette applied tightly to plasma membrane

Patch of membrane pulled from cell Patch of membrane placed in aqueous solution

Micropipette Electrodes

Electronics to hold transmembrane potential (V-) constant and measure current flowing acrossmembrane function.The 11-47 Electrical measurements of ion-channel FIGURt flow of ions is by measuring the of an ion channel estimated "activity" through it, using the patch-clamptechnique A finely drawn-out and negative is pressed pipette(micropipette) againstthe cell surface, pressure sealbetweenpipetteandmemin thepipetteformsa pressure brane.As the pipetteis pulledawayfrom the cell, it pullsoff a tiny (whichmaycontainoneor a few ion channels) Afpatchof membrane solution,the patchin an aqueous ter placingthe pipetteand attached researcher can measurechannelactivityas the electriccurrentthat solutionIn of the pipetteandthe aqueous flowsbetweenthe contents potential practice, a circuitis setup that"clamps"the transmembrane must flow to maintain that given value and measures the current at a can researchers currentdetectors, this voltageWith highlysensitive measure the currentflowingthrougha singleion channel,typicallya Thetraceshowingthe currentas a functionof time few picoamperes (in milliseconds) revealshow fastthe channelopensand closes,how the V- at difit opens,andhow longit staysopen.Clamping frequently ferentvaluespermitsdeterminationof the effectof membranepotenof channelfunction. tial on theseparameters

The structure of a Potassium channel from the bacterium StreptomycesI iuida ns, determined crystallographicallybY RoderickMacKinnonin 1998, providesmuch insight into the way ion channelswork. This bacterialion channelis related in sequenceto all other knov,n K+ channelsand servesas the prototype for such channels, K+ includingthe voltage-gaLed MacKinnon Roderick channelofneurons.Amongthe membersof this protein family, the similaritiesin sequenceare greatestin the "poreregion,"which contains the ion selectivityfllter that allowsK+ (radius 1.33A) to pass10,000timesmorereadilythan Na* (radius0.95A)at a rate (about 105ions/s)approachingthe theoretical Iimit for unrestricted diffusion. The K+ channel consistsof four identical subunits that spanthe membraneand form a cone within a cone surrounding the ion channel, with the rnedeend of the doubte cone facing the extracellularspace (Fig. I f-48). Each subunithas two transmembranea helices as well as a third, shorter helix that contributes to the pore region.The outer coneis formedby one of the transmembranehelicesof each subunit' The inner cone, formed by the other four transmembraneheLices,surrounds the ion channel and cradles the ion selectivityfilter. Both the ion speciicity and the high flux through from what we know of the channelare understandable and outer plasma inner the At the channel'sstructure.

Biological Membranes andTransport

Frq

(a)

(b)

Backbone carbonyl oxygensform cage that frts K+ precisely, replacing waters of hydration sphere

Outside

Alternating K+ sites (blue or green) occupied Helix dipole stabilizes K*

sphere.Further stabilizationis provided by the short helicesin the pore regionof eachsubunit,with the partial negativechargesof their electric dipolespointed at K" in the channel.About two-thirds of the way through the membrane,this channelnarrowsin the region of the selectivity fllter, forcing the ion to give up its hydrating water molecules.Carbonyloxygen atoms in the backboneof the selectivityfllter replacethe water molecules in the hydrationsphere,forminga seriesof perfectcoordination shellsthrough which the K+ moves.This favorable interactionwith the f,lter is not possiblefor Na+, which is too small to make contact with all the potential oxygen ligands. The preferential stabilization of K+ is the basis for the ion selectivity of the fllter, and mutations that changeresiduesin this part of the protein eliminate the channel'sion selectivity.The K+-binding sites of the fllter are flexible enoughto collapseto fit any Na* that enters the charmel, and this conformational changeclosesthe channel. There are four potentialK+-bindingsitesalongthe selectivityfilter, each composedof an oxygen "cage" that providesligandsfor the K- ions (Fig. 11-49). In the crystal structure, two K+ ions are visible within the selectivity fllter, about 7.5 A apart,and two water molecules occupy the unfilled positions. K+ ions pass through the fllter in singleflle; their mutual electrostatic repulsion most likely just balancesthe interaction of eachion with the selectivityfllter and keepsthem moving. Movementof the two K+ ionsis concerted:first they occupypositions1 and 3, then they hop to positions2 and 4 (Fig. 11-48c).The energeticdifferencebetween

K* with hydrating water molecules (c) FfGURE 11-48 The K+ channel of Streptomyces lividans. (pDB lD 1BL8)(a) Viewedin the planeof the membrane, the channelconsists of eighttransmembrane helices(two from eachof four iclentical subunits),forminga conewith itswideendtowardtheextracellular space Theinnerhelicesof the cone(lightercolored)linethe transmembrane channel,andtheouterhelicesinteract with the lipidbilayerShortseg_ mentsof eachsubunitconverge in theopenendof theconeto makea selectivity filter.(b)Thisview,perpendicular to the planeof the mem_ brane,showsthe foursubunits arranged arounda centralchanneljust wide enoughfor a singleK+ ion to pass.(c) Diagramof a K+ channel in crosssection,showingthe structural featurescriticalto function. (SeealsoFig.11-49 )

membrane

surfaces, the entryways to the channel have several negatively charged amino acid residues, which presumably increase the local concentration of cations such as K+ and Na+. The ion path through the membrane begins (on the inner surface) as a wide, waterfllled channel in which the ion can retain its hvdration

FIGURE 11-49 K+ bindingsitesin the selectivitypore of the K+ channel.(PDBlD 1J95)Carbonyloxygens(red)ofthe peptidebackbonein the selectivity filterprotrudeintothechannel,interacting with andstabilizinga K* ion passingthrough.Theseligandsare perfectlypositionedto interactwith eachof four K* ions,but not with the smaller Na- ions.Thispreferential interaction with K+ is the basisfor the ion selectivity The mutualrepulsionbetweenK* ionsresultsin occupation of only two of the four K* sitesat a time (bothgreenor both blue) and counteracts the tendencyfor a lone K+ to staybound in one site. Thecombinedeffectof K* bindingto carbonyloxygensand repulsion betweenK- ionsensures thateachion keepsmoving,changingposi.l tionswithin 10 to 00 ns,andthatthereareno largeenergybarriers to ion flow throughthe membrane.

1 1 . 3S o l u t e T r a n s p o r t a ( r o s s M [ Oeomr b ] ranes thesetwo conflgurations(1, 3 and2,4) is very small;energetically,the selectivitypore is not a seriesofhills and valleysbut a flat surface,which is ideal for rapid ion movementthrough the channel.The structure of the channelseemsto havebeen optimizedduring evolution to give maximal flow rates and high specificity. Voltage-gated K- channelsare more complexstructures than that iliustratedin Figure Il-48, but they are variationson the sametheme. For example,the mammalian voltage-gatedK+ channelsin the Shaker family have an ion channel like that of the bacterral channel shownin Figure 11-48,but with additionalprotein domainsthat sensethe membranepotential,move in responseto a changein potential,and in movingtrigger (c)

(a)

the openingor closingof the K+ channel(Fig. 1f-50)' The critical transmembranehelix in the voltage-sensing domain of Shaker K* channels contains four Arg residues;the positivechargeson these residuescause the helix to moverelativeto the membranein response to changesin the transmembraneelectrical fleld (the membranepotential). Cells also have channelsthat specifically conduct Na* or Caz*,and excludeK*. In eachcase,the abilityto discriminateamongcationsrequiresboth a cavity in the binding site of just the right size (neither too large nor the ion and the precisepositoo small)to accommodate of carbonyl oxygensthat can cavity tioning within the This fit can be achieved shell. replacethe ion'shydration Voltage sensor

Outside

Inside

View from inside face Closed

(b)

St)

11-50 Structuralbasisfor voltagegatingin the K+ channel. FIGURE (PDBlD 2A79)Thiscrystalstructureof the Kv1.2-82 subunitcomplex to thatshown from rat brainshowsthe basicKt channel(corresponding .l.l-48) to makethe channel with the extramachinerynecessary in Fig. helical sensitive to gatingby membranepotential:four transmembrane The entirecomplex, of eachsubunitand four p subunits. extensions to the viewed(a) in the planeof the membraneand (b) perpendicular is represented membraneplane(asviewedfrom outsidethe membrane), .l-48, with eachsubunitin a differentcolor;eachof thefour asin Figure'l p subunits In (b),each iscoloredlikethesubunitwithwhichit associates. (red) to is numbered, Sl 56.55 and helixof onesubunit transmembrane itsell and arecomparaform channel the 56 from eachof four subunits helicesof eachsubunitin Figure11-48. 51 bleto thetrvotransmembrane the highly helices. The54 helixcontains to 54 arefourtransmembrane

and is believedto be thechiefmovingpartof the Arg residues conserved mechanism.(c) A schematicdiagramof the voltagevoltage-sensing gatedchannel,showingthe basicpore structure(center)and the extra 54,theArg-containing thatmakethechannelvoltage-sensitive; structures helix, is orange Forclarity,the B subunitsare not shownin this view' exerts electricalpotential(insidenegative) Normally,the transmembrane the cytosolic toward in 54, chains Arg side a pull on positivelycharged and with the pull is lessened, side.When the membraneis depolarized the extoward is drawn 54 potential, membrane of the reversal complete coupledto opening side (d)Thismovementof 54 is physically tracellular and closingof the K* channel,which is shownherein its open and AlthoughK+ is presentin the closedchannel,the closedconformations. K- passage' porecloseson the bottom,nearthe cytosol,preventing

aI

410

Biological Membranes andTransport

with moleculessmallerthan proteins; for example,valinomycin (Fig. 11-45) can provide the preciseflt that gives high speciflcity for the binding of one ion rather than another. Chemistshave designedsmall molecules with very high specificity for binding of Li+ (radius 0.60A), Na+ (radius0.9bA), K* (radius1.BBA), or Rb* (radius 1.48A). The biologicalversions,however-the channel proteins-not only bznd, spectficallybut conductions acrossmembranesinagated, fashion.

6ated lon(hannels Are(entral inNeuronal Function Virtually all rapid signalingbetween neurons and their targettissues(suchas muscle)is mediatedby the rapid opening and closing of ion channelsin plasmamem_ branes.For example,Na+ channelsin neuronalplasma membranessensethe transmembraneelectrical gradient and respondto changesby openingor closing.These voltage-gatedion channelsare typically very selective for Na* over other monovalent or divalent cations (by factors of 100 or more) and have very high flux rates (>107ions/s).Closedin the restingstate,Na- channels are opened-activated-by a reduction in the membrane potential; they then undergo very rapid inactivation. Within millisecondsof opening,a channelcloses and remains inactive for many milliseconds.Activation followed by inactivation of Na+ channelsis the basisfor signalingby neurons (seeFig. lZ-28). Another very well-studied ion channelis the nicotinic acetylcholine receptor, which functions in the passageof an electrical signalfrom a motor neuron to a muscle fiber at the neuromuscularjunction (signaling the muscleto contract).Acetylcholinereleasedby the motor neuron dffuses a few micrometersto the plasma membrane of a myocy'te,where it binds to an acetylcholinereceptor.This forces a conformationalchangein the receptor,causingits ion channelto open.The resultrng inward movementof positively chargedions into the myoclte depolarizesits plasmamembraneand triggers contraction. The acetylcholinereceptor allows Na+, Caz+,and K+ to pass through its channelwith equal ease,but other cationsand all anionsare unableto pass. Movementof Na+ through an acetylcholinereceptor ion channelis unsaturable(its rate is linear with respecrro extracellular[Na*]) andvery fast-about 2 x 107ions/s under physiologicalconditions.

o ,// cH3-c'(

?",

o-cH2-cHzrN_CHg

i", Acetylcholine The acetylcholine receptor charmel is typical of many other ion channels that produce or respond to electrical signals: it has a "gate" that opens in response to stimulation by a signal molecule (in this case acetylcholine) and an intrinsic timing mechanism that closes the gate aft,er a

split second.Thus the acetylcholinesignalis transientan essentialfeature of all electricalsignalconduction. Based on similaritiesbetween the amino acid sequences of other ligand-gatedion channelsand the acetylcholinereceptor,neuronalreceptorchannelsthat respond to the extracellular signals 7-aminobutyric acid (GABA), glycine, and serotonin are grouped together in the acetylcholinereceptor superfamily,and probablysharethree-dimensional structure and gating mechanisms.The GABA4 and glycine receptors are anion channelsspecifi,cfor Cl- or HCO3 whereasthe serotoninreceptor,like the acetylcholinereceptor,is cation-speciflc. Another classof ligand-gatedion channelsrespond to i,ntracel|ular ligands:3',5'-cyclic guanosinemononucleotide (cGMP) in the vertebrate eye, cGMP and cAMPin olfactoryneurons,and ATP and inositol 1,4,btrisphosphate(IP3) in many cell types. These channels are composedof multiple subunits,eachwith six transmembranehelical domains.We discussthe signaling functionsofthese ion channelsin Chapter12. Table 11-6 showssome transportersdiscussedin other chaptersin the context of the pathwaysin which they act.

(anHave Defective lonChannels Severe (onseq Physiological uences The importance of ion channels to physiological processesis clear from the effects of mutations in

speciflcion-channelproteins (Table 11-7, Box 11-3). Geneticdefectsin the voltage-gatedNa+ channelof the myocy'teplasmamembraneresult in diseasesin which musclesare periodically either paralyzed(as in hyperkalemic periodic paralysis) or stiff (as in paramyotonia congenita).Cystic fibrosis is the result of a mutation that changesone amino acid in the protein CFTR,a Clion channel;the defectiveprocesshere is not neurotransmissionbut secretionby various exocrine gland cellswith activitiestied to Cl- ion fluxes. Manynaturally occurringtoxins act on ion charurels, and the potency of these toxins further illustrates the rmportance of normal ion-channelfunction. Tetrodotoxin (produced by the puffer fish, Sphaero,id,es rubri,pes) and saxitoxin (produced by the marine dinoflagellateGonyaulaa, which causes"red tides") act by bindingto the voltage-gated Na+ channelsofneurons and preventing normal action potentials. Puffer flsh is an ingredient of the Japanesedelicacyfugu, which may be prepared only by chefs speciallytrained to separate succulent morsel from deadly poison. Eating shellflsh that have fed on Gonyaular can also be fatal; shellf,sh are not sensitiveto saxitoxin, but they concentrateit in their muscles,which becomehighly poisonousto organisms higher up the food chain. The venom of the black mamba snake contains dendrotoxin, which interferes with voltage-gatedK+ channels.T\-rbocurarine. the active

s r] a rctr o sMse m b r a n e[+r I 1 . 35 o l u tTer a n s p o

TYansportsystemand location

Figurenumber Role

Adenine nucleotideantiporter of mitochondrial inner membrane

19-28

Imports substrateADP for oxidative phosphorylation,and exports product ATP

Acyl-carnithe/carnitine transporter of mitochondrial inner membrane

r /-o

Imports fatty acidsinto matrix for B oxidation

Pi-H- sy'rnporterof mitochondrialinner membrane

h , , +

t9-28

Malate-a-ketoglutaratetranspofter of mitochondrial irmer membrane

19-29

SuppliesP1for oxidativephosphoryIation Shuttlesreducing equivalents(as malate) from matrix to c1'tosol

Glutamate-aspartate transporter of mitochondrial inner membrane

1.9-29

Completesshuttling begunby malate-a-ketoglutarateshuttle

Citrate transporter of mitochondrialinner membrane

2r-\0

Py'ruvatetransporter of mitochondrialinner membrane Fatty acid transporter of myoc5,teplasma membrane

2r-t0

Providescltosolic citrate as sourceof acetyl-CoA for lipid s5'nthesis Is part of mechanismfor shuttling citrate from matrix to cytosol

1.7-3

Imports fatty acidsfor fuel

ComplexI, III, and ry proton transportersof mitochondrialinner membrane

19-16

mechanismin Acts as energy-conserwing oxidativephosphorylation,converting electron flow into proton gradient

Thermogenin(uncouplerprotein), a proton pore of mitochondrialirurer membrane

19-34,23-35

Allows dissipationof proton gradient in mitochondriaas meansof thermogenesis and/or disposalof excessfuel

Cltochrome b/complex, a proton transporter of chloroplastthylakoid

19-59

Bacteriorhodopsin,a iight-driven proton pump

19-66

FoFl ATPase/ATPsynthaseof mitochondrialinner membrane,clrloroplastthylakoid, and bacterial plasmamembrane P,{riose phosphateantiporter of chloroplastinner membrane

19-64

Acts as proton pump, driven by electron flow throughthe Z scheme;sourceofproton gradient for photoslrrthetic ATP slmthesis Is light-driven sourceof proton gradient for AIP slr-rthesisin halophilicbacterium Interconvertsenergyof proton gradient and ATP during oxidativephosphorylationand photophosphoryIation Exports photosyntheticproduct from stroma; imports P1for ATP synthesis Exports secretedproteins through plasma membrane Ttansportsinto ER proteins destinedfor plasma membrane,secretion,or organelles

2.0.-1:n 2,0-16

Bacterialprotein transporter

2744

Protein translocaseof ER

27-38

Nuclearpore protein translocase LDL receptor in animal celi plasmamembrane

gv

2r42

Shuttlesproteins betweennucleusand c''toplasm Imports, by receptor-mediatedendocytosis,lipid carrying particles

Glucosetransporter of animal cell plasma to membrane;regulatedby insulin

\2-16

Increasescapacityofmuscle and adiposetissueto take up excessglucosefrom blood

IP3-gated Ca"- channel of endoplasmic reticulum

12-10

cGMP-gated Caz+ channei ofretinal rod and cone ceils

12-36

Allows signalingvia changesof cytosolic Ca2+ concentratron Allows signalingvia rhodopsinlinked to cAMP phosphodiesterase in vertebrate eye

Voltage-gated Na- channel of neuron

t2-25

-

^

9!

-

component of curare (used as an arrow poison in the Amazon region), and two other toxins from snake venoms, cobrotoxin and bungarotoxin, block the acetylcholine receptor or prevent the opening of its ion

At

Createsaction potentialsin neuronalsignal transmission

channel.By blocking signalsfrom nervesto muscles,all thesetoxins causeparalysisand possiblydeath' On the positive side, the extremely high affinity of bungaroloxin for the acetylcholinereceptor (Ka - 10-15u) has

B i o t o g i cMaet m b r a naensdT r a n s p o r t

[ot{

Ion ehannel

Affeetedgene

Disease

Nat (voltage-gated, skeietal muscle)

SCN4A

Hyperkalemicperiodic paralysis(or paramyotoniacongenita)

Na+ (voltage-gated, neuronal)

SCNlA

Generalizedepilepsywith febrile seizures

Na+ (voltage-gated, cardiac muscle)

SCNSA

Long QT s5mdrome3

ua- (neuronal)

CACNAlA

Familialhemiplegicmigraine

Caz+(voltage-gated,retina)

CACNAlF

Congenitalstationarynight blindness

Caz+(potycystin-1)

PKDl

Polycystickidney disease

K* (neuronal) K+ (voltage-gated,neuronal)

KCNQ4

Dominant deafness

KCNQz

Benignfamilial neonatalcomrlsions

Nonspeciflccation (cGMP-gated,retinal)

CNCGl

Retinitis pigmentosa

Acetylcholinereceptor (skeletalmuscle)

CHRNAl

Congenitalmyasthenicsyndrome

cl-

CFTR

Cysticflbrosis

^

,+

.

proved useful experimentally:the radiolabeledtoxin was used to quantify the receptor during its purification. r

concentrationto the side with lower. Others transport solutesagainstan electrochemical gradient;this requrresa sourceof metabolic energy. Carriers,Iike enz;rmes,show saturationand stereospeci-flcity for their substrates.Tfansport via these systemsmay be passiveor active. Primary activetransport is driven by AIP or electron-transfer reactions;secondaryactive transport is driven by coupledflow of two solutes,one of which (often H+ or Na*) flows down its electrochemicalgradient as the other is pulled up its gradient.

Tetrodotoxin

HzN HN

Saxitoxin

2Cr-

HsCO o-Tubocurarine chloride

SUMMAR 1Y 1 . 3 S o l u tTer a n s p o a rctr o s s Membranes r

Movementof polar compoundsand ions across biologicalmembranesrequirestransporterproteins. Sometransporterssimply facilitate passivediffusion acrossthe membranefrom the side with higher

The GLUTtransporters,suchas GLUTI of erythrocyles,carry glucoseinto cells by facilitated diffusion,Thesetransportersare uniporters, carrying only one substrate.Syrnporterspermit simultaneouspassageof two substancesin the samedirection;examplesare the lactosetransporter of E. coLi,,driven by the energy of a proton gradient (lactose-H*symport),and the glucosetransporter of intestinalepithelialcells,driven by a Na* gradient (glucose-Na+ s;.'rnport). Antip orters mediate simultaneouspassageof two substancesin opposite directions;examplesare the chloride-bicarbonate exchangerof erythrocytesand the ubiquitous Na-K- ATPase. In animal cells,Na+K+ ATPasemaintainsthe differencesin cytosolicand extracellular concentrationsof Na+ and K+, and the resulting Na+ gradientis usedas the energysourcefor a variety of secondaryactivetransportprocesses. The Na-K* MPase of the plasmamembraneand the Ca2* transportersof the sarcoplasmicand endoplasmicreticulum (the SERCApumps) are examplesof P-typeMPases;they undergo reversiblephosphorylationduring their catalytic cycle. F-t;pe ATPaseproton pumps (ATP synthases)are central to energy-conserving

F u r t h e r R e a [d. i, n{ s mechanismsin mitochondriaand chloroplasts. V-type ATPasesproduce gradientsof protons acrosssomeintracellular membranes,including plant vacuolarmembranes. ABC transporterscarry a variety of substrates (including many drugs) out of cells,using ATP as energysource. Ionophoresare lipid-solublemoleculesthat bind specificions and carry them passivelyacross membranes,dissipatingthe energyof electrochemical ion gradients. Watermovesacrossmembranesthrough aquaporins. Someaquaporinsare regulated;somealsotransport glycerolor urea. Ion channelspror,rdehydrophilic pores through which selections can diffuse,moving down their electrical or chemicalconcentrationgradients; they characteristicallyare unsaturable,have very high flux rates, and are highly speciflcfor one ion. Most are voltage-or ligand-gated. The neuronalNa+ channelis voltage-gated, and the acetylcholinereceptorion channelis gated by acetylcholine,which triggers conformational changesthat open and closethe transmembrane nath.

Reading Further Composition

and Architecture

of Membranes

Boon, J.M. & Smith, B.D. (2002) Chemicalcontrol of phospholipid distribution acrossbilayermembranesMed Res Reu 22,251-281. Intermediatelevel review of phospholipidaslrrrnetry and factors that influenceit. Dowhan, W. (1997) Molecularbasisfor membranephospholipidsdiversity:why are there so many lipids?Annu Reu Bi,ochem 66, 199-232 Ediden, M. (2002) Lipids on the frontrer: a century of ceil-membrane Bi'o\.4,414-418. bilayers Nat.Reu Mol. CeLL Short review of how the notion of a lipid bilayer membranewas developedand confimed. Haltia, T. & Freire, E. (1995) Forcesand factors that contribute to the structural stabiiity of membraneproteins.Bzochi,m Bi'ophys Acta 124L,295-322. Gooddiscussionof the secondaryand tertiary structuresof membraneproteins and the factors that stabilizethem Von Heiine, G. (2006) Membraneprotein topology.Nat Reu MoI CeIIBi,oI 7, 909-918 White, S,H., Ladokhin, A.S., Jayasinghe, S., & Hristova, K. (2001)Howmembranesshapeproteinstructure J. Bi'oLChem 276, 32,395-32,398 Brief, intermediate-levelreview of the forces that shapetransmembranehelices Wimley, W.C. (2003) The versatileB barrel membraneprotein Curr Opi,n Struct Bi'oL 13, 1-8. Intermediatelevel review. Memtrrane Dynamics

KeyTerms Terms in bold are d,efi,nedi,n tlrc glossary. fluid mosaic model 373 mieelle 374 bilayer 374 integral proteins 375 peripheral proteins 375 amphitropic proteins 375 hydropathy index 378 B barrel 379 gel phase 381 liquid-disorderedstate 381 liquid-orderedstate 381 flippases 382 floppases 383 scramblases 383 FBAP 383 microdomains 384 ra.ffts 384 caveolin 386 caveolae 386 fusion proteins 387 SNARES 388 simple diffusion 390 membrane potential

(v^) 390

electrochemical gtadient 390 electrochemical potential 390 facilitated diffusion 390 passivetransport 390 transporters 391 carriers 391 channels 391 electroneutral 395 cotransport systems 395 antiport 395 symport 395 uniport 395 active transport 395 electrogenic 396 P-typeATPases 396 SERCApump 397 F-typeATPases 399 ATP synthase 399 V-type ATPases 399 ABC transporters 400 ionophores 404 aquaporins (AQPs) 404 ion channel 406

Arnaout, M.A., Mahalingam, 8., & Xiong, J.-P. (2005) integrin structure, allostery,and bidirectionalsignalingAnnu Reu CeIl Deu BioL 21,381-410. Brown, D.A. & London, E. (1998) Functionsof lipid rafts in biologDeu Bi'oL 14, 111-136. ical membranes. Annu Reu. CzLL Daleke, D.L. (2007) Phospholipidflippases.J. Bi,oI Chem.282, 82r-825. Intermediate-levelreview. Deveaux, P.F.,Lopez-Montero' I.' & Bryde, S. (2006) Proteins involvedin lipid translocationin eukaryotic cells.Chem Phgs Lipids l4l, Il9-132. Didier, M., Lenne, P.-F.,Rigneault' H.' & He' H.-T (2006) Dlrramicsin the plasmamembtane:how to combinefluidity and order EMBO J 25, 3446-3457 Intermediatelevel review of studiesof membranedynamics,with fluorescentand other probes Edidin, M. (2003) The state of lipid rafts: from model membranesto cells.Annu Reu Bi,ophys Bi,om,olStruct. 82, 257-283 Advancedreview. Frye, L.D. & Ediden, M. (1970) The rapid intermixing of cellsurfaceantigensafter formation of mouse-humanheterokaryons J. CeLL Sci. 7,319-335 The classicdemonstrationof membraneprotein mobi-lity. Grafram, T.R. (2004) Flippasesand vesicle-mediatedprotein transport Tiends CeLIBi'oI14,670-677 Intermediatereview of flippasefunction Jahn, R. & Scheller, R.H. (2006) SNAREs-enginesfor membrane fusion NaI Reu CeILMoLBi'ol 7,631-643 Excellentintermediate-levelreview of the role of SNAREsin membranefusion, and the fusion mechanismitself Janmey, P.A. & Kunnunen, P.K.J. (2006) Biophysicalpropertiesof lipids and d1namicmembranes Ttends CeILBi'ol 16, 538-546

f

--l

lo14)

B i o l o g i cMael m b r a naensdT r a n s p o r t

Linder, M.E. & Deschenes, R.J. (2007) Patmitoylation:policing proteinstabilityandtrafflcNat Reu Mol CeLIBi,oL8.74-84 Marguet, D., Lenne, P.-F.,Rigneault, H., & He, H.-T. (2006) DVnamrcsin the plasmamembrane:how to combinefluiditv and order EMBO J.25,3446 3457 Intermediatelevel rer,rewof the methodsand results of studies on molecularmotronsin the membrane Mayer, A. (2002) Membranefusion in eukarvotic cells Annu Reu Cell Deu BzoL 18, 289-314 Advancedreview of membranefusion.with emnhaslson the consewedgeneralfeatures Palsdotti4 H. & Hunte, C. (2004) Lipids in membraneprotein structures Btochim Biophgs Acta 1666,2-18 Parton, R.G. (2003) Caveolae-from ultrastructure lo molecular mechanismsNat Reu MoL Cell Biot 4, 162-167. A concisehislorrcalreview of caveolae.caveolin.and rafts. Parton, R.G. & Simons, K. (2007)The multiplefacesof caveolae Nat, Reu Mol CellBioL 8, 185-194 Phillips, S.E., Ilincent, P., Rizzieri, K.E., Schaaf, G., & Bankaitis, V.A. (2006) The diversebiologicalfunctions ofphosphatidylinositoltransfer proteins in eukaryotes Crit Reu Biochem Mol Bi,oL 4L,2149 Advancedreview of the role of theseprotetnsin lipid signaling and membranetrafficking Pomorski, T., Holthuis, J.C.M., Herrmann, A., & van Meer, G. (2004) Ttacking down lipid flippasesand their biologicalfunctions.J CelISci 117, 805-813 Sprong, H., van der Slulis, P., & van Meer, G. (2001)How proteins move lipids and lipids move proteins Nat Reu MoL CeUBi,ot 2,504-513 intermediatelevel review Thmm, L.IC (ed.). (2005)Propin-Lzpzd Interactrons From Membratrc Domains to Cell:LtlnrNetutorks,Wiley-VCH,Weirrheim,Genruny. van Deurs, 8., Roepstorff, K., Hommelgaard, A,M., & Sandvig, K. (2003) Caveolae:anchored,multifunctionalplatformsin the lipid ocean Ttends CeLlBioI 13, 92-100 Yeagle, P.L. (ed.). (2004) The Stntcture oJBiologi,cat Mernbranes,2ndedn,CRCPress,Inc , BocaRaton,FL Zimmerberg, J. & Kozlov, M.M. (2006) How proteins procluce cellularmembranecurvature Nat Reu MoL CettBioL 7 , g-Ig T[ansporters Abramson, J., Smirnova, I., Kasho, V., Verner, G., Kaback, H.R,, & Iwata, S. (2003) Structure and mechanismof the lactose permeaseof Escheri,chza coLi,Sci,ence301, 610-615. Fujiyoshi, Y., Mitsuoka, K., de Groot, B.L., Philippsen, A., Grubmiiller, H., Agre, P., & Engel, A. (2002) Structure ancl functlonof waterchannels.Cutr. Opi,n Stntct Bi,oL lZ, 509-515 Jorgensen, P.L.,Hfrkansson,K.O., & Karlish, S.J.D. (2008) Structure and mechanismof Na,K-ATPase: functional sites and their interactionsAnnu Reu Physiol 65,812 849 Kjellbom, P., Larsson, C., Johansson, I., Karlsson, M., & Johanson, U. (1999) Aquaporinsand water homeostasisin plants Tiends Plant Sci, 4,308 314 Intermediatelevel renew. Kiihlbrandt, W. (2004) Biology,structure and mechanismof p-type ATPasesNat Reu MoL CeIIBioI 8,282 295 Intermediatelevel review,very well illustrated Mueckler, M. (1994) Facllitativeglucosetransporters.Ezr .l Br,oche'm219,713-725 Schmitt, L. & Tbmp6, R. (2002) Structure and mechanismof ABC transporters Cum Opi,n Stntct BioL 12,T54-760.

Stokes, D.L. & Green, N.M. (2003) Structure and function of the calciumpump Annu Reu Bi,ophgs Bi,omol Struct 32,445468 Advancedrelrew Sui, H., Han, B.-G., Lee, J.K., Walian, P., & Jap, B.K. (2001) Structural basisof water-specifictranspofi through the AQPI water channel Nature 414, 872-878 High-resolutionsolution of the aquaporinstructure by x-ray ^--.^|-ll ^^-^-L,, LrJ JLdrulir alI

r.y.

Toyoshirna, C. & Mizutani, T. (2004) Crystalstructure of the calclumpump with a bound ATP analogueNature 43O,529-535 Toyoshima, C., Nomura, H., & Tbuda, T. (2004) Lumenalgating mechanismrevealedin calciumpump crystal structureswith phosphateanalogsNature 432, 361-368. The supplementarymatenalsavailablewith the onfineversion of tlus article include an excellentmovie of the putative gating mechanism Watson, R.T. & Pessin, J.D. (2006) Bridgingthe GAPbetrveen insulin signalirrgand GLUT4translocation Trends Biochem Sci, 3r,215 222 intermediatelevel reuew of the regulationof glucosetranspoft through GLUT4 Ion Channels Ashcroft, F.M. (2006). From moleculeto malady.Nature 440, 440447 A short review of the many known casesin which geneticdefects in ion channelslead to diseasein humans Doyle, D.A., Cabral, K.M., Pfuetzner, R.A., Kuo, A., Gulbis, J.M., Cohen, S.L., Chait, 8.T., & MacKinnon, R. (1998)The structure of the potassiumchannel:molecularbasisof K+ conduction and selecfivity.Sctence 28O,69-77 The flrst crystal structure of an ion channelis described Edelstein, S.J. & Changeux, J.P. (1998) Allosteric transitionsof the acetylcholinereceptor Adu Prot Chem 51,121-184 Advanceddiscussionof the conformationalchangesinduced by acetylcholine. Gadsby, D.C., Vergani, P., & Csanady, L. (2006) The ABC protein turned chloride channelwhosefailure causescvstic flbtosis Nature 440,477-483 This is one of sevenexcellentreviewsofion channelsDublished togefher in this issueof Nature Gouaux, E. & MacKinnon, R. (2005) Principlesof selectiveion transportin channelsand pumps.Science310, 1461-1465. Short review of the architecturalfeaturesof channelsand oumps that give eachprotein its ion speciflcity. Guggino, W.B. & Stanton, B.A. (2006) New insightsinto cystic fibrosis:molecularswitchesthat regulate CFTR.Nature Reu Molec CeIIB'i,oI7,426-436 Hille, B. (2001)Ion Channels of Erci,tab\e Membranes,Srd edn, SinauerAssociates,Sunderland,MA Intermediate-leveltext emphasizingthe function of ion channels. Jiang, Y., Lee, A., Chen, J., Ruta, V., Cadene,M., Chait, 8.T., & MacKinnon, R. (2003)X-ray structureofavoltage-dependent K+ charurelNature 423, 33-4I King, L.S., Kozono, D., & Agre, P, (2004) From structure to dlsease:the evolvingtale of aquaporinbiology.Nat Reu MoL Cell Bi,ot 5, 687-698 Intermediatelevelrenew of the loca.lizatlon of aquaporinsin mammaliantissuesand the effectsof aquaporindefectson physiology. Lee, A.G. & Ea"st,J.M. (2001) What the s[ructure of a calcium pump tells us aboutits mechanismBiochemJ.356, 665-683 Long, S.8., Camptrell, E.B., & MacKinnon, R. (2005) Crystal structure of a mammalianvoltage-dependentShaker famtlyK+ channelSctence309, 897-902

Problems415

Long, S.B., Campbell,8.8., & MacKinnon, R. (2005) Voltage sensor of Kv1 2: structural basis of electromechanical coupling Science 309, 903 908 These two articles by Long and coauthors describe the structural studies that led to models for voltage sensing and gating in the Kchannel Miyazawa, A., Fqiiyoshi, Y., & Unwin, N. (2003) Structure and gating mechanism of the acetylcholine receptor pore Na,ture 423, 949 955 Intermediatelevel rer'rew. Nehet E. & Sakmann, B. (1992) The patch clamp technique Sci ,4m (March) 266,44 5I Clear description of the electrophysiologrcalmethods used to measure the activity of single ion channels, by the Nobel Prize urnning developers of this technique Sheppard, D.N. & Welsh, M.J. (1999) Structure and function of the CFTR chloride channel PLtysiol Reu 79, S23 546 One of 1I reviews in this journal issue on the CFTR chloride channel; the rer.rews cover struclure, activity, regulation, bioslnthesis, and pathophysiology Shi, N., Ye, S., Alam, A., Chen, L., & Jiang, Y. (2006) Atomic structure of a Na+- and K*-conducting channel Nature 440, 427-129 Crystallographic study of an ion channel that admits both Naand K*, and the structural explanation for this dual specificity. Tombola, F., Pathak, M.M., & Isacoff, E.Y. (2006) How does voltage open an ion channel'lAnnu Reu CeLlDeu BioI 22,23 52 Advanced review of the mechanisms of voltage gating of ion channels Yellen, G. (2002) The voltage-gated potassrum channels and their relatives Nature 419. 35-42

Problems the Cross-Sectional Area of a Lipid 1. Determining Molecule When phospholipids are layered gently onto the surface of water, they orient at the air-water interface with their head groups in the water and their hydrophobic tails in the air. An experimentai apparatus (a) has been devised that reduces the surface area available to a layer of lipids. By measuring the force necessary to push the lipids together, it is possible to determlne when the molecules are packed tightly in a continuous monolayer; as that area is approached, the force needed to further reduce the surface area increases sharpiy (b) How would you use thls apparatus to determine the average area occupied by a single lipid molecule in the monolayer? Force applied here to compress

monola:er

lf @

r-T

i I

.__*..-

__---tr

(a)

of one ery'throcyte. They obtained the data shown ln the table Were these investigators justifled in concluding that "chromocytes leq,'throcytes] are covered by a layer of fatty substances that is two molecules thick" (i.e , a lipid bilayer)?

Animal

Volume of packed cells (mt)

Total surface area oflipid Number monolayer ofcells (per mm3) from cells (m2)

62

Total surface axea ofone cell (pm2)

98

40

8,000,000

Sheep

10

9,900,000

60

29.8

Human

1

4,740,000

092

qql

Dog

Source: Data from Gorter, E & Grendel, F (1925) On bimolecular layers of lipoids on the chromocytes of the blood I Exp Med 41, 439-443.

3. Number of Detergent Molecules per Micelle When a small amount of the detergent sodium dodecyl sulfate (SDS; Na+CH3 (CHz) 11OSO3 ) is dissolved in water, the detergent ions enter the solution as monomeric species. As more detergent ls added, a concentration is reached (the critical micelle concentration) at which the monomers associateto form micelles The critical micelle concentration of SDS is 8.2 mrin The micelles have an average particle weight (the sum of the molecular weights of the constituent monomers) of 18,000. Calculate the number of detergent molecules in the average micelle. 4. Properties of Lipids and Lipid Bilayers Lipid bilayers formed between two aqueous phases have this important property: they form two-dimensionai sheets, the edges of which close upon each other and undergo self-sealing to form vesicles (liposomes). (a) What properties of lipids are responsible for this property of bilayers? ExPlain (b) What are the consequences of this property for the structure of biological membranes? 5. Length of a Fatty Acid Molecule The carbon-carbon bond distance for single-bonded carbons such as those in a saturated fatty acyl chain is about 1.5 A. Estimate the length of a single molecuie of palmitate in its fully extended form If two molecuies of palmitate were placed end to end, how would their total length compare with the thickness of the lipid bilayer in a biological membrane?

40

The Dependence of Lateral Diffusion 6. Temperature experiment described in Figure 11-17 was performed at 37 "C' oC, what effect would If the experiment were carried out at 10 you expect on the rate of diffusion? Why?

?so >rn

4-"

x ,^ E

(b)

2. Evidence for a Lipid Bilayer In 1925, E Gorter and F. Grendel used an apparatus llke that described in Problem 1 to determlne the surface area of a lipid monolayer formed by lipids extracted from erythrocytes of several animal species. They used a microscope to measure the dimensions of individua1 cells, from whlch they calculated the average surface area

t.4 1.0 0.6 Area (nmz/molecule)

7. Synthesis of Gastric Juice: Energetics Gastric juice (pH 1.5) is produced by pumping HCI from biood plasma (pH 7.4) into the stomach Calculate the amount of free energy

-

l

a le m b r a n e a sn dT r a n s p o r t [ 4 1 6 _ ] B i o l o g i cM

required to concentrate the Ht in 1 L of gastric juice at 3Z .C. Under cellular conditions, how many moles of ATp must be hydrolyzed to provide thls amount of free energy? The freeenergy change for ATP hydrolvsis under cellular conditions is about -58 kJ/mol (as explained in Chapter 13). Ignore the effects of the transmembrane electrical potential.

sphlngomyelin. Although the phospholipid components of the membrane can diffuse in the fluid bilayer, this sidedness is preserved at all times. How? 15. Membrane Permeability At pH 7, tryptophan crosses a lipid bilayer at about one-thousandth the rate of indole, a closely related compound:

8. Energetics of the Na*K* ATpase For a typical vertebrate celi with a membrane potential of -0.020 V (inside negative), what is fhe free-energy change for transporting 1 mol of Na- from the cell into the blood at 32 "C? Assume the concentration of Na- inside the cel1is 12 mlr and that in blood Dlasma is 145 mu. 9. Action of Ouabain on Kidney Tlssue Ouabain speciflcally inhibits the NatK* MPase actlvity of animal tissues but is not known to inhibit any other enzyrne. When ouabain is added to thin slices of tiving kidney tissue, it inhibits oxygen consumption by 660/oWhy? What does this observation tell us about the use of respirat,oryenergy by kidney tissue? 10. Energetics of Symport Suppose you determined experimentally that a cellular transport system for glucose, driven by synport of Na*, could accumulate glucose to concentrations 25 times greater than in the external medium, while the external [Na*] was only 10 times greater than the intracellular [Na-]. Would this violate the laws of thermody,namics? If not, how could you explain this observation? ll. Location of a Membrane Protein The following obseruations are made on an unknown membrane protein, X. It can be extracted from disrupted er5,.throc1'te membranes into a concen_ trated salt solution, and it can be cleaved into fragments by proteo$tic erz),rnes. Tfeatment of e4ttrocytes with proteo\tic enz)rynesfollowed by disruption and extraction of membrane components yields intact X. However, treatment of eryrthrocy,te "ghosts" (which consist of just plasma membranes, produced by disrupting the cells and washing out the hemoglobin) with proteo\rtic eru5.'rnesfollowed by disruption and extraction yields extensively fragmented X What do these obserwations indicate about the location of X in the plasma membrane? Do the proper_ ties of X resemble those of an integral or peripheral membrane protein? 12. Membrane Self-sealing Cellular membranes are selfsealing-if they are punctured or disrupted mechanically, they quickly and automatically reseal What properties of mem_ branes are responsible for this important feature? 13. Lipid Melting Temperatures Membrane lipids in tissue samples obtained from different parts of the leg of a reindeer have different fatty acid compositions. Membrane lipids from

Suggestan explanationfor this observation. 16. Water Flow through an Aquaporin A human erythrocyte has about 2 x 105AQP-I monomersIf water moiecules flow through the plasma membraneat a rate of b x 108per AQP-1tetramerper second,and the volumeof an erythrocyte is 5 x 10 11mL, how rapidly could an e4throc5,te halve its volume as it encounteredthe high osmolarity (1 u) in the interstitial fluid of the renal medulla?Assumethat the en'throcyte consistsentirelyof water 17. Labeling the Lactose Ttansporter A bacteria"llactose transporter,which is higtrly speciflcfor lactose,containsa Cys residuethat is essentialto its transport activity.Covalentreaction of N-ethylmaleimide (NEM) with this Cys residue irreversibly inactivatesthe transporter A high concentrationof lactosein the medium preventsinactivationby NEM, presunabtyby stericallyprotectingthe Cysresidue,which is h or near the lactose-bindingsite Youknow nothing elseabout the transporter protein Suggestan experimentthat might allow you to determinethe M. of the Cys-containing transporterpolypeptide. 18. Predicting Membrane Protein Topology from Sequence You have cloned the gene for a human erythrocy,te protein, which you suspectis a membraneprotein. From the nucleotidesequenceof the gene,you know the aminoacid sequence.From this sequencealone,how would you evaluate the possibilitythat the protein is an integral protein? Suppose the protein proves to be an integral protein, either type I or type II. Suggestbiochemicalor chemicalexperimentsthat might allow you to deternine which tlpe it is. 19. Intestinal Uptake of Leucine Youare studyingthe uptake of l-leucine by epithelialcells of the mouse intestine. Measurementsof the rate of uptake of lleucine and severalof its analogs,with and without Na* in the assaybuffer, yield the results given in the table. What can you conciude about the properties and mechanismof the leucine transporter?Would you expect L-leucineuptake to be inhibited by ouabain? Uptakein presenceof Na+

tissue near the hooves contain a larger proportion of unsaturated fatty acids than those from tissue in the upper leg What is the signiflcance of this observation?

Substrate

V^

14. Flip-Flop Diffusion The inner leaflet (monotayer) of the human erythrocyte membrane consists predominantly of phosphatidylethanolamine and phosphatidylserine The outer

l-Leucine

420

l-Leucine

310

L-Valine

225

leaflet consists predominantly of phosphatidylcholine and

4(rur) 0.24 47 0.31

Uptakein absenceof Na*

Z^*

I(, (rur) 0.2 ^o

19

0.31

P r o b l e mfs$ l

20. Effect of an Ionophore on Active Thansport Consider the leucine transporter describedin Problem 19. Would 7^n* and/orK, changeif you addeda Na- ionophoreto the assaysoIulion containingNa ' ? Explain. 21. Surface Density of a Membrane Protein E coli can be inducedto make about 10,000copiesof the lactosetransporter (M, 31,000)per cell.Assumelhal E. coli,is a cylinder1 pm in diameter and 2 pm long. \\{hat fraction of the plasma membranesurfaceis occupiedby the lactosetransporter molecules?Explain how you arrived at this conclusion. 22. Use of the Helical Wheel Diagram A helicalwheel is a two-dimensionalrepresentationof a helix, a view alongits central axis (seeFig. 1l-29b; seealsoFig. 4-4d). Usethe helical wheel diagram below to determine the distribution of amino acid residues in a helical segment with the sequence-ValAsp-Arg-Val-Phe-Ser-Asn-Val-Cys-Thr-His-Leu-Lys-ThrLeu-Gln-Asp-Lys1

(c) Go to the Protein Data Bank (wwwrcsb.org) Use the PDB identifier lDEP to retrieve the data pagefor a portion of the B-adrenergicreceptor (one type of epinephrine receptor) isolatedfrom a turkey. Using Jmol to explore the structure,predict whether this portion of the receptor is Iocated within the membrane or at the membrane surface. Explain. (d) Retrievethe datafor a portion of anotherreceptor,the acetylcholinereceptor of neurons and myocytes,using the PDB identifler 1,{11.As in (c), predict where this portion of the receptor is locatedand explain your answer. If you havenot used the PDB, seeBox 4-4 (p. 129) for more information

Problem DataAnalysis 25. The Fluid Mosaic Model of Biological Membrane Structure Figure 11-3 showsthe currently acceptedfluid mosaic model of biological membrane structure. This model was presented in detail in a review article by S. J' Singerin 1971.In the article, Singerpresentedthe three models of membranestructure that had been proposedby that time:

What can you say about the surfaceproperties of this helix?How would you expectthe helix to be orientedin the tertiary structure of an integral membraneprotein? 23. Molecular Species in the E. coli Membrane The plasma membrane of E coli, is about 75% protein and 25o/o phospholipid by weight How many moiecules of membrane lipid are presentfor eachmoleculeof membraneprotein?Assume an averageprotein M, of 50,000and an averagephospholipid M, of 750. What more would you need to know to estimatethe fractionof the membranesurfacethat is covered by lipids?

Biochemistry ontheInternet 24. Membrane Protein Topology The receptor for the hormone epinephrinein animai cells is an integral membrane protein (M, 64,000)that is believedto havesevenmembranespanrungreglons. (a) Show that a protein of this size is capableof spanning the membraneseventimes. (b) Given the amino acid sequenceof this protein, how would you predict which regions of the protein form the helices? membrane-spanning

C Model. This was the A. The Davson-Danielli-Robertson most widely acceptedmodelin 1971,when Singer'sreviewwas published.In this model, the phospholipidsare arrangedas a bilayer. Proteins are found on both surfacesof the bilayer, attached to it by ionic interactions between the charged head groups of the phospholipidsand charged groups in the proteins. Crucially,there is no protein in the interior of the bilayer' B. The BensonLipoprotein Subunit Model. Here, the proteins are $obular and the membraneis a protein-lipid mixture The hydrophobic tails of the lipids are embeddedin the hydrophobicparts of the proteins The lipid head groups are exposedto the solvent There is no lipid bilayer.

rt 1 B _ _B l i o l o g i cMael m b r a naensdT r a n s p o r t f C The Lipid-Globular Protein Mosaic Model. This is the model shownin Figure 11-3 The lipids form a bilayer and proteins are embeddedin it, someextending through the bilayer and others not. Proteins are anchored in the bilayer by hydrophobic interactions between the hydrophobic tails of the lipids and hydrophobicportions of the protein. For the data givenbelow,considerhow eachpiece of information aligns with each of the three models of membrane structure. Which model(s) are supported,which are not supported, and what reservationsdo you have about the data or their interpretation?Explain your reasoning (a) When cellswere fixed, stainedwith osmiumtetroxide, and examjned in the electron microscope,they gave images Iike that in Figure 11-1: the membranesshowed a "railroad track" appearance,with two dark-staininglines separatedby a light space. (b) The thicknessof membranesin cellsfixed and stained in the sameway was found to be 5 to 9 nm. The thicknessof a "naked"phospholipidbilayer,without protehs, was4 to 4.b nm. The thicknessof a singlemonolayerof protehs wasabout 1 nm. (c) In Singer'swords: "The averageamino acid composition of membraneproteins is not distinguishablefrom that of soluble proteins In particular,a substantialfraction of the residuesis hydrophobic"(p. 165). (d) As describedin Problems 1 and 2 of this chapter,researchershad extracted membranesfrom celis, extracted the lipids, and comparedthe areaof the lipid monolayerwith the area of the original cell membrane.The interpretation of the results was complicatedby the issue illustrated in the graph of Problem 1: the area of the monolayerdependedon how hard it was pushed.With very light pressures,the ratio of

monolayer area to cell membranearea was about 2 0. At higher pressures-thought to be more like those found in cells-the ratio was substantiallylower. (e) Circular dichroism spectroscopyuses changesin polarizationof W light to makeinferencesabout protein secondary structure (seeFig. 4-9). On average,this techniqueshowed that membraneproteins havea large amount of a helix and little or no B sheet.This finding was consistentwith most membrane proteins haviry a globular structure (f) PhospholipaseC is an enzy'rnethat removesthe polar head group (including the phosphate)from phosphoiipids In severalstudies,treatment of intact membraneswith phospholipase C removed aboul 70o/oof the head groups without disrupting the "railroad track" structure of the membrane. (g) Singer describeda study in which "a glycoprotein of molecularweight about 31,000in human red blood cell membranesis cleavedby tryptic treatment of the membranesinto solubleglycopeptidesof about 10,000molecularweight, while the remaining portions are quite hydrophobic" (p. 199). Tl'ypsin treatment did not causegross changesin the membranes,which remainedintact Singer'sreview also included many more studies in this area In the end,though,the data availablein 1971did not conclusivelyprove Model C was correct. As more data have accumulated,this model of membranestructure hasbeen accepted by the scientific communitv Reference Singer, S.J. (1971) The molecular organization of biological membranes In ,Slrr,r,ctureand Functi,on of Bi,o\ogi,cal Membrones (Rothfield, L.I., ed ), pp 145-222, Academic Press, Inc., New York

When I firstenteredthe studyof hormoneaction,some25 yearsago, therewas a widespreadfeelingamongbiologiststhat hormoneaction c o u l d n o t b e s t u d i e dm e a n i n g f u l l yi n t h e a b s e n c eo f o r g a n i z e dc e l l structure.However,as I reflectedon the historyof biochemistry,it seemedto me therewas a real possibilitythat hormonesmiqht act at t h e m o l e c u l a rl e v e l . -Earl W.Sutherland,NobelAddress,1971

Biosignaling 12.1 General Features ofSignal Transduction 419 12.2 GProtein-Coupled Receptors andSecond Messengers 423 12.3 ReceptorTyrosine Kinases439 (yclases, 12.4 Receptor Guanylyl cGMP, andProtein Kinase G 445 12.5 Multivalent Adaptor Proteins and Membrane Rafts 446 12.6 Gated lon(hannels 449 12.7 Integrins:Bidirectional(ellAdhesion Receptors455 12.8 Regulation ofTransciption bySteroid Hormones456 12.9 Signaling inMicroorganisms andPlants 457 12.10 Sensory Transduction inVision, 0lfaction, and Gustation461 12.11Regulation ofthe(ellCycle byProtein Kinases469 12.120nrogenes,Tumor5uppressor Genes,and (ellDeath 473 Programmed he ability of cells to receive and act on srgnals from beyond the plasma membrane is fundamental to life. Bacterial cells receive constant inDut from rnern-

brane proteins that act as information receptors, sampling the surroundingmedium for pH, osmoticstrength, the availabilityof food, oxygen,and light, and the presence of noxiouschemicals,predators,or competitorsfor food. Thesesignalselicit appropriateresponses,such as motion toward food or awayfrom toxic substancesor the formation of dormant spores in a nutrient-depleted

medium. In multicellular organisms, cells with different functions exchange a wide variety of signals. Plant cells respond to growth horrnones and to variations in sunlight. Animal cells exchange information about the concentrations of ions and $ucose in extracelluLar fluids, the interdependent metaboLic activities taking place in different tissues, and, in an embryo, the correct placement of cells during development. In all these cases, the signal represents'inJormnti,on that is detected by specific receptors and converted to a cellular response, which always involves a chem'ical process. This conversion of information into a chemical change, signal transduction, is a universal property of living cells.

.'12.1 Features Transduction General ofSignal Signaltransductionsare remarkablyspecificand exquisitely sensitive.Specificity is achievedby precisemolecular complementaritybetween the signal and receptor molecules(Fig. 12-la), mediatedby the samekinds of weak (noncovalent)forces that mediate enzymesubstrateand antigen-antibodyinteractions.Multicellular organismshave an additional level of speciflcity, becausethe receptorsfor a givensignal,or the intracelIular targets of a given signalpathway,are present only hormone,for in certaincell types.Thyrotropin-releasing example,triggersresponsesin the cells of the anterior pituitary but not in hepatocy'tes, which lack receptors for this hormone.Epinephrinealtersglycogenmetabolism in hepatocytesbut not in adipocytes;in this case, both cell types have receptors for the hormone, but whereashepatocy'tescontainglycogenand the $ycogenmetabolizingenzymethat is stimulatedby epinephrine, adipoc;,'tescontain neither. Three factors account for the extraordinary sensitivity of signaltransducers:the high affinity of receptors for signal molecules,cooperativity (often but not always) in the ligand-receptorinteraction, and amplification of the signal by enzyme cascades.The affinity

F"l

Fttl

Biosisnatins

Signal (a) Specificity

(c) Desensitization/Adaptation

Signal molecule frts binding site on its complementary receptor; other signals do not frt.

Receptor activation triggers a feedback circuit that shuts off the receptor or removes it from the cell surface.

J

----/)I

Response

(b) Amplification

(d) Integration

When enzymes activate enzJ[nes, the number of affected molecules increases geometrically in an enzJrme cascade.

When two signals have opposite effects on a metabolic characteristic such as the concentration ofa second messenger X, or the membrane potential V-, the regulatory outcome results from the integrated input from both receptors.

rlllJlrll Net AlXl or V-

Enzyme

J

Response

FIGURE 12-1 Fourfeaturesof signal-transducing systems.

betweensignal(ligand) and receptorcan be expressed asthe dissociationconstantK6,usually10-t0 M orlessmeaningthat the receptordetectspicomolarconcentrations of a signalmolecule.ReceptorJigandinteractions are quantified by Scatchardanalysis,which yields a quantitativemeasureof affinity (fr-j and the number of ligand-bindingsitesin a receptorsample(Box 12-1). CooperativiQr in receptor-ligandinteractionsresults in largechangesin receptoractivationwith smallchanges in ligand concentration(recall the effect of coopeiativity on oxygenbindingto hemoglobin;seeFig. 5-12). Amplification by enzyme cascades results when an enzyrne associatedwith a signalreceptoris activatedand,in turn, catalyzesthe activationof manymoleculesof a secondenzyrne,each of which activatesmany moleculesof a third enzyrne,and so on (Fig. 12-1b).Suchcascades can produce amplificationsof severalordersof magnitudewithin milliseconds.The responseto a signalmust alsobe terminated suchthat the downstreameffectsaxein propoftion to the strength of the originat stimulus. The sensitivity of receptor systemsis subject to modifi.cation.When a signalis present continuously,desensitization of the receptorsystemresults(Fig. 12-1c); when the stimulus falls below a certain threshold, the systemagainbecomessensitive.Think of what happens to your visual transductionsystemwhen you walk from bright sunlight into a darkened room or from darkness into the light. A final noteworthy feature of signal-transducing systemsis integration (Fig. 12-1d), the ability of the systemto receivemultiple signalsand produce a unified responseappropriateto the needs of the cell or organism. Different signaling pathways conversewith each other at severallevels,generatinga wealth of interactions that maintain homeostasisin the cell and the organism.

Oneofthe revelationsofresearchon signalingis the remarkabledegreeto which signalingmechanismshave been conservedduring evolution.Although the number of different biologicalsignals(Table 12-1) is probablyin the thousands,and the kinds of responseselicited by these signalsare comparablynumerous,the machinery for transducingall of these signalsis built from about 10 basictypes of proteh components. In this chapter we examine some examplesof the major classesof signaiingmechanisms,Iooking at how they are integrated in speciflcbiologicalfunctions such as the transmissionof nerve signals;responsesto hormones and growth factors; the sensesof sight, smell, and taste; and control of the cell cycle.Often, the end result of a signalingpathway is the phosphorylationof a few specifi.ctarget-cellproteins,which changestheir activities and thus the activities of the cell. Throughout our discussionwe emphasizethe conseryationof fundamental mechanismsfor the transductionof biological signalsand the adaptationof thesebasicmechanismsto a wide range of signalingpathways. Weconsiderthe moleculardetailsof severalrepresentative signal-transductionsystems,classifiedaccording

Antigens Cell surfaceglycoproteins/ oligosaccharides Developmentalsignals Extracellularmatrix nnmnnnantq

Growth factors Hormones

Light Mechanicaltouch Neurotransmitters Nutrients Odorants Pheromones Tastants

Transduction 12.1General Features ofSignal forl

The cellular actions of a hormone begin when the hormone (ligand, L) binds specrficallyand tightly to its protein receptor (R) on or in the target cell. Binding is mediated by noncovalent interactions (hydrogen-bonding, hydrophobic, and electrostatic) between the complementary surfaces of ligand and receptor Receptor-ligand interaction brings about a conformational change that alters the biological activity of the receptor, which may be an enz;.'rne,an enzyrne regulator, an ion channel, or a regulator of gene expression. Receptor-ligand binding is described by the equation

Fl c) d d

tr

(a)

Total hormoneadded,tl,l + tRLl

R+Li-RL Receptor

Ligand

Receptor-ligand complex

This binding,like that of an enzymeto its substrate,depends on the concentrationsof the interactingcomponentsand can be describedby an equilibriumconstant: d

+Lg

R Receptor

Ligand

-

ft r

lRLl

RL

lr

Receptor-ligand complex

k'1

uxo "" ffi: n_i: whereK. is the associationconstantandKa is the dissociationconstant. Like enzyme-substratebinding, receptor-ligand bindingis saturableAs more ligandis addedto a flxed amount of receptor,an increasingfraction of receptor moleculesis occupiedby ligand (Fig. la). A roughmeasure of receptor-ligandaffinity is given by the concentration of ligand needed to give half-saturationof the receptor. Using Scatchard analysis of receptorJigand binding,we can estimateboth the dissociationconstant K6 and the number of receptor-bindingsitesin a given preparation.Whenbindinghasreachedequilibrium,the total number of possiblebinding sites,B-.,, equalsthe number of unoccupiedsites, representedby [R], plus the numberof occupiedor ligand-boundsites,[RL];that is, B*u" : [R] + [RL] The numberof unboundsitescan be expressedin terms of total sites minus occupied sites:[R] : B-,, - tRLl The equilibriumexpressioncan now be written Ku:

tRL] lLl(B-.* - tRLl)

lr

ti

(b)

Bound hormone, [RL]

1 Scatchard interaction. A radioFIGURt analysis of a receptor-ligand conlabeledligand(L)-a hormone,for example-isaddedat several centrationsto a fixed amountof receptor(R),and the fractionof the the receptorhormoneboundto receptoris determinedby separating hormonecomplex(RL)fromfreehormone. (a)A plot of [RL]versusILI + tRLl(totalhormoneadded)is hyperbolic,risingtowarda maximumfor [RL]as the receptorsitesbenonspecificbinding To control for nonsaturable, come saturated. hormonesbind nonspecifically to the lipid bilayer, sites(eicosanoid is alsonecesseriesof bindingexperiments for example),a separate sary.A largeexcessof unlabeledhormoneis addedalongwith the dilutesolutionof labeledhormone.Theunlabeledmoleculescompete site with the labeledmoleculesfor specificbindingto the saturable binding.Thetruevaluefor but notfor the nonspecific on the receptor, nonspecific bindingfrom specificbindingis obtainedby subtracting t o t a lb i n d i n g . (b) A linearplot of tRLl/[L]versus[RL]givesK6and B-u, for the complex Comparetheseplotswith thoseof Vsverreceptor-hormone .l/[S] for an enzyme-substrate complex(seeFig sus[S]and 1/V6versus 6-12, Box 6-1).

Rearrangingto obtain the ratio of receptor-boundIigand to free (unbound)ligand,we get lBoundl: 'Ill : KutB^u*-lRLl)

rFreel =j1-._- ,;;; f\d

From this slope-interceptform of the equation,we can see that a plot of [bound ligand]/[freeligand] versus [bound ligand] shouldgive a straight line with a slope of

-K^

(-l/Ki

and an intercept

on the abscissa of Bmax,

the total number of binding sites (Fig. 1b). Hormonee to ligand interactions typically have K6 values of l0 ri 10 M,coffespondingto very tight binding. Scatchardanalysisis reliablefor the simplestcases, when but as with Lineweaver-Burkplots for enz5,rrnes, plots protein, the deviate the receptor is an allosteric from linearity.

7r1 B i o s i g n a l i n g G protein100 nlr). Calciumlevelsdrop duringillumination,becausethe steady-state[Ca"-] in the outer segment is the result of outward pumping of Caz* through the Na+-Ca2*exchangerof the plasmamembrane (seeFig. 12-36) and influx ofCaz* through open cGMP-gatedchannels.In the dark, this produces a [Ca'*] of about 500 nu-enough to inhibit cGMPsynthesis. After brief illumination, Caz* entry slows and [Ca'*] declines (step @). The inhibition of guanylyl cyclaseby Caz* is relieved,and the cyclaseconverts GTP to cGMP to return the system to its prestimulus state(step @). Rhodopsinitselfalsoundergoeschangesin response to prolongedillumination.The conformationalchangeinduced by light absorptionexposesseveralThr and Ser residues in the carboxyl-terminal domain. These residuesare quickly phosphorylatedby rhodopsin kinase (step @ in f'8. 12-38), which is functionally and structurally homologousto the B-adrenergickinase @ARK) that desensitizesthe B-adrenergicreceptor (Fig. l2-8). The Ca'*-binding protein reeoverin inhibits rhodopsinkinaseat high [Ca'*], but the inhibition is relieved when [Ca2+]drops after illumination, as described above.The phosphorylatedcarboxyl-terminal domainof rhodopsinis bound by the protem arrestin l, preventing further interaction between activated rhodopsinand transducin.Arrestin 1 is a closehomolog of arrestin 2 (Baru;Fig. 12-8). On a relatively long time scale(secondsto minutes),the alI-trans-retinalof an excitedrhodopsinmoleculeis removedandreplacedby 11ces-retinal,to produce rhodopsin that is ready for anotherround of excitation (step @ in Fig. 12-38).

(one(ellsSpecialize Vision inColor Color vision involves a path of sensorytransduction in cone cells essentiallyidentical to that describedabove, but triggered by slightly different light receptors.Three types ofcone cellsare specializedto detectJightfrom different regionsof the spectrum,using three related photoreceptorproteins (opsins).Each cone cell expresses only one kind of opsin,but eachtype is closelyrelatedto rhodopsinin size,amino acid sequence,and presumably three-dimensionalstructure. The djfferencesamongthe opsins,however,are great enoughto place the chromophore, 11-czs-retinal,in three slightly different environments,with the result that the three photoreceptors have different absorption spectra (Fig. 12-39). We discriminatecolors and hues by integrating the output from the three types of conecells,eachcontainingone of the threephotoreceptors.

100 90 80 70 r 60 a 50 d 40 € 30 20 10 0

400

450

500

550

600

650

Wavelength (nm) 12-39 Absorptionspectraof purified rhodopsinand the red, FIGURE green,and blue receptorsof conecells.Thespectra,obtainedfrom inpeakat about420, 530, dividualcone cellsisolatedfrom cadavers, rhodopsinis at about for absorption maximum nm, and the and 560 for humansisabout380 to thevisiblespectrum 500 nm. Forreference, 7 5 0n m .

Color blindness, such as the inability to distinguish red from green, is a fairly corunon, genetically inherited trait in humans.The various tlpes of color blindness result from different opsin mutations' One form is due to loss of the red photoreceptor;affected individualsare red- dichromats (they see only two primary colors). Otherslack the green pigment and are green- dichromats. In some cases,the red and green photoreceptorsare presentbut have a changed amino acid sequencethat causesa change in their absorption spectra, resulting in abnormal color vision. Dependingon which pigment is altered,suchindividuals are red-anomalous trichromats or green-anomalous trichromats. Examination of the genes for the visual receptorshas allowedthe diagnosisof color blindnessin a famous "patient" more than a century after his death (Box 12-4)!r

Mechanisms Use andGustation 0lfaction Vertebrate System totheVisual 5imilar The sensory cells that detect odors and tastes have much in colrtmonwith the rod and cone cells. Olfactory neurons have long thin cilia extending from one end of the cell into a mucouslayer that overlaysthe cell. These cilia present a Iarge surfacearea for interaction with olfactory signals.The receptors for olfactory stimuli are ciliary membraneproteins with the familiar GPCRstructure of seven transmembranea helices. The olfactory signal can be any one of the many volatile compounds for which there are speciflcreceptorproteins.Our ability to discriminateodors stemsfrom hundreds of different olfactory receptorsin the tongue and nasalpassages and from the brain's ability to integrate input from

ll"l

Biosignaling

The chemist John Dalton (of atomic theory fame) was color-blind. He thought it probable that the yitreous humor of his eyes (the fluid that fllls the eyeball behind the lens) was tinted blue, unlike the colorlessfluid of normal eyes. He proposed that after his death, his eyes should be dissected and the color of the vitreous humor determined. His wish was honored. The day after Dalton's death in Juiy 1844, Joseph Ransome dissected his eyes and found the vitreous humor to be perfectly coloriess. Ransome, Iike many scientists, was reluctant to throw samples away. He placed Dalton's eyes in a jar of preservative, where they stayed for a century and a half. Then, in the mid-1990s, molecular biologists in England took small samples of Dalton's retinas and extracted DNA. Using the known gene sequences for the opsins of the red and green light receptors, they ampiified the relevant sequences(using techniques described

in Chapter9) and determinedthat Daltonhad the opsin gene for the red photopigmentbut lacked the opsin genefor the greenphotopigment.Dalton was a green dichromat.So,150yearsafter his death,the experiment Dalton started-by hypothesizingabout the causeof his color blindness-was finally finished

different types of olfactory receptors to recognize a "hybrid" pattern, extending our range of discrimination far beyond the number of receptors. The oifactory stimulus arrives at the sensory cells by diffusion through the air. In the mucous layer covering the olfactory neurons, the odorant molecule binds di-

rectly to an olfactoryreceptoror to a specfficbindingprotein that carriesthe odorantto a receptor(F'ig. 12-40). Interaction between odorant and receptor triggers a change in receptor conformationthat results in the replacementof bound GDPby GTP on a G protein,Guy, analogousto transducinand to G. of the B-adrenergic

FIGURE I Dalton'seyes

Olfactory neuron

Cilia

R) Odorant (O) arrives at the mucous layer and binds directly to an olfactory receptor (OR) or to a binding protein (BP) that carries it to the OR. (h. w/

t

. r: r:.]::r':!li:lt:l,itltr;l:.1:if:.r.

r=l

@ Activated OR catalyzes GDP-GTP exchange onaGprotein(Go11), causing its dissociation into o and B-y.

i

I'

Dendrite

Axon

Air

@

v (6j

G.-GTP activates adenylyl cyclase, which catalyzes cAMP synthesis, raising [cAMP].

Mucous layer

cAMP-gated cation channels open. Ca2+ enters,raising internal [Qs2+1.

il

->

ATP o\

c1-

:,,,1 ,..t.;:: ::,.,:., 1 ":;:...:.,, 1, Ciliary membrane

GTP

Goyhydrolyzes GTP to GDP, shutting itself off. PDE hydrolyzes cAMP. Receptorkinase phosphorylates OR. inactivaring it. Odorant is removedby metabol.ism.

@ Ca2*reducesthe affrnity ofthe cation channel for cAMP, lowering the sensitivity ofthe system to odorant

\g-l

gu2+-gated chloride channels open. EfIlux ofCl depolarizes the cell, triggering an electrical signal to the brain.

F I G U R E l 2 - 4M 0 o l e c u l a r e v e n t soolff a c t i o n . T h e s e i n t e r a c t i o n s o c c u r i n t h e coillfiaacotfo r v r e c e p t o r c e l l s .

andGustation inVision,0lfaction, Transduction Sensory 12,10 7U') then activatesadenylylcyclase system.The activatedGo11 cAMP from of the crliary membrane,which s1'nthesizes ATP,raisingthe local [cAMP].The cAMP-gatedNa- and Ca2* charmelsof the ciliary membrane open, and the influx of Na* and Ca2+producesa small depolarization calledthe reeeptor potential. If a sufflcientnumber of odorant moleculesencounter receptors,the receptor potential is strong enoughto causethe neuron to fire an action potential. This is relayed to the brain in several stagesand registersas a speciflcsmell.AII these events occurwithin 100to 200ms. When the olfactory stimulus is no longer present, the transducingmachinery shuts itself off in several returns [cAMP]to the ways.A cAMPphosphodiesterase prestimuluslevel. Go6hydrolyzesits bound GTPto GDP, thereby inactivating itself. Phosphorylation of the receptor by a specific kinase prevents its interaction by a mechanismanalogousto that used to with Go11, desensitizethe B-adrenergicreceptor and rhodopsin. destroyed And lastly, some odorants are enz)..rnatically by oxidases. The senseof taste in vertebratesreflectsthe activity of gustatory neurons clusteredin taste buds on the surfaceof the tongue.In these sensoryneurons, GPCRsare coupledto the heterotrimericG protein gustducin (very similar to the transducin of rod and cone cells). Sweet-tastingmoleculesare those that bind receptorsin "sweet"tastebuds.Whenthe molecule (tastant) binds,gustducinis activatedby replacement of bound GDP with GTP and then stimulates cAMP production by adenylyl cyclase.The resulting elevationof [cAMP] activatesPKA, which phosphorylates K- channelsin the plasma membrane,causing them to close. Reducedefflux of K- depolarizesthe

cell (Fig. L2-4L). Other taste buds specializein detecting bitter, sour, salty,or umami (savory) tastants, using variouscombinationsof secondmessengersand ion channelsin the transductionmechanisms.

Features Several Share Systems oftheSensory 6F(Rs Systems Signaling Hormone of withGP(Rs We have now looked at severalt5,pesof signaling systems (hormone signaling,vision, olfaction, and gustation) in which membrane receptors are coupled to through G prosecondmessenger-generatingenzJ,Tnes teins. As we haveintimated, signalingmechanismsmust have arisen early in evolution;genomicstudieshave revealed hundreds of genes encoding GPCRsin vertebrates,arthropods(Drosophi'Laand mosquito),and the roundworm Caenorhabdzti'selegans. Even the common baker's yeast SaccharornAcesuses GPCRsand G proteins to detect the opposite mating type. Overall patterns have been conserved,and the introduction of variety has given modern organisms the ability to respondto a wide range of stimuli (Table l2-8)' Of the approximately29,000genesin the human genome,as many as 1,000encodeGPCRs,including hundredsfor olfactory stimuli and many "orphan receptors"for which the natural ligand is not yet known. AII well-studied transducirg systemsthat act through heterotrimericG proteins sharesomeconunonfeatures, which reflecttheir evolutionaryrelatedness(Fig. L242). The receptors have seventransmembranesegments,a domain (generallythe loop betweentransmembraneheIices 6 and 7) that interacts with a G protein, and a carboxyl-terminalcy[oplasmicdomainthat undergoesreversiblephosphorylationon severalSer or Thr residues. Basolateral membrane

Apical membrane

GTP

o

uur

Sweet-tasting molecule (S) binds to sweet-tastereceptor(SR), activating the G protein gustducin (Ggust).

@ Gustducin n subunit activates adenylyl cyclase (AC) of the apical membrane, raising [cAMP].

Taste ceII

mechanismfor sweettastants. tIGURE12-41 Transduction

F'4

Biosignaling

Acetylcholine(muscarinic) Adenosine

Follicle-stimulating hormone (FSH)

Angiotensin

GABA (7-aminobutynicacid)

Orytocin Platelet-activatingfactor Prosta$andins

AIP (extracellular)

Glucagon

Bradykinin

Glutamate

Calcitonin

Somatostatin

Cannabinoids

Growth hormone-releasing hormone (GHRH) Histamine

Tastants

Catecholamines

Leukotrienes

Thyrotropin

Llght

Thyrotropin-releasing hormone (TRH)

Cholecystokinin Corticotropin-releasing factor (CRF)

Secretin Serotonin

Luteinizinghormone (LH)

CyclicAMP (Di,ctgostelium discoideum)

Melatonin

Vasoactiveintestinal peptide

Odorants

Vasopressin

Dopamine

Opioids

Yeastmating factors

The ligand-bindingsite (or, in the caseof light reception, the light receptor)is buried deepin the membraneand includesresiduesfrom severalof the transmembranesegments. Ligand binding (or fuht) inducesa conformational changein the receptor,exposinga domainthat caninteract with a G protein. HeterotnmericG proteins activate or inhibit effectoren4{nes (adenylylcyclase,pDE, or pLC), which changethe concentrationof a secondmessenger (cAMP,cGMP,IP3,or Caz*).In the hormone-detecting systems, the final output is an activated protein kinase that regr-rlatessomecellular processby phosphorylatinga pro_ tein criticalto that process.In sensoryneurons,the outout

asopressrn Epinephrine

I

Light

is a changein membranepotential and a consequentelectrical signalthat passesto another neuron in the pathway connectingthe sensorycell to the brain. All these systemsself-inactivate.Bound GTp is converted to GDPby the intrinsic GTPaseactivity of G proteins, often augmentedby GTPase-activating proteins (GAPs) or RGSproteins (regulatorsof G-proteinsrgnating; see Fig 12-5). In somecases,the effector enzymes that are the targetsof modulationby G proteinsalsoserve as GAPs.The desensitizationmechanism involvingphosphorylation of the carboxyl-terminalregion followed by arrestinbindingis widespread,and may be universal.

j

J

[*-]

t"^i

!

(G",)

[e-c i

LAC]

trDEl

J

JlcAMPl

I J J€@

J J

tlcAup

t*) \=_\

iRhi

'gi

( c 'l_ ,)

Sweet tastant

Odorants

I

+ JlcGMPl

I J

il r ,,1.l

1@ vlr D

C12+,Na+

FIGURE 12-42 Common featuresof signalingsystemsthat detect hormones,light, smelts,and tastes.CpCRsprovidesignalspecificity, and their interactionwith C proteinsprovidessignalamplification. Heterotrimeric C proteinsactivateeffectorenzymes:adenylylcy_ clase(AC),phospholipase C (PLC),and phosphodiesterases (pDEs) thatdegradecAMPor cCMp.Changes in concentration of the second messengers (cAMP,cCMp, lpr) resultin alterations of enzymaticac_ tivitiesby phosphorylation or alterations in the permeability(p) of

lG"'f !4c

J J

1[rP3]

I I

ltcaupl

I J

Gil G*") \-t

LecI

I

+ ttcAMPl ,.-------I ( P K A )I

-d€

']:j:1_l.,Ji:'j: IP"^"*

'fps.z*,Na*

lD vr

K+

surfacemembranes to Ca2+,Na+, and K*. Theresultingdepolariza_ tion or hyperpolarization of the sensorycell (the signal)passes throughrelayneuronsto sensorycentersin the brain. In the best_ studiedcases,desensitization includesphosphorylation of the recep_ tor and binding of a protein (arrestin)that interruptsreceptor_C proteininteractions. VR is the vasopressin receptor;B_ARis the p_ adrenergic receptor. Other receptorand C-proteinabbreviations are as usedin earlierillustrations.

1 2 . 1 1R e g u l a t ioof nt h eC e lCl y c lbeyP r o t e iKni n a s e [sa t t ]

1Y 2 . 1 0 S e n s oTr rya n s d u c ti ino n SUMMAR Vision 0 ,l f a c t i oann, d Gu s t a t i o n Vision,olfaction,and gustationin vertebrates employGPCRs,which act throughheterotrimeric G proteinsto changethe 7- of a sensoryneuron.

r

In rod and conecellsof the retina,light activates rhodopsin,which activatesthe G protein transducin. The freed a subunit of transducin activatesa which lowersIcGMP] cGMPphosphodiesterase, ion chamelsin and thus closescGMP-dependent the outer segmentof the neuron.The resulting of the rod or conecell carriesthe hyperpolarization signalto the next neuron in the pathway,and eventuallyto the brain.

r

In olfactory neurons,olfactory stimuli, acting through GPCRsand G proteins,trigger either an increasein [cAMP] (by activatingadenylylcyclase) or an increasein [Ca'*] (by activatingPLC). These affection channelsand thus secondmessengers the Z*.

r

r

r

GustatoryneuronshaveGPCRsthat respondto tastantsby altering levels of cAMP,which changes 7- by gating ion channels There is a high degreeof conservationof signaling proteinsand transductionmechanismsacross signalingsystemsand acrossspecies.

by oftheCellCycle 12.11Regulation

Kinases Protein One of the most dramatic manifestations of signaling pathways is the regulation of the eukaryotic cell cycle. During embryonic growth and later development, cell division occurs in virtually every tissue. In the adult organism most tissues become quiescent. A cell's "decision" to divide or not is of crucial importance to the organism. When the regulatory mechanisms that limit cell dMsion are defective and cells undergo unregulated division, the result is catastrophic-cancer. Proper cell division requires a precisely ordered sequence of biochemical events that assures every daughter cell a full complement of the molecules required for life. Investigations into the control of cell division in diverse eukaryotic cells have revealed universal regulatory mechanisms. Signali4g mechanisms much like those discussed above are central in determinurg whether and when a cell undergoes cell division, and they also ensure orderly passagethrough the stages of the cell cycle.

Stages The(ell(ycleHasFour Cell division accompanyingmitosisin eukaryotesoccurs in four well-definedstages(Fig. 12*43). In the S (synthesis) phase,the DNA is replicatedto producecopies

G2 Phase No DNA synthesis RNA and protein synthesis continue.

M Phase Mitosis (nuclear division) and cytokinesis (cell division) yield two daughter cells.

G0 Phase Terminally differentiated cells withdraw from cell cycle indefrnitely.

GO Reentry point A cell returning from G0 enters at early Gl phase.

G1 Phase RNA and protein synthesis. No DNA synthesis. S Phase DNA synthesis doubles the amount of DNA in the cell RNA and protein also synthesized.

point Restriction A cell that passes this ooint is committed lo pass into S phase.

(in hours) of the cellcycle.Thedurations 12-43Eukaryotic FI6URE typical. are shown but those vary, fourstages forboth daughter cells. Inthe G2 phase (G indicates the gap between divisions), new proteins are synthesized and the cell approximately doubles in size In the M phase (mitosis), the maternal nuclear envelope breaks down, paired chromosomes are pulled to opposite poles of the cell, each set of daughter chromosomes is surrounded by a newly formed nuclear envelope, and cytokinesis pinches the cell in half, producing two daughter cells (see Fig.24-25).In embryonic or rapidly proliferating tissue, each daughter cell divides again, but only after a waiting period (G1). In cultured animal cells the entire process takes about 24 hours' After passing through mitosis and into Gl, a cell either continues through another division or ceasesto divide, entering a quiescent phase (G0) that may last hours, days, or the lifetime of the cell. When a cell in G0 begins to divide again, it reenters the division cycle through the G1 phase. Differentiated cells such as hepatocytes or adipocytes have acquired their specialized function and form; they remain in the G0 phase' Stem cells retain their potential to divide and to differentiate into any of a number of cell tYPes.

Oscillate Kinases Protein ofCyclin-Dependent Levels The timing of the cell cycle is controlled by a family of protein kinases with activities that change in response to celIular signals. By phosphorylating speciflc proteins at precisely timed intervals, these protein kinases orchestrate the metabolic activities of the cell to produce orderly cell division. The kinases are heterodimers with a regulatory subunit, cyclin, and a catalytic suburLit,cyclindependent protein kinase (CDK). In the absence of

l;'4

Biosignaling

terminal helix Glu51

r

*F

.

CDK2 (inactive)

FIGURE 12-44 Activationof cyclin-dependent protein kinases(CDKs)by cyclin and phosphorylation.CDKs,a familyof relatedenzymes,areactiveonly when associated with cyclins,anotherproteinfamily.Thecrystalstructure of CDK2with and withoutcyclinreveals the basisfor this activation. (a) Withoutcyclin (pDB lD IHCK), CDK2 foldsso rhatone segmenti theT loop,obstructs the bindingsitefor proteinsubstrates and thusinhibitsprorern kinaseactivityThebindingsitefor ATPis alsoneartheT loop.(b) Whencyclinbinds(pDB lD l FlN),it forcesconformational changes thatmovetheT loopawayfromtheactivesiteand reorientan amino-terminal helix,bringinga residuecriticalto catalysis into the ac1Clus1) tive site (c) Phosphorylation of a Thr residuein the T loop producesa negatively charged residuethat is stabilized by interaction with threeArg residues, holdingCDK in its active (PDBlD l lsl. conformation

Phosphorylated

yclin subunit

Thrloo

Aro15o

( Glu51

J:

b '^b-

r CDK2 (inactive) cyclin, the catalytic subunit is virtually inactive. When cyclin binds, the catalytic site opens up, a residue essen_ tial to catalysis becomes accessible (Fig. lZ_44), and the activity of the catalytic subunit increases 10,000-folcl. Animal cells have at least 10 different cyclins (desig_ nated A, B, and so forth) and at least 8 CDKs (CDKI through CDK8), which act in various combinations at speciflc points in the cell cycle. plants also use a family of CDKs to regulate their cell division in root and shoot meristems, the principal tissues in which dinsion occurs. In a population of animal cells undergoing s5mchro_ nous division, some CDK activities show striking oscilla_ tions (Fig. 12-45). These oscillations are the result of four mechanisms for regulating CDK activity: phospho_ rylation or dephosphorylation of the CDK, controlled degradation of the cyclin subunit, periodic syrthesis of CDKs and cyclins, and the action of specific CDKinhibiting proteins. In general, active CDKs enable a cell to enter a stage of cell division. Regulation of CDKs by phosphorylation The activity of a CDK is strikingly affected by phosphorylation and dephosphorylation of two critical residues in the protein (Fig. f2-46a). phosphorylation of Tlr15 near the amino terminus renders CDK2 inactive; the @fy, residue is in the ATP-binding site of the kinase, and the

i

CDK2 (active)

h

@

Time FIGURE l2-45 Variationsin the activitiesof specificCDKsduringthe cell cycle in animals.Cyclin E-CDK2activitypeaksnear the C1 phase-Sphaseboundary,when the activeenzymetriggerssynthesis of enzymesrequiredfor DNA synthesis (seeFig.12-48).CyclinA_CDK2 activityrisesduringthe S and C2 phases, thendropssharplyin the M phase,ascyclinB-CDK.Ipeaks negatively charged phosphate group blocks the entry of

ATP.A speciflcphosphatase(a pTpase) dephosphorylates this @t" residue,permitting the binding of ATp. Phosphorylationof Thr160in the "T loop',of CDK, catalyzedby the CDK-activatingkinase,forces the T loop out of the substrate-binding cleft, permitting substrate binding and catalytic activity (seeFig. l2-44c).

i ni n a s e s 1 7 ' l 1 2 . 1 1R e g u l a t ioofnt h eC e lCl y c lbeyP r o t e K

(a)

,€\ \9/ Cyclin-CDK complexforms, but phosphorylation on Tw15 blocksATP-binding site; still inactive.

/6) \!)

CycIin synthesis leads to its accumulation.

@) PlosphoryIation of Thr16o-in T loop and removal of Tyrl" phosphoryl group activates cyclin-CDK manyfold.

':-. P

A

\4, CDK phosphorylates phosphatase,which activatesmore CDK.

an No cyclin present; CDK is inactive.

tll-----

\\lllll/

Phosphatase ) Phosphatase 1 Attttrrlr-" I

CDK (b)

G\ CDK nhosohorvIates I t-__)

i;. \-------DBiF, ".;l;',,.,s

DBRP triggers addition ofubiquitin molecules to cyclin by ubiquitin ligase.

@ Cyclin is degraded by proteasome, Ieaving CDK inactive.

and proteolyof CDK by phosphorylation tl6UR[ i?-46 Regulation at thetimeof miproteinkinaseactivated sis.(a)Thecyclin-dependent tosis (the M-phaseCDK) has a "T loop" that can fold into the rn the T loop is phosphorylated, site When Thr160 substrate-binding the CDK site,activating the loop movesout of the substrate-binding

One circumstance nism is the presence which leads to arrest protein kinase (called

that triggers this control mechaof single-strand breaks in DNA, of the cell cycie in G2. A speciflc Rad3 in yeast), which is activated

by srngle-strandbreaks, trrggers a cascadeleading to the inactivation of the PTPase that dephosphorylates Tlr'o of CDK The CDK remains inactive and the cell is arrested in G2 The cell cannot divide until the DNA is repaired and the effects of the cascadeare reversed. Highly speciflc Controlled Degradation of Cyclin and precisely timed proteolytic breakdown of mitotic cyclins regulates CDK activity throughout the cell cycle. Progress through mitosis requires first the activation then the destruction of cyclins A and B, which activate the catalytic subunlt of the M-phase CDK. These cyclins contain near their amino terminus the sequence -Arg-Thr-Ala-Leu-GIy-Asp-Iie-GIy-Asn-, the "destruction box," which targets them for degradation. (This usage of "box" derives from the common practice, in

itsown complextriggers manyfold(step@).(b)Theactivecyclin-CDK box recognizing of DBRP(destruction by phosphorylation inactivation protein;step@) DBRPand ubiquitinligasethenattachseveralmoleby it for destruction culesof ubiquitin(U)to cyclin(step@), targeting proteolyticenzymecomplexes(step@.)). proteasomes,

diagrammrng the sequence of a nucleic acid or protein' of enclosing within a box a short sequence of nucleotide

or amino acid residues with some speci-fi-cfunction. It does not imply any three-dimensional structure ) The protein DBRP (destruction box recognizing protein) recognizesthis sequence and initiates the process of cyclin degradation by bringing together the cyclin and another protein, ubiquitin. Cyclin and activated ubiquitin are covalently joined by the enzyrne ubiquitin ligase (Fig. 12_46b). Several more ubiquitin molecules are then appended, providing the signal for a proteoll'tic en4''rne complex, or proteasome, to degrade cyclin. What controls the timing of cyclin breakdorm? A feedback loop occurs in the overall process shown in Figure 12-46.lncreased CDK activity activates cyclin proteolysis. Newly synthesized cyclin associates with and activates CDK, which phosphorylates and activates DBRP. Active DBRP then causes proteolysis of cyclin' The lowered cyclin Ievel causes a decline in CDK activity, and the activity of DBRP also drops through slow,

--t

472

Biosignaling

constantdephosphorylationand inactivationby a DBRp phosphatase.The cyclin level is ultimately restored by synthesisof new cyclinmolecules. The role of ubiquitin and proteasomesis not limited to the regulationof cyclin; aswe shall seein Chapter22, both alsotake part in the turnoverof cellularproteins,a processfundamentalto cellularhousekeeping. Regulated Synthesis of CDKs and Cyclins The third mechanismfor changingCDK activity is regulation of the rate of syrrthesisof cyclin or CDK or both. For example,cycJinD, cyclin E, CDKZ,and CDK4are synthesizedonly when a specifictranscriptionfactor,E2F, is presentin the nucleusto activatetranscriptionof their genes.Symthesis of E2F is in turn regulatedby extracellular signals such as growth factors and cy,tokines (developmentalsignalsthat induce cell division), compounds found to be essentialfor the division of mammalian cells in culture. They induce the synthesisof specific nuclear transcription factors essentialto the production of the enzyrnesof DNA syrrthesis.Growth factors trigger phosphorylationof the nuclear proteins Jun and Fos,transcription factors that promote the synthesis of a variety of gene products,including cyclins, CDKs,and E2F.In turn, E2F controlsproductionof several enzymesessentialfor the synthesisof deoxynucleotidesand DNA, enablingcellsto enter the S phase (Fig. r2-47). Growth factors, cytokines

Ii MAPK

* cascade

I

Phosphorylation of Jun and Fos in nucleus

Cyclins, CDKs

Transcription factor E2F I transcriptional | ""got"tiot V Enzymes for DNA synthesis

Passagefrom Gl to S phase FIGURE 12-47 Regulationof cetl divisionby growthfactors.The path from growthfactorsto cell divisionleadsthroughthe enzymecascaoe that activatesMAPK;phosphorylation of the nucleartranscriptionfac_ torsJunand Fos;and the activityof the transcription factorE2F,which promotessynthesisof severalenzymesessentialfor DNA synthesis

Inhibition of CDKs Finally,specificprotein inhibitors bind to and inactivatespeci-flcCDKs.One suchprotein is p21,which we discussbelow. Thesefour control mechanismsmodulatethe activity of speciflc CDKs that, in turn, control whether a cell will divide,differentiate,becomepermanentlyquiescent,or begin a new cycle of division after a period of quiescence.The detailsof cell cycle regulation,such as the number of different cyclins and kinasesand the combinations in which they act, differ from speciesto species, but the basic mechanismhas been conservedin the evolution of all eukaryoticcells.

(DKs (ellDivision Regulate byPhosphorylating (ritical Proteins We have examinedhow cells maintain close control of CDK activity, but how does the activity of CDK control the cell cycle?The list of target proteins that CDKsare known to act upon continuesto grow,and much remains to be learned. But we can see a generalpattern behind CDK regulationby inspectingthe effect of CDKson the structures of lamin and myosin and on the activity of protein. retinoblastoma The structure of the nuclear envelopeis maintained in part by highly organizedmeshworksof intermediate fi.lamentscomposedof the protein lamin. Breakdownof the nuclear envelopebefore segregationof the sister chromatidsin mitosis is partly due to the phosphorylation of lamin by a CDK, which causeslamin fllamentsto depolynerize. A secondkinasetarget is the ATP-drivencontractile machinery (actin and myosin) that pinches a dividing cell into two equalparts during cy'tokinesis.After the division, CDK phosphorylatesa small regulatory subunit of myosin, causingdissociationof myosin from actin filamentsand inactivatingthe contractilemachinery.Subsequent dephosphorylationallows reassemblyof the contractile apparatusfor the next round of cytokinesis. A thfud and very important CDK substrate is the retinoblastoma protein, pRb; when DNA damageis detected,this protein participatesin a mecharLism that arrestscelldivisionin Gl (FiS. 1248). Namedfor the retinal tumor cell line in which it was discovered,pRb functions in most,perhapsall, celltypesto regulatecelldivisionin responseto a variety of stimuli. UnphosphorylatedpRb binds the transcriptionfactorE2F;whileboundto pRb,EZF cannot promotetrarscription of a group of genesnecessaryfor DNA synthesis(the genesfor DNA po\nnerasea, ribonucleotidereductase,and otherproteins;seeChapter2b). Irt this state,the cell cyclecannotproceedfrom the Gl to the S phase,the step that commitsa cell to mitosisand cell division. The pRb-E2F blocking mecharLismis relieved when pRb is phosphorylatedby cyclin E-CDK2, which occurs in responseto a signalfor cell divisionto proceed. Whenthe protein kinasesATM and ATR detect damageto DNA (signaledby the presenceof the protein MRN

l e a t h[ t t { 1 2 . 1 20 n c o g e n e s , T 5u ump0prr e s s o r G e n ePsr,oagnrda m mCeedlD

Double-strand break in DNA

cells. When the damage is too severe to allow effective repair, this same machinery triggers a process (apoptosis, described below) that leads to the death of the cell, preventing the possible development of a cancer.

Intact DNA

1Y 2 . 1 1 R e g u l a t ioofnt h eC e l l SUMMAR C y c lbeyP r o t e iKni n a s e s r

Progressionthroughthe cell cycleis regulatedby the protein kinases(CDKs),which cyclin-dependent act at speciflcpoints in the cycle,phosphorylating key proteins and modulatingtheir activities.The catal1,'ticsubunit of CDKsis inactive unless associatedwith the regulatory cyclin subunit.

r

The activity of a cyclin-CDKcomplex changes during the cell cycle through differential synthesis of CDKs,speciflcdegradationof cyclin, phosphorylationand dephosphorylationof critical residuesin CDKs,and bindingof inhibitoryproteins to speciflccyclin-CDKs.

t. I

t\ I

)-

Inactir.e

/"""

/PRb"

i''i"l'Yi'

\. y,7r. ",: Inactive

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synthesis I Y Passage from G 1t o S

Cell division blocked by p53

Cell division occurs normally

tIGURE12-48 Regulationof passagefrom G1 to S by phosphorylaof genes factorE2Fpromotestranscription tion of pRb.Transcription The retinoblastoma to DNA synthesis. for certainenzymesessential it and preventing protein,pRb,can bind E2F(lowerleft),inactivating of pRbby CDK2prevents transcription of thesegenesPhosphorylation alE2F,andthe genesaretranscribed, it frombindingand inactivating a lowingcell division.Damageto the cell'sDNA (upperleft)triggers CDK2,blockingcell division.Whenthe series of eventsthatinactivate proteinMRN detectsdamageto the DNA, it activatestwo proteinkiand activatethe trannases,ATM and ATR,and they phosphorylate of another scriptionfactorp53 Active p53 promotesthe synthesis protein,p21,an inhibitorof CDK2 Inhibitionof CDK2stopsthe phosto bindand inhibitE2F phorylation of pRb,whichtherefore continues genes essential to cell divisionare not tranWith E2Finactivated, scrrbedand cell divisionis blocked.When DNA hasbeenrepaired, and thecell divides. thisseriesof eventsis reversed.

at a double-strandbreak site), they phosphorylatep53, activatingit to serveas a transcriptionfactor that stimuof the proteinp21 (Fig. 12-48).This latesthe sy'nthesis protein inhibits the protein kinase activity of cyclin E-CDK2. In the presenceof p21, pRb remainsunphosphorylatedand boundto E2F,blockingthe activity of this transcriptionfactor, and the cell cycle is arrestedin Gl. This givesthe cell trme to repair its DNA before entering the S phase,thereby avoidingthe potentially disastrous transfer of a defectivegenometo one or both daughter

,'12,12 Genes, Suppressor 0ncogenes,Tumor Death Cell andProgrammed Ttmors and cancer are the result of uncontrolled cell division. Normally, cell division is regulated by a family of extracellular growth factors, proteins that cause resting cells to divide and, in some cases,differentiate' The result is a precise balance between the formation of new cells (such as skin cells that die and are replaced every few weeks, or white blood cells that are replaced every few days) and cell destruction. When this balance is disturbed by defects in regulatory proteins, the result is sometimes the formation of a clone of cells that divide repeatedly and without regulation (a tumor) until their presence interferes with the function of normal tissues-cancer. The direct cause is almost always a genetic defect in one or more of the proteins that regulate cell division. In some cases,a defective gene is inherited from one parent; in other cases, the mutation occurs when a toxic compound from the environment (a mutagen or carcinogen) or high-energy radiation interacts with the DNA of a single cell to damage it and introduce a mutation. In most casesthere is both an inherited and an environmental contribution, and in most cases,more than one mutation is required to cause completely unregulated division and full-blov'n cancer.

for oftheGenes Forms AreMutant 0ncogenes (ell (ycle the ThatRegulate Froteins Oncogenes were originally discovered in tumorcausing viruses, then later found to be derived

from genesin the animalhost cells,proto-oncogenes, which encodegrowth-regutatingproteins.During a viral infection,the host DNA sequenceof a proto-oncogeneis sometimescopiedinto the viral genome,where

474

Biosignaling

it proliferates with the virus. In subsequent viral infection cycles, the proto-oncogenes can become defective by truncation or mutation. Viruses, unlike animal cells, do not have effective mechanisms for correcting mistakes during DNA replication, so they accumulate mutations rapidly. When a virus carrying an oncogene infects a new host cell, the viral DNA (and oncogene) can be incorporated into the host cell's DNA, where it can now interfere with the regulation of cell division in the host cell. In an alternative, nonviral mechanism, a single cell in a tissue exposed to carcinogens may suffer DNA damage that renders one of its regulatory proteins defective, with the same effect as the oncogenic mechanism: failed regulation of cell division. The mutations that produce oncogenes are genetically dominant; if either of a pair of chromosomes contains a defective gene, that gene product sends the signal "divide" and a tumor will result. The oncogenic defect can be in any of the proteins involved in communicating the "divide" signal. Oncogenes discovered thus far include those that encode secreted proteins, growth factors, transmembrane proteins (receptors), cytoplasmic proteins (G proteins and protein kinases), and the nuclear transcription factors that control the expression ofgenes essential for cell division (Jun, Fos). Some oncogenesencode surface receptors with defective or missing signal-binding sites, such that their intrinsic Tlr kinase activity is unregulated. For example, the oncoprotein ErbB is essentially identical to the normal receptor for epidermal growth factor, except that ErbB lacks the amino-terminal domain that normally binds EGF (Fig. 12-49) and as a result senclsthe Extracellular

space

EGF-binding domain

r

Tyrosine kinase dornain EGF-binding site empty; tyrosine kinase is inactive Norrnal

-/. '//l\\-

\

Binding of EGF actrvates tyrosine kinase.

EGF receptor

l\\\ Tyrosine kinase is constantly active ErbB protein

FIGURE12-49 Oncogene-encoded defective ECF receptor. The produ c t o f t h e e r b B o n c o g e n e ( t h e E r b B p r o t e i n ) i s a t r u n c a t e dv e r s i o n o f t h e n o r m a l r e c e p t o rf o r e p i d e r m a lg r o w t h f a c t o r ( E C F ) .l t s i n t r a c e l l u l a r d o m a i n h a s t h e s t r u c t u r en o r m a l l y i n d u c e d b y E C F b i n d i n g , b u t t h e p r o t e i n l a c k s t h e e x t r a c e l l u l a rb i n d i n g s i t e f o r E C F U n r e g u l a t e d b y E C F ,E r b Bc o n t i n u o u s l ys i g n a l sc e l l d i v i s i o n .

"divide"signalwhetherEGFis presentor not. Mutations in erbB2, the gene for a receptor 1)rr kinase related to ErbB, are commonly associatedwith cancers of the glandularepitheliumin breast,stomach,and ovary.(F or an explanationof the use of abbreviationsin naming genesand their products,seeChapter25.) The prominent role playedby protein kinasesin signalingprocessesrelatedto normaland abnonnalcell division hasmadethem a prime target in the developmentof drugs for the treatment of cancer (Box 12-5). Mutant forms of the G protein Ras are corrrmonin tumor cells. The ras oncogeneencodesa protein with normal GTP bindingbut no GTPaseactivity.The mutant Rasprotein is therefore alwaysin its activated (GTP-bound)form, regardlessof the signalsarrivingthrough normalreceptors. The result can be unregulatedgrowth. Mutationsin rns areassociated with 30%to 50%of lungandcoloncarcmomasand more than 90o/o of pancreaticcarcinomas.r

Defects inCertain 6enes Remove lrlorrnal (ell Restraints on Division Ttrmor suppressor genes encodeproteinsthat normally restrain cell division.Mutation in one or more of these genes can Iead to tumor formation. Unregulated growth due to defective tumor suppressor genes, unlike that due to oncogenes, is genetically recessive; tumors form only 1f botlz chromosomes of a pair contain a defective gene. This is because the function of these genes is to prevent cell division, and if either copy of the gene for such a protein is normal, the normal inhibition of dir.rsionwill take place In a person who inherits one correct copy and one defective copy, every cell begins with one defective copy of the gene. If any one of those 1012somatic cells undergoes mutation in the one good copy, a tumor may grow from that doubly mutant cell. Mutations in both copies of the genes for pRb, p53, or p21 yield cells in which the normal restraint on cell clivision is lost and a tumor forms. Retinoblastoma occurs in children and causesblindness if not surgically treated. The cells of a retinoblastoma have two defective versions of the fib gene (two defective alleles). Very young children who develop retinoblastoma commonly have multiple tumors in both eyes. These children have inherited one defective copy of the ftb gene, which is present in every cell; each tumor is derived from a single retinal cell that has undergone a mutation in its one good copy of the fib gene (A fetus with two mutant alleles in every cell is nonviable.) People wrth retinoblastoma who survive childhood also have a high incidence of cancers of the lung, prostate, and breast later in life. A far less likely event is that a person born with two good copies of the fib gene will have indepenclent mutations in both copies in the same cell. Some individuals do develop retinoblastomas later in childhood, usually with oniy one tumor in one eye These individuals were presumably born with two good copies (alleles) of fib in

l e a t h| 4 7 5 | u popr r e sG s oern easn, dP r o g r a m mCeedlD 12.12 0 n c o g e n e s , TS um

Whena singlecell divideswithout any regulatory limitation, it eventuallygivesrise to a clone of cells so large that it interferes with normal physiologicalfunctions (Fig. 1). This is cancer,a leadingcauseof death in the developedworld, and increasinglyso in the developing world. In all types of cancer,the normal regulationof cell divisionhasbecomedysfunctionaldue to defectsin one or more genes.For example,genesencodingproteins that normally send intermittent signalsfor cell division become oncogenes,producing constitutively active signalingproteins; or genes encodingproteins that normally restrain cell division (tumor suppressor genes)mutate to produceproteinsthat lack this braking function. In many tumors, both kinds of mutation haveoccurred. Many oncogenesand tumor suppressorgenesencodeprotein kinasesor proteinsthat act in pathwaysupto streamfrom proteinkinases.It is thereforereasonable protein kinasescould hope that specificinhibitors of prove valuablein the treatment of cancer.For example, a mutant form of the EGF receptoris a constantlyactive receptor Tlr kinase (RTK), signaling cell division whetherEGF is presentor not (seeFig. 12-49).In about 30%of all womenwith invasivebreastcancer,a mutation in the receptorgeneHER2/neuyields an RTK with activity increasedup to 100-fold.AnotherRTK,vascular endothelial growth factor receptor (VEGF-R), must be activatedfor the formation of new blood vessels(angiogenesis)to prolede a solid tumor with its own blood supply,and inhibition of \IEGF-R might starvea tumor of essentialnutrients. NonreceptorTlr kinasescan also mutate, resulting ir constant signalingand unregulated cell division.For example,the oncogeneAbl (from the with acutemyeloid leukemiavirus) is associated ,Abelson leukemia,a relativelyrare blooddisease(-5,000 casesa year in the United States).Anothergroup of oncogenes

encode unregulated cyclin-dependentprotein kinases. In each of these cases,specificprotein kinaseinhibitors might be valuablechemotherapeuticagentsin the treatment of disease.Not surprisingly,huge efforts are under way to develop such inhibitors. How should one approachthis challenge? Protein kinasesof all types show striking conservation of structure at the activesite. All sharewith the prototrcical PKA structure the featuresshown n Frgure2: two lobesthat enclosethe active site, with a P loop that helps to align and bind the phosphorylgroupsof ATP,an activationloop that movesto open the active site to the protein substrate,and a C helix that changesposition as the enzyme is activated, bringing the residues in the substrate-bindingcleft into their binding positions. The simplest protein kinase inhibitors are ATP analogsthat occupy the ATP-binding site but cannot serve as phosphorylgroup donors. Many such compoundsare known,but their clinicalusefuhessis limited by their lack of selectivity-they inhibit virtually all protein kinases and would produce unacceptableside (continued on nent page)

P loop C helix

Activation loop PD318088 Catalytic loop

Carboxylterminal lobe

divisionof a singlecell in thecolonledto a pri1 Unregulated FIGURE cancersareseen to the liver.Secondary marycancerthat metastasized aswhitepatchesin thisliverobtainedat autopsy

featuresof the activesiteof proteinkinases(PDB 2 Conserved FIGURE lobessurround and carboxyl-terminal lD 159l).The amino-terminal the activesiteof the enzyme,nearthe catalyticloop and the sitewhere ATPbinds.The activationloop of this and manyother kinasesunderthen movesawayfrom the activesiteto expose goesphosphorylation, cleft,which in this imageis occupiedby a spethe substrate-binding in the PD318088.TheP loop is essential enzyme, of this cific inhibitor for ATP aligned be correctly must also helix C and the of ATR binding bindingand kinaseactivity.

\rd

Biosisnatins

effects. More selectivity is seen with compounds that fill part of the ATP-binding site but also interact outside this site, with parts of the protein unique to the target protein kinase. A third possible strategy is based on the fact that although the active conformations of all protein kinases are similar, their inactive conformations are not. Drugs that target the inactive conformation of a specific protein kinase and prevent its conversion to the active form may have a higher specificity of action. A fourth approach employs the great specificity of antibodies. For example, monoclonal antibodies (p. 173) that bind the extracellular portions of specific RTKs could eliminate the receptors' kinase activity by preventing dimerization or by causing their removal from the cell surface. In some cases, an antibody selectively binding to the surface of cancer cells could cause the immune system to attack those cells. Between 1998 and mid-2006, only eight new drugs were approved in the United States for use in cancer therapy: five small molecules and three monoclonal antibodies, each having shown efflcacy in clinical trials. For example, imatinib mesylate (Gleevec; Fig. 3a), one of the small molecule inhibitors, has proved nearly 100% effective in bringing about remission in patients mth early-stage chronic myeloid leukemia. Erlotinib (Tarceva; Fig. 3b), which targets EGF-R, is effective against advanced non-small-cell lung cancer (NSCLC). Because many cell-division signaling systems involve more than one protein kinase, inhibitors that act on several protein kinases may be useful in the treatment of cancer. Sunitinib (Sutent) and sorafenib (Nexavar) target several protein kinases, including VEGF-R and PDGF-R. These two drugs are in clinical use for patients with gastrointestinal stromal tumors and advanced renal cell carcinoma, respectively. Tlastuzumab (Herceptin), cetuximab (Erbitux), and bevacizumab (Avastin) are monoclonal antibodies that target HER2/neu, EGF-R, and VEGF-R, respectively; all three drugs are in clinical use for certain types of cancer. At least a hundred more compounds are in preclinical trials. Among the drugs being evaluated are some obtained from natural sources and some produced by synthetic chemistry. Indirubin is a component of a Chinese herbal preparation traditionally used to treat certain leukemias; it inhibits CDK2 and CDKS. F lavopiridol (Fig. 3d), a synthetic analog of an alkaloid extracted from the stem bark of the Indian plant Amoora rohi,tuka, is a general CDK inhibitor. With several hundred potential anticancer drugs heading toward clinical testing, it is realistic to hope that some will prove more effective or more target-specific than those now in use.

(a) Imatinib (Gleevec) bound to Abl

(b) Erlotinib (Tarceva) bound to EGF-R

(c) AtP boundto CDK2

(d) Flavopiridol bound to CDK2

NH z-l

(

Y//

\-

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\-) \-N'-

Imatinib (Gleevec)

o OH 'N-

I Erlotinib (Tarceva)

FIavopiridol

FIGURE 3 Someproteinkinaseinhibitors now in clinicaltrialsor clinical use,showingtheirbindingto thetargetprotein.(a)lmatinibbindsto theAbl oncogene kinaseactivesite(PDBlD l lEp);it occupiesboththe ATP-binding siteand a regionadjacentto thatsite.(b) Erlotinibbinds to the activesiteof ECF-R(PDBtD 1M17) (c), (d) Flavopiridol is an inhibitorof the cyclin-dependent kinaseCDK2;shownnereare normal ATPbinding(c) at the activesire(PDBlD 1S9l)and flavopiridol binding(d),whichprevents the bindingof ATp(pDBtD 2A4L).

l e a t h| 4 7 7 | s oe rn easn, dP r o g r a m mCeedlD 1 2 . 1 20 n c o g e n e s , I uSmuoprp r e sG

every cell, but both -Rballeles in a single retinal cell have undergone mutation, Ieading to a tumor. After about age three, retinal cells stop dividing, and retinoblastomas at later ages are quite rare. Stability genes (also called caretaker genes) encode proteins that function in the repair of major genetic defects that result from aberrant DNA replication, ionizing radiation, or environmental carcinogens. Mutations in these genes lead to a high frequency of unrepaired damage (mutations) in other genes, including proto-oncogenesand tumor suppressor genes, and thus to cancer. Among the stability genes are ATM (see Fig. J2-aD; the XP gene family, in which mutations lead to xeroderma pigmentosum; and the BRCAI genes associated with some t;,pes of breast cancer (see Box 25-I). Mutations in the gene for p53 also causetumors; in more than 90% of human cutaneous squamous cell carcinomas (skin cancers) and in about 50% of all other human cancers, p53 is defective. Those very rare indileduals who inheri,t one defective copy of p53 commonly have the LiFraumeni cancer syndrome, with multiple cancers (of the breast, brain, bone, blood, Iung, and skin) occurrirLg at high frequency and at an early age. The explanation for multiple tumors in this case is the same as that for Rb mutations: an individual born with one defective copy of p53 n every somatic cell is Iikely to suffer a second p53 mutation in more than one cell during his or her lifetime. In summary then, three classesof defects can contribute to the development of cancer: oncogenes, in which the defect is the equivalent of a car's accelerator pedal being stuck dov,n, wrth the engine racing; mutated tumor suppressor genes, in which the defect leads to the equivalent of brake failure; and mutated stability genes, with the defect leading to unrepaired damage to the cell's replication machinery, the equivalent of an unskilled car mechanic. Mutations in oncogenesand tumor suppressorgenes do not have an all-or-none effect. In some cancers, perhaps in all, the progression from a normal cell to a malignant tumor requires an accumulation of mutations (sometimes over several decades),none of which, alone, is responsible for the end effect. For example, the development of colorectal cancer has several recognizable stages,each associatedwith a mutation (FiS. 12-50). If an epithelial cell in the colon undergoes mutation of both copies of the tumor suppressor geneAPC (adenomatous polyposis coli), it begins to divide faster than normal and produces a clone of itself, a benign polyp (early adenoma) For reasons not yet known, the APC mutation resr-rltsin chromosomal instability, and whole regions of a chromosome are lost or rearranged during cell dhrsion Ttus instability can lead to another mutation, commonly in ras, that converts the clone into an intermediate adenoma. A third mutation (often in the tumor suppressor gene DCC) Ieads to a late adenoma. Only when both copies of p53 become defective does this cell mass become a carcinoma-a malignant, Iife-threatening tumor. The full sequence therefore requires at least

NormaI colorectal epithelium

l-"

t

Early adenoma Intermediate adenoma

L-,e, 1 pcc )

Advanced adenoma ---l

por

i

Colorectal carcinoma

Tumor suppressor gene Oncogene Unknown

status

12-50 Fromnormalepithelialcell to colorectalcancer.ln the FIGURE geneAPC lead colon,mutationsin both copiesof the tumorsuppressor (earlyaderapidly multiply too that cells of epithelial to benignclusters mutation a second APC undergoes in defective noma) lf a cell already to an intergives rise cell mutant ras, the doubly proto-oncogene in the forminga benignpolyp.Whenoneof thesecellsunadenoma, mediate genesDCC andp53, dergoesfurthermutationsin the tumorsuppressor in Senesnotyet mutations tumorsform.Finally, aggressive increasingly lead to a malignanttumor and finallyto a metastatic characterized tumorthat can spreadto othertissues.Most malignanttumorsof other probablyresultfrom a seriesof mutations suchasthis. tissues

sevengenetic "hits": two on each of three tumor suppressorgenes (,4PC,DCC, and p53) and one on the proto-oncogener.I,s.There are probablyseveralother routes to colorectalcancer as well, but the principle that full malignancy results only from multiple mutations is likely to hold true for all of them. When a polyp is detected in the early adenomastage and the cells containing the flrst mutations are removed surgically, late adenomasand carcinomaswill not develop;hence the importanceof early detection.Cellsand organisms, too, have their early detection systems'For example, the ATM and ATR proteins describedin Section12.11 can detectDNA damagetoo extensiveto be repairedeffectively. They then trigger, through a pathway that includesp53,the processof apoptosis,in which a cell that hasbecomedangerousto the organismkills itself.r

(ellSuicide lsProgrammed Apoptosis Many cells can precisely control the time of their ov,n death by the processof programmed cell death, or apoptosis (app'-a-toe'-sis;from the Greek for "dropping off," as in leavesdropping in the fall)' One trigger for apoptosisis irreparabledamageto DNA' Programmed cell death also occurs during the developmentof an

o78- Biosignaling J, embryo,when somecellsmust die to give a tissueor organ its final shape. Carving fingers from stubby limb budsrequiresthe preciselytimed deathof cellsbetween developingfinger bones. During developmentof the nematodeC elegansfrom a fertilizedegg,exactly l3l cells (of a total of 1,090somaticcells in the embryo) must undergoprogranuneddeathin order to construct the adult body Apoptosisalsohasrolesin processesother than development.If a developrngantibody-producing cell generates antibodies against a protein or glycoprotein normailypresentin the body,that cell undergoesprogrammed death in the thymus gland-an essential mechanism for eliminating anti-self antibodies. The monthly sloughingof cells of the uterine wall (menstruation) is anothercaseof apoptosismediatingnormalcell death.The droppingof leavesin the fall is the result of apoptosisin specificcells of the stem. Sometimescell suicideis not programmedbut occursin responseto biologicalcircumstancesthat threatenthe rest of the organism. For example, a virus-infected cell that dies beforecompletionof the infectioncyclepreventsspread ofthe virusto nearbycells.Severestressessuchasheat, hyperosmolarity,W light, and garTrmairradiation also trigger cell suicide;presumablythe organismis better off with any aberrant,potentially mutated cells dead. The regulatory mechanismsthat trigger apoptosis involve someof the sameproteins that regulatethe cell cycle. The signal for suicide often comesfrom outside, through a surface receptor. Titmor necrosis factor (TNF), producedby cells of the rmmunesystem,interactswith cellsthroughspeci_fic TNF receptors.Thesereceptors have TNF-bindingsites on the outer face of the plasmamembraneand a "death domain" (-80 amino acidresidues)that carriesthe self-destructsignaltlLrough the membraneto cytosolic proteins such as TRADD (?NF receptor-associated deathdomain) (Fig. t2-51). Another receptor, Fas, has a similar death domain that allows it to interact with the cytosolicprotein FADD (flas-associateddeath domain), which activatesthe cytosolic proteasecaspase8. This enzyrnebelongsto a family of proteasesthat participate in apoptosis;all are synthesizedas inactive proenzyrnes,all have a critical Cysresidueat the active site, and all hydrolyzetheir target proteinson the carboxyl-terminalside of specificAsp residues(hencethe name caspase,from Cys andAsp). When caspase8, an "initiator" caspase,is activated by an apoptotic signalcarried through FADD, it further self-activatesby cieavingits own proenz).rneform. Mitochondriaare one target of active caspase8. The proteasecausesthe releaseof certain proteins contained betweenthe inner and outer mitochondrialmembranes: c;,tochromec (Chapter 19) and several,,effector"caspases.Cytochromec bindsto the proenz5.rne form of the effectorenzymecaspase9 and stimulatesits proteolytic activation. The activated caspase9 in turn catalyzes wholesale destruction of cellular proteins-a major

DNase

I

*

Cell death

FIGURE 12-51 Initialeventsof apoptosis. Receptors in the plasmamembrane(Fas, TNF-R1)receivesignals fromoutsidethecell (theFasligandor tumornecrosis factor(TNF),respectively). Activatedreceptors fosterinteractionbetweenthe "deathdomain"(anB0 aminoacid sequence) of Fas orTNF-R'landa similardeathdomainin thecytosolic proteins FADDor TRADD.FADDactivates a cytosolicprotease, caspase 8, thatproteolytically activates othercellularproteases. proteases TRADDalsoactivates Theresultingproteolysis is a primaryfactorin cell death

causeof apoptoticcell death.One speciflctarget of caspase action is a caspase-activated deoxyribonuclease. In apoptosis,the monomericproducts of protein and DNA degradation(aminoacidsand nucleotides)are releasedin a controlledprocessthat allowsthem to be taken up and reused by neighboringcells. Apoptosis thus allows the organismto eliminate a cell that is unneeded or potentially dangerouswithout wasting its components.

SUMMAR 1Y 2 . 1 2 0 n c o g e nTeus m , or S u p p r e sG s oern easn, d P r o g r a m m( e ldlD e a t h r

Oncogenes encodedefectivesignalingproteins.By continuallygiving the signalfor cell divrsion,they leadto tumor formation.Oncogenes are genetically dominant and may encodedefectivegrowth factors, receptors,G proteins,protein kinases,or nuclear regulatorsof transcription.

r

Ttrmor suppressorgenesencoderegulatoryproteins that normally inhibit cell division;mutations in thesegenesare geneticallyrecessivebut can lead to tumor formatiorL.

f_-__l

F u r t h eRre a d i n gI 4 7 9 )

Canceris generallythe result of an accumulationof mutationsin oncogenesand tumor suppressor genes. Whenstabilitygenes,which encodeproteins necessaryfor the repair of geneticdamage,are mutated, other mutations go unrepaired,including and tumor suppressor mutationsin proto-oncogenes genesthat can Ieadto cancer. Apoptosiscan be triggered by extracellularsignals such as TNF, acting through plasmamembrane receptors.

KeyTerms

responseregulator 458 receptorlikekinase (RLK) 460 rhodopsin 463 opsin 463 transducin 463 rhodopsinkinase 465 arrestin L 465 receptor potential 467 cyclin 469 cyclin-dependent Protein kinase(CDK) 469

471 ubiquitin proteasome 47L growth factors 472 retinoblastomaprotein (pRb) 472 oncogenes 473 proto-oncogenes 473 tumor suppressor genes 474 programmedcell death 477 apoptosis 477

Reading Further

Terms i,n bold are defined i,n the glossary cooperativitV 420 ampliication 420 enzJrmecascade 420 desensitization 420 Scatchardanalysis 421 G protein-coupled receptors (GPCRs) 423 secondmessenger 423 guanosinenucleotide-binding protein 423 proteins 423 G agonists 423 423 antagonists B-adrenergicreceptors 423 stimulatory G protein (G,) 424 GTPaseactivator proteins (GAPs) 426 regulatorsof G protein signaling(RGSs) 426 guanosinenucleotideexchangefactors (GEFs) 426 adenylylcyclase 426 protein cAMP-dependent krnase(proteinkinaseA; PKA) 427 consensus sequence 429

inhibitory G protein (G,) 43r adaptor proteins 431 AKAPs (A kinase anchoring proteins) 431 phospholipaseC (PLC) 432 inositol1,4,5-trisphosphate (lPa) 432 proteinkinaseC (PKC) 433 green fluorescent protein (GFP) -134 fluorescence resonance anard\/

frqnqfer

(FRET) 435 calmodulin(CaM) 436 Ca2* /caimodulin-depenctent proteinkinases(CaM kinases) 437 receptor tyrosine kinase (RTK) 43e SH2 domain 439 small G proteins 440 MAPKs 440 443 cltokine guanosine3',5'-cyclic (cyclic monophosphate GMP;cGMP) 445 nrrelie nrrclentidp protein cGMP-dependent phosphodiesterase 430 kinase (protein kinaseG; PKG) 445 B-adrenergicreceptorkinase nicotinic acetylcholine 431. @ARK) (Barr; receptor 453 B-arrestin integrin 455 arrestin 2) 43I hormone response G protein-coupled element (HRE) 456 receptor kinases (GRKs) 431 two-component signaling systems 457 cAMPresponseelement hindino nrotein receptor histidine (OREB) 43r kinase 457

General Cohen, P. (2000) The regulationof protein function bv multisite phosphorylation-a 25 year tpdate Ttends Bzochem Sci 26, 596-601 Histoncalaccountof protein phosphorylation Giepmans, B.N.G., Adams, S.R., Ellisman, M.H., & Tbien' R.Y. (2006) The fluorescenttoolbox for assessingprotein location and finction Sci,enceBl2, 217-224. A short, intermediatelevel review of FRET. Pawson, T. & Scott, J.D. (2005) Protein phosphorylationin signali4g-50 years and cor-urtingTlends Biochem Scz.3O,286-290 G Protein-Coupled

Reeeptors (GPCRs)

Beene, D.L. & Scott, J.D. (2007) A-kinaseanchoringproteins take shape.Curr Opin CelI Bi,o, 19, 192-198. Birnbaumer, L. (2007) The discoveryof signaltransductionby G proteins:a personalaccountand an overviewof the initial findings and contributionsthat led to our presentunderstanding.Bzochi'm Biophg s. Acta 1768, 756-77| Cooper, D.M.F. & Crossthwaite' A.J. (2006) Higher-orderorganization and regulationof adenylylcyclases Ttends Pharrnacol Sci 27,42643r DeWire, S.M., Ahn, S', Lefkowitz, R.J., & Shenoy, S.K. (2007) €-arrestinsand cell signaltng.Annu Reu Phgsi'o\.69' 483-510 Advancedreview;one of five reviewson arrestinsin this issue Escrib6, P.V.(2007) G protein-coupledreceptors,signalingmechanismsand pathophysiologicalrelevance.Bi'ochi,m Bi,ophys Acta Biomembr 1768,747. The editorial introduction to a seriesof 20 paperson GPCRs Fredriksson, R, & Schitith, H.B. (2005) The repertoire of G-protein-coupledreceptorsin fully sequencedgenomesMoI Phat-mncol 67,7414-1425. Hannm,H.E. (1998) The many facesof G protein signaling..l Biol Chem 273,669-672. Introduction to a seriesof short reviewson G proteins Kim, C., Xuong, N.-H., & Tbylor, S.S. (2005) Crystaistructure of a complexbetweenthe catalyticand regulatory (RIa) subunitsof PKA Sci,ence307, 690-696 Kobilka, B,K. (2006) G protein coupledreceptor structure and activation.Bi,ochi'm BiophEs Acta 1769, 794-807 Lefkowitz, R.J. (2007) Introduction to specialsectionon arrestins. Annu Reu Physi'ol 69 This introducesflve excellentadvancedreviewson the roles of arrestin

[480]

Biosignating

Pinna, L.A. & Ruzzene, M. (1996) How do protein krnasesrecognize their substrates? Bzochim Bio12tt7,1s Acta IBl.4, lgl-225 Advanced review of the factors, including consensussequencesr that give protein klnases their specificity

Changeux, J.-P. & Edelstein, S.J. (2005) AJlostericmechanisms of srgnaltransduction Sctence3O8, 1424-1428 Short, intermediatelevel descriptionof receptor allostery,using the acetylcholinereceptor as example.

Rehmann, H., Wittinghofer, A., & Bos, J.L. (2002) Capturing cyclic nucleotides in action: snapshots from crystallographic studies Nat Reu Mol Cell.Biol 8,63-73

Tombola, F., Pathak, M.M., & Isacoff, E.Y. (2006) How does voltageopen an ion channel?Annu Reu CeLL Deu B,i,oI 22,28-b2.

Schoeneberg, T., Hofreiter, M., Schulz, A., & Roempler, H. (2007) Learning from the past: evolution of GpCR functrons ?rends Phat"mo.coLSci 28, \17-12I Yeagle, P.L. & Albert, A.D. (2006) G-protein coupled receptor structure Br,ocLtirn Bioph.ys Acta 1768,808 824 Zhang, J., Campbell, R.E., Tlng, A.y., & Tbien, R.y. (2002) Creating new fluorescent probes for cell blology. Nat Reu MoI CeILBioL 3, 906-918 The basis for techniques such as those descrrbed in Box 12-3 Receptor

Enzymes

Hubbard, S.R. & Miller, W.T. (2002) Receptor tvrosine krnases: mechanisms of activation and signaling Czr,m Opin CeIt Biot lg, 117-),23 Maures, T.J., Kurzer, J.H., & Carter-Su, C. (2007) SH2B1 (SH2B) and JAK2: a multifunctional adaptor protein and kinase made for each other Trends EndocrhrcL Metab l8, BB 45 Saltiel, A.R. & Pessin, J.E. (2002) Insulin signaling pathways in time and space Tiends Bi,oche.m Sci 12,6b-TI Short, intermediatelevel review. Shields, J.M., Pruitt, K., McFall, A., Shaub, A., & De4 C.J. (2000) Understanding Ras: it ain't over'trl it,s over Ttend,s Cett BioL 1o,147 154 Intermediatelevel renew of the monomeric G protein Ras Tlganis, T. & Bennett, A.M. (2002) Protein tyrosine phosphatase function: the substrate perspective Bi,ochem J 4OZ, I-fi Zqjchowski, L.D. & Robbins, S.M. (2002) Lipid rafts and little caves: compartntentalized signalling in membrane microdomains Eur J. Bi,ochem 269,737-Tb2 Proteins

and Membrane

Rafts

Bhattacharyya, R.P., Rem6nyi, A., yeh,8.J., & Lim, W.A. (2006) Domains, motifs, and scaffolds: the role of modular interactions in the evolutron and wlring of cell signaling circtits Annu Reu Btochem 75,655-680 Advanced review of rnodular signaling proteins Pawson, T. (2007) Dynamrc control of signaling by modular adaptor proteins Cun Opin CeLlBioL 19, l1Z-116 Smith, F.D., Langetrerg, L,K., & Scott, J.D. (2006) Anchorecl cAMP signaling: onward and upward A short history of compartmen_ talized cAMP signal transduction. Eur J. CeLLBi,ot gb, 591-69g Short, intermediatelevel review introducing an entire journal is_ sue on the subject of AIiAPs and cAMp signaling Smith, F.D., Langeberg, L.K., & Scott, J.D. (2006) The where's and when's of kinase anchoring Tlends Biochem Sci, Bl-, JI6_BZJ. Intermediate review of AKAps Receptor

Ion Channels

See also Chapter

Chin, D. & Means, A.R. (2000) Calmodulin:a prototypical calcilrm receptor TtrendsCeILBioI lO, 322-328 Thkahashi,A., Camacho,P.,Lechleiter, J.D., & Herman, B. (1ggg) Measurement of intracellularcalcfumPhysia| Reu 79, l08g-112b Advancedreview of methodsfor estimatingintracellularCaz+ levelsin real time. Integrins

Gartrers, D.L., Chrisman, T.D., Wiegn, p., Katafuchi, T., AIbanesi, J.P., Bielinski, V., Barylko, B., Redfield, M.M., & Burnett, J.C., Jr. (2006) Membrane guanylyl cyclase receptors: an tpdate TrendsEndocri,noL Metab 17,251 2b8

Adaptor

Calcium Ions in Signaling Berridge, M.J., Lipp, P., & Bootman, M.D. (2000)The versatility and uruversalityof calciumsignalin1 Nat Reu Mol CelIBi,ol l,ll-ZI. Intermediatereview.

77, Further

Reading,

Ion Channels.

Ashcroft, F.M. (2006) From molecule to malady. Nature 44O, 440-447 Short, intermediatelevel review of human diseasesassociated w r t h d e l e e t si n i o n c h a r r n e l s .

Delon, I. & Brolvn, N.H. (2007) Integrins and the actin cy'toskeleton. Curr. Oyti'n CeIIBi,oL 19,43-50 Steroid Hormone Receptors and Action Biggins, J.B. & Koh, J.T. (2007) Chemicalbiologyof steroid and nuclearhormonereceptorsCum Opin Chem Bi,ot.11,gg-lf0 HaIl, J.M., Couse, J.F., & Korach, K.S. (2001) The multifaceted mechanismsof estradioland estrogenreceptor signaling J Bzol Ch.e'm276, 36,869-36,872 Brief, intermediate-levelreview. Signaling in Plants and Bacteria Assmann, S.M. (2005) G proteinsgo green:a plant G protein signalingFAQ sheet Science 3lO,71,-73 Bakal, C.J. & Davies, J.E. (2000) No longer an exclusiveclub: eukaryoticsignalingdomainsin bacteria Ttends CeILBi,ol. 10. B2-S8 Intermediatereview. Becraft, P.W (2002) Receptorkinasesignalingin plant development.Annu Reu CeILDeuBi.oL18,163-192 Advancedreview. Chen, Y.F.,Dtheridge, N., & Schaller, G.E. (200b) Ethylene signaltransductionAnn BotanA95, 901-91b Intermediatelevel review. Ferreira, F.J. & Kieber, J.J. (2005) Cytokinin signa\ng.Cum. Opin PLardBioL 8, 518-525 Hwang, I., Chen, H.-C., & Sheen,J. (2002)Tho-componentcircuitry nArabidopsis c1'tokininsignaltransduction.Natttre 4lB, B8J-B89 Li, J. & Jin, H. (2007) Regulationof brassinosteroidsignaling TtrendsPlant Sci, 12,37-41. Liu, X., Yue, Y, Li, B., Nie, Y.,Li, W., Wu, W.-H.,& Ma, L. (2007) A G protein-coupledreceptoris a plasmamembrane receptor for the plant hormoneabscisicacid. Sci,enceBlE. 17t2-1216 McCarty, D.R. & Chory, J. (2000) Conservationanclinnovationin plant signalingpathways.CeII 103,201-209 Meijer, H.J.G. & Munnik, T. (2003) Phospholipid-based signaling rnplants Annu Reu PIantBi,oI 54.265-306 Advancedreview. Nakagami, H., Pitzschke, A., & Hirt, H. (200b) EmergingMAp kinasepathwaysin plant stresssignaling Tfends plant Sci 10, 339-346 Paciorek, T. & Friml, J (2006) Aruin signaling J. CeIISci.. ll9, 1199-1202 Tlchtinsky, G., Vanoosthuyse, V., Cock, J.M., & Gaude, T. (2003) Maklnginroadsinto plant receptor khase signallingpathways Tlends Plant Sci 8. 231-237.

Problems i +ot l Vert, G., Nemhauser, J.L,, Geldner, N., Hong, F., & Chory, J. (2005) Molecularmechanismsof steroidhormonesignallingin planLsAnnu Reu CellDeu BioL 2L,I77-201. \lsion, Olfaction, and Gustation Baylor, D. (1996) Ho,&photonsstart vision Proc Natl Acad Sci" usA 93, 560-565 One of six short reviewson vision in this journal issue. Margolskee, R.F. (2002) Molecularmechanismsof bitter and sweet tastetransductionJ. Bi,ol Chem 277,14 Menon, S.T.,Han, M., & Sakmar,T.P.(2001)Rhodopsin:structural basisofmolecularphysiology. Physi,olReu 81,1659 1688 Advancedreview Mombaerts, P. (2001)The humanrepertoireofodorantgenes Annu Reu Genom Hum Genet,2,493-510 andpseudogenes Advancedrenew Cell Cycle and Cancer Bartek, J. & Lukas, J. (2007) DNA damagecheckpoints:from initlation to recoveryor adaptation Cun Opin Cell B'iol 19,238-245 Bublil, E.M. & Yarden, Y. (2007) The EGF receptor family: spearheadinga merger of signalingand therapeutics.Cun Opin CellBiol Ig,124-134 Chau, B.N. & Wang, J.Y.J. (2003) Coordinatedregulationof life anddeathbyRBNor Reu Cam,cer!,130 138. receptors Dorsam, R.T. & Gutkind, J.S. (2007)G-protein-coupled and cancerNature Reu Cancer 7,79 94 Kinzler, K.Iry.& Vogelstein, B. (1996) Lessonsfrom hereditary colorectalcancerCell 87,159-170 Evidencefor multistep processesin cancerdevelopment. Levine, A.J. (1997) p53, the cellulargatekeeperfor growth and division Cell 88, 323-331 Interrnediatecoverageof the function of protein p53 in the normal cell cycle and in cancer Obaya, A.J. & Sedirry,J.M. (2002) Regulationof cyclln-Cdk actMty in mammaliancells CeLl MoI Li,JeSci 59,126-142 Sher4 C.J. & McCormick, F. (2002) The RB and p53 pathwaysin cancer Cancer CelI 2, 102-112

in this chapter about hormone action, interpret each of the experimentsdescribedbelow. Identify substanceX and indiofthe results. catethe signiflcance (a) Addition of epinephrine to a homogenateof normal liver resulted in an increasein the actMty of glycogenphosphorylase.However,when the homogenatewas first centrifuged at a high speed and epinephrine or glucagon was added to the clear supernatant fraction that contains phosphorylase,no increasein the phosphorylaseactivity occurred. (b) When the particulate fraction from the centrifugation in (a) was treated with epinephrine, substanceX was produced. The substancewas isolated and puri-fled.Unlike epinephrine, substanceX activated giycogen phosphorylase when added to the clear supernatantfraction of the centrifuged homogenate. (c) SubstanceX was heat-stable;that is, heat treatment did not affect its capacity to activate phosphorylase (Hint: Would this be the caseif substanceX were a protein?) Substance X was nearly identicai to a compound obtained when pure ATP was treated with barium hydroxide. (Fig. 8-O wiil be helpful ) 2. Effect of Dibutyryl cAMP versus cAMP on Intact Cells The physiologicaleffects of epinephrineshouldin principle be mimicked by addition of cAMP to the target cells' In practice, addition of cAMP to intact target cells elicits only a minimal physiologicalresponse.Why? When the structuraliy related derivative dibutyryl cAMP (shown below) is added to intact cells,the expectedphysiologicalresponseis readily apparent. Explain the basis for the difference in cellular responseto these two substances.Dibutyryl cAMP is widely used in studiesof cAMP function.

\-,"",,,.".

Apoptosis Anderson, P. (1997) Kinasecascadesregulatingentry into apoptosis Microbiol MoI Biol Reu 61,33-46 Ashkenazi, A. & Dixit, V.M. (1998) Death receptors:signalingand modulationScience281, 1305-1308 This and the papersby Green& Reedand Thornberry & Lazebnik(below) are in an issueofScierzcedevotedto apoptosis Duke, R.C., Ojcius, D.M., & Young,J.D.-E. (1996)Cellsuicidein Scz Am 275 (December),80-87 hea.lthand disease. Green, D.R. & Reed, J.C. (1998)Mitochondriaand apoptosis. Science281, 1309-1312 Jacobson, M.D., Weil, M., & Raff, M.C. (1997)Programmedcell death in animaldevelopment CelI 88,347-354 Lawen, A. (2003) Apoptosis-an introduction Bi,oessays25, 888-896 Thornberr5r, N.A. & Lazebnik, Y. (1998) Caspases:enemieswithin Science281, 1312

Problems 1. Hormone Experiments in Cell-Free Systems In the 1950s,Earl W. Sutherland,Jr., and his colleaguescarriedout pioneeringexperimentsto elucidatethe mechanismof action of epinephrine and glucagon. Given what you have learned

-cll2-'o H

o:P-O I o(

O. tc-(cHolocH" DibutyrYlcAMP

{N6,02'-Dibutyryl adenosine 3',5'-cyclic monophosphate)

3. Effect of Cholera Toxin on Adenylyl Cyclase The gram-negative bacterltm Vibri'o cholercLeproducesa protein, choleratoxin (M. 90,000),that is responsible for the characteristicsymptoms of cholera: extensive loss of body water and Na" through continuous,debilitating diarrhea.If body fluids and Na* are not replaced,severedehydration results; untreated, the disease is often fatal. When the choleratoxin gainsaccessto the humanintestinal tract it binds tightly to specific sites in the plasma membrane of the epithelial cells lining the small intestine,

[.trl

Biosignating

causing adenylyl cyclaseto undergo prolonged activation (hoursor days). (a) What is the effect of choleratoxin on tcAMpl in the intestinal cells? (b) Based on the information above, suggesthow cAMp normally functions in intestinal epithelialcells (c) Suggesta possibletreatment for cholera.

13. Nonhydrolyzable GTP Analogs Many enzJ,mes can hydrolyzeGTPbetweenthe p and 7 phosphates.The GTPanalog 5'-triphosphate(Gpp(NH)p), shownbeB,7-imidoguanosine Iow, cannot be hydrolyzed between the B and 7 phosphates.

o

4. Mutations in PKA Explain how mutations in the R or C subunit of cAMP-dependentprotein kinase (pKA) might lead to (a) a constantiyactivePKA or (b) a constantlyinactivepKA. 5. Therapeutic Effects of Albuterol The resprratory slmptoms of asthma result from constriction of the bronchiand bronchiolesof the lungs,causedby contraction of the smoothmuscleof their walls. This constriction can be reversedby raising [cAMP] in the smooth muscle Explain the therapeuticeffects of albuterol,a B-adrenergicagonist taken (by inhalation) for asthma Would you expect this drug to have any side effects?How might one designa better drug that did not havetheseeffects? 6. Termination of Hormonal Signals Signais carried by hormonesmust eventuallybe terminated.Describeseveral different mechanismsfor signaltermination 7. Using FRET to Explore Protein-protein Interactions in Vivo Figure 12-8 showsthe interaction between B-arrestin and the B-adrenergicreceptor. How would you use FRtrT (seeBox 12-3) to demonstratethis interactionin living cells?Which proteins would you fuse? Which wavelengths would you use to illuminate the cells, and which would you monitor?What would you expectto observeif the interactionoccurred?If it did not occur?How might you explain the failure of this approachto demonstratethis interaction? 8. EGTA Iqi ection EGTA (ethyleneglycol-bis( p-aminoethyl ether)-N*V*V'fl'-tetraacetic acid) is a chelating agent with high affinity and speciicity fot Ca2*. By microinjecting a cell with an appropriateCa2*-EGTAsolution,an experimentercan preventcytosoiic[Ca2*]from risingabovel0-7 u. How would EGTA microinjectionaffect a cell'sresponseto vasopressln (seeTable 124)? To glucagon? 9. Amplification of Hormonal Signals Describe all the sourcesof amplificationin the insulin receptor system.

ooo

ilHilll o-i-*-i-o-Pt-o-7>

ooo OH OH Gpp(NH)p (B,7-imidoguanosine 5'-triphosphate) Predict the effect of microinjection of Gpp(NH)p into a myoclte on the cell'sresponseto B-adrenergicstimulation. 14. Use of Toxin Binding to Purify a Channel protein a-Bungarotoxinis a powerful neurotoxin found in the venom of a poisonoussnake(Bungaru,s multi,ci,nctus).It binds with high specificityto the nicotinic acetylcholinereceptor (AChR) protein and preventsthe ion channelfrom opening.This interaction was usedto purify AChRfrom the electric orAanof torpedoflsh. (a) Outline a strategyfor using a-bungarotoxincovalently bound to chromatographybeadsto purify the AChR protein. (Hint: SeeFig. 3-17c.) (b) Outline a strategy for the use of [125l]a-bungarotoxin to purify t he AChRprotein 15. Resting Membrane Potential A variety of unusual invertebrates,including giant clams,mussels,and polychaete worms, live on the fringes of deep-seahydrothermal vents, wherethe temperatureis 60'C (a) The adductor muscle of a giant clam has a resting membranepotential of -95 mV Given the intracelluiar and extracellularionic compositionsshownbelow,would you have predicted this membranepotential?Why or why not?

Concenhation(nM) Ion

Intracellular

Extracellular

Na*

50 400 2l 0.4

440 20 560 10

l\

10. Mutations in ros How would a mutation in ros that leads to formation of a Ras protein with no GTpaseactivity affect a cell'sresponseto insulin? ll. Differences among G Proteins Comparethe G proteins G", which acts in transducingthe signalfrom B-adrenergic receptors,and Ras.Whatpropertiesdo they share?How do they differ?What is the functionaldifferencebetweenG, and G1? 12. Mechanisms for Regulating protein Kinases Identify eight generaltypes of protein kinasesfound in eukaryotic cells, and explain what factor is d,i,recttgresponsiblefor activating eachtype.

(-;a-

(b) Assumethat the adductor muscle membraners permeableto only one of the ions listed above Which ion could determinethe Z*? 16. Membrane Potentiats in Frog Eggs Fertilzation of afrog oocyteby a spermcell triggersionic changessimilarto thoseobservedin neurons (during movementof the action potential) and initiatesthe eventsthat result in cell divisionand development of the embryo. Oocfies can be stimulated to divide

P r o b l e m[-"] s without fertilization,by suspendingthem in 80 mlt KCI (normal pond water contains9 mu KCI). (a) Calculatehow much the changein extracellular [KCl] changesthe resting membranepotential of the oocyte. (Hint: Assumethe oocytecontains120mu K+ and is permeableonly to K* ) Assumea temperatureof 20'C. (b) When the experiment is repeatedin Ca2+-freewater, elevated[KCI] has no effect. What doesthis suggestabout the mechanismof the KCi effect? 17. Excitation Thiggered by Hyperpolarization In most neurons, membrane depolarizati,on leads to the opening of voltage-dependention channels,generation of an action potential, and ultimately an influx of Caz+,which causesrelease of neurotransmitter at the axon terminus. Devise a cellular strategy by which hgperpolari,zati,onfil rod cells could produce excitation of the visual pathway and passageof visual signalsto the brain. (Hint: The neuronal signalingpathway in higher organismsconsists of a series of neurons that relay information to the brain (see Fig 12-35). The signal releasedby one neuron can be either excitatory or inhibitory to the following, postsynaptic neuron.) 18. Genetic "Channelopathies" There are many genetic diseasesthat result from defects in ion channels. For each of the following, explain how the moleculardefect might lead to the symptomsdescribed. (a) A loss-of-functionmutation in the geneencodingthe a subunitofthe cGMP-gated cationchannelofretinal conecells Ieadsto a completeinability to distinguishcolors. (b) Loss-of-functionallelesof the gene encodingthe a subunit of the ATP-gatedK* channel shown in Figure 23-29 iead to a condition known as congenital hyperinsulinismpersistentlyhigh levels of insulin in the blood. (c) Mutationsaffectingthe B subunit of the ATP-gatedKchannelthat prevent ATP binding lead to neonataldiabetespersistently low ievels of insulin in the blood in newborn babies 19. Visual Desensitization Oguchi's diseaseis an inherited form of nisht blindness.A-ffectedindividuals are slow to recovervision after a flash ofbright light againsta dark background,such as the headlightsof a car on the freeway. Suggest what the molecular defect(s) might be in Oguchi'sdisease.Explain in molecuiarterms how this defect would accountfor night blindness 20. Effect of a Permeant cGMP Analog on Rod Cells An analog of cGMP, 8-Br-cGMq will permeate cellular membranes,is only slowlydegradedby a rod cell'sPDE activity,and is as effective as cGMP in opening the gated channej in the cell'souter segment.If you suspendedrod cellsin a buffer containing a relatively high [8-Br-cGMP],then illuminated the cells while measuringtheir membranepotential, what would you obserue? 21. IIot and Cool Taste Sensations The sensationsof heat and cold are transduced by a group of temperature-

gated cation channels.For example, TRPV1, TRPV3, and TRPMS are usually closed, but open under the following 'C; and c o n d i t i o n s :T R P V 1 a t > 4 3 ' C ; T R P V 3 a t > 3 3 TRPMSaI Fez+ NO2 + H2O NO; + 2H* + 2e- --------+ Cytochrome-f (Fe3*) + e ------->

0.816 0.77r 0.421

cytochrome/(Fe'*) Fe(CN)8Fe (CD3- (ferricyanide) + e- ------> -------> Cytochromeo,3(Fe3*) + e

0.365 0.36

cytochromeo3 (Fe2*1 02 + 2H* -t 2e- ----->H2O2 . .a+u],tocnromeo (r'e- J t € ------+

U.JD

cytochromea (Fe2*) Cytochromec(Fe3*1I e ---->

0.29

c (Fe2*) cy'tochrome (Fe3*) * e -------> Cytochromecr

0 254

c, (Fe2*) cyl.ochrome (Fe3+1 + e -------> Cy'tochromeb

0.22

0.295

0.077 0.045 0.031 ------->Hz pH (at standardconditions, 0-,r 0.000 2H* + 2e-0.015 Crotonyl-CoA+ 2H* + 2e --> but5'ryl-CoA -0.166 malatezOxaloacetatez + 2H+ + 2e -------> -0.185 lactateP;.ruvate- + 2H+ + 2e- ------> -0.197 Acetaldehyde+ 2H+ + 2e- --> ethanol -0.219* FAD + 2H+ + 2e- ------+FADH2 b (Fez') cytochrome ubiquinol + H2 tlbiquinone + 2H+ + 2e- -------> succinate2Fumaratez- + 2H+ + 2e- ------+

Glutathione+ 2H+ + 2e ----> 2 reducedglutathione s + 2H+ * 2e ------>H2S

-0.23

dihydrolipoic acid Lipoic acid + 2H* + 2e- -------> NADH NAD" + H* + 2e- -------+ ----> NADPH NADP* + H* + 2e-

-0.320

Acetoacetate+ ZH' + Z€ ----+ p-hydroxybutyt'ate

-0.243 -0.29 -0.324

-0.346

a-Keto$utarate * CO2+ 2H- + 2e- --> isocitrate 2H+ + 2e- ------>Hz(at pH 7) (Fe'*) Ferredoxin (F"t*) + e- ------->ferredoxin

-0.38 -0.414 -0/32

andMolecular of Biochemistry Source:DatamostlyfromLoach,R.A,(1976) In Handbook ' andChenicalData,voll, pp.122-130, G.D.,ed.),Physical edn(Fasman, Biotogy,3td FL BocaRaton, CRCPress, + Thisis thevalueforfreeFAD;FADboundto a specific (e.9.,succinate dehyflavoprotein environment. on its protein E'' thatdepends hasa different drogenase)

free-energychangefor any oxidation-reductionreaction from the values of E'o in a table of reduction potentials ofreactingspecies. (Table13-7) andthe concentrations

al

aa l c t iT o ynp e s ! S t 0 1 B i o e n e r g eat incdsB i o c h e m iRc e

I

W0RKED EXAMPLE 13-3 (alculation of46'oand involve electron transfers.For example,in many organ46ofaRedox Reaction isms, the oxidation of glucose supplies energy for the

Calculate the standard free-energy change, AG'o, for the reaction in which acetaldehyde is reduced by the biological electron carrier NADH: Acetaldehyde+ NADH * H+ ->

ethanol + NAD*

Then calculate the actual free-energy change, AG, when [acetaldehyde] and [NADH] are 1.00 M, and [ethanol] and [NAD*] are 0.100 rr,r.The relevant half-reactions and their,O'' values are: (1) Acetaldehyde+ 2H* + 2e- ------+ ethanol E'' : -0.197Y (2) NAD* + 2H* * 2e ----+ NADH + H+ E'": -0.320Y Remember that, by convention, A,E'ois E'" of the electron acceptor minus E'o of the electron donor. Solution: Because acetaldehyde is accepting electrons (n : 2) from NADH, LE'o : -0.I97 V - (-0.320 \D : 0.123V Therefore, AG'o: -nJ L,E'' : -2(96.5kJN.mol)(0.123V) : -23.7kJlmol This is the free-energy change for the oxidation-reduction reaction at25 "C and pH 7, when acetaldehyde,ethanol, NAD*, and NADH are all present at 1.00 lr concentrations. To calculate AG when [acetaldehyde] and INADHI are 1.00M, and [ethanol] and [NAD+] are 0.100 u, we first determine E for each reductant, using Equation 13-5: llacetaldehyde:.8-

-

^ t RT.

_ln

nd

[acetaldehYde] -_

_

lethanolj

0'026v ' 1'00 ttoroo 2 : -0.197V + 0.013 : -0.167V V (2.303) = -0.197-'' *

^ ^^ RT, rlre-olr: L- + -r" 0 . 3 2 C : ' '. *'

INAD ]

INADIII 0 . 0 2 6v ' 0 . 1 0 0 ttr-oo 2 : - 0 . 3 2 0V + 0 . 0 1 3V ( - 2 . 3 0 3 )= - 0 . 3 5 0V

From this we can calculate A-U.then use Eouation 13-6 to calculate AG: LE : A.G: : :

-0.167V - (-0.350)V : 0.183V -nJ A,E -2(96.5 kJ/V . molX0.183V) -35.3 kJ/mol

This is the actual free-energy change at the speci_fied concentrations ofthe redox oairs.

Cellular Oxidation ofGlurose to(arbon Dioxide (arriers Requires 5pecialized Eleetron The principles of oxidation-reduction energetics described above apply to the many metabolic reactions that

production of ATP. The complete oxidation of glucose: + 602 ------) c6H12o6 6co2 + 6H2o has a AG'o of -2,840 kJ/mol.This is a much larger releaseof free energythan is required for ATP symthesisin cells(50 to 60 kJ/mol;seeWorkedExample13-2). Cells convert glucoseto CO2not in a single, high-energyreleasingreaction but rather in a seriesof controlled reactions,someof which are oxidations.The free energy releasedin theseoxidationstepsis of the sameorder of magnitudeas that required for ATP synthesisfrom ADP, with someenergyto spare.Electronsremovedin these oxidation steps are transferredto coenzymesspecialized for carrying electrons,such as NAD+ and FAD (describedbelow).

AFew Types of(oenzymes andProteins Serve asUniversal Hectron Carriers The multitude of enz;rmesthat catalyzecellular oxidations channelelectronsfrom their hundredsof different substratesinto just a few types of universalelectron carriers. The reduction of these carriers in catabolic processesresultsin the conservationof free energyreleased by substrate oxidation. NAD, NADfl FMN, and FAD are water-solublecoenzymesthat undergo reversible oxidation and reduction in many of the electron-transferreactionsof metabolism.The nucleotides NAD and NADP move readily from one enz),rneto another; the flavin nucleotidesFMN and FAD are usually very tightly bound to the enzymes,called flavoproteins, for which they serveas prosthetic groups.Lipid-soluble quinonessuch as ubiquinone and plastoquinoneact as electroncarriersand proton donorsin the nonaqueous environmentof membranes.Iron-sulfur proteins and cytochromes,which have tightly bound prosthetic groups that undergo reversibleoxidation and reduction, also serve as electron carriers in many oxidation-reduction reactions.Someof theseproteinsare water-soluble, but others are peripheral or integral membraneproteins (seeFig. 11-6). We concludethis chapterby describingsomechemical features of nucleotide coenz).rnesand some of the enz).rnes(dehydrogenasesand flavoproteins) that use them. The oxidation-reduction chemistry of quirones, iron-sulfur proteins, and cytochromesis discussedin Chapter19.

NADH andNADPH ActwithDehydrogenases asSoluble Elertron Carriers Nicotinamide adenine dinucleotide (NAD; NAD* in its oxidized form) and its close analog nicotinamide adenine dinucleotide phosphate (NADP;NADP+ when oxidized) are composed of two nucleotides joined through their phosphate groups by a phosphoanhydride bond

Reacti0ns 0xidation-Reduction 13.4Biological I sr z ]

oo c\

t?

I O:P-O I o I O:P-O I o_

CIlr. -o_-

\

H

I

i1

i

i .-c-

NH,

NH,

or

L\

N-

R A side

R

+ II

I

H

B side

NADH

H

(reduced)

OH NHo

t-

N

CH,, -/

--\N

(-) NN"

Adenine

1.0 g 0.8 € 0.6 q

< 0.4 In NADP* this hydroxyl group is esterifred with phosphate. (a)

0.2 0.0

220

240

260

280 300 320 340 Wavelength (nm)

360

380

(b) F I G U R1E3 - 2 4 N A D a n d N A D P .( a ) N r c o t i n a m i daed e n i n ed i n u cleotide,NAD*, and its phosphorylated analogNADP* undergoreductionto NADH and NADPH,accepting a hydrideion (twoelectrons andoneproton)froman oxidizable substrate. Thehydrideion is added to eitherthe front (theA side)or the back(the B side)of the planar nicotinamide ring (seeTable13-B) (b) The UV absorption spectraof

ring producesa NAD+ and NADH Reductionof the nicotinamide bandwith a maximumat 340 nm.Theproducnew,broadabsorption reactioncan be convetion of NADH duringan enzyme-catalyzed at of the absorbance nientlyfollowedby observingthe appearance tcm ') 340 nm (molarextinctioncoefficiente3a,J:6,200v

(Fig.

oxidized

f 3-2{t). Because the nicotinamide ring resembles pyridine, these compounds are sometimes called

pyridine nucleotides. The vitamin niacin is the source of the nicotinamide moiety in nicotinamide nucleotides. Both coenzymes undergo reversible reduction of the nicotinamide ring (Fig. 13-24). As a substrate molecule undergoes oxidation (dehydrogenation), giving up two hydrogen atoms, the oxidized form of the nucleotide (NAD+ or NADP+) accepts a hydride ion (:H-, the equivalent of a proton and two electrons) and is reduced (to NADH or NADPH). The second proton removed from the substrate is released to the aqueous solvent. The half-reactions for these nucleotide cofactors are NAD* + 2e * 2H+ -----+NADH + H* NA-DP* r 2e- * 2H* -----+NADPH + H* Reduction of NAD+ or NADP+ converts the benzenoid ring of the nicotinamide moiety (with a fixed positive charge on the ring nitrogen) to the quinonoid form (with no charge on the nitrogen). The reduced nucleotides absorb Iight at 340 nm; the oxidized forms do not (Fig. J3-2ab); this difference in absorption is used by biochemists to assay reactions involving these coenzymes. Note that the plus sign in the abbreviations NAD- and NADP- doesnot indicate the net charge on these molecules (in fact, both are negatively charged); rather, it indicates that the nicotinamide ring is in its

form, with

a positive

charge on the nitrogen

atom. In the abbreviations NADH and NADPH, the "H" denotes the added hydride ion. To refer to these nucleotides without specifying their oxidation state, we use NAD and NADP The total concentration of NAD+ + NADH in most tissues is about 10-5 trt; that of NADP+ + NADPH is 6 about 10 lt. In many cells and tissues, the ratio of NAD+ (oxidized) to NADH (reduced) is high, favoring hydride transfer from a substrate lo NAD* to form NADH. By contrast, NADPH is generally present at a higher concentration than NADP+, favoring hydride [ransfer from NADPH to a substrate. This reflects the specializedmetabolic roles of the two coenzy'rnes:NADgenerally functions in oxidations-usually as part of a catabolic reaction; NADPH is the usual coenzyme in reductions-nearly always as part of an anabolic reaction. A few enz;rmescan use either coenzyme, but most show a strong preference for one over the other. Also, the processesin which these two cofactors function are segregated in eukaryotic cells: for example, oxidations of fuels such as pyruvate, fatty acids, and c-keto acids derived from amino acids occur in the mitochondrial matrix, whereas reductive biosynthetic processes such as fatty acid synthesis take place in the cytosol. This functional and spatial specialization allows a cell to maintain two distinct pools of electron carriers, with two distinct functions.

l"'-l B i o e n e r g eat ni cdsB i o c h e m iRceaal c t iTo ynp e s More than 200 enzymes are known to catalyze reactions in which NAD* (or NADP+) accepts a hydride ion from a reduced substrate, or NADPH (or NADH) donates a hydride ion to an oxidized substrate. The general reactions are AHz + NAD* ---+ A + NADH + H* A + NADPH * H* ------+AH2 + NADP* where AH2 is the reduced substrate and A the oxidized substrate. The general name for an enzyme of this type is oxidoreductase; they are also commonly called dehydrogenases.For example, aicohol dehydrogenasecatalyzes the first step in the catabolism of ethanol, in which ethanol is oxidized to acetaldehyde: CHgCH2OH* NAD* -----+ CHBCHO + NADH + H* Ethanol Acetaldehyde Notice that one of the carbon atoms in ethanol has lost a hydrogen; the compound has been oxidized from an alcohol to an aldehyde (refer again to Fig. 13-22 for the oxidation states of carbon). When NAD+ or NADP+ is reduced, the hydride ion could in principle be transferred to either side of the nicotinamide ring: the front (A side) or the back (B side), as represented in I'igure I3-24a. Studies with isotopically Iabeled substrates have shown that a given enzyme catalyzes either an A-t;pe or a B-type transfer, but not both. For example, yeast alcohol dehydrogenaseand lactate dehydrogenaseofvertebrate heart transfer a hydride ion to (or remove a hydride ion from) the A side of the nicotinamide ring; they are classed as type A dehydrogenases to distinguish them from another group of enz),rnes that transfer a hydride ion to (or remove a hydride ion from) the B side of the nicotinamide ring (Table 13-8). The specificityfor one side or another can be very striking; Iactate dehydrogenase, for example, prefers the A side over the B side by a factor of 5 x 107! The basis for this preference lies in the exact positioning of the enzyme groups involved in hydrogen bonding with the -CONH2 group of the nicotinamide.

tIGURE 13-25 The Rossman fold.Thisstructural motifis founclin rhe (a) lt consists NAD-bindingsiteof manydehydrogenases of a pairof structurally similarmotifs,eachhavingthreeparallelp sheets andtwo a helices(9-a-9-o-9.(b) The nucleotide-binding domainof the en(derivedfrom PDBlD 3LDH)with NAD zyme lactatedehydrogenase (ball-and-stick structure) boundin an extended conformation through hydrogenbondsand saltbridgesto the pairedF-"-P-"-F motifsof the Rossman fold (shadesof green).

Most dehydrogenases that use NAD or NADP bind the cofactorin a conservedprotein domain calledthe Rossmannfold (named for Michael Rossmann,who deduced the structure of lactate dehydrogenaseand flrst describedthis structuralmotif). The Rossmannfold typically consistsof a six-strandedparallelp sheetand four associated a helices(Fig. f 3-25). The associationbetweena dehydrogenase and NAD or NADPis relativelyloose;the coenzlirnereadilydffuses from one enzyrneto another,acting as a water-soluble

Enz5rme

Coenzyme

Stereochemieal specificityfor nicotinamide ring (Aor B)

Textpage(s)

Isocitrate dehydrogenase

NAD+

A

a-Ketoglutaratedehydrogenase Glucose6-phosphatedehydrogenase

NAD-

B

625

NADP+

B

560

Malatedehydrogenase

NAD+

Glutamatedehydrogenase Glyceraldehyde3-phosphatedehydrogenase

NAD+ or NADP+

B

680

NAD+

B

Lactate dehydrogenase

NAD+

A

547

Alcohol dehydrogenase

NAD*

A

547

624

628

i ocnt i o n[ su t t - - l 1 3 . 4B i o l o g i cOaxl i d a t i o n - R e d uRcet a

carrier of electronsfrom one metaboliteto another.For example,in the production of alcohol during fermentation of glucoseby yeast cells, a hydride ion is removed ($ycerfrom glyceraldehyde3-phosphateby one enz)..rne aldehyde3-phosphatedehydrogenase, a type B en4'rne) and transferredto NAD+ The NADH produced then leavesthe enzS.rne surface and diffuses to another en(alcoholdehydrogenase, 25.'rne a bpe A enz;.'rne),which transfers a hydride ion to acetaldehyde,producing ethanol: (1) Glyceraldehyde3-phosphate+ NAD+ -----> + NADH + H* 3-phosphoglycerate (2) Acetaldehyde+ NADH + H+ -----+ethanol + NAD+ Sum: Glyceraldehyde3-phosphate* acetaldehyde-----+ 3-phosphoglycerate+ ethanol Notice that in the overall reaction there is no net production or consumption of NAD+ or NADH; the coen4mes function catall'tically and are recycled repeatedly wrthout a net change in the concentration of NAD+ + NADH.

Dietary Deficiency theVitamin Form of ofNiacin, NAD andNADB Causes Pellagra As we noted in Chapter6, and will discussfurther are dein the chaptersto follow,most coenzy'rnes pyridinewe from vitamins. The rived the substances call like rings of NAD and NADP are derivedfrom the vrtamin niacin (nicotinic acid;Fig. 13-26), which is synthesized from tryptophan. Humans generally cannot synthesize sufficientquantitiesof niacin,and this is especiallyso for individualswith dietslow in tryptophan (maize,for example, has a low tryptophan content). Niacin deficiency, which affects all the NAD(P)-dependentdehydrogenases,causesthe serioushumandiseasepellagra(Italian for "rough skin") and a related diseasein dogs, black-

o

glucose6-phosphate GlucoseG-phosphate-----+fructose6-phosphate Using the AG'" valuesin Table 13-4, calculatethe equilibrium constant,K'.r, for the sum of the two reactions: Glucosel-phosphate ------+fructose6-phosphate 10. Effect of IATPI/[ADP] Ratio on Free Energy of Ilydrolysis of ATP Using Equation 13-4, plot AG against ln Q (mass-actionratio) aI 25 "C for the concentrations of ATR ADq and P1in the table below. AG'' for the reaction is -30 5 kJ/mol.Usethe resultingplot to explainwhy metabolism is regulatedto keep the ratio [ATP]/[ADP]high.

(mM) Concentration ATP ADP p.

5 0.2 10

2.2 12.L

I 4.2 I4.T

0.2 D.U

14.9

1n

11. Strategy for Overcoming an Unfavorable Reaction: ATP-Dependent Chemical Coupling The phosphorylation of glucoseto glucose6-phosphateis the initial step in the

.524l

B i o e n e r g eat incdsB i o c h e m iRc e aa l c t iT o ynp e s

catabolismof giucose The direct phosphorylationof glucose by P1is describedby the equation Glucose* Pi ---+ glucose6-phosphate+ HzO AG'" : 13.8kJ/mol (a) Calculatethe equilibrium constantfor the abovereaction at 37 'C. In the rat hepatocytethe physiologicalconcentrations of glucose and P1are maintained at approximatelv 4.8 mrr,t.What is the equilibrium concentrationof glucose O-phosphate obtainedby the directphosphorylation ofglucose by P;? Does this reaction representa reasonablemetabolic stepfor the catabolismof glucose?Explain. (b) In principle,at least,oneway to increasethe concentration ofglucose 6-phosphateis to drive the equilibriumreaction to the right by hcreasingthe intracellularconcentrationsof glucose and P, Assuminga fixed concentrationof p1at 4 8 mrr,t, how high would the intracellularconcentrationof glucosehave to be to give an equilibrium concentrationof glucose6-phosphate of 250 p.rtt(the normal physiologicalconcentration)? Would this route be physiologicallyreasonable,given that the maximum solubility of glucoseis less than 1 u? (c) The phosphorylationof glucosein the cell is couplecl to the hydrolysisof ATP; that is, part of the free energyof ATp hydrolysisis usedto phosphoryiateglucose: (1)

Glucose+ P; ----+ glucose6-phosphate+ H2O AG'": 13.8kJ/mol (2) ATP + HzO--> ADP + Pr AG,.: -80.5 kJ/mot -----+ Sum: Glucose+ ATP glucose6-phosphate+ ADp CalculateK'"nat 37 'C for the overallreaction For the ATpdependentphosphorylationof glucose,what concentrationof glucoseis neededto achievea2b0 pt,tintracellularconcentration of glucoseO-phosphatewhen the concentrationsof ATp and ADP are 3.38 mM and 1.32mrra,respectively?Does this couplingprocessprovide a feasibleroute, at least in principle, for the phosphorytationof glucosein the cell?Explain (d) Although coupling ATP hydrolysis to glucose phosphorylation makes thermodynamicsense,we have not yet specifiedhow this coupling is to take place. Given that cou_ pling requires a commonintermediate,one conceivableroute is to use ATP hydrolysis to raise the intracellular concentration of Pr and thus drive the unfavorablephosphorylationof $ucose by P1 Is this a reasonableroute? (Think about the solubility products of metabolicintermecliates.) (e) The ATP-coupledphosphorylationof glucose is cat_ alyzed in hepatocytes by the enz).rneglucokinase This enzyrne birds ATP and glucoseto form a glucose-ATp-enz).rne complex,and the phosphoryigroup is transferred directly from ATP to glucose.Explain the acivantages of thrs route. 12. Calculations of AG'o for ATp-Coupled Reactions From data in Table 13-6 calculatethe AG,. value for the following reactions (a) Phosphocreatine+ ADP -----+creatine * ATp (b) ATP + fructose -+ ADp * fructose 6-phosphate 13. Coupling ATP Cleavage to an Unfavorable Reaction To explore the consequencesof coupling ATp hydrolysis

under physiologicalconditions to a thermodynamicallyunfavorablebiochemicalreaction,considerthe hypotheticaltransformation X -+ I for which AG'' : 20 kJ/mot. (a) What is the ratio [!/[Xl at equilibrium? (b) SupposeX and Y participatein a sequenceof reactions during which ATP is hydrolyzedto ADP and Pr.The overallreactionis X + ATP + HrO -----+ y + ADp + pi Calculate[Y]/[X]for this reaction at equilibrium. Assumethat the temperatureis 25'C and the equilibriumconcentrationsof ATP,ADq and P, are 1 u. (c) We know that [ATP],[ADP],and [P1]are?zo,1 M under physiologicalconditions Calculate[Y]/[X]for the ATP-coupled reactionwhen the valuesof [ATP],[ADPI,and [P1]are those found in rat myocytes(Table 13-5). 14. Calculations of AG at Physiological Concentrations Calculatethe actual,physiologicalAG for the reaction Phosphocreatine+ ADP -----+creatine + ATP aL37'C, as it occursin the cytosolofneurons,with phosphocreatineat 4.7 mv.,creatineat 1.0mu, ADP at 0.73mlr, and ATPat26mrra. 15. Free Energy Bequired for ATP Synthesis under Physiological Conditions In the cytosol ofrat hepatocytes, the temperatureis 37'C and the mass-action ratio, e, is tATP] : 5.33x 102v-1 tADpltpJ Calculatethe free energy required to sy'nthesizeATp in a rat hepatocj,'te. 16. Chemical Logic In the glycoly'ticpathway,a six-carbon sugar (fructose 1,6-bisphosphate)is cleaved to form two three-carbon sugars, which undergo further metabolism (see Fig. 14-5). In this pathway,an isomerizationof glucose 6-phosphateto fructose 6-phosphate(shovmbelow) occurs two steps before the cleavagereaction (the intervening step is phosphoryiationof fructose C-phosphateto fructose 1,6bisphosphate (p. 532)). H

n" ,ro

I

C

H-C-OH

I

I

H-C-OH

I

HO-C-H

I

i,ilrr:l)lrollr

\o5(l

C:O I HO-C-H

I

H-C-OH

H-C-OH

H-C-OH

H-C-OH

I

I cHroPoi

GlucoseG-phosphate

I

I cHroPoS

Fructose 6-phosphate

What does the isomerization step accomplish from a chemical perspective? (Hint: Consider what might happen if the C-C bond cleavagewere to proceed without the preceding isomerization.)

lcrl

Problems

17. Enzymatic Reaction Mechanisms I Lactate dehydrogenase is one of the many enzymes that requlre NADH as coenzyne. It catalyzes the conversion of pyruvate to lactate:

o'r...ro 'C

NA-DH+ H

lr

ox ,o .C

NAD

HO*C-H

C:O

II

CH,

CHA l-Lactate

Pyruvate

Draw the mechanism of this reaction (show electron-pushing arrows) (Hint: This is a common reaction throughout metabolism; the mechanism is similar to that catalyzed by other dehydrogenases that use NADH, such as alcohol dehydrogenase ) 18. Enzymatic Reaction Mechanisms II Biochemical reactions often look more complex than they really are. In the pentose phosphate pathway (Chapter 14), sedoheptulose 7-phosphate and glyceraldehyde 3-phosphate react to form er54hrose4-phosphate and fructose 6-phosphate in a reaction catalyzed by transaldolase

ofATP,ADP,and P1are 3.5, 1 50, physioiogical concentratlons and 5.0mtt, respectively. (b) A 68 kg (150 lb) adult requiresa caloric intake of 2,000kcal (8,360kJ) of food per day (24 hours). The food is ATP, metabolizedand the free energy is used to s5,'nthesize which then provides energyfor the body'sdaily chemicaland mechanicalwork. Assumingthat the efflciency of converting food energyinto ATP is 50%, calculatethe weight of ATP used by a human adult in 24 hours. What percentageof the body weightdoesthis represent? (c) Although adults slnthestze latge amounts of AIP daily, their bodyweight,structure,and compositiondo not changesigniicantly durhg this period. Explain this apparentcontradiction 20. Rates of Tirrnover of y and B Phosphates of ATP If a small amount of ATP labeledwith radioactivephosphorusin the terminatposition,[y-"p]etp, is addedto a yeastextract, 32Pactivity is found in P1within a few minabout half of the utes, but the concentrationof ATP remainsunchanged'Explaln. If the sameexperimentis carried out using ATP labeled 32Pdoesnot with 32Pin the centralposition,IB-3'PIATRthe appearin P1within such a short time Why? 21. Cleavage of AfP to AMP and PP1during Metabolism Synthesisof the activatedform of acetate(acetyl-CoA)is carprocess: ried out in an ATP-dependent

CH"OH C:O HOCH

Acetate + CoA + ATP -----+acetyl-CoA+ AMP + PPi

H-C_OH r(rH

-\,/

H-C-OH r+l H-C-OH

C

|

H-C-OH

cH2oPo32Sedoheptulose 7-phosphate

cH2oPo32 Glyceraldehyde 3-phosphate

CH,OH

o.H \\ // t,

I

C:O I HO-C-H

H-C-OH

I

H-C-OH

I

CH2OPO32 Erythrose 4-phosphate

H-C-OH +l

H-C-OH

I CH2OPOB2 Fructose 6-phosphate

Draw a mechanism for this reaction (show electron-pushing arrows) (Hint: Take another look at aldol condensations, then consider the name of this enzy'rne.) 19. Daily ATP Utilization by Human Adults (a) A total of 30 5 kJ/mol of free energy is needed to slmthesize ATP from ADP and P1when the reactants and products are at 1 u concentrations and the temperature is 25 "C (standard state). Because the actual physiologlcal concentrations of ATP, ADP, and P1are not 1 u, and the temperature is 37 "C, the free energy required to synthesize ATP under physiological conditions is different from AG'o. Calculate the free energy required to synthesize ATP in the human hepatocy'te when the

(a) The AG'o for hydrolysis of acetyl-CoAto acetateand CoAis -32.2 kJ/moland that for hydrolysisof ATP to AMP and PP1is -30.5 kJ/mol. CaiculateAG'' for the AlP-dependent synthesisof acetyl-CoA (b) Almost all cells contain the enz;nne inorganic pywhich catalyzesthe hydrolysisof PP1to P1. rophosphatase, What effect doesthe presenceof this enz;nnehaveon the s1nthesis of acetyl-CoA?ExPlain. 22. Energy for H+ Pumping The parietal cells of the stomach llning contaln membrane"pumps" that transport hydrogen ions from the cytosol (pH 7.0) into the stomach, contributing to the acidity of gastricjuice (pH 1 0). Calculate the free energy required to transport 1 mol of hydrogenions through these pumps. (Hint: See Chapter 11) Assumea of 37'C. temperature 23. Standard Reduction Potentials The standard reductlon potentlal, E'", of any redox pair is definedfor the half-cell reaction: Oxidizing agent + n electrons -+

reducing agent

The E'" valuesfor the NAD+/NADHand pyruvate/lactateconjugateredox pairsare -0.32 V and -0.19 V, respectively (a) Which redox pair has the greater tendency to lose electrons?Explain. (b) Which pair is the stronger oxidizing agent?Explail (c) Beginning with 1 M concentrationsof each reactant and product at pH 7 and 25 "C, in which direction will the followingreactionproceed? Py,ruvate + NADH + H+ .-

lactate + NAD+

[.'u]

B i o e n e r g eat incdsB i o c h e m iRc e aa l c t iT o ynp e s

(d) What is the standardfree-energychange(AG,") for the conversionof pyruvateto lactate? (e) Whatis the equilibriumconstant((o) for this reaction? 24. Energy Span ofthe Respiratory Chain Electrontransfer in the mitochondrialrespiratorychainmay be representecl by the net reactionequation NADH + H+ + iO, =-

H2O + NAD*

(a) CalculateM'' for the net reactionof mitochondrial electrontransfer.UseE'' valuesfrom Table1B-2. (b) CalculateAG'' for this reaction. (c) How manyATPmoleculescantheoreti,collgbe generatedby this reactionifthe free energyofATP synthesisunder cellular conditionsis 52 kJ/mol?

and bioci,tin (a small zwitterionic molecule) moved in only one direction between two particular tlpes of glia (nonneuronal cells of the nelvous system). Dye injected into astrocy'tes would rapidly pass into adjacent astrocytes, oligodendrocy'tes, or Mtiller cells, but dye injected into oligodendrocy'tes or Milller cells passed slowly if at all into astrocy'tes.A11of these cell g,pes are comected by gap junctions. Although it was not a centrai point of their article, the authors presented a molecular model for how this unidirectional transport might occur, as shown in their Figure 3:

25. Dependence ofElectromotive Force on Concentrations Calculatethe electromotiveforce (in volts) registered by an electrodeimmersedin a solutlon containingthe following mixturesof NAD- and NADHat pH Z 0 and 25.C, with referenceto a half-cellof E'" 0 00 V (a) I 0 mvrNAD+ and 10 mu NADH (b) 1 0 mu NAD* and 1.0mn NADH (c) 10mrr NAD* and I 0 mu NADH 26. Electron Affinity of Compounds List the following in order of increaslngtendencyto acceptelectrons:a-ketoglutarate * CO2(yieldingisocltrate);oxaloacetate; 02; NADp+. 27. Direction of Oxidation-Reduction Reactions Which of the followingreactionswould you expectto proceedin the directionshown,understandardconditions,in the presenceof the appropriateenzyrnes? (a) Malate + NAD* ------+ oxaloacetate+ NADH * H+ (b) Acetoacetate+ NADH * H+ ------> B-hydroxybutyrate* NAD* (c) Pyruvate + NADH f H+ ---+ lactate + NAD+ (d) Pytuvate + B-hydroxybutyrate_------> lactate* acetoacetate (e) Malate + pyruvate------+ oxaloacetate* lactate (f) Acetaldehyde* succinate-+ ethanol + fumarate

DataAnalysis Problem 28. Thermodynamics Can Be Thicky Thermod5'namics is a challengingarea of study and one with many opportunitiesfor confusion.An interestlngexampieis found in an article by Robinson,Hampson,Munro,and Vaney,publishedin Sc,i,ence in 1993.Robinsonand colleaguesstudiedthe movementof small moleculesbetweenneighboringcells of the nervoussystem through cell-to-cellchannels(gapjunctions) They founclthat the dyesLucifer yellow (a small,negativelychargecimolecule)

(A)

Astrocyte

Oligodendrocyte

(B)

Astrocyte

Oligodendroclte

The flgure legend reads: "Model ofthe unidirectional diffusion of dye between coupled oligodendrocytes and astrocytes, based on differences in connection pore diameter. Like a flsh in a flsh trap, dye molecules (black circles) can pass from an astrocl'te to an oligodendrocyte (A) but not back in the other direction (B) " Although this article clearly passed review at a wellrespectedjournal, severalletters to the editor (1994) followed, showing that Robinson and coauthors'modei vioiated the second law of thermodyrramics. (a) Explain how the model violates the second law. Hint: Consider what would happen to the entropy of the system if one started with equal concentrations of dye in the astrocyte and oligodendrocyte connected by the "flsh trap" type of gap junctions. (b) Explain why this model cannot work for small molecules, although it may allow one to catch flsh. (c) Explain why a flsh trap does work for flsh (d) Provide two plausible mechanisms for the unidirectional transport of dye molecules between the cells that do not violate the second law of thermodvnamics. References Lettersto the editor (7994) Sci,ence 265, 10lZ-1019 Robinson, S.R.,Hampson, E.C.G.M.,Munro, M,N., & Vaney,D.L (1993) Unidirectional coupling of gap junctions between neuroAlia Science262,10721074

The problem of alcoholic fermentation,of the origin and natureof change,which converted and apparentlyspontaneous that mysterious g i n e , s e e m st o h a v ee x t h e i n s i p i dj u i c eo f t h e g r a p ei n t o s t i m u l a t i n w erted a fascinationover the minds of naturalphilosophersfrom the very earliesttimes. -Arthur Harden,Alcoholic Fermentation, 7923

and GIuconeogenesis, Glycolysis, Pathway Phosphate thePentose 528 14.1 Glycolysis forGlycolysis543 Pathways 14.2 Feeder Anaerobic Conditions: ofPyruvate under 14.3 Fates 546 Fermentation 551 14.4 Gluconeogenesis Oxidation558 Pathway ofGlucose Phosphate 14.5 Pentose lucose occupiesa central position in the metabolism of plants,animals,and many microorganisms. It is relatively rich in potential energy,and thus a good fuel; the completeoxidationof glucoseto carbon dioxide and water proceedswith a standardfree-energy changeof -2,840 kJ/mol.By storingglucoseas a high molecularweight pol;rmersuchas starchor glycogen,a cell can stockpile large quantities of hexoseunits while maintaining a relatively low c;'tosolic osmolarity.When energydemandsincrease,glucosecanbe releasedfrom these intracellularstoragepol)rmersand used to produce ATP either aerobicallyor anaerobically. Glucoseis not only an excellent fuel, it is also a remarkably versatile precursor, capableof supplying a huge array of metabolic intermediatesfor biosl'nthetic coLi,can obreactions.A bacteriumsuchas,Uscheri'chict' glucose the carbon skeletonsfor every amino tain from acid, nucleotide,coen4.'rne,fatty acid, or other metabolic intermediateit needsfor growth. A comprehensive studyof the metabolicfatesof glucosewould encompass hundredsor thousandsof transformations.In animals and vascularplants,glucosehas four major fates:it may desbe usedin the synthesisof complexpolysaccharides tined for the extracellularspace;stored in cells (as a polysaccharide or assucrose);oxidizedto a three-carbon compound (pyruvate) via glycolysisto provide ATP and

metabolicintermediates;or oxidized via the pentose phosphate(phosphogluconate)pathway to yield ribose 5-phosphatefor nucleic acid synthesisand NADPH for reductivebiosyntheticprocesses(FiS. 14-1 ). Organismsthat do not have accessto glucosefrom other sourcesmust make it. Photosyntheticorganisms make glucose by first reducing atmosphericCO2 to trioses,then convertingthe triosesto glucose.Nonphotosyntheticcellsmakeglucosefrom simplerthree- and four-carbonprecursorsby the processofgluconeogenesis, effectively reversing glycolysis in a pathway that usesmany of the glycolytic enzyrnes. In this chapter we describethe individual reactions gluconeogenesis, and the pentosephosglycolysis, of phate pathway and the functional signiflcanceof each pathway.We alsodescribethe variousmetabolicfates of the pyruvateproducedby glycolysis.They include the fermentations that are used by many organismsin anaerobicnichesto produceATP and that are exploited industrialtyas sourcesof ethanol,lactic acid,and other Extracellular matrix and cell wall polysaccharides

GlYcogen, starch, sucrose

\/

synthesisof

\

/

oxidationvia

s'iorace

/

\

;:il*:13'

/

ilil$;"on*on^7 / Ribose 5-phosphate 14-1 Major pathwaysof glucoseutilization.Althoughnot the FIGURE only possiblefatesfor glucose,thesefour pathwaysarethe mostsignificantin termsof theamountofglucosethatflowsthroughthemin most cells.

Grr)

F"l

G l y c o l yG s i lsu, c o n e o g e naensdtihse, p e n t o speh o s p h aptaet h w a y

commerciallyusefulproducts.And we look at the path_ waysthat feed varioussugarsfrom mono-, di-, and polysaccharidesinto the glycol;,tic pathway.The discussion of glucosemetabolismcontinuesin Chapter 1b, where we use the processesof carbohydratesynthesisand degradationto illustrate the manymechanismsby which organismsregulate metabolic pathways.The biosynthetic pathwaysfrom glucoseto extracellularmatrix and cell wall polysaccharides and storagepolysaccharides are discussedin Chapter20.

14JlGlycolysis In glycolysis (from the Greek glykys, ,,sweet" or "sugar,"andlgsi,s,"splitting"),a moleculeof glucoseis degradedin a series of enzl'rne-catalyzedreactions to yield two molecules of the three-carbon compound pyruvate. During the sequentialreactions of glycolysis, some of the free energyreleasedfrom glucoseis conservedin the form of ATP and NADH Glycolysiswas the first metabolicpathwayto be elucidatedand is probably the best understood.From EduardBuchner'sdiscovery in 1897of fermentationin brokenextractsof yeastcells until the elucidation of the whole pathway in yeast (by Otto Warburgand Hansvon Euler-Chelpin)and in muscle (by Gustav Embden and Otto Meyerhof) in the 1930s,the reactionsofglycolysisin extractsofyeastand musclewere a majorfocusof biochemicalresearch.The philosophicalshift that accompaniedthese discoveries wasannouncedby JacquesLoebin 1906: Through the discovery of Buchner, Biology was relieved of another fragment of mysticism. The splitting up of sugarinto CO2and alcoholis no more the effect of a "vital principle" than the splitting up of canesugarby invertase.The history of this problem is instructive, as it warns us againstconsidering problemsas beyond our reach becausethey have not yet found their solution. The development of methods of enz1nnepurif,ca_ tion, the discoveryand recognitionof the importanceof coenzyrressuch as NAD, and the discoveryof the piv_ otal metabolicrole of ATP and other phosphorylated

Hansvon Euler-Chelpin, 1873-1964

CustavEmbden, 1874-1933

compoundsall cameout of studiesof glycolysis.The glycolytic enzyrnesof many specieshave long since been purifled and thoroughly studied. Glycolysisis an almost universalcentral pathway of glucosecatabolism,the pathwaywith the largestflux of carbon in most cells. The glycoly'ticbreakdown of glucose is the sole source of metabolic energy in some mammaliantissues and cell types (erythrocytes, renal medulla,brain, and sperm, for example).Someplant tissuesthat are modifledto storestarch (suchaspotato tubers) and some aquatic plants (watercress, for example) derive most of their energy from glycolysis; many anaerobicmicroorganismsare entirely dependent on glycolysis. Fermentation is a generalterm for the anaerobi,c degradationof glucoseor other organicnutrients to obtain energy,conservedasATP.Becauseliving organisms first arosein an atmospherewithout oxygen,anaerobic breakdownof glucoseis probably the most ancient biological mechanism for obtaining energy from organic fuel molecules.And asgenomesequencingof a wide variety of orgarLismshas revealed,some archaeaand some parasiticmicroorganisms lack one or more of the enzyrnes of glycolysisbut retain the core of the pathway;they presumablycarry out variant forms of glycolysis.In the corrse of evolution,the chemistryof this reactionsequencehas beencompletelyconserued; the $yco$tic enz;rmesof vertebrates are closely similal, in amino acid sequenceand three-dimensionalstructure, to their homologsin yeast and spinach.Glycolysisdjffers amongspeciesonly in the detailsof its regulationand in the subsequentmetabolic fate of the pyruvate formed. The thermodlnamic principles and the types of regulatory mechanismsthat govern $ycolysis are corrmon to all pathwaysof cell metabolism. The glycolytic pathway,of central imporfancein itseJf,can alsoserveasa modelfor manyaspectsof the pathwaysdiscussedthroughoutthis book. Before examiningeachstep of the pathwayin some detail, we take a look at glycolysisas a whole.

AnOverview:Glycolysis Has TwoPhases

The breakdownof the six-carbonglucoseinto two molecules of the three-carbonpyruvate occurs in l0 steps, the first 5 of which constitute the preparatorg phase (Fig. l4-2a). In thesereactions,gucoseis first phosphorylatedat the hydroxyl group on C-6 (step @;. fire o-$ucose 6-phosphatethus formed is converted to l-fructose 6-phosphate (step @), which is again phosphorylated, this time at C-1, to fleld n-fructose 1,6-bisphosphate (step @). For both phosphorylations, ATPis the phosphoryl group donor.As all sugarderivaOtto Meyerhof, 1884-.1 951 tives in glycolysisare the D isomers,we

is 1 4 . 1G l y c o l y s[rrr] 6

(a)

Preparatory

Glucose

phase

Phosphorylation of glucose and its conversion to glyceraldehyde 3-phosphate

first primins [) reaclon

HOH

Glucose 6-phosphate

1l ll

e

rAr ll \?

ll

/6\

tI tl

\7

Hexokinase Phosphohexose lSOmerase

Fructose 6-phosphate Phospho@ 1l'uctokinase-1

second priming reaction

(3J

cHr-O-@

Fructose 1,6-bisphosphate

Glyceraldehyde 3-phosphate + Dihydroxyacetone phosphate a-\ !D'

@ Aldolase /}' €,

Trrose phosphate lsomerase

//o ^ eFo-cH,-9H-c\ OHH

@-o-cur-c-cH,oH o Payoff phase

Glyceraldehyde 3-phosphate (2)

(FFo-cs,-c

t-""o'H

3"

@ Glyceraldehyde 3-phosphate

1,3-Bisphosphoglycerate(2)

1lr 2

firstArF-

forming reaction 61 lI v (substratelwel ll\ phosphorylatioo) ll,

dehydrogenase

-q

Phosphoglycerate kinase

3-Phosphoglyc er ate (2)

1l

@il It

2-Phosphoglycerate(2)

1t

O - [ lb zn,o Phosphoenolpyruvate (2) secondATPf:*p-eI:"fi.'"

lf 6b) [, (subgfiare-revel l\ phosphoqdf,tion)

"

2

CH"-CH-C(

J" J

'o-

I (ry

Phosphoglycerate mutase @ Enolase

,o cH,:f-c\ do-

I (ry

2

J

Pyruvate

Oxidative conversion of glyceraldehyde 3-phosphate to pymvate and the coupled formation ofATP and NADH

(2)

//o cH3-c-c\ do-

FIGURE 14-2 The two phasesof glycolysis.Foreach moleculeof gluphase(a), two moleculesof cosethat passesthroughthe preparatory 3-phosphate are formed;both passthroughthe payoff glyceraldehyde (b). phase Pyruvateisthe end productof the secondphaseof glycolysis. Foreachglucosemolecule,hvo ATPare consumedin the preparatory phaseand fourATPareproducedin the payoffphase,givinga netyield

of two ATPper moleculeof glucoseconvertedto pyruvate'The numberedreactionstepsare catalyzedby the enzymeslistedon the right, to the numberedheadingsin the text discussion' and alsocorrespond hereat @ ha' Keepin mind that eachphosphorylgroup,represented (-PO3_)' two negativecharges

f-I

s i lsu, c o n e o g e naensdtihs e, p e n t o speh o s p h aptaet h w a y i 5 3 0 _ l G l y c o l yG will usually omit the n designation except when emphasizing stereochemistry. Fructose 1,6-bisphosphate is split to yield two three-carbon molecules, dihydroxyacetone phosphate and glyceraldehyde 3-phosphate (step @); this is the ,,ly_ sis" step that gives the pathway its name. The dihydroxyacetone phosphate is isomerized to a second molecule of glyceraldehyde 3-phosphate (step @), ending the first phase of glycolysis. From a chemical perspective, the isomerization rn step @ is critical for setting up the phosphorylation and C-C bond cleavage reactions in steps @ and @, ur detailed later. Note that two molecules of ATP are invested before the cleavage of $ucose into two three-carbon pieces; there wrll be a good return on this investment To summarize: in the preparatory phase of glycolysis the energy of ATP is invested, raising the freeenergy content of the intermediates, and the carbon chains of all the metabolized hexoses are converted to a conunon product, glyceraldehyde 3-phosphate. The energy gain comes rn the pagolf phase of glycolysis (Fig. 14-2b). Each molecule of glyceraldehyde 3-phosphate is oxidized and phosphorylated by inorganic phosphate (not by ATp) to form 1,3-bisphospho_ glycerate (step @). Energy is then released as the two molecules of 1,3-bisphosphoglycerateare converted to two molecules of pyruvate (steps @ through @;. nluctr of this energy is conserved by the coupled phoiphorylation of four molecules of ADp to ATp The net yield is two molecules of ATP per molecule of glucose used, be_ cause two molecules of ATp were invested in the preparatory phase. trnergy is also conserved in the pay_ off phase in the formation of two molecules of the electron carrier NADH per molecule of glucose. In the sequential reactions ofglycolysis, three types of chemical transformations are particularly noteworthy: (1) degradation ofthe carbon skeleton ofglucose to yield pyruvate; (2) phosphorylation of ADp to ATp by compounds with high phosphoryl group transfer poten_ tial, formed during glycolysis; and (B) transfer of a hy_ dride ion to NAD*, forming NADH. Fates of Pguvate With the exception of some interesting variations in the bacterial realm, the pyruvate formed by glycolysis is further metabolized via one of three catabolic routes. In aerobicorganisms or tissues, under aerobic conditions, glycolysis is only the first stage in the complete degradation of glucose (Fig. f 4-3) Pyruvate is oxidized, with loss of its carboxyl group as CO2,to yield the acetyl group of acetyl-coenz).rneA; the acetyl group is then oxidized completely to CO2 by the citric acid cycle (Chapter 16). The electrons from these oxidations are passed to 02 through a chain of carriers in mitochondria, to form H2O. The energy from the electron-transfer reactions drives the slmthesis of ATp in mitochondria (Chapter t9). The second route for pyruvate is its reduction to lactate via lactic acid fermentation. When vigorously

2 Ethanol + 2CO2 Fermentation to ethanol in yeast

Animal, plant, and many microbial cells under aerobic conditions FIGURE 14-3 Threepossiblecatabolicfatesofthe pyruvateformedin glycolysis.Pyruvatealsoservesas a precursorin manyanabolicreactions,not shownhere.

contractingskeletalmuscle must function under lowoxygen conditions (hypoxia), NADH cannot be reoxidized to NAD+, but NAD+ is required as an electron acceptor for the further oxidation of pyruvate. Under theseconditionspytuvateis reducedto lactate,accepting electronsfrom NADH and thereby regeneratingthe NAD+ necessaryfor glycolysisto continue.Certaintissuesand cell types (retina and erythrocytes,for example) convert glucose to lactate even under aerobic conditions,and lactate is also the product of glycolysis under anaerobicconditions in some microorganisms (Fis. 14-3). The third major route of pyruvate catabolismleads to ethanol.In someplant tissuesand in certain invertebrates,protists,and microorganisms suchasbrewer'sor baker's yeast, pyruvate is converted under hypoxic or anaerobicconditions to ethanol and CO2,a process called ethanol (alcohol) fermentation (Fig. 14-g). The oxidation of pyruvate is an important catabolic process,but pyruvatehas anabolicfatesas well. It can, for example,providethe carbonskeletonfor the synthesis of the amino acid alanineor for the syrrthesisof fatty acids.We return to these anabolicreactionsof pyruvate in later chapters. ATP and NADH Formation Coupled to Glycolysis During glycolysissomeof the energyof the glucosemolecule is conservedin ATP, while much remains in the product, pyruvate. The overall equationfor glycolysisis Glucose + 2NAD* + 2ADp -f 2pi---> 2 pyruvate + 2NADH + 2H+ + 2ATp + 2H2O

(14-1)

14.1Glycolysis L53! For eachmoleculeof glucosedegradedto pyruvate,two moleculesof ATP are generatedfrom ADP and P1,and two moleculesof NADH are produced by the reduction of NAD+ The hydrogen acceptor in this reaction is NAD* (see Fig. 13-24), bound to a Rossmarurfold as shovmin Figure 13-25.The reduction of NAD* proceeds by the enz;rmatictransfer of a hydride ion (:H-) from the aldehyde group of glyceraldehyde3-phosphateto the rucotinamidering of NAD*, yreldingthe reduced coenzy'rneNADH. The other hydrogenatom of the substrate moleculeis releasedto the solutionas H+. We can now resolve the equation of glycolysisinto two processes-the conversionof glucoseto pyruvate, which is exergonic: 2 pyruvate+ 2NADH+ 2H* (74-2) Glucose+ 2NAD+-----+ AGi": -146 kJ/mol and the formation of ATP from ADP and Pi, which is endergonic: 2ADP + 2Pi ---> 2ATP + 2}l2O

(14-3)

LGf :2(30.5 kJ/mol): 61.0kJ/mol The sum of Equationsl4-2 and 14-3 givesthe overall standardfree-energychangeof glycolysis,AGi': aG;": aci'+ LGi": -146 kJ/mol+ 61kJ/mol : -85 kJ/mol Under standard conditions,and under the (nonstandard) conditionsthat prevail in a cell, glycolysisis an essentially irreversibleprocess,driven to completionby a Iargenet decreasein free energy. Energy Bemaining in Pyruvate Glycolysisreleases only a small fraction of the total availableenergy of the glucosemolecule;the two moleculesof pyruvateformed by glycolysisstill contain most of the chemicalpotential energyof glucose,energythat can be extractedby oxidative reactions in the citric acid cycle (Chapter 16) and oxidativephosphorylation(Chapter19). Importance of Phosphorylated Intermediates Each of the nine glycolytic intermediatesbetween glucose and pyruvateis phosphorylated(Fig. 14-2). The phosphorylgroupsseemto havethree functions. 1. Becausethe plasmamembranegenerallylacks transportersfor phosphorylatedsugars,the phosphorylated glycolS,ticintermediatescannot leave the cell. After the initial phosphorylation,no further energyis necessaryto retain phosphorylated intermediatesin the cell, despitethe large difference in their intracellular and extracellular concentrations. 2. Phosphorylgroupsare essentialcomponentsin the enzymaticconservationof metabolicenergy.Energy releasedin the breakageof phosphoanhydride bonds (such as those in ATP) is partially conserved

in the formation of phosphateesterssuch as glucose 6-phosphate.High-energyphosphatecompounds and formed in glycolysis(1,3-bisphosphoglycerate phosphoenolpyruvate)donatephosphorylgroups to ADP to form ATP. 3. Binding energyresulting from the binding of phosphate groups to the active sites of enzymeslowers the activationenergyand increasesthe specificity of the enzymaticreactions(Chapter6). The phosphate groups of ADP,ATP,and the glycolytic intermediatesform complexeswith Mgz+,and the substratebinding sites of many glycolytic enz)'rnes for theseMgz* complexes.Mostglyare specifi.c require Mg'* for activity. enzyrnes coly'tic

ofGlycolysis Phase ThePreparatory ATP Requires In the preparatoryphaseof glycolysis,two moleculesof ATP are invested and the hexose chain is cleavedinto two triosephosphates.The realizationthat phosphorylated, hexoseswere intermediatesin glycolysiscame slowly and serendipitously.In 1906,Arthur Harden and William Young tested their hypothesis that inhibitors of proteolytic enzymeswould stabilize the glucosefermenting enzyrnesin yeast extract. They addedblood serum (known to contain inhibitors of proteolytic en2:lrnes)to yeast extracts and observedthe predicted stimulationof glucosemetabolism.However,in a control experimentintended to show that boiling the serumdestroyed the stimulatory activity, they discovered that boiled serumwasjust as effectiveat stimulatingglycolysis! Careful examinationand testing of the contents of the boiled serum revealedthat inorganicphosphatewas responsiblefor the stimulation. Harden and Youngsoon discoveredthat glucoseaddedto their yeastextractwas converted to a hexose bisphosphate(the "HardenYoung ester," eventually identified as fructose 1,6-bisphosphate).This was the beginning of a long series of investigationson the role of organic esters and anhydrides of phosphate in biochemistry,which has led to our current understandingof the central role of phosphoryl group transfer in biologY'

ArthurHarden, .l 865-1940

WilliamYoung, 187B-1942

-

l

phosphate pathway Gluconeogenesis, andthepentose ' 5321 Glycolysis, @ Phosphorylation of Glucose In the first step of glycolysis,glucoseis activatedfor subsequentreactions by its phosphorylationat C-6 to yield glucose 6-phosphate, with ATP as the phosphoryl donor:

isomerization of glucose6-phosphate, an aldose,to fructose 6-phosphate,a ketose: 6

cHroPo3cHroPoS-

5

+/a

4

H

HO

H

OH H ) , OH

cH2oH

ivlg-

-

tKH

plro.pholrt,xo-'c l s o t n e ta s c

Fructose 6-phosphate

AG'": 1.TkJ/mol

GlucoseG-phosphate

This reaction, which is irreversible under intracellular conditions,is catalyzedby hexokinase. Recallthat ki_ nasesare enzymesthat catalyzethe transfer of the ter_ minal phosphoryl group from ATp to an acceptor nucleophile(see Fig. 13-20). Kinasesare a subclassof transferases (seeTable6-3). The acceptorin the caseof hexokinaseis a hexose,normally D-glucose,although hexokinasealso catalyzesthe phosphorylationof other conunonhexoses,suchas D-fructoseand l-mannose,in sometissues. _Hexokinase,like many other kinases,requires Mg2+for its activity,becausethe true substrateof the enzymeis not ATpa- but the MgATp2- complex (see Fig. 13-12).Mg2+shieldsthe negativechargesof the phosphorylgroups in ATp, making the terminal phos_ phorus atom an easiertarget for nucleophilicattack by an -OH of glucose.Hexokinaseundergoesa profound changein shape,an inducedfit, when it binds glucose; two domainsof the protein move about g A closer to each other when ATP binds (see Fig. 6-22). This movementbrings bound ATp closer to a molecule of glucosealso bound to the enzymeand blocks the ac_ cess of water (from the solvent), which might other_ wise enter the active site and attack (hydrolyze) the phosphoanhydridebonds of ATp. Like the other nine enzymesof glycolysis,hexokinaseis a soluble,cytoso_ lic protein. Hexokinaseis present in nearly all organisms. The human genome encodesfour different hexoki_ nases (I to IV), all of which catalyzethe same reac_ tion. TWo or more enzymes that catalyzethe same reaction but are encoded by different genes are calledisozymes (seeBox Ib_Z). One of the isozymes present in hepatocytes,hexokinaseIV (also called glucokinase),differs from other forms of hexokinase in kinetic and regulatoryproperties,with important physiological consequencesthat are described in Section15.3. @ Conversion ofGlucose G-phosphate to Fructose 6-Phosphate Theenz}rmephosphohexoseisomerase firhosphoglucose isomerase) catalyzesthe reversible

2

OH

Glucose 6-phosphate

aG'' : -16.7 kJ/mol

Ho

The mechanismfor this reaction involvesan enediol intermediate(Fig. f4-4). The reactionproceedsreadily in either direction,as might be expectedfrom the rela_ tively small changein standardfree energy.This isomerization has a critical role in the overall chemistry of the glycolytic pathway,as the rearrangementof the carbonyl and hydroxyl groupsat C-l and C-2 is anecessary prelude to the next two steps.The phosphorylationthat occursin the next reaction(step @) requiresthat the group at C-l fust be convertedfrom a carbonylto an alcohol,and in the subsequentreaction (step @; cleav_ ageofthe bondbetweenC-3and C-4requiresa carbonyl groupat C-2 @. a97). @ fhosphorylation of Fructose 6-phosphate to Fructose 1,G-Bisphosphate In the secondof the two primrngreactionsof glycolysis,phosphofructokinase-l (PFK-I) catalyzesthe transfer of a phosphoryl group from ATPto fructose6-phosphate to yield fructose 1,6bisphosphate: SHroPor,-

o.._ dHr-ou

'Ku so)' H\-lloH

ATP

A-DP

phospholi tLctokillsc 1IrFK-I)

I

6

Fructose 6-phosphate

cH2oPoS-

o

'KH uo

dH,-opoa* 2

OH

Fructose1,6-bisphosphate LG'": -L4.2kJ/mol

KEYC0NVENTI0N: Compounds that contain two phos_ phate or phosphoryl groups attached at different positions in the molecule are named bisphosphates (or bi,sphospho compounds); for example, fructose 1,6-bisphosphateand 1,3-bisphosphoglycerate.Com_ pounds with two phosphates linked together as a py_ rophosphoryl group are named d,i,phosphates; for example, adenosine diphosphate (ADp). Similar rules apply for the naming of tri,sphosphates (such

1 4 . 1G l y c o l y Issi s E

6cHroeo!Fructose 6-phosphate

Glucose 6-phosphate

uKn Ho

Dissociation and closing of the ring

Binding and opening of the ring

H.

H\ B3--

ri-iC-oH Phosphohexose isonerase

HO3CH

HnJou ,l

H'COH

uJHroeo!

H*

\ Proton abstraction by active-site Glu (B:) Ieads to cis-enediol formation.

reaction. isomerase 14*4 The phosphohexose MECHANISM FIGURE (steps Theringopeningandclosingreactions @ and @) arecatalyzed omittedherefor simplicHis residue, by mechanisms by an active-site ity.Theproton(pink)initiallyat C-2 is mademoreeasilyabstractable by electronwithdrawalby the adjacentcarbonyland nearbyhydroxyl

seep 432)andtrzphosas inositol 1,4,5-trisphosphate; phates (suchas adenosinetriphosphate,ATP).r The enzymethat formsfructose1,6-bisphosphate is calledPFK-1 to distinguishit from a secondenzyme (PFK-2) that catalyzesthe formation of fructose in a sepafrom fructose6-phosphate 2,6-bisphosphate rate pathway (the roles of PFK-2 and fructose 2,6in Chapter15).The PFK-l are discussed bisphosphate reaction is essentiallyirreversibleunder cellular conditions, and it is the first "commrtted"step in the glycolytic pathway;glucose6-phosphateand fructose 6-phosphatehave other possiblefates, but fructose is targetedfor glycolysis. 1,6-bisphosphate Somebacteriaand protists and perhapsall plants that uses pyrophosphate have a phosphofructokinase (PP), not ATP,as the phosphorylgroup donor in the synthesisof fructose1,6-bisphosphate: Fructose6-phosphatei- PP,

I

M8', * P1 fructose1,6-bisphosphate -2.9 : kJ/mol AG''

Phosphofructokinase-l is subject to complex allosteric regulation; its activity is increased whenever the cell's AIP supply is depleted or when the AIP breakdoum products, ADP and AMP (particularly the latter), accumulate. The enz5rmeis inhibited whenever the cell has ample ATP and is well supplied by other fuels such as fatty acids. In some organisms,fructose 2,6-bisphosphate

B: Iil-C-OH C:O

H+

CH I HOCH

@

r4) H

'o'PH

Btr{ \_-.11

1

2

OH

3l H

OH

HOH

lcH2oH

/ @

I

HCOH

HOCH

I

HCOH

General acid catalysis by same Glu facilitates formation of fructose 6phosphate.

HCOH

t"

cH2oPoicis-EnedioI intermediate

HCOH

t"

cH2oPoi-

group. After its transferfrom C-2 to the active-siteClu residue (a weak acid),the protonis freelyexchangedwith the surrounding from C-2 in step@ is not solution;that is, the protonabstracted to C-1 in step@. is added that one the same essarily Mechanism lsomerase Phosphohexose

(not to be confusedwith the PFK-I reactionproduct, is a potent allostericactivator fructose 1,6-bisphosphate) of PFK-1. Ribulose5-phosphate,an intermedratein the pentosephosphatepathwaydiscussedlater in this chapter, also activates phosphofructokinaseindirectly. The muitiple layersof regulationof this step in glycolysisare discussedin greaterdetail in Chapter15. @ Cleavage of Fructose 1,6-Bisphosphate The enzlrne fructose 1,G-bisphosphatealdolase, often called simply aldolase, catafyzesa reversiblealdol condensais cleaved tion (seeFig. 13-4).Fructose1,6-bisphosphate to yield two di-fferenttriosephosphates,glyceraldehyde 3-phosphate, an aldose,and dihydroxyacetone phosphate, a ketose: ri^1

HHO 4l

13

Fructose 1,6-bisphosPhate

Dihydroxyacetone phosphate

Glyceraldehyde 3-PhosPhate

AG'' = 23.8 kJ/mol

-phosphate pathway Gluconeogenesis, andthePentose [ 534I Glycolysis, Thereare two classesof aldolases. ClassI aldolases, found in animalsand plants, use the mechanismshown in Figure 14-5. ClassII enz;rmes, in fungi and bacteria, do not form the Schiffbaseintermediate.Instead,a zinc ion at the active site is coordinated with the carbonyl oxygen at C-2; LheZn21 polarizesthe carbonylgroup and stabilizesthe enolate intermediate created in the C-C bond cleavagestep. Although the aldolasereaction has a strongly positive standard free-energychange in the direction of fructose 1,6-bisphosphate cleavage,at the lower concentrationsof reactantspresent in cells the actual freeenergy change is small and the aldolasereaction is readily reversible.We shall seelater that aldolaseacts in the reversedirectionduring the processof gluconeogenesis(seeFig. 14-16).

of the Thiose Phosphates Only @Interconversion one of the two triose phosphates formed by aldolase, glyceraldehyde 3-phosphate, can be directly clegraded in the subsequent steps of glycolysis. The other product, dihydroxyacetone phosphate, is rapidly and reversibly converted to glyceraldehyde 3-phosphate by the fifth enzyme of the glycolytic sequence, triose phosphate isomerase:

Dihydroxyacetone phosphate

Glyceraldehyde B-phosphate

AG'':

7.5 kJ/mol

cH2oPo32o qHroPOs2HHO ,l

OH Fmctose 1,6-bisphosphate

Binding and opening of the ring

Protonated Schiff base Hzo

f,

I

) @

a) uJHroeo!-

Active-site Lys attacks substrate carbonyl,leading to formation of tetrahedral intermediate.

HCOH

JH,oeo3-

Aldolase

Rearrangement leads to formation ofprotonated Schiff base on enzyme; electron delocalization facilitates subsequent steps.

H Lys-N: H

CH"OPOS l" C:O

nJoH

CH"OPOS H lLys-N*-Q :A-

:B-

Schiffbase is hydrolyzed in reverse ofSchiff base formation.

H Lys-

I

/;\

HO-C-H I -B:

H-B-

Isomerization

Protonated Schiffbase

ME(HANISM FIGURE 14-5 The classI aldolasereaction.The reaction shownhereis the reverseof an aldol condensationNotethatcleavage betweenC-3 and C-4 dependson the presenceof the carbonylgroup

_

cH2oPoS :A-

C ll

c..--

@

H

C-C bond cleavage (reverse of aldoi condensation) leads to release of first product.

@

l" cHzoPo;-

\?,

-B:

r6on

t^ ', cH2oPo6i

releesod

I cH2oH

H-A-

-""

Glyceraldehyde 3-phosphate firsL H,, preduct_ 'C lrO

Dihydroxyacetone phosphate

Proton exchange with solution restores enzyme.

cH2oPo3'- l

H

H -B-H

H H-B

Covalent enzymeenamine intermediate

a t C - 2 A a n d B r e p r e s e n ta m i n o a c i d r e s i d u e st h a t s e r v e a s g e n e r a l a c i d ( A ) o r b a s e( B ) .

1 4 . 1G l y c o l y sfLi .sr"' ;1l

Fructose 1,6-bisphosphate

tqnr-o*@ 'c:o 'l

no:J-n H-rJ-oH g-!c-on Derived from glucose carbon 1

ol

"cttr-o-@

1 ^A gH,-o--(D H-q:o

Derived from glucose carbon 1

ll

2

C:O

J

cHzoH

H-C-OH

cHr-o-@

Dihydroxyacetone phosphate

K

o

Glyceraldehyde 3-phosphate

l)llosl)llilto

Derived from glucose carbons 4or3 5or2 6or 1

ttjC:o

n-Glyceraldehyde 3-phosphate

"t H.=C-OH "l "cH2-o--(B) .t Subsequent reactions of glycolysis

tsotll('1 us('

(a)

(b)

14-6 Fateof the glucosecarbonsin the formationof glycerFIGURE (a)Theoriginof thecarbonsin thetwo three-caraldehyde3-phosphate. reactions. bon productsof the aldolaseand triosephosphateisomerase 3-phosphate The end productof the two reactionsis glyceraldehyde is (two molecules)(b) Eachcarbonof glyceraldehyde 3-phosphate derivedfrom eitherof two specificcarbonsof glucose.Note that the

differs 3-phosphate numberingof the carbonatomsof glyceraldehyde glyceraldehyde In it is derived. which from glucose the that of from the mostcomplexfunctionalgroup(thecarbonyl)is spec3-phosphate, experi. Thisnumberingchangeis importantfor interpreting ifiedasC-.1 ments with glucosein which a singlecarbon is labeledwith a

The reaction mechanism is similar to the reaction pro-

@ Oxiaation of Glyceraldehyde 3-Phosphate to l,3-Bisphosphoglycerate The first step in the payoff phaseis the oxidation of glyceraldehyde3-phosphateto 1,3-bisphosphoglycerate, catalyzed by glyceraldehyde 3-phosphate dehYdrogenase:

isomerasein step@ of glycomotedby phosphohexose Iysis (Fig. l4-4). After the triose phosphateisomerase reaction,the carbonatoms derivedfrom C-1, C-2, and C-3 of the starting glucoseare chemicallyindistinguish(FiS. 1a-6); ablefrom C-6,C-5,and C-4,respectively the two "halves"of glucosehave both yielded glyceraldehyde3-phosphate. This reaction completesthe preparatoryphase of glycolysis.The hexosemoleculehas been phosphoryIatedat C-l and C-6and then cleavedto form two mole3-phosphate. culesof glyceraldehyde

(SeeProblems6 and 9 at the end of this chapter') radioisotope.

NAD*

NADH + H* /

glvceraltlehydc 3lhosphate clehvdrogenase

Glyceraldehyde 3-phosphate

Inorganic phosPhate

?

ATP andNADH Yields Phase ofGlycolyris ThePayoff

II

The payoff phase of glycolysis (Fig. 14-2b) includes the energy-conserving phosphorylation steps in which some of the chemical energy of the glucose molecule is conserved in the form of ATP and NADH. Remember that one molecule of glucose yields two molecules of glyceraldehyde 3-phosphate, and both halves of the glucose molecule follow the same pathway in the second phase of glycolysis. The conversion of two molecules of glyceraldehyde 3-phosphate to two molecules of pyruvate is accompanied by the formation of four molecules of ATP from ADP. Howevet, the net yield of ATP per molecule of glucose degraded is only two, becausetwo ATP were invested in the preparatory phase of glycolysis to phosphorylate the two ends of the hexose molecule.

o* ,o*f-- oqdI

HCOH

l"_

CH,OPO; 1,3-Bisphosphoglycerate AG'': 6.3 kJ/mol

This is the flrst of the two energy-conservingreactions of glycolysis that eventually lead to the formation of ATP. The aldehyde group of glyceraldehyde3-phosphate is oxidized,not to a free carboxyl group but to a carboxylic acid anhydride with phosphoric acid. This

p e n t o speh o s p h aptaet h w a y L 5 3 6 _ l G l y c o l y sGi lsu, c o n e o g e naensdtihse,

CH.OPO?_

,l

.NAD*

INAD+

Glyceraldehyde 3-phosphate

',, Glyceraldehyde 3-phosphate dehydrogenase

c iCys

H H" s

I cys

Formation of enzJmresubstratecomplex.The active-siteCys has a reducedpK. (5.5 instead of8) when NAD* is bound, and is in the more reactive, thiolate form.

I I 611 | I

Acovalentthiohemiacetal linkage forms betweenthe substrateand the -Sg.oop ofthe Cys residue.

J

CHt

I HCOI I

The covalentthioester linkage betweenthe substrate and enzyme undergoesphosphorolysis (attack by P1)releasing the secondproduct, 1,3-bisphosphoglycerate.

c-I

oP( 1,3-Bisphospl

I cvs

' - , . sI I cys

fhe NADH product leaves the

ilHf#ff:fJi:?oso5u.o'

ME(HANlsM FIGURE 14-7 Theglyceraldehyde 3-phosphate dehydrogenase reaction.

type of anhydride,calledan acyl phosphate, hasa very high standardfree energyof hydrolysis(AG'. = _4g.8 kJ/mol;seeFig. 13-14,TabletB-6). Muchof the free energy of oxrdation of the aldehydegroup of glyceraldehyde 3-phosphateis conservedby formation of the acyl phosphategroup at C-1 of 1,3-bisphosphoglycerate. Glyceraldehyde3-phosphateis covalentlybound to the dehydrogenaseduring the reaction (FiS. f4-Z). The aldehydegroup of glyceraldehyde3-phosphatereacts with the -SH group of an essentialCysresiduein the active site, in a reaction analogousto the formation of a hemiacetal(seeFig. 7-b), in this caseproducinga thi,ohemiacetal.Reaction of the essential Cys residue with a heavymetal such as Hg2* irreversiblyinhibits the enzlirne. The amount of NAD+ in a cell (-10-s u) is far smallerthan the amount of glucosemetabolizedin a few minutes. Glycolysiswould soon cometo a halt if the NADH formed in this step of glycolysiswere not continuouslyreoxidizedand recycled.We return to a discussionof this recycling of NAD+ later in the chapter. @ Phosptroryl Tbansfer from l,B-Bisphosphoglycerate to AI)P The enz;..rnephosphoglycerate kinase transfers the high-energyphosphoryl group from

the carboxyl group of l,3-bisphosphoglycerateto ADp, forming ATP and 3-phosphoglycerate:

o

o\ ,,o-i-ocdt-+

lHCOH

t"

CH,OPO; 1,3-Bisphosphoglycerate

ox ,roc Hiou

l" cH2oPos-

3-Phosphoglycerate

ATP AG'' = -18.5 kJ/mol

pr

1 4 . 1G l y c o l y s i s

Notice that phosphoglycerate kinase is named for the reverse reaction, in which it transfers a phosphoryl group from ATP to 3-phosphoglycerate Like all enzymes, it catalyzes the reaction in both directions. This enzyrne acts in the direction suggestedby its name during gluconeogenesis(see Fig. 14-16) and during photosynthetic CO2assimilation (see Fig. 20-4) In glycolysis, the reaction it catalyzesproceeds as shov,'nabove, in the direction of ATP synthesis. Steps @ and @ of glycolysis together constitute an energy-coupling process in which 1,3-bisphosphoglycerate is the common intermediate; it is formed in the first reaction (which would be endergonic in isolation), and its acyl phosphate group is transferred to ADP in the second reaction (which is strongly exergonic). The sum of these two reactionsis Glyceraldehyde3-phosphate+ ADP + Pi + NAD+ + 8-phosphoglycerate+ ATP + NADH + Ht AG'": -72.2kJ/mol Thus the overall reaction is exergonic Recall from Chapter 13 that the actual free-energy change, AG, is determined by the standard free-energy change, AG'o, and the mass-action ratio, Q, which is the ratio fproducts]/[reactants](see Eqn 13-4). For step @

groupbetweenC-2and C-3 of glycerate;Mgz* is essential for this reaction:

2-Phosphoglycerate

3-Phosphoglycerate

LG'': 4'4kJ/mol The reactionoccursin two steps(Fig. 14-8). A phosphoryl group initially attached to a His residue of the mutase is transferred to the hydroxyl group at C-2 of forming 2,3-bisphosphoglycerate S-phosphoglycerate, group at C-3 of 2,3-BPGis phosphoryl (2,3-BPG).The then transferred to the same His residue, producing and regeneratingthe phosphorylated 2-phosphoglycerate

Phosphoglycerate

mutase

A G : A G , "+ , 8 ? I n Q :.J(''Jlflln

,

[1,3-bisphosphoglycerate][NADH]

Notice that [H+] is not includedin Q In biochemicalcalculations,[H+] is assumedto be a constant(10-7 u), and this constantis includedin the definitionof AG'" fu. 491). ratio is lessthan 1.0,its natuWhenthe mass-action ral Iogarithmhasa negativesign.Step @, by consuming keeps the product of step@ (t,3-Uispnosphoglycerate), relatively low in the steady [1,3-bisphosphoglycerate] stateand therebykeepsQ for the overallenergy-coupling processsmall.WhenQ is small,the contributionof ln Q can make AG strongly negative.This is simply another way of showinghow the two reactions,steps@ and @, are coupledthrougha commonintermediate. The outcome of these coupled reactions,both reversibleunder cellular conditions,is that the energyreIeasedon oxidationofan aldehydeto a carboxylategroupis conservedby the coupledformationof AIP from ADP and Pi.The formation of ATPby phosphorylgrouptransferfrom is referredto a substratesuch as 1,3-bisphospho$ycerate asa substrate-level phosphorylation, to distinguishthis mechanismfrom respiration-linked phosphorylation. Substratelevel phosphorylationsinvolve soluble en4,.rnes in and chemicalintermediates(1,3-bisphosphoglycerate ttus case). Respiration-linkedphosphorylations,on the other hand, involve membrane-boundenz}.'rnesand transmembranegradientsof protons(Chapter19). @ Conversion of 3-Phosphoglycerate to 2Phosphoglycerate The enzyme phosphoglycerate mutase catalyzesa reversibleshift of the phosphoryl

PhosPhorYl transfer occurs between an active-site His and C-2 (OH) ofthe substrate' A second active-site His acts as general base catalYst. .

-ooc r,A H-C-O-PO3

I

N,

'' H H2C-O

\zN

2.3-Bisphosphoglycerate\

jz,s--Bpcr

Hi'

I

HN-{

/

_His yI transfer from C-3 rstrateto the frrst e His. The second e His acts as general ;yst. His

-ooc n-i-o I

H2C-O 2-Phospho{

I t

_His

mutasereaction. 14-8 Thephosphoglycerate FIGURE

Frrl G l y c o l y sGilsu,c o n e o g e naensdtihse, P e n t o speh o s p h aptaet h w a y enzyme.Phosphoglycerate mutaseis initially phosphorylated by phosphoryltransfer from 2,3-BpG,which is required in small quantitiesto initiate the catalytic cycle and is continuouslyregeneratedby that cycle.

In this substrate-levelphosphorylation,the product pyruvate first appearsin its enol form, then tautomerizes rapidly and nonenz;.'rnatically to its keto form, which predominatesat pH 7:

@ Oetrydration of 2-Phosphoglycerate to phosphoenolpyruvate In the second glycolytic reaction that generatesa compoundwith high phosphorylgroup transfer potential (the first was step @), enolase promotesreversibleremovalof a moleculeof waterfrom 2-phosphoglycerateto yield phosphoenolpyruvate (PEP):

o.o *c'

o.\\ ,/.o

Hro

C

t/

H-C-OPO32-

.-

I

I c-oPo3z-

('nolits(l

dr,

HO-CH, 2-Phosphoglycerate

Phosphoenolpyruvate LG'"=7.5 kJ/mol

The mechanismof the enolasereactioninvolvesan enolic intermediatestabilizedby Mg2* (seeFig. 6-23). The reactron converts a compound with a relatively low phosphorylgrouptransferpotential(AG,. for hydrolysis of 2-phosphoglycerate is - 12.6kJ/mol)to onewith high phosphorylgroup transferpotential (AG,. for pEp hy_ drolysisis -61.9 kJ/mol)(seeFig. tB-13, Tablet3-6). @ tlanster of the Phosphoryl Group from phosphoenolpyr.uvate to ADP The last step in glycolysis is the transfer of the phosphoryl group from phospho_ enolpyruvate to ADP, catalyzed,by pyruvate kinase, which requiresK+ and either MA2+or Mnz*

ox ,'oC

I I

c:o CHe Pyruvate (keto form)

The overall reaction has a large, negativestandardfreeenergy change,due in large part to the spontaneous conversionofthe enolform ofpyruvateto the keto form (see Fig. 13-13). About half of the energyreleaseclby PEP hydroiysis(AG'' : -61.9 kJ/mol) is conseruedin the formation of the phosphoanhydridebond of ATp (AG'' : -30.5 kJ/mol), and the rest (-8I.4 kJ/mol) constitutesa large driving force pushingthe reactiontoward ATP synthesis.We discussthe regulation of pyruvate kinasein Chapter15.

The0verall Balanre Sheet Shows a Net6ainofATP We can now construct a balancesheet for glycolysisto accountfor (1) the fate of the carbonskeletonof glucose,(2) the input of P, and ADP and output of ATp,and (3) the pathway of electronsin the oxidation-reduction reactions. The left-hand side of the following equation showsall the inputs of ATP,NAD+, ADp, and p1(consult Fig. I4-2), and the right-hand side showsall the outputs (keepin mind that eachmoleculeof glucoseyieldstwo moleculesof pyruvate): Glucose+ 2ATP+ 2NAD++ 4ADp-r 2pi----> 2 pyruvate+ 2ADP+ 2NADH+ 2IJ++ 4ATp + 2H2O Cancelingout commonterms on both sidesof the equation givesthe overall equationfor glycolysisunder aerobic conditions:

Phosphoenolp5mrvate

-

Md..

Kt

ADP

] pr r Lrr.rt,

Jr'"",. ox

,oc I c:o + I

Glucose+ 2NAD++ 2A_DP * 2Pi---> 2 pyruvate+ 2NADH+ 2H+ + 2ATp+ 2H2O The two moleculesof NADH formed by glycolysisin the cytosolare, under aerobicconditions,reoxidizedto NAD+ by transfer of their electronsto the electrontransfer chain,which rn eukaryoticcellsis locatedin the mitochondria.The electron-transferchainpassesthese electronsto their ultimatedestination,02: 2NADH + 2If+ * 02 --+

CHa Pyruvate

ATP

LG'": -31.4 kJ/mol

2NAD+ + 2Ii'2O

Electron transfer from NADH to 02 in mitochondria provides the energy for synthesis of ATp by respirationlinked phosphorylation (Chapter 19). In the overall glycolytic process, one molecule of glucose is converted to two molecules of pyruvate (the pathway of carbon). TWo molecules of ADp and two of Pi are converted to two molecules of ATp (the pathway

1 4 . 1G l y c o l y slGt'] is

of phosphorylgroups).Four electrons,as two hydride ions,are transferredfrom two moleculesof glyceraldehyde 3-phosphateto two of NAD+ (the pathway of electrons).

lsunder TightRegulation Glycolysis During his studies on the fermentation of glucose by yeast,Louis Pasteurdiscoveredthat both the rate and the total amount of glucoseconsumptionwere many times greater under anaerobicthan aerobic conditions. Later studies of muscle showed the same large difference in the rates of anaerobicand aerobicglycolysis. The biochemicalbasis of this "Pasteur effect" is now clear. The ATP yield from glycolysisunder anaerobic conditions (2 ATP per molecule of glucose) is much smaller than that from the complete oxidation of glucoseto CO2under aerobicconditions(30 or 32 AIP per glucose;see Table 19-5). About 15 times as much gluasaerocosemust thereforebe consumedanaerobically bically to yield the sameamount of ATP. The flux of glucosethrough the glycolytic pathway is regulatedto maintainnearly constantATP levels (as well as adequatesuppliesof glycolytic intermediates that serve biosynthetic roles). The required adjustment in the rate of glycolysisis achievedby a complex interplay among ATP consumption,NADH regeneration, and allostericregulationof severalglycolytic enzymes-including hexokinase,PFK-I, and pyruvate kinase-and by second-to-secondfluctuations in the concentrationof key metabolitesthat reflect the cellular balancebetweenATP productionand consumption. On a slightly longer time scale,glycolysisis regulated by the hormonesglucagon,epinephrine,and insulin, and by changesin the expressionof the genesfor several glycolytic enzymes.An especiallyinterestingcase of abnormalregulationof glycolysisis seenin cancer. The GermanbiochemistOtto Warburgfirst observedin 1928that tumors of nearly all types carry out glycolysis at a much higher rate than normal tiss:ue,euen when orygen 'is aua'ilable. This "Warburg effect" is the basisfor severalmethodsof detectingand treating cancer(Box 14-1). Warburg is generally considered the preeminent biochemrstof the first half of the twentieth century. He made seminalcontributionsto many other areas of biochemistry including respiration, photosynthesis,and the enzymology of intermediarymetabolism. Beginning rn 1930, Warburgand his associatespurif,ed and crystallizedsevenof of glycolysis.They the enz}.'rnes developedan experimentaltool that revolutioruzedbiochemical studies of oxidative metaboOtto Warburg, lism: the Warburgmanometer, 1883-1970

which directly measuredthe oxygenconsumptionof tissuesby monitonng changesin gas volume, and thus allowed quantitative measurement of any enzyme with oxidaseactivity. Tfained in carbohydratechemistryin the laboratory of the great Emil Fischer (who won the Nobel Prize in Chemistry in 1902), Warburg himself won the Nobel Prize in Physiologyor Medicinein f931. Severalof Warburg'sstudentsand colleaguesalsowere awardedNobel Prizes:Otto Meyerhof in 1922,Hans Krebs and Fritz Lipmannin 1953,and HugoTheorellin 1955.Meyerhof's laboratory provided training for Lipmann, and for several other Nobet Prize winners: SeveroOchoa (1959), Andre Lwoff (1965),and GeorgeWald(1967).

Mellitus 1 Diabetes inType lsDeficient Uptake Glucose The metabolismof glucosein mammalsis limited by the rate of glucose uptake into cells and its phosphorylationby hexokinase.Glucoseuptake from the blood is mediated by the GLUT family of glucose fransporters (see Table 11-3). The transporters of hepatocytes(GLUTI, GLUT2) and of brain neurons (GLUT3) are always present in plasmamembranes.In contrast,the main glucosetransporter in the cells of skeletal muscle, cardiac muscle, and adipose tissue (GLUT4) is sequesteredin small intracellularvesicles and movesinto the plasmamembraneonly in response to an insulin signal (Fig. L4-g). We discussedthis insulin signaling mechanism in Chapter 12 (see Fig. f2_I6). Thus in skeletalmuscle,heart, and adiposetissue,glucoseuptakeand metabolismdependon the normal releaseof insulin by pancreaticB cells irt response to elevatedbloodglucose(seeFiC.23-27)' Individuals with type 1 diabetes mellitus (also calledinsulin-dependentdiabetes)havetoo few B cells and cannotreleasesufflcientinsulin to trigger glucose uptakeby the cellsof skeletalmuscle,heart, or adipose tissue.Thus, after a meal containirLgcarbohydrates,glucose accumulatesto abnormally high levels in the blood, a conditionknown as hyperglycemia'Unableto take up glucose,muscle and fat tissue use the fatty acidsof storedtriacylglycerolsas their principalfuel. In the liver, acetyl-CoAderivedfrom this fatty acid breakdown is converted to "ketone bodies"-acetoacetate and p-hydroxybutyrate-which are exported and carried to other tissuesto be used as fuel (Chapter 17). These compoundsare especiallycritical to the brain, which uses ketone bodies as alternative fuel when glucose is unavailable.(Fatty acids cannot pass through the blood-brainbarrier and thus are not a fuel for brain neurons.) In untreatedtype I diabetes,overproductionof acetoacetateand F-hydroxybutl'rateleadsto their accumulation in the blood, and the consequentlowering of blood pH produces ketoacidosis, a life-threatening condition. Insulin injection reversesthis sequenceof events:GLUT4 moves into the plasmamembranesof

[r*'] G l y c o l y sGilsu,c o n e o g e naensdtihse, P e n t o sPeh o s p h aptaet h w a y

In many types of tumors found in humans and other animals,glucoseuptake and glycolysisproceed about 10 times faster than in normal, noncanceroustissues. Most tumor cells grow under hypoxic conditions (i.e., with limited oxygen supply) because,at least initially, they lack the capillary network to supply sufficient oxygen.Cancercells locatedmore than 100to 200p.m from the nearestcapillariesmust dependon glycolysis alone (without further oxidation of pyruvate) for much of their ATP production. The energy yield (2 ATP per glucose)is far lower than can be obtainedby the complete oxidation of pyruvate to CO2 in mitochondria(about30 ATP per glucose;Chapter19). So, to make the same amount of ATp, tumor cells must take up much more glucosethan do normal cells,converting it to pyruvateand then to lactate as they recycle NADH. It is likely that two early steps in the transformationof a normal cell into a tumor cell are (1) the changeto dependenceon glycolysisfor ATp production,and (2) the developmentof toleranceto a low pH in the extracellularfluid (causedby releaseof the end product of glycolysis,lactic acid). In general, the more aggressivethe tumor, the greater is its rate of glycolysis. This increasein glycolysisis achievedat least in part by increased synthesis of the glycoly'tic enzyrnes and of the plasma membranetransporters GLUT1 and GLUTS(see Table 1i-3) that carry glucoseinto cells. (Recall that GLUT1 and GLUT3 are not dependent on insulin.) The hypoxia-inducible transcription factor (HIF-l) is a protein that acts at the level of mRNA syrrthesisto stimulate the production of at least eight glycolytic enz)..rnes and the glucose transporters when oxygensupply is limited (Fie. 1). Wth the resulting high rate of glycolysis,the tumor cell can survive anaerobic conditions until the supply of blood vesselshas caught up with tumor growth. Another protein induced by HIF_ 1 is the peptide hormone \EGF (vascular endothelial growth factor), which stimulatesthe outgrowth of blood vessels(angiogenesis)toward the tumor.

FIGURE 1 The anaerobicmetabolism of glucosein tumorcellsyields far lessATP (2 per glucose)than the completeoxidationto CO2 that takesplacein healthycellsunderaerobicconclitions (-30 ATpperglucose),so a tumor cell mustconsumemuch more glucoseto produce the sameamount of ATP.Clucosetransporters and most of the gly_ colyticenzymesareoverproduced in tumors.Compounds thatinhibit hexokinase, glucose6-phosphate dehydrogenase, or transketolase blockATPproductionby glycolysis, thusdeprivingthe cancercell of energyand killingit

There is alsoevidencethat the tumor suppressor protein p53, which is mutated in most types of cancer (p. 477), controls the synthesis and assemblyof mitochondrial proteins essentialto the passageof electronsto 02. Cellswith mutant p53 are defective in mitochondrial electron transport and are forced to rely more heavily on glycolysis for ATP production (Fie. 1). This heavierrelianceof tumors than of normal tissue on $ycolysissuggestsa possibilityfor anticancertherapy: inhibitors of $ycolysismight target and kill tumors by depleting their supply of ATP. Three inhibitors of hexokinase have shown promise as chemotherapeuticagents: 2-deoxyglucose,lonidamine, and 3-bromopyruvate.By

!

.. exoklnase 8*

8'-8'

- - 2-DeoxYglucose Lonidamine - - 3-Bronop,mvate 6-Amino nicotinic

acid

+

I .l''xlniyn"

I

3 COz

14.1Glycolysis [t*t]

Ho

ATP .g>

\/

AllP

o P o I oHO

VOH HO'r

[18F]2-FIuoro-2-deoxyglucose (FdG)

18F

[18F]6-Phospho-2-fluoro-2-deoxyglucose (6-Phospho-FdG)

trapsthe FdC in by hexokinase FIGURE 2 Phosphorylation of 18F-labeled 2-fluoro-2-deoxyglucose 18F. whereitspresence can be detectedby positronemissionfrom cells(as6-phospho-Fdc),

preventingthe formationof glucose6-phosphate,these compounds not only deprive tumor cells of glycolytically produced ATP but also prevent the formation of pentosephosphatesvia the pentosephosphatepathway,which alsobeginswith glucose6-phosphate.Without pentosephosphates,a cell cannot synthesizethe nucleotidesessentialto DNA and RNA symthesisand thus cannot grow or divide. Another anticancer drug alreadyapprovedfor clinicaluse is imatinib (Gleevec), tyrosinekidescribedin Box l2-5.h inhibits a speci-fic nase,preventingthe increasedsynthesisof hexokinase normally triggered by that kinase.The thiamine analog oxythiamine,which blocks the action of a transketolase-likeenzyrnethat convertsxylulose5-phosphateto glyceraldehyde3-phosphate(Fig. 1), is in preclinical trials as an antitumor drug.

The high $ycolytic rate in tumor cells alsohas diagnostic usefulness.The relative rates at which tissues take up glucosecan be used in some casesto pinpoint the location of tumors. In positron emissiontomography (PET), individualsare injected with a harmless,isotopically labeled glucose analog that is taken up but not metabolizedby tissues.The labeled compoundis 2-fluoro-2-deoxyglucose(FdG), in which the hydroxyl group 18F(Fig. 2). This at the C-2 of glucoseis replacedwith compound is taken up via GLUT transporters and is a good substratefor hexokinase,but it cannot be converted to the enediolintermediatein the phosphohexose isomerasereaction (see Fig. 14-4) and therefore The extent of its accuaccumulatesas 6-phospho-FdG. and phosphoryof uptake its rate mulation dependson 10 or more is typically lation, which as noted above Decay of tissue. normal in times higher in tumors than 18Fyieldspositrons(two per 18Fatoml that can be detected by a series of sensitive detectors positioned around the body, which allows accurate localization of accumulated6-phospho-Fdc(Fig. 3). tissueby positronemissiontomogra3 Detectionof cancerous FIGURE phy (PET). Theadult malepatienthad undergonesurgicalremovalof a The imageon the left,obprimaryskincancer(malignantmelanoma). (CTscan),showsthe lotomography tainedby whole-bodycomputed panelis a PETscanafter The central bones. and soft tissues cationof the t8F-labeled2-fluoro-2-deoxyglucose (FdC) the patienthad ingested Darkspotsindicateregionsof highglucoseutilization.As expected'the brainand bladderare heavilylabeled-the brain becauseit usesmost 18Fof the glucoseconsumedin the body,and the bladderbecausethe is excretedin the urine.When the intensityof labeled6-phospho-FdC the label in the PETscanis translatedinto falsecolor (the intensityinon from greento yellowto red)and the imageis superimposed creases of the (right) the bones in cancer reveals the CT scan,the fusedimage upperspine,in the liver,and in someregionsof muscle'all the resultof from the primarymalignantmelanoma. cancerspreading

542

G l y c o l yG s i lsu, c o n e o g e naensdtihse, P e n t o sp eh o s p h aptaet h w a y

Primarv defectin diabetes

a 6 e

Plasma membrane

.:)

e

I

X' i''

Yancreas ^ w(rsecretes insulin

€o

GLIJT4

.-----x--------PKB I

.Hexo_tGalactitol

in this disorder are relatively mild, The other s5.'rnptoms and strict limitation of galactosein the diet greatly diminishestheir severity. galactosemiais more seriTfansferase-deficiency ous; it is characterizedby poor growth in childhood, speechabnormality,mental deflciency,and liver damage that may be fatal, evenwhen galactoseis withheld from galactosemialeads to the diet. Epimerase-deficiency when dietary galacsevere is less but s1'rnptoms, similar controlled. I is carefully tose D-Mannose,releasedin the digestion of various polysaccharidesand glycoproteins of foods, can be phosphorylatedat C-6by hexokinase: Mg'*

Mannose + ATP -----+ mannose 6-phosphate + ADP

Mannose6-phosphateis isomerizedby phosphomannose isomerase to neld fructose6-phosphate,an intermediateof glycolysis.

F-'l

G l y c o l y sGi lsu, c o n e o g e naensdtihse, P e n t o sPeh o s p h aPtaet h w a y

Phosphorolfiiccleavageof a glucoseresiduefrom an end of the polS.nner, forming $ucose l-phosphate, is catalyzedby glycogenphosphorylase or starch phosphorylase. Phosphoglucomutase then converts the glucose1-phosphateto glucose6-phosphate, which can enter glycolysis.

Galactose 1-phosphate

cH2oH

Hq/it

t^ .4-U

OHH

\T

UDP-galactose

O-UDP

- - . . 1l . l r r , - r

NS* ,

+H-1

Lrr,,-r, I l-,,,,,r'r',.,.,

Ingestedpolysaccharides and disaccharides are convertedto monosaccharides by intestinal hydrolytic enzyrnes,and the monosaccharides then enter intestinalcellsand are transportedto the liver or other tissues. A variety of o-hexoses,includingfructose, galactose,and mannose,can be funneledinto glycolysisEachis phosphorylatedand converted to glucose6-phosphate, fructose6-phosphate, or fructose1-phosphate. Conversionof galactose1-phosphate to glucose I -phosphateinvolvestwo nucleotide derivatives: UDP-galactose and UDP-glucose. Geneticdefects in any of the three enz)rrnesthat catalyze conversionof galactoseto glucose1-phosphate result in galactosemiasof varflng severity.

14.3Fates ofPyruvate under Anaerobic Conditions: Fermentation o-lupFl -gltrtosc nlct ls(

UDP-glucose

H

o-lutF.l

HO HOH

FIGURE 14-12 Conversionof galactoseto glucose1-phosphate. The conversionproceedsthrougha sugar-nucleotide derivative,UDp'l-phosphate galactose, whichis formedwhengalactose displaces glucose1-phosphate from UDP-glucose. UDp-galactose isthenconverted by UDP-glucose 4-epimerase to UDp-glucose, in a reactionthat involvesoxidationof C-4 (pink)by NAD*, then reductionof C-4 by NADH; the resultis inversionof the configuration at C-4.The UDpglucoseis recycledthroughanotherroundof the samereactionThe net effectof this cycle is the conversionof galactose1-phosphateto .l glucose -phosphate; thereis no net productionor consumption of UDP-galactose or U DP-glucose

S U M M A R1Y4 . 2 F e e d ePra t h w a fyosr Gl y c o l y si s r

Endogenousglycogenand starch,storageforms of glucose,enter glycolysisin a two-stepprocess.

Under aerobic conditions, the pyruvate formed in the final step of glycolysisis oxidized to acetate (acetylCoA), which entersthe citric acid cycleand is oxidized to CO2and H2O.The NADHformedby dehydrogenation of glyceraldehyde3-phosphateis ultimately reoxidized to NAD+ by passageof its electronsto 02 in mitochondrial respiration.Under hypoxic (low-oxygen) conditions, however-as in very active skeletalmuscle, in submergedplant tissues,solid tumors, or in lactic acid bacteria-NADH generatedby glycolysiscannot be reoxidizedby 02. Failureto regenerateNAD* would leave the cell with no electronacceptorfor the oxidationof glyceraldehyde3-phosphate,and the energy-yielding reactionsof glycolysiswould stop.NAD* must therefore be regeneratedin someother way. The earliest cells lived in an atmospherealmost devoid of oxygenand had to developstrategiesfor deriving energyfrom fuel moleculesunder anaerobicconditions. Most modern organismshaveretained the ability to continually regenerate NAD+ during anaerobic glycolysis by transferring electronsfrom NADH to form a reduced end product suchas lactateor ethanol.

Pyruvate lstheTerminal Electron Acceptor in [actic AcidFermentation When animal tissues cannot be supplied wrth sufficient oxygen to support aerobic oxidation of the pyruvate and NADH produced in glycolysis, NAD+ is regenerated

'14.3 Fates ofPyruvate under Anaerobic Conditions: Fermentation F*f from NADH by the reduction of pyruvate to lactate. As mentionedearlier,sometissuesand cell types (such as erythrocy[es,which haveno mitochondriaand thus cannot oxidize pyruvate to CO2)produce lactate from glucose even under aerobicconditions.The reduction of py'ruvatein this pathway is catalyzedby lactate dehydrogenase,which formsthe L isomerof lactate atpHT: oo

,,o

fc:o

lx* \-/

t,-

cH"

or..

NADH+ H-

?

- Ho-c-H I CH,

rrr.'fL rir'lt\11t1,il,'nit\1,

"

,,o-

Pytuvate

l-Lactate

LG'": -25.1kJlmol The overall equilibrium of the reaction strongly favors Iactate formation, as shown by the Iarge negative standard free-energy change. In glycolysis, dehydrogenation of the two molecules glyceraldehyde of 3-phosphate derived from each molecule of glucose converts two molecules of NAD* to two of NADH. Because the reduction of two molecules of pyruvate to two of lactate regenerates two molecules of NAD+, there is no net change in NAD+ or NADH: Glucose

2Pyruvate

\

2NADH \

\

Yeast and other microorganismsferment glucose to ethanoland CO2,rather than to lactate.Glucoseis converted to pyruvate by glycolysis,and the pyruvate is convertedto ethanoland CO2in a two-stepprocess:

ox ,,oc I

TPP, I "M6" 2 / >*

C:O

I

/

pvnr\ ale

CHt Pyruvate

N ADH + H*

Coz

rlecar borvlitse

1

I \

oo ,," c I

CHe Acetaldehyde

NAD- OH I -/,

^1."i."f dehydrrrgennse

CHq

t-

CHs Ethanol

In the flrst step, pyruvate is decarboxylated in an irreversible reaction catalyzed by pyruvate decarboxylase. This reaction is a simple decarboxylation and does not involve the net oxidabion of pyruvate. Pyruvate decarboxylase requires Mg"- and has a tightly bound coenzJrme,thiamine pyrophosphate, which is discussed below. In the second step, acetaldehyde is reduced to ethanol through the action of alcohol dehydrogenase, with the reducing power furnished by NADH derived from the dehydrogenation of glyceraldehyde 3-phosphate. This reaction is a well-studied case of hydride transfer from NADH (FiS. 14-13). Ethanol and CO2are thus the end products of ethanol fermentation, and the overall equation is Glucose+ 2ADP f 2Pi -----+ 2 ethanol + 2CO2+ 2ATP + 2HzO

2NAD*

/ t/

Ethanol lstheRedueed Product in[thanol Fermentation

I t 2Lactate

The lactate formed by active skeletal muscles (or by erythrocytes) can be recycled; it is carried in the blood to the liver, where it is converted to glucose during the recovery from strenuous muscular activity. When lactate is produced in large quantities during vigorous muscle contraction (during a sprint, for example), the acidification that results from ionization of lactic acid in muscle and blood limits the period of vigorous activity. The best-conditioned athletes can sprint at top speed for no more than a minute (Box 14-2). Although conversion of glucose to lactate includes two oxidation-reduction steps, there is no net change in the oxidation state of carbon; in glucose (C6H12O6)and lactic acid (C3H6O3),the H:C ratio is the same. Nevertheless, some of the energy of the glucose molecule has been extracted by its conversion to lactate-enough to give a net yield of two molecules of ATP for every glucose molecule consumed. Fermentation is the general term for such processes,which extract energy (as ATP) but do not consume oxygen or change the concentrations of NAD+ or NADH. Fermentations are carried out by a wide range of organisms, many of which occupy anaerobic niches, and they yield a variety of end products, some of which f,nd commercial uses.

As in lactic acid fermentation, there is no net change in the ratio of hydrogen to carbon atoms when glucose (H:C ratio : 12/6 : 2) is fermented to two ethanol a n d t w o C O 2 ( c o m b i n e dH : C r a t i o : 1 2 / 6 : 2 ) . I n a l l

R

H,a

llH .\-t.' ll I

\fi2 NAD*

|

R

'NHz+

I CH.-C-OH

t!

-bthanol ,

reaction. FISURE 1,1-13The alcohol dehydrogenase MFCHANISM Mechanism Alcohol Dehydrogenase $

Gluconeogenesis, andthePentose Phosphate Pathway L54B_]Glycolysis, fermentations,the H:C ratio of the reactantsand products remainsthe same. Pyruvate decarboxylaseis present in brewer's and baker's yeast (Saccharomyces cereui,s'iae)and in all

other organismsthat ferment glucoseto ethanol,including someplants. The CO2produced by p;,'ruvatedecarboxylation in brewer's yeast is responsiblefor the characteristic carbonation of champagne.The ancient

Most vertebratesare essentiallyaerobicorgarusms;they convert glucoseto p;rmvateby gJycolysis,then use moIecularoxygento oxidizethe pyruvatecompletelyto CO2 and H2O.Anaerobiccatabolismof $ucose to lactate occurs during short bursts of extrememuscularactivity,for examplein a 100 m sprint, during which oxygencannot be carriedto the musclesfast enoughto oxidizepyruvate. Instead,the musclesuse their stored$ucose (glycogen) as fuel to generateAIP by fermentation,with lactate as the end product. In a sprint, lactatein the bloodbuildsup to high concentrations.It is slowlyconvertedbackto glucose by gluconeogenesis in the liver in the subsequent rest or recoveryperiod, during which oxygen is consumed at a gradually diminishing rate until the breathurg rate returns to normal. The excessoxygen consumedin the recoveryperiod representsa repaymentof the oxygen debt.This is the amountof oxygenrequiredto supply AIP for gluconeogenesis during recoveryrespiration,in order to regeneratethe glycogen"borrowed" from liver and muscleto carry out intensemuscularactivity in the sprint. The cycle of reactionsthat includesglucoseconversionto lactatein muscleand lactateconversionto $ucosein liver is called the Cori cycle, for Carl and Gerty Cori, whose studiesin the 1930sand 1940sclarified the pathwayand its role (seeBox 15-4). The circulatory systemsof most small vertebrates can carry oxygen to their musclesfast enoughto avoid having to use muscleglycogenanaerobically.For example, migrating birds often fly great distancesat high speedswithout rest and without incurring an oxygen debt. Many running animalsof moderatesize alsomaintain an essentiallyaerobic metabolismin their skeletal muscle.However,the circulatory systemsof larger animals, including humans,cannot completelysustainaerobicmetabolismin skeletalmusclesoverlong periodsof intense muscular activity. These animals generally are slow-movingunder normal circumstancesand engagein intense muscular activity only in the gravest emergencies,becausesuchburstsof activity requirelongrecovery periodsto repaythe oxygendebt. Alligatorsand crocodiles,for example,are normally sluggishanimals.Yet when provoked they are capable of lightning-fast charges and dangerouslashings of their powerful tails. Suchintensebursts of activity are short and must be followed by long periods of recovery. The fast emergencymovementsrequire lactic acid fermentation to generateATP in skeletal muscles.The

stores of muscle glycogen are rapidly expended in intense muscular activity, and lactate reachesvery high concentrationsin myocytes and extracellular fluid. Whereasa trained athlete can recover from a i00 m sprint in 30 min or less, an alligator may require many hours of rest and extra oxygen consumption to clear the excesslactatefrom its blood and regeneratemuscle glycogenafter a burst of activity. Other large animals,such as the elephant and rhinoceros,have similar metaboliccharacteristics,as do diving mammalssuchaswhalesand seals.Dinosaursand other huge,now-extinct animalsprobablyhad to depend on lactic acid fermentationto supply energyfor muscuIar activity, followed by very long recoveryperiods during which they were mlnerable to attack by smaller predators better able to use oxygen and thus better adaptedto continuous,sustainedmuscularactivity. Deep-seaexplorationshaverevealedmany speciesof marineJifeat great oceandepths,where the oxygenconcentrationis near zero.For example,the primitive coelacanth, a Iargeflsh recoveredfrom depths of 4,000m or more off the coast of South Africa, has an essentially anaerobicmetabolismin virtually all its tissues.It converts carbohydratesto lactate and other products, most of which must be excreted. Somemarine vertebratesferment glucoseto ethanoland CO2in orderto generateATP.

14.3Fates ofPyruvate under Anaerobic Conditions: Fermentation lEat] art of brewingbeer involvesseveralenz;rmaticprocesses in addition to the reactions of ethanol fermentation (Box 14-3) In baking,CO2releasedby pyruvate decarboxylasewhen yeast is mixed with a fermentablesugar causesdoughto rise. The enz}.'rne is absentin vertebrate tissuesand in other organismsthat carry out lactic acid fermentation. Alcoholdehydrogenase is presentin many organisms that metabolizeethanol,including humans.In the liver it catalyzesthe oxidation of ethanol, either ingestedor

producedby intestinal microorganisms,with the concomitant reduction of NAD+ to NADH. In this case,the reactionproceedsin the direction oppositeto that involvedin the production of ethanolby fermentation.

Beer brewing was a science learned early in human history, and later refined for larger-scale production. Brewers prepare beer by ethanol fermentation of the carbohydrates in cereal grains (seeds) such as barley, carried out by yeast glycolJ,'tic enz5.nnes.The carbohydrates, Iargely poiysaccharides,must first be degraded to disaccharides and monosaccharides. In a process called malting, the barley seeds are allowed to germinate until they form the hydrolytic enzy'rnesrequired to break down their polysaccharides,at which point germination is stopped by controlled heating. The product is malt, which contains enzlrnes that catalyze the hydrolysis of the B linkages of cellulose and other cell wall polysaccharides of the barley husks, and enz;.'rnessuch as a-amylase and maltase. The brewer next prepares the wort, the nutrient medium required for fermentation by yeast cells. The malt is mixed with water and then mashed or crushed. This allows the enzSrmesformed in the malting process to act on the cereal polysaccharides to form maltose, glucose, and other simple sugars, which are soluble in the aqueous medium. The remaining cell matter is then separated,and the liquid wort is boiled with hops to give flavor. The wort is cooled and then aerated. Now the yeast cells are added. In the aerobic wort the yeast grows and reproduces very rapidly, using energy obtained from available sugars. No ethanol forms during this stage, because the yeast, amply supplied with oxygen, oxidizes the pyruvate formed by glycoiysis to CO2 and H2O via the citric acid cycle. When all the dissolved oxygen in the vat of wort has been consumed, the yeast cells switch to anaerobic metabolism, and from this point they ferment the sugars into ethanol and CO2. The fermentation process is controlled in part by the concentration of the ethanol formed, by the pH, and by the amount of remaining sugar. After fermentation has been stopped, the cells are removed and the "raw" beer is ready for final processing. In the flnal steps of brewing, the amount of foam (or head) on the beer, which results from dissolved proteins, is adjusted. Normally this is controlled by proteoly'tic enzymes that arise in the malting process. If these

enzymesact on the proteinstoo long,the beer will have very little head and will be flat; if lhey do not act long enough,the beer will not be clearwhen it is cold.Sometimes proteoll'tic enz}rmesfrom other sourcesare added to controlthe head. Much of the technologydevelopedfor large-scale production of alcoholic beveragesis now fuding application to a wholly different problem: the production of ethanol as a renewablefuel. With the continuing depletion of the known storesof fossilfuels and the rising cost of fuel for internal combustionengines,there is increasedinterestin the useof ethanolasa fuel substitute or extender.The principal advantageof ethanolas a fuel is that it can be produced from relatively i,neapens'iue and renewabla resourcesrich in sucrose,starch, or cellulose-starch from corn or wheat, sucrose from beets or cane,and cellulosefrom straw,forest industry waste, or municipal solid waste. $pically, the raw material (feedstock) is first converted chemicallyto monosaccharides, then fed to a hardy strain of yeastin fermenter(Fig. 1). The fermentation an industrial-scale can yield not only ethanolfor fuel but also side products suchas proteinsthat canbe usedas animalfeed.

Thiamine Pyrophosphate Carries "Active A(etaldehyde" 6roups The pyruvatedecarboxylasereactionprovidesour flrst encounter with thiamine pyrophosphate

fermentations to oroducebiofueland other FIGURE 1 lndustrial-scale of liters productsaretypicallycarriedout in tanksthathold thousands of medium.

["'] (a)

G l y c o l yG s i lsu, c o n e o g e naensdtihse, P e n t o P s eh o s p h aPtaet h w a y

thiazolium rrng

---H-

oo il

tl

P-O-P-O

oo Thiamine pyrophosphate (TPP)

Hydroxyethyl thiamine pyrophosphate

ME(HANISMFIGURE 14-14 Thiamine pyrophosphate (TPP)and its role in pyruvatedecarboxylation. (a) TPPis Thereactive thecoenzymeformof vitaminBr (thiamine). carbonatomin thethiazolrum ringof TPPisshownin red. In the reactioncatalyzedby pyruvatedecarboxylase, two of the threecarbonsof pyruvatearecarriedtransientlyon TPP in the form of a hydroxyethyl,or "active acetaldehyde,"group(b), which is subsequently released as acetaldehyde.(c) The thiazoliumring of TPP stabilizes carbanionintermediates by providingan electrophilic (electron-deficient) structureinto which the carbanion electronscan be delocalizedby resonance. Structures with this property,oftencalled"electronsinks,"play a role in many biochemicalreac ns-here, facilitating carbon-carbonbondcleavage. ThiaminePyrophosohate Mechanism

resonmce stabilization

(TPP) (FiS. 14-f4), a coenzl'rne derived from vitamin 81. Lack of vitamin 81 in the human dret leads to the condition known as beriberi, characterized by an accumulation of body fluids (swelling), pain, paralysis, and ultimately death. r Thiamine pyrophosphate plays an important role in the cleavageofbonds adjacent to a carbonyl group, such as the decarboxylation of a-keto acids, and in chemical rearrangements in which an activated acetaldehyde group is transferred from one carbon atom to another (Table 14-1). The functional part of TPP, the thiazolium ring, has a relatively acidic proton at C-2. Loss of this proton produces a carbanion that is the active speciesin

TPP-dependentreactions(Fig. 1,4-14).The carbanion readily addsto carbonylgroups,and the thiazoliumring is thereby positionedto act as an "electronsink" that greatly facilitates reactionssuch as the decarboxylation catalyzedby pyruvate decarboxylase.

(ommon Fermentations AreUsed toProduce Some Chemicals Foods andlndustrial Our progenitorslearned millennia ago to use fermentation in the productionandpreservationof foods.Certain microorganismspresent in raw food products ferment the carbohydratesand yield metabolic products that give the foods their characteristicforms, textures, and tastes. Yogurt, already known in Biblical times, is produced when the bacteriumLactobaczllusbulgari,cus ferments the carbohydratein milk, producing lactic acid;the resulting drop in pH causesthe milk proteins to precipitate,producingthe thick texture and sour taste

14.4Gluconeogenesis lttl

Enzyme

Pathway(s)

Plruvate decarboxylase

Ethanol fermentation

Bondcleaved

Bondformed

,r/o

Rr_C..

H

,o

// R'-C.,

PJ,'ruvate dehydrogenase a-Ketoglutaratedehydrogenase

Synthesisof acetyl-CoA Citric acid cycle

Ttansketolase

Carbon-assimilation reactions Pentosephosphatepathway

S-CoA

ooH ill

R3-C-C-R4

R3-C-C-R5

I

H

of unsweetenedyogurt. Another bacterium,Propionibacteri,um freudenrei,chi,i,, ferments milk to produce propionicacid and CO2;the propionicacid precipitates milk proteins,and bubblesof CO2causethe holescharacteristicof Swrsscheese.Manyother foodproductsare pickles,sauerkraut,sausage, the resultof fermentations: soy sauce,and a variety of nationalfavorites,such as kimchi (Korea), tempoyak (lndonesia),keflr (Russia), dahi (India), and pozol(Mexico).The drop in pH associated with fermentationalsohelpsto preservefoods,because most of the microorganismsthat cause food spoilagecannot grow at low pH. In agriculture,plant byproductssuchas corn stalksare preseruedfor use as animal feed by packing them into a large container (a silo) with limited accessto air; microbialfermentation producesacidsthat lower the pH. The silagethat results from this fermentationprocesscan be kept as animal feed for long periodswithout spoilage. In 1910 Chaim Weizmann(later to become the first president of Israel) discoveredthat the bact erium CLost r ?,dLum acet obut y ri cutn ferments starch to butanol and acetone.This discoveryopenedthe field of industrial fermentations,in which somereadily available material rich in carbohydrate (corn starchor molasses, for example)is suppliedto a pure culture of a specificmicroorganism,which fermentsit into a product of greater commercial value. The ethanolusedto make"gasohol"is producedby microbial fermentation,as are formic, acetic,propionic,butyric, and succinic acids, and glycerol, methanol, isopropanol,butanol,and butanediol.Thesefermentations are generallycarried out in huge closedvats in which temperatureand accessto air are controlledto favor the multiplication of the desiredmicroorganism and to exclude contaminatingorganisms.The beauty of industrial fermentationsis that complicated,multistep chemicaltransformationsare carried out in high yields and with few side products by chemicalfactories that reproduce themselves-microbial cells. For some industrial fermentations,technology has been

ooH ilt I

H

developedto immobilizethe cells in an inert support, to passthe starting material continuouslythrough the bed of immobilizedcells, and to collect the desired product in the effluent-an engineer'sdream!

nder 1Y 4 . 3 F a t eosf P y r u v aut e SUMMAR C o n d i t ions: Anaerobic Fermentation r

The NADH formed in glycolysismust be recycledto regenerateNAD+,which is requiredas an electron acceptorin the first step of the payoffphase.Under aerobicconditions,electronspassfrom NADHto 02 in mitochondrialrespiration.

r

Underanaerobicor hypoxicconditions,many organismsregenerateNAD+ by transferring electronsfrom NADH to pyruvate,forming lactate. Otherorganisms,suchasyeast,regenerateNADby reducingpyruvateto ethanoland CO2.In these anaerobicprocesses(fermentations),there is no net oxtdationor reduction of the carbonsof glucose.

r

A variety of microorganismscan ferment sugarin freshfoods,resultingin changesin pH, taste,and texture, and preservingfood from spoilage.Fermentationsare usedin industry to producea wide variety of commerciallyvaluableorganiccompoundsfrom inexpensivestarting materials.

14.4Gluconeogenesis The centralrole of glucosein metabolismaroseearlyin evolution, and this sugar remains the nearly universal fuel and building block in modern organisms,from microbesto humans.In mammals,some tissuesdepend almostcompletelyon glucosefor their metabolicenergy. For the human brain and nervoussystem,as well as the erythrocytes,testes,renal medulla,and embryonictissues,glucosefrom the blood is the sole or major fuel source.The brain alonerequiresabout 120g ofglucose

f

l

Gluconeogenesis, andthePentose Phosphate Pathway [552] Glycolysis, each day-more than half of all the glucose stored as glycogenin muscleand liver. However,the supply of glucosefrom thesestoresis not alwayssufficient;between mealsand duringlongerfasts,or aftervigorousexercise, glycogenis depleted.For thesetimes,organismsneeda methodfor synthesizingglucosefrom noncarbohydrate precursors This is accomplishedby a pathway called gluconeogenesis ("new formation of sugar"), which convertspyruvate and related three- and four-carbon compounds to glucose. Gluconeogenesis occurs in all animals, plants, fungi, and microorganisms.The reactions are essentially the samein all tissuesand all species.The important precursorsof glucosein animalsare three-carbon compoundssuch as lactate,pyruvate,and glycerol,as well as certainaminoacids(Fig. 14-15). In mammals, Blood

Other monosaccharides

Sucrose

Glucose6-phosphate

Animals

Phosphoenol pyruvate

Pyruvate A I I Lactate

Glucogenic Glycerol amino acids

t I

Triacylglycerols

3-Phospho-

ate

COz frxation

FIGURE 14-15 Carbohydratesynthesisfrom simple precursors.The pathwayfrom phosphoenolpyruvate to glucose6-phosphate is common to the biosynthetic conversionof manydifferentprecursors of carbohydratesin animalsand plants The path from pyruvateto phosphoenolpyruvate leadsthroughoxaloacetate, an intermediate of the citric acidcycle,whichwe discuss in Chapter16.Anycompoundthatcanbe convertedto eitherpyruvateor oxaloacetate can thereforeserveasstartrng materialfor gluconeogenesis. Thisincludesalanineand aspartate, which are convertibleto pyruvateand oxaloacetate, respectively, and other amino acidsthat can alsoyield three-or four-carbonfragments, the so-calledglucogenicaminoacids(Tat:le14-4; seealsoFig..l8-l 5). Plantsand photosynthetic bacteriaare uniquelyableto convertCO2to carbohydrates, usingthe glyoxylatecycle(p. 639)

gluconeogenesis takesplace mainly in the liver, and to a lesser extent in renal cortex and in the epithelial cellsthat line the insideof the smallintestine.The glucoseproducedpassesinto the blood to supply other tissues.After vigorous exercise,lactate produced by anaerobicglycolysisin skeletalmuscle returns to the liver and is convertedto glucose,which movesback to muscle and is convertedto glycogen-a circuit called the Cori cycle (Box 14-2; seealsoFig. 23-20).In plant seedlings,stored fats and proteins are converted,via paths that include gluconeogenesis, to the disaccharide sucrosefor transport throughout the developing plant. Glucoseand its derivativesare precursorsfor the synthesisof plant cell walls,nucleotidesand coenzymes,and a variety of other essentialmetabolites.In many microorganisms,gluconeogenesis starts from simple organic compoundsof two or three carbons, such as acetate, lactate, and propionate, in their growth medium. Although the reactionsof gluconeogenesis are the samein all organisms,the metaboliccontext and the regulation of the pathway differ from one speciesto another and from tissueto tissue.In this sectionwe focus on gluconeogenesis as it occursin the mammalianliver. In Chapter 20 we show how photosynthetic organisms use this pathway to convert the primary products of photosynthesisinto glucose,to be storedas sucroseor starch. Gluconeogenesis and glycolysisare not identical pathways running in opposite directions, although they do shareseveralsteps(Fig. 14-f6); 7 of the 10 enzymaticreactionsof gluconeogenesis are the reverseof glycolyticreactions.However,three reactions of glycolysis are essentiallyirreversible in vivo and cannot be used in gluconeogenesis: the conversionof glucoseto glucose6-phosphateby hexokinase,the phosphorylationof fructose 6-phosphateto fructose 1,6-bisphosphate by phosphofructokinase-1, and the conversion of phosphoenolpyruvateto pyruvate by pyruvatekinase(Fig. 14-16).In cells,thesethree reactions are characterizedby a large negativefree-energy change,whereasother glycolyticreactionshavea AG near 0 (Table I4-2). In gluconeogenesis, the three irreversiblestepsare bypassedby a separateset of enzymes,catalyzingreactionsthat are sufficiently exergonic to be effectivelyirreversiblein the direction of glucosesynthesis.Thus,both glycolysisand gluconeogenesisare irreversibleprocessesin cells. In animals, both pathwaysoccur largelyin the cytosol,necessitating their reciprocaland coordinatedregulation.Separate regulation of the two pathwaysis brought about through controls exerted on the enzymatic steps uniqueto each. We begin by consideringthe three bypass reac(Keep in mind that "bypass" tions of gluconeogenesis. refers throughout to the bypassof irreversibleglycolytic reactions.)

14.4 Gluconeogenesis Ftrl

Glycolyticreaetionstep

AG" (kJ/mol) AC (kJ/mol)

(1) Glucose+ AIP --+ glucose6-phosphate+ ADP @ Glucose6-phosphate-- fructose G-phosphate * ADP @ Fructose6-phosphate* ATP-----+fructose1,6-bisphosphate i* @ Fructose 1,6-bisphosphate glyceraldehyde3-phosphate

dihydroxyacetonephosphate*

phosphateiglyceraldehyde3-phosphate @ OinyOroxyacetone l,3-bisphosphoglycerate+ NADH + H+ @ Glyceraldehyde3-phosphate* Pi + NAD+ r+ ADP r3-phosphoglycerate* ATP @ t,S-nisptrosphoglycerate 3-Phosptroglycerate 2-phosphoglycerate r@ phosphoenolpy'mvate* H2O @ 2-enosphoglycerate;------+pyruvate * ATP Phosphoenolpytuvate + AIIP @

Gluconeogenesis Glucose

P1 g l u c , ' s e6 - p h o s p L r e t a " e

Hzo

Glucose6-phosphate

(2) 1,3-Bisphosphoglycerate

tl (2)ADP-.l,,7>(2)ADP (2)ArP.4l*,r,ot" 'll

(2) 3-Phosphoglycerate

I

'll

(2) 2-Phosphoglycerate

1^.1 LA.L

_ L'.2

23.8

-6to0

t.D

6.3

0to4 -2to2

- 18.8

0to2

4.4

0 to 0.8 0 to 3.3

_41

A

-ro.i

free-energy change, as definedin Chaptert3 (pp.491-4921. Note:AG'' is thestandard of glycolytic fromthe actualconcentrations AG is the free-energy changecalculated present in erythrocytes, at pH 7.Theglyunderphysiological conditions intermediates equations in gluconeogenesis areshownin red.Biochemical bypassed colyticreactions (p 501). for H or charge balanced arenotnecessarily

(2)GDP

{l

pguvate

-

a

0to25

is The flrst of the bypassreactionsin gluconeogenesis the conversion of pyruvate to phosphoenolpyruvate (PEP). This reactioncannotoccurby simplereversalof the pyruvate kinase reaction of glycolysis (p. 538), which has a large, negative free-energy change and is therefore irreversible under the conditionsprevailingin intact cells (Tablel4-2, stepQp). Instead,the phosphorylation of pyruvate is achievedby a roundabout sequence of reactions that in eukaryotes requires enzymesin both the cytosol and mitochondria.As we shall see,the pathwayshown in Figure 14-16 and describedin detail here is one of two routes from pyruvate to PEP; it is the predominant path when pyruvate or alanine is the glucogenicprecursor. A secondpathway, describedlater,predominateswhen lactateis the glucogemcprecursor. Pyruvate is first transported from the cytosol into mitochondria or is generatedfrom alaninewithin mitochondria by transamination,in which the a-amino group is transferred from alanine (leavingpyruvate) to an a-keto carboxylic acid (transaminationreactions are discussedin detail in Chapter18). Then pyruvate carboxylase, a mitochondrialenz;rrnethat requiresthe

(2) Glyceraldehyde 3-phosphate

(2)ADP

t.7

-oD

(onversion Requires to Phosphoenolpyruvate ofPyruvate Reactions TwoExergonic

lf

rf

-16.7

(2) Phosohoenolovruvate "

1 cur

r kin;,:"

\pnp

Ft

kinase

(2)ATP (2) Pyruvate

GTP

and gluconeogene14-16 Opposingpathwaysof glycolysis FIGURE of glycolysisareon the leftside,in red; sisin rat liver.Thereactions is on the right,in blue. the opposingpathwayof gluconeogenesis shown here are The major sitesof regulationof gluconeogenesis discussedlater in this chapter,and in detail in Chapter15. Figure 'l producedin routefor oxaloacetate an alternative 4-1 9 illustrates mitochondria

uuo l

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coenzymebiotin, convertsthe pyruvateto oxaloacetate (Fig. 14-17): Pyruvate+ HCOt + ATP ------> oxaloacetate+ A-DP+ Pi Q4-4) The carboxylation reaction involves biotin as a carrier of activated bicarbonate, as shown in Figure l4-18; the reaction mechanism is shown in Figure 16-16. (Note that HCO| is formed by ionization of carbonic acid formed from CO2 + HzO.) HCOt is phosphorylated by ATP to form a mixed anhydride (a carboxyphosphate); then biotin displaces the phosphate in the formation of carboxybiotin. Pyruvate carboxylase is the first regulatory enzlirne in the gluconeogenic pathway, requiring acetyl-CoA as a positive effector. (Acetyl-CoA is produced by fatty acid

Bicarbonate

oxidation (Chapter 17), and its accumulationsignalsthe availabilityof fatty acids as fuel.) As we shall see in Chapter16 (see Fig. 16-15), the py'uvate carboxylase reaction can replenishintermediatesin another central metabolicpathway,the citric acid cycle.

jJ

Pyruvate carboxylase 1

Site 1

Pyruvate

o- 9

lt

+ CH"-C-C HO-C \"\

//o

Long biotinyl-Lys tether moves CO2 from site 1 to site 2.

oo

. P) r u\ llto cllt lt)\\ ltsc

,' i I \

a>

+'

I

o. I

NIP

"+

riotin

'.)

Lys

ADP + Pi

Oxaloacetate (a) Site 2

Oxaloacetate

o.. ? c ,.c ooo-

o

o-

GTP

o-Poi t" cHr:c-cooPhosphoenolpyruvate ft) FIGURE 14-17 Synthesisof phosphoenolpyruvate from pyruvate. (a) In mitochondria,pyruvateis convertedto oxaloacetate in a biotinrequiringreactioncatalyzedby pyruvatecarboxylase(b) In the cytosol, oxaloacetateis convertedto phosphoenolpyruvate by pEp carboxykinase. The CO2 incorporatedin the pyruvatecarboxylasereaction is lost hereas CO2 The decarboxylationleadsto a rearrangement of electronsthat facilitatesattackof the carbonyloxygenof the pyruvatemoietyon the y phosphateof CTP

.//o

cH2-c\

Oxaloacetate

FIGURE 14-18 Role of biotin in the pyruvatecarboxylasereaction. The cofactorbiotin is covalentlyattachedto the enzymethroughan amide linkageto the e-aminogroup of a Lys residue,forminga biotinyl-enzyme. The reactionoccursin two phases, which occurat two differentsitesin theenzyme.At catalyticsite1, bicarbonate ion is convertedto CO2at the expenseof ATP.ThenCO2 reactswith biotin, formingcarboxybiotinyl-enzyme. The long arm composedof biotin and the Lysside chain to which it is attachedthen carry the CO2 of carboxybiotinyl-enzyme to catalyticsite 2 on the enzymesurface, whereCO2 is releasedand reactswith the pyruvate,formingoxaloacetateand reSenerating the biotinyl-enzymeThegeneralroleof flexible armsin carryingreactionintermediates betweenenzymeactivesitesis described in Figure16-17,andthe mechanistic detailsof the pyruvate carboxylase reactionareshownin Figure16-16. Similarmechanisms occur in otherbiotin-dependent carboxylationreactions,suchasthose (seeFig 17-1 i) and acetylcatalyzedby propionyl-CoAcarboxylase (seeFig 2.1-.1 CoA carboxylase ).

14.4Gluconeogenesis tGtt] Becausethe mitochondrialmembranehasno transporter for oxaloacetate, beforeexport to the cy'tosolthe oxaloacetateformed from pyruvatemust be reducedto malate by mitochondrialmalate dehydrogenase, at the expenseof NADH: Oxaloacetate + NADH + H* i-

l,-malate + NAD* (14-5)

The standard free-energychangefor this reaction is quite high, but under physiologicalconditions (including a very low concentrationof oxaloacetate)AG : 0 and the reaction is readily reversible.Mitochondrial malatedehydrogenase functionsin both gluconeogenesis and the citric acid cycle,but the overallflow of metaboIitesin the two processes is in oppositedirections. Malateleavesthe mitochondrionthrough a specific transporterin the inner mitochondrialmembrane(see Fig 19-30), and in the cltosol it is reoxidizedto oxaloacetate,with the productionof cy'tosolicNADH: Malate + NAD*

-----+ oxaloacetate + NADH + H-

(14-6)

There is a logic to the route of these reactions through the mitochondrion. The [NADH]/[NAD+]ratio in the cytosolis 8 x 10-4, about 105times lower than in mitochondria.BecausecytosolicNADH is con(in the conversionof 1,3sumedin gluconeogenesis bisphosphoglycerateto glyceraldehyde3-phosphate; Fig. 14-16),glucosebiosynthesiscannotproceeduriless NADH is available.The transport of malatefrom the mitochondrionto the c1'tosoland its reconversionthere to oxaloacetateeffectively moves reducing equivalentsto the cy'tosol,wherethey are scarce.This path from pyruvateto PEPthereforeprovidesan importantbalancebetween NADH produced and consumedin the cytosol duringgluconeogenesis. A secondpyruvate + PEP bypasspredominates when lactateis the glucogenicprecursor(Fig. 14-19). This pathwaymakesuse of lactateproducedby glycolysis in ery'throcy'tesor anaerobicmuscle, for example, and it is particularly important in Iargevertebratesafter vigorousexercise(Box 14-2). The conversionof lactate

The oxaloacetate is then convertedto PEPby phosphoenolpyruvate carboxykinase (Fig 14-17). This - -

t+

,

Mg"--dependent reaction requires GTP as the phosphoryl group donor: Oxaloacetate+ GTP +

PEP + CO2+ GDP

(14-7)

The reaction is reversible under intracellular conditions; the formation of one high-energy phosphate compound (PEP) is balanced by the hydrolysis of another (GTP). The overall equation for this set ofbypass reactions, the sum ofEquations l4-4 through 14-7, is Pyruvate + ATP + GTP + HCO3 ------+ PEP + ADP + GDP + Pi + CO2 (14_8) AG'' : 0.9kJ/mol TWo high-energy phosphate equivalents (one from ATP and one from GTP), each yielding about 50 kJ/mol under cellular conditions, must be expended to phosphorylate one molecule of pyruvate to PEP. In contrast, when PEP is converted to pyruvate during glycolysis, only one ATP is generated from ADP. Although the standard freeenergy change (AG'') of the two-step path from pyruvate to PEP is 0.9 kJ/mol, the actual free-energy change (AG), calculated from measured cellular concentrations of intermediates, is very strongly negative (-25 kJ/mol); this results from the ready consumption of PEP in other reactions such that its concentration remains relatively Iow. The reaction is thus effectively irreversible in the cell. Note that the CO2 added to pyruvate in the pyruvate carboxylase step is the same molecule that is lost in the PEP carboxykinase reaction (Fig. 14-17b). This carboxylation-decarboxylation sequence represents a way of "activating" pyruvate, in that the decarboxylation of oxaloacetate facilitates PEP formation. In Chapter 2l we shall see how a similar carboxylation-decarboxylation sequence is used to activate acetyl-CoA for fatty acid biosynthesis (see Fig. 2l-l).

cyLosoltc

PEP r rrrboxvkrnase

Oxaloacetate

t

+H+

"l;,;;:1,; d"hv,lr,,g,.nrre l\

I Malate '

'

. . 1' , .

,:.

r,i' ir

.,1

':" . 1' .l ' , ' l t : . l " , l '

Lactate 14-19 Alternativepathsfrom pyruvateto phosphoenolpyruFIGURE vate. The relativeimportanceof the two pathwaysdependson the for of lactateor pyruvateand the cytosolicrequirements availability when Thepathon the rightpredominates NADH for gluconeogenesis lactateis the precursor,becausecytosolicNADH is generatedin the reactionand doesnot haveto be shuttledout of lactatedehydrogenase (see the mitochondrion text)

L 5 5 6]

G l y c o l yG s i lsu, c o n e o g e naensdtihse, P e n t o sPeh 0 s p h aPtaet h w a y

to pyruvate in the cytosol of hepatocytes yields NADH, and the export of reducing equivalents (as malate) from mitochondria is therefore unnecessary.After the pyruvate produced by the lactate dehydrogenasereaction is transported into the mitochondrion, it is converted to oxaloacetate by pyruvate carboxylase, as described above. This oxaloacetate,however, is converted directly to PEP by a mitochondrial isoz;rme of PEP carboxykinase, and the PEP is transported out of the mitochondrion to continue on the gluconeogenic path. The mitochondrial and cy[osolic iso4nnes of PEP carboxykinase are encoded by separate genes in the nuclear chromosomes, providing another example of two distinct enzymes caLalyzingthe same reaction but having different cellular locations or metabolic roles (recall the iso4,'rnesof hexokinase).

(onversion ofFructose 1,6-Bisphosphate toFructose 6-Phosphate lstheSecond Bypass The second glycolytic reaction that cannot participate in gluconeogenesis is the phosphorylation of fructose 6phosphate by PFK-1 (Table 14-2, step @). Becausethis reaction is highly exergonic and therefore irreversible in intact cells, the generation of fructose 6-phosphate from fructose 1,6-bisphosphate(Fig. 14-16) is catalyzed by a different enzyme, Mgz*-clependent fructose 1,6bisphosphatase (FBPase-l), which promotes the essentially irreversible hydrolysi,s of the C-l phosphate (zof phosphoryl group transfer to ADP): Fructose1,6-bisphosphate * H2O -> fructose6-phosphate+ P1 AG'' : -16.3 kJ/mol FBPase-l is so named to distinguish it from another, similar enzyrne (FBPase-2) with a regulatory role, which we discuss in Chapter 15.

Conversion ofGlucose 6-Phosphate toGlucose lstheThird Bypass The third bypassis the finalreactionofgluconeogenesis, the dephosphorylation of glucose6-phosphateto yield glucose(Fig. 14-16). Reversalof the hexokinasereaction (p. 532) would require phosphorylgroup transfer from glucose6-phosphateto ADP,forming ATP,an energeticallyunfavorablereaction (Table 14-2, step@;. ffre reaction catalyzedbyglucose 6-phosphatase doesnot require synthesisof ATP; it is a simple hydrolysis of a phosphateester: Glucose6-phosphate+ HrO -----+glucose* P; AG'" : -13.8 kJ/mol This Mg2+-activated enz).rneis found on the lumenal side of the endoplasmic reticulum of hepatocytes, renal cells, and epithelial cells of the small intestine (see Fig. 15-28), but not in other tissues, which are therefore unable to supply glucose to the blood. If other tissues had glucose 6-phosphatase, this enzyme's activity would hydrolyze the glucose 6-phosphate needed within those tissues for glycolysis. Glucose produced by gluconeogenesisin the liver or kidney or ingested in the diet is delivered to these other tissues, including brain and muscle, through the bloodstream.

Gluconeogenesis lsEnergetically Expensive, butEssential The sum of the biosynthetic reactions leading from pyruvateto free bloodglucose(Table14-3) is 2 Pyruvate+ 4ATP+ 2GTP+ 2NADH+ 2}J++ 4H2O---+ glucose + 4ADP+ 2GDP+ 6pi + 2NAD* (14-g) For eachmoleculeof glucoseformedfrom pyruvate,six high-energyphosphategroupsare required,four from ATP and two from GTP.In addition. two molecules of

Pyruvate + HCOt f ,'tTP-------+ oxaloacetate+ ADP + pi Oxaloacelate' + GTP ,- ptrcsphoenolpynrvat,e + (lO2 + GDP Phosphoenolpyruvate+ H2O3 2-phosphoglycerate 2-Phosphoglycerate 3-phosphoglycerate i3-Phosphoglycerate +ATP- .- l,3-bisphosphoglycerate +ADp

Xz xz xz x2 xz

l,3-Bisphosphoglycerate+ NADH + H+ - .- glyceraldehyde3-phosphate+ NAD+ + pi Glyceraldehyde3-phosphateidihydroxyacetonephosphate Glyceraldehyde 3-phosphate+ dihydroxyacetone phosphater-------+ Fructose1,O-bisphospirale liuctose d-phosphate* p,

xz

fructose1,6-bisphosphate

Fructose6-phosphateiglucose6-phosphate GlucoseO-phosphate * HrO ------+ glucose* P; Sum: 2 FyTuvate+ 4ATP + 2GTP + 2NADH + 2H+ + 4HzO------->glucose + 4ADp + 2GDp + 6pj + 2NAD-

Note:ThebypaSsreactionsareinred;a|IothelIeactionsarereversib|eStepsofglyco|ySiS.ThefigureSattherightindicatethatthereaction three-carbonprecUrsorSarereqUiredtomakeamo|ecU|eofg|ucose.ThereactionsreqUiredtorep|acethecytoso|icNADHconSumedintheg|ycera|dehyde3-phoS nasereaction(theconVersionoflactatetopyruVateinthecytoso|orthetranspoofr thissummary. Biochemical equations arenotnecessarily balanced for H andcharge(p.501).

14.4Gluconeogenesis [tttl NADH are requiredfor the reductionof two molecules of 1,3-bisphosphoglycerate. Clearly,Equation 14-9 is not simplythe reverseof the equationfor conversionof glucoseto pyruvate by glycolysis,which would require only two moleculesof ATP:

amino groups in liver mitochondria, the carbon skeletons remaining (pyruvate and a-ketoglutarate,respectively) are readilyfunneledinto gluconeogenesis.

Glucose+ 2ADP+ 2Pi + NAD* ---> 2 pyruvate+ 2ATP+ 2NADH+ 2H+ + 2Il2O

No net conversionof fatty acids to glucoseoccurs in mammals.As we shallseein Chapter17,the catabolism of most fatty acids yields only acetyl-CoA.Mammals cannot use acetyl-CoAas a precursor of glucose,because the pyruvate dehydrogenasereaction is irreversible and cells have no other pathway to convert acetyl-CoAto py'ruvate.Plants,yeast,and many bacteria do havea pathway (the glyoxylatecycle;seeFig. 16-20) for convertingacetyl-CoAto oxaloacetate,so these organismscan use fatty acids as the starting material for gluconeogenesis. This is important during the germinafor tion of seedlings, example;beforeleavesdevelopand photosynthesiscan provide energyand carbohydrates, the seedlingrelieson storedseedoilsfor energyproduction and cell wall biosynthesis. Although mammals cannot convert fatty acids to carbohydrate,they can use the smallamount of glycerol producedfrom the breakdovmoffats (triacylglAcerols) Phosphorylationof glycerol by for gluconeogenesis. glycerol kinase,followedby oxidation of the central carphosphate,an intermedibon, yields dihydroxyacetone in liver. ate in gluconeogenesis As we rmll seein Chapter21, glycerolphosphateis an essentialintermediate in triacylglycerol synthesis in adipocytes,but these cells lack glycerol kinase and so cannot simply phosphoryIateglycerol.Instead, carry out a truncatedversionof gluconeogeadipocy'tes nesis,knornmas glyceroneogenesis:the conversionof pyruvate to dihydroxyacetonephosphatevia the early followedby reduction of reactionsof gluconeogenesis, phosphateto glycerolphosphate the dihydroxyacetone (seeFig. 2l-21).

The synthesisof glucosefrom pyruvateis a relativelyexpensiveprocess Much of this high energycostis necessary to ensure the irreversibility of gluconeogenesis. Under intracellularconditions,the overall free-energy changeof glycolysisis at least -63 kJ/mol.Under the same conditionsthe overall AG of gluconeogenesis is -16 kJ/mol.Thus both glycolysisand gluconeogenesis are essentiallyirreversibleprocessesin cells.

(itricAcid (ytleIntermediates and5ome Amino Acids AreGlucogenic The biosyntheticpathwayto glucosedescribedaboveallowsthe net synthesisof glucosenot only from pyruvate but alsofrom the four-.five-.and six-carbonintermediatesof the citric acid cycle (Chapter16). Citrate,isocitrate, o-ketoglutarate,succinyl-CoA,succinate,fumarate, and malate-all are citric acid cycle intermediatesthat can undergooxidationto oxaloacetate(seeFig. 16-7). Someor all of the carbonatomsof most aminoacidsderived from proteinsare ultimatelycatabolizedto pyruvate or to intermediatesof the citric acid cycle. Such amino acids can therefore undergonet conversionto giucoseand are said to be glucogenic (Table 14-4). Alanine and glutamine, the principal moleculesthat transportaminogroupsfrom extrahepatictissuesto the liver (see Fig 18-9), are particularlyimportant glucogenic amino acids in mammals.After removal of their

(onvert (annot toGlucose Fatty Acids Mammals

Regulated AreReciprocally andGluconeogenesis Glycolysis Pyruvate Alanine Cysteine Glycine Serine Threonine T?yptophan* a-Ketoglutarate Arginile Giutamate Giutamine Histidine Proline

Succinyl-CoA Isoleucine* Methionine Threonine Valine Fumarate Phenylalanine* T5'rosine* Oxaloacetate Asparagine Aspartate

Ifglycolysis (the conversion ofglucose to pyruvate) and gluconeogenesis (the conversion of pyruvate to glucose) were allowed to proceed simultaneously at high rates, the result would be the consumption of ATP and the production of heat. For example, PFK-I and FBPase-L catalyze opposing reactions: ATP + fructose6-phosphate ADP + fructose1,6-bisphosphate * H2O Fructose1,6-bisphosphate fructose6-phosphate+ P1 The sum of these two reactions is

Note:Alltheseaminoacidsareprecursors of bloodglucose or liverglycogen, because theycanbe converted to pyruvate or citricacidcycleintermediates 0fthe 20 common aminoacids,onlyleucine andlysineareunableto furnishcarbonfor netglucose synthesis, *These (seeFig.18-21). aminoacidsarealsoketogenic

ATP + H2O -----+ADP + Pi * heat These two enz;.'rnatic reactions, and several others in the two pathways, are regulated allosterically and by

F"l

G l y c o l yG s i lsu, c o n e o g e naensdtihse, P e n t o sPeh o s p h aPtaet h w a y

covalent modiflcation (phosphorylation). In Chapter 15 we take up the mechanisms of this regulation in detail. For now, sufflce it to say that the pathways are regulated so that when the flux of glucose through glycolysis goes up, the flux of pyruvate toward glucose goes down, and VIce versa.

monophosphate pathway; Fig. 14-20).In this oxidative pathway,NADP+ is the electron acceptor,yielding NADPH. Rapidlydividing cells, such as those of bone marrow)skin, and intestinal mucosa,and those of tumors,usethe pentoseribose5-phosphateto makeRNA, DNA, and such coenz;.'rnes as ATP,NADH, MDH2, and nnpnT\mo

SUMMAR 1Y 4.4 Gluconeogenesis r

Gluconeogenesis is a ubiquitousmultistepprocess in which glucoseis producedfrom lactate,pyruvate, or oxaloacetate, or any compound(includingcitric acid cycleintermediates)that canbe converted to one of theseintermediates.Sevenof the steps in gluconeogenesis are catalyzedby the same enzyrnesusedin glycolysis;theseare the reversible reacttons.

r

Three irreversiblestepsin glycolysisare blpassed by reactionscatalyzedby gluconeogenicenz;,rnes: (1) conversionofpyruvate to PEPvia oxaloacetate, catalyzedby pyruvate carboxylaseand PEP carboxykinase;(2) dephosphorylationof fructose 1,6-bisphosphate by FBPase-1; and (3) dephosphorylation of glucose6-phosphate by glucose6-phosphatase.

r

Formationof one moleculeof glucosefrom pyruvate requires 4 ATP,2 GTP,and 2 NADH; it is expensive.

r

In mammals,gluconeogenesis in the liver,kidney, and smallintestineprovidesglucosefor useby the brain,muscles,and efihrocytes.

r

Pyruvatecarboxylaseis stimulatedby acetyl-CoA, increasingthe rate of gluconeogenesis when the cell hasadequatesuppliesof other substrates (fatty acids) for energyproduction.

r

Animals cannot convert acetyl-CoAderived from fatty acidsinto glucose;plantsand microorganisms can.

r

Glycolysisand gluconeogenesis are reciprocally regulatedto preventwastefuloperationof both pathwaysat the sametime.

14.5Pentose Phosphate Pathway of Glucose Oxidation In most animal tissues, the major catabolic fate of glucose 6-phosphate is glycol5,tic breakdown to pyruvate, much of which is then oxidized via the citric acid cycle, ultimately leading to the formation of ATp. Glucose G-phosphate does have other catabolic fates, however, which lead to specialized products needed by the cell. Of particular importance in some tissues is the oxidation of glucose 6-phosphate to pentose phosphates by the pentose phosphate pathway (also called the phosphogluconate pathway or the hexose

A

In other tissues,the essentialproduct of the pentose phosphatepathway is not the pentosesbut the electrondonorNADPH,neededfor reductivebiosynthesis or to counterthe damagingeffectsof oxygenradicals. Tissuesthat carry out extensivefatty acid synthesis (liver, adipose,lactating manunarygland) or very active synthesisof cholesteroland steroid hormones(liver, adrenal glands,gonads)require the NADPH providedby this pathway.Erythrocytes and the cells of the lens and corneaare directly exposedto oxygenand thus to the damagingfree radicals generatedby oxygen. By maintaining a reducing atmosphere(a high ratio of NADPH to NADP+ and a high ratio of reduced to oxidized glutathione), such cells canprevent or undo oxidativedamageto proteins,Iipids,and other sensitivemolecules.In erythrocytes,the NADPH produced by the pentose phosphatepathwayis so important in preventing oxidative damagethat a genetic defect in glucose6-phosphate dehydrogenase,the flrst enzyrneof the pathway, can have seriousmedicalconsequences (Box I4-4). I Nonoxidative phase

Oxidative phase Glucose 6-phosphate

Ribose 5-phoephate

I

I

Nucleotides, coenzymes, DNA, RNA FIGURE 14-20 Ceneralschemeof the pentosephosphatepathway. NADPHformedin the oxidativephaseis usedto reduceglutathione, CSSC(seeBox 144\ and to supportreductivebiosynthesis. Theother productof the oxidativephaseis ribose5-phosphate, which servesas a precursor for nucleotides, coenzymes, andnucleicacids.In cellsthat are not usingribose5-phosphate for biosynthesis, the nonoxidative phaserecycles six molecules of the pentoseintofivemolecules of the hexoseglucose 6-phosphate,allowing continued productionof (in six cycles)to CO2 NADPHand converting glucose6-phosphate

s ex i d a t i o [rrr] n o yf G l u c oO 1 4 . 5P e n t o P s eh o s p h aPtaet h w a

Favabeans,an ingredient of falafel,havebeen an important food sourcein the Mediterraneanand Middle East since antiquity. The Greek phllosopherand mathematician Pythagorasprohibited his followers from dining on fava beans,perhapsbecausethey make many people sick with a condition calledfavism,which can be fatal. In favism, erylhrocytes begin to lyse 24 to 48 hours after ingestion of the beans, releasingfree hemoglobininto the blood. Jaundice and sometimeskidney failure can result. Similar symptomscan occur with ingestionof the antimalarial drug primaquine or of sulfa antibiotics, or following exposure to certain herbicides.These symptoms have a geneticbasis:glucoseG-phosphate dehydrogenase(G6PD) deflciency,which affects about 400 million people worldwide. Most G6PD-deflcientindividuals are asymptomatic;only the combination of G6PD deflciency and certain environmentalfactors produces the clinical manifestations. Glucose 6-phosphate dehydrogenase catalyzes the first step in the pentose phosphate pathway (see Fig. L4-21), which produces NADPH. This reductant, essentialin many biosynthetic pathways,also protects cells from oxidative damage by hydrogen peroxide (HzOz)and superoxidefree radicals,hrghly reactiveoxidants generatedas metabolic byproducts and through the actions of drugs such as primaquine and natural products such as divicine-the toxic ingredient of fava beans.During normal detoxi-fication,H2O2is converted to H2Oby reduced glutathione and glutathione peroxidase,and the oxidizedglutathione is convertedback to the reduced form by glutathionereductaseand NADPH (Frg.1) H2O2is alsobroken down to H2Oand O2by catalase,which alsorequiresNADPH.In G6PD-deflcientindividuals, the NADPH production is diminished and detoxification of H2O2is inhibited. Cellular damageresults:lipid peroxidationleadingto breakdovmof erythroc;,te membranesand oxidationof proteins and DNA. The geographicdistribution of G6PD deflciency is instructive. Frequenciesashigh as25o/ooccur in tropical Africa, parts of the Middle East, and SoutheastAsia, areaswhere malariais most prevalent.In addition to such epidemiologicalobservations,in vitro studies show that growth of one malaria parasite, Plasmodi,um Jalci'pav\)n'r,is inhibited in G6PD-deflcienterythrocytes.The parasite is very sensitive to oxidative damage and is

killed by a level of oxidative stressthat is tolerable to a G6PD-deflcienthuman host. Becausethe advantageof resistanceto malaria balancesthe disadvantageof lowered resistanceto oxidative damage,natural selection sustainsthe G6PD-deficientgenotypein human populations where malaria is prevalent. Only under overwhelming oxidative stress,causedby drugs,herbicides, or divicine, doesG6PDdeficiencycauseseriousmedical problems. An antimalarialdrug such as primaquineis believed to act by causing oxidative stress to the parasite' It is ironic that antimalarial drugs can causehuman illness through the samebiochemicalmechanismthat provides resistanceto malaria.Divicine also acts as an antimalarial drug, and ingestionof fava beansmay protect against malaria. By refusing to eat falafel, many Pythagoreans with normal G6PD activity may have unwittingly increasedtheir risk of malaria!

Pentose The 0xidative Phase Produces Phosphates andNADPH

an intramolecularesform 6-phosphoglucono-6-Iactone, ter. NADP+ is the electron acceptor,and the overall equilibriumlies far in the direction of NADPHformation. The lactoneis hydrolyzedto the free acid 6-phosphogluconate by a specific lactonase, then 6-phosphogluconate undergoes oxidation and decarboxylation by

The first reaction of the pentosephosphatepathway (Fig. 14-2f ) is the oxidationof glucose6-phosphate by glucose G-phosphate dehydrogenase (G6PD) to

Hydroxyl free radical

Oxidative damage to lipids, proteins, bNA

r\rur NA.DP* \

Glucose o-phosphate

fl NAIr.t,tl NADPH + HT

\./

/

6-PhosPho^e,luco3e. glucono-d-lactone dehydrogmase

I Roleof NADPH and glutathionein protectingcells against FIGURE Reducedglutathione(CSH)protects highlyreactiveoxygenderivatives. the cell by destroyinghydrogenperoxideand hydroxylfree radicals. of CSH from its oxidized form (CSSC)requiresthe Regeneration reaction' dehydrogenase NADPHproducedin the glucose6-phosphate

rEuolG l y c o l y sGi lsu, c o n e o g e naensdtihse, P e n t o Ps eh o s p h aPtaet h w a y

I Glucose 6-phosphate

I cHroPoSl,- NADp* t/

glLl(os(. Irrsl)llltr, (( (l ogi IlLst

I Mg2+ l\

NeoPu+n

l\

J

6-Phosphoglucono-6-lactone

| ,- H'o l/

Jt*'*

o.\\ /'o-

CH2OH

C

I

HCOH I HOCH

6-Phosphogluconate

I

HCOH

lC:O tl

H-C-OH | H_C_OH I

I HCOH I CH,OPO; -

TheNonoxidative Phase Recycles Pentose Phosphates toGlucose 6-Phosphate In tissues that require primarily NADPH, the pentose phosphatesproducedin the oxidativephaseof the pathway are recycled into glucose 6-phosphate.In this nonoxidative phase, ribulose 5-phosphate is first epimerizedto xylulose5-phosphate:

I cHroPo3-,

Glucose G-phosphate + 2NADP* + H2O -----+ ribose 5-phosphate + CO2 + 2NADPH + 2H+

The net result is the productionof NADPH,a reductant for biosyntheticreactions,and ribose 5-phosphate,a precursorfor nucleotideslmthesis.

I

I tLt' t

6-phosphogluconate dehydrogenase to form the ketopentoseribulose5-phosphate;the reactiongenerates a secondmoleculeof NADPH. (This ribulose 5-phosphate is important in the regulation of glycolysis and gluconeogenesis, as we shall seein Chapter15.) Phosphopentose isomerase convertsribulose 5-phosphate to its aldoseisomer,ribose5-phosphate. In sometissues, the pentosephosphatepathwayendsat this point, and its overall equationis

cH2oPo32 Ribulose 5-phosphate

CH,OH

t'

C:O .

- hosprlare r ) l ):ton": nplnerrrsc

HO-C-H I H_C_OH I

cH2oPo32Xylulose 5-phosphate

NADP*

[g'*

-9 '>

NADPH +H* COz

CHOOH

t-

C:O

I I HCOH I cH2oPo; HCOH

o-Ribulose 5-phosphate

CHO

I

HCOH I

o-Ribose HCOH --5-phosphate I HCOH I cH2oPoSFIGURE 14-21 Oxidativereactionsofthe pentosephosphatepathway. Theend productsareribose5-phosphate, CO2,and NADpH.

Then, in a series of rearrangementsof the carbon skeletons(Fig. 14-22), six five-carbonsugarphosphates are converted to five six-carbon sugar phosphates, completing the cycle and allowing continued oxidation of glucose 6-phosphatewith production of NADPH. Continued recycling leads ultimately to the conversionof glucose6-phosphateto six CO2.TWoenzymes unique to the pentosephosphatepathway act in these interconversionsof sugars:transketolaseand transaldolase.Thansketolase catalyzesthe transfer of a two-carbonfragmentfrom a ketosedonor to an aldose acceptor (Fig. 74-23a). In its first appearance in the pentose phosphate pathway, transketolase transfers C-l and C-2 of xylulose 5-phosphateto ribose 5-phosphate,forming the seven-carbonproduct sedoheptulose7-phosphate (Fig. I4-23b). The remaining three-carbonfragment from xylulose is glyceraldehyde3-phosphate. Next, transaldolase cata\yzesa reaction similar to the aldolasereactionof glycolysis:a three-carbonfragment is removedfrom sedoheptulose 7-phosphateand condensedwith glyceraldehyde3-phosphate,forming

Oxidation[tut] of Glucose Pathway Phosphate 14.5Pentose

oxidative reactions of pentose phosphate pathway

\ Ribose 5-phosphate

Fructose ---.> Glucose , - ^ , 6-phosphate 6-phosphate

Sedoheptulose 7-phosphate

t

I PhusPhohexose lsomerase I

Xylulose 5-phosphate

Glyceraldehyde 3-phosphate

Erythrose 4-phosphate

Fructose 6-phosphate

t iTffilf;.,"5C

3C

aldolase

f

4C

6C

t l;H:,1*:"'n*" S-phosphate

Glyceraldehyde 3-phosphate (b)

(a)

to five hexoses(6C).Notethatthis involvestwo setsof the interconversionsshown in (a). Everyreactionshown here is reversible;unidirectional arrowsare usedonly to makeclearthe directionof the reactions during continuousoxidationof glucose6-phosphate'In the lightthe directionof thesereacindependentreactionsof photosynthesis, (seeFig.20-10). tionsis reversed

FIGURE 14-22 Nonoxidative reactionsof the pentose phosphate pathway.(a) Thesereactionsconvertpentosephosphatesto hexose (seeFig.14-21)to conphosphates, allowingthe oxidativereactions tinue.Transketolase and transaldolase arespecificto this pathway;the pathways. otherenzymes alsoservein theglycolyticor gluconeogenic (b) A schematicdiagramshowingthe pathwayfrom six pentoses(5C)

Aldose acceptor

Ketose donor

(a)

CH,OH

t-

o. .H \\ ,/ C I

CH,OH

t-

HO-C-H

I

H-C-OH

C:O

H-C-OH

-

H-c-oH I

H_C-OH

O -"H

I

I

HO-C-H H-C-OH I cH2oPo3'Xylulose 5-phosphate

C:O

TPP transketolase

H-C-OH

I cHroPoi

CH2OPO3'-

Glyceraldehyde 3-phosphate

Ribose 5-phosphate

I

./ (_;

H-C-OH T

H-C-OH I

cHroPoS-

Sedoheptulose 7-phosphate

ft) (a)Thegeneralreactioncatalyzedby transkeFIGURE 14-23 Thefirst reactioncatalyzedby transketolase. TPP,from a ketosedonor tolaseisthe transferof a two-carbongroup,carriedtemporarilyon enzyme-bound and a seven-carbon phosphate (b) a triose to phosphates pentose to an aldoseacceptor. Conversionoftwo 7-phosphate. sugarphosphate, sedoheptulose

F.{

G l y c o l yG s i lsu, c o n e o g e naensdtihse, P e n t o sPeh o s p h aPtaet h w a y

c-o HO H

CH'OH

t-

C-H

u: t,

O H -\",

C-OH

HO-C-H

OH \//

HCOH

H

C

I

HCOH+

H-C-OH

tr ansalclollsc

H-C

Sedoheptulose 7-phosphate

I

H-C-OH

OH

I cH2oPo;

I

cH2oPoS

cHroPo;

I

H-C-OH

C-OH

I

cH2oPo32-

Erythrose 4-phosphate

Glyceraldehyde 3-phosphate

Fructose 6-phosphate

FIGURE 14-25 The second reaction catalyzedbv transketolase.

CH,OH

CH,OH

o*

C:O HO-i] H--C

H OH

I' c-o I

,rt C

O H -\,,,

I

H-C-OH +l H-C-OH

crHroPoj XyIuIose 5-phosphate

fructose 6-phosphate and the tetrose erythrose 4-phosphate (FiS. f4-24). Now transketolase acts again, forming fructose 6-phosphate and glyceraldehyde 3-phosphate from erythrose 4-phosphate and xylulose 5-phosphate (Fig. 14-25). tho molecules of glyceraldehyde 3-phosphate formed by two iterations of these reactions can be converted to a molecule of fructose 1,6bisphosphate as in gluconeogenesis(Fig. 14-16), and finally FBPase-l and phosphohexose isomerase convert fructose 1,6-bisphosphate to glucose 6-phosphate. Overall, six pentose phosphates have been converted to five hexose phosphates (Fig. 14-22b)-the cycle is now complete! Tfansketolase requires the cofactor thiamine py'rophosphate (TPP), which stabilizesa two-carbon carbanion in this reaction (Fig. f4-26a), just as it does in the pyruvate decarboxylase reaction (Fig i4-14). Tfansaldolase uses a Lys side chain to form a Schiff base with the carbonyl group of its substrate, a ketose, thereby stabilizing a carbanion (Fig. 14-26b) that is central to the reaction mechanisrn. The process described in Figure 14_21is knornn as the oxidative pentose phosphate pathway. The first and third steps are oxidations with large, negative standard free-energy changes and are essentially irreversible in the cell. The reactions of the nonoxidative part of the pentose phosphate pathway (Fig. 14-22) are readily reversible and thus also provide a means of converting hexose phosphates to pentose phosphates As we shall see in Chapter 20, a process that converts hexose phosphates to pentose phosphates is crucial to the photosynthetic assimilation of CO2 by plants. That pathway, the reductive pentose phosphate pathway, is essentially

HO-C-H

I

H-C-OH

TPP tr nrtsheLolrrsr:

I cHroPoS

H

C-OH

cH2oPoi

Erythrose 4-phosphate

Glyceraldehyde 3-phosphate

+l

H-C-OH cH2oPo32 Fructose 6-phosphate

(a) Tfansketolase OH HOH2C-C

I

I

'-*ilR

stabilization

R' TPP (b) Tfansaldolase CH2OH

cH2oH

Protonated Schiffbase FIGURE 14-26 Carbanionintermediates stabilizedby covalentinteractionswith transketolase (a)Theringof TPPstabilizes and transaldolase. the carbanionin the dihydroxyethyl groupcarriedby transketolase; see Fig 1a44 for thechemistry of TPPaction.(b) In thetransaldolase reaction,the protonatedSchiffbaseformedbetweenthe e-aminogroupof a Lyssidechainand the substrate stabilizes the C-3 carbanionformed afteraldol cleavage

the reversal of the reactions shown in Figure 14-22 and employs many of the same enzyrnes All the enz).rnes in the pentose phosphate pathway are located in the cytosol, Iike those of glycolysis and most of those of gluconeogenesis.In fact, these three pathways are connected through several shared intermediates and enzymes. The glyceraldehyde 3-phosphate formed by the action of transketolase is readily convefted

Oxidation ofGlucose Pathway Phosphate 14.5Pentose [tutl to dihydroxyacetonephosphateby the glycolyticenz).rne triose phosphateisomerase,and thesetwo triosescan be joined by the aldolaseas in gluconeogenesis, forming Alternatively,the triose phosfructose 1,6-bisphosphate. phatescanbe oxidizedto p5,ruvateby the glycolyticreactions. The fate of the trioses is determinedby the cell's relativeneedsfor pentosephosphates,NADPH,and ATP.

lsExacerbated by Wernicke-Korsakoff Syndr0me a Defect inTransketolase Wernicke-Korsakoffsyndrome is a disorder caused by a severe deficiency of thiamine, a component of TPP. The syndrome is more common amongpeoplewith alcoholismthan in the generalpopulation, becausechronic, heavy alcohol consumption interferes with the intestinal absorptionof thiamine. The syndromecan be exacerbatedby a mutationin the genefor transketolasethat resultsin an enzymewith a Ioweredaffinity for TPP-an affinity one-tenththat of the normal enzyme. This defect makes individuals much more sensitiveto a thiamine deficiency:even a moderatethiamine deficiency(tolerablein individuals with an unmutatedtransketolase)can drop the level of TPP belowthat neededto saturatethe enzyme.The result is a slowingdown of the whole pentosephosphate pathway.In peoplewith Wernicke-Korsakoff syndrome this results in a worseningof symptoms,which can include severememory loss,mental confusion,and partial paralysis.r

hetween Glytolysis lsPartitioned Glucose 6-Phosphate Pathway Phosphate andthePentose

needsof the cell and on the concentrationof NADP+ in the cytosol.Without this electronacceptor,the first reaction of the pentosephosphatepathway (catalyzedby G6PD) cannot proceed.When a cell is rapidly converting NADPH to NADP+ in biosynthetic reductions, the level of NADP+ rises, allostericallystimulatingG6PD and thereby increasingthe flux of glucose6-phosphate through the pentosephosphatepathway Whenthe demandfor NADPHslows,the I drops,the pentosephosphatepathwayslows,and glucose6-phosphateis insteadusedto fuel glycolysis.

S U M M A R1Y4 . 5 P e n t o sPeh o s P h a t e P a t h w aoyf G l u c o s e 0xidation r

r

r

Whether glucose6-phosphateenters glycolysisor the pentose phosphatepathway dependson the current Glucose

I

't, Glucose 6-phosphate pentose phosphate pathway

glYcolYsis , ott

H

6-Phosphogluconolactone

NaOPU {i-'.--

)

Pentose phosphates 14-27 Roleof NADPH in regulatingthe partitioningof gluFIGURE cose 6-phosphatebetween glycolysisand the pentosephosphate pathway.When NADPH is forming fasterthan it is being used for and glutathionereduction(seeFig. 14-20), INADPHI biosynthesis pathway. risesand inhibitsthe firstenzymein the pentosephosphate glycolysis. for is available glucose 6-phosphate result, more As a

r

The oridati,ue pentosephosphatepathway (phosphogluconatepathway,or hexose monophosphatepathway) brings about oxidation at C-1ofglucose6-phosphate, and decarboxylation reducingNADP+ to NADPH and producing pentose phosphates. NADPHprovidesreducingpower for biosynthetic reactions,and ribose5-phosphateis a precursor for nucleotideand nucleic acid synthesis.Rapidly growing tissuesand tissuescarrying out active biosynthesisof fatty acids,cholesterol,or steroid hormonessendmore glucose6-phosphatethrough the pentosephosphatepathwaythan do tissues with lessdemandfor pentosephosphatesand reducingpower. The first phaseofthe pentosephosphatepathway consistsoftwo oxidationsthat convertglucose 6-phosphateto ribulose5-phosphateand reduce NADP+to NADPH.The secondphasecomprises nonoxidativestepsthat convert pentosephosphates which beginsthe cycle to glucose6-phosphate, agaln. In the secondphase,transketolase(with TPP as cofactor) and transaldolasecatalyzethe interconversionof three-,four-,flve-,six-, and sugars,with the reversibleconversion seven-carbon phosphatesto flve hexose pentose of six reactionsof phosphates.In the carbon-assimilating the catalyze enzymes photosl'nthesis, the same phosphate pentose process, reduct'iue the reverse pathway:conversionofflve hexosephosphatesto six pentosephosphates. that lowersits A geneticdefectin transketolase Wernicke-Korsakoff affinity for TPP exacerbatesthe syndrome. Entry of glucose6-phosphateeither into glycolysis or into the pentosephosphatepathway is largely determinedby the relative concentrationsof NADP+ and NADPH.

5 6 4 I G l y c o l y sGi lsu, c o n e o g e naensdtihse, p e n t o speh o s p h aptaet h w a y

KeyTerms Terms i,n bold are defined in the glossarg glycolysis 528 mutases 544 fermentation 528 isomerases b44 lacticacidfermentation 530 lactoseintolerance S4b hypoxia 530 galactosemia S4S ethanol (alcohol) thiamine ppophosphate fermentation 530 (Tpp) 549 isozymes 532 gluconeogenesis b1z acyl phosphate 536 biotin bS4 substrate-level pentose phosphate phosphorylation 537 pathway bb8 respiration-linked phosphogluconate phosphorylation pathway 5b8 537 phosphoenolpyruvate hexose monophosphate (PEP) 538 pathway bb8

FurtherReading General Fruton, J.S, (1999) Protei.ns, Genes, and, Enzyrnes: The Interpl,aA of Chemistry and Biology, Yale University press, New Haven Thrs text includes a detailed historical account of research on glycolysis Glycolysis Boiteux, A. & Hess, B. (1981) Design of glycolysis philos Tians R Soc Lond Ser BBioI Sci, 298,b-22 Intermediatelevel review of the pathwav and the classic view of its control Dandekar, T., Schuster, S., Snel, B., Hu;men, M., & Bork, p. (1999) Pathway alignment: application to the comparative analysrs of glycolytic enzymes Bi,ochem J. g4g,lIE_124 Intermediatelevel tion of glycolysis

review of the bioinformatic view of the evolu_

Dang, C.V. & Semenza, G.L. (lggg) Oncogemc alteratrons of metabolism Tlends Bi,ochem Sci, 24,68_72. Brief review of the molecular basis for increased glycolysis in tumors Erlandsen, H., Abola, E.E., & Stevens, R.C. (2000) Combining structural genomics and enzymology: completing the picture rn metabollc pathways and enzyme active sites Cun Opzn Struct Biol 1O,719-730 Intermediatelevel review of the structures of the glycol5,tic enzlTnes Gatenby, R.A, & Gilties, R.J. (2004) Why do cancers have high aerobic glycolysis?Nat Reu Cancer 4, ggl_ggg Hardie, D.G. (2000) Metabolic control: a new solution to an old problem Curr Bi,oL f0, RZ5Z-R259 Harris, A.L. (2002) Hypoxia-a key regulatorv factor in tumour gro\e'th N0, Reu Cancerz,SS-47 Heinrich, R., Melendez-Hevia, E., Montero, F., Nuno, J,C., Stephani, A., & Waddell, T.D. (1999) The structural clesrgn of glycolysis: an evolutionary approach. Biochem Soc Ttans 27. 294-298 Keith, B. & Simon, M.C. (2002) Hypoxia-inducible factors, stem cells, and cancer CelL l2g,46E .472 Intermediate-level review.

Knowles, J. & Albery, Iry.J, (1977) Perfection in enz1,.mecatalysis: the energetics of triose phosphateisomerase Acc Chem.Res 10. 1 0 5 - 1 11 Kresge, N., Simoni, R.D., & Hill, R.L. (2005) Otto Fritz Meyerhof and the elucidation of the glycolytic pathway. J. BioL Chem.280,3 Brief review of classic papers, which are also available on[ne Kritikou, E. (2006) p53 turns on the energy switch. Nntr Reu MoI CeLlBi,oL 7,552-553 Pelicano, H., Martin, D.S., Zu, R-H., & Huang, p. (2006) Glycolysis inhibition for anticancer treatment Oncogene 25,46554646 Intermedratelevel renew Phillips, D., Blake, C.C.F., & Watson, H.C. (eds). (1981) The Enz)rynesof Glycolysis: Structure, Activity and Evolution phzlos Tlans R Soc Lond SerBBi,oL 9ci,293,\-214. A collection of excelient rer,rews on the enzymes of glycolysis, written at a level challenging but comprehensible to a beginmng student of biochemistry Plaxton, W.C. (1996) The organizatron and regulation ofplant glycolysts Annu Reu Plant Physi,ol Plant Mot BioL 47,I8b-214 Very helpful review of the subcellular localization of glycolytic enzymes and the regulation of glycolysis in plants Rose, I. (1981) Chemistry of proton abstraction by glycoly,tic enzS,mes (aldolase, isomerases, and pyruvate kinase) Phzlos Tlans R Soc Lond Ser B Bi,oL Sci 293,131,144 Intermediate-level review of the mechamsms of these enzvmes Shirmer, T. & Evans, P.R. (1990) Structural basis for the allosteric behavior of phosphofructokinase Nature 348, 140-14b Smith, T.A. (2000) Mammalian hexokinases and their abnormal expressionincancer Br J. Bi,omed Sci, 52,120-lZ8 A review of the four hexokinase isozymes of mammals: their properties and tissue distributions and their expressron during the development of tumors Feeder

Pathways

fbr Glycolysis

Elsas, L.J. & Lai, K. (1998) The molecular biology of galactosemia Genet Med 1,40-48. Novelli, G. & Reichardt, J.K. (2000) Molecular basis of disorders of human galactose metabolism: past, present, and future Mol Genet Metab 71,62 65 Petry, K.G. & Reichardt, J.K, (1998) The fundamental importance of human galactose metabolism: lessons from genetics and biochem_ istry Tlends Genet 14,98-102 Van Beers, E.H., Buller, H.A., Grand, R.J., Einerhand, A.WC,, & Dekker, J. (1995) Intestinal brush border glycohydrolases: structure, function, and development Crit Reu Biochem MoI BioL 3O,197-262 Fermentations Demain, A.L,, Davies, J.E., Atlas, R.M., Cohen, G., Hershberger, C.L., Hu, W.-S., Sherman, D,H., Willson, R.C., & Wu, J.H.D. (eds). (1999) Manua\ oJ Industriat Microbi,ology and Br,otechnologg, Amefican Soclety for Microbiology, Washington, DC Classic introduction to all aspects of industrial fermenranons Liese, A., Seelbach, K., & Wandrey, C. (eds). (2006) Ind,ustrial BiotransJormalions, John Wrley & Sons, New york The use of microorganisms in industry for the s5,rrthesisof valuable products from inexpensive startlng materials Gluconeogenesis Gerich, J.E., Meyer, C., Woerle, H.J., & Stumvoll, M. (2001) Re_ nal gluconeogenesis: its importance in human glucose homeostasts Di,abetes Care 24, 382-391

Problems565-l L J

Inlermediatelevel

review of the contribution of kidnev tissue to

ol r rnnnonopncciq

Gleeson, T. (1996) Post-exerciselactatemetabolism:a comparative re!'rewof sites,pathways,and regulation Annu Reu Phgsiol 68, 565-581 and relatedaspects Hers, H.G. & Hue, L. (1983)Gluconeogenesis of glycolysis.Aznu Reu Bzochem 52, 617-653 Matte, A., Thri, L.W, Goldie, H., & Delbaere, L.T.J. (1997) carboxykinase Structure and mechanismof phosphoenolpyruvate J B'ioLChem 272,8105-8108 Oxidative Pentose Phosphate Pathway Chayen, J., Howat, D.W., & Bitensky, L. (1986) Cellularbiochemdehydrogenase istry of glucose6-phosphateand 6-phosphogluconate activities Cell Bi,ochem Funct 4,249-253 Horecker, B.L. (1976) Unravelingthe pentosephosphatepathway InRefi,ectionson Bi,ochemi,stry(Kornberg,A., Cornudella,L , Horecker,B L , & Oro,J , eds),pp 65-72,PergamonPress,Inc , Oxford. Kletzien, R.F., Harris, P.K., & Foellmi, L.A. (1994) Glucose a "housekeeping"enzyrnesubjectto 6-phosphatedehydrogenase: regulationby hormones,nutrients, and oxidant stress. tissue-speci-flc FASEBJ, 8, 174 I81 An rntermediatelevelreview. Kresge, N., Simoni, R.D., & Hill, R.L. (2005) BernardL Horecker's contributionsto elucidatingthe pentosephosphatepalhway J. Bi,ol Chem 28O,26 Brief review of ciassicpapers,which are alsoavailableonline Ltzzato, L., Mehta, A., & Vulliarny, T. (2001) Glucose6-phosphate dehydrogenasedeficiency.In The Metaboli,cand Molecular Basesof Inh.eritedDiseose,Sth edn (Scriver,C R , Sly,WS , Childs,B., Beaudet, A L , Valle,D., Kinzler,K W, & Vogelstein,B , eds), pp 4517-4553, McGraw-HillInc.,New York. The four-volumetreatisein which this afticle appearsis filled with fascinatinginformation about the clinical and biochemicalfeaturesof hundredsof inherited diseasesof metabolism Martini, G. & Ursini, M.V. (1996) A new leaseon life for an old enzyme.BioE ssays 18, 631-637 reviewof glucose6-phosphatedehydroAn intermediate-level genase,the effectsof mutationsin this enz).rnein humans,and the effectsof knock-outmutalionsin mice Notaro, R., Afolayan, A., & Luzzatto, L. (2000) Humanmutations in glucose6-phosphatedehydrogenasereflect evolutionaryhistory FASEBJ. L4,485-494 Wood, T. (1985) The PentosePhosphatePathway, AcademicPress, inc . Orlando.FL Wood, T. (1986) Physiologicalfunctionsof the pentosephosphate pathway.CeLlBiochem Funct 4, 24I-247

Problems 1. Equation for the Preparatory Phase of Glycolysis Write balancedbiochemicalequationsfor all the reactionsin the catabolismof glucoseto two moleculesof glyceraldehyde 3-phosphate(the preparatory phase of glycolysis),including the standardfree-energychangefor eachreaction.Then write the overallor net equationfor the preparatoryphaseof glycolysis,with the net standardfree-energychange. 2. The Payoff Phase of Glycolysis in Skeletal Muscle In working skeletal muscle under anaerobicconditions, glycer-

aldehyde 3-phosphateis converted to pyruvate (the payoff phase of glycolysis),and the pyruvate is reduced to lactate. Write balancedbiochemicalequationsfor all the reactionsin this process,with the standardfree-energychangefor eachreaction. Then write the overall or net equation for the payoff phaseof glycolysis(with lactateas the end product), including the net standardfree-energychange. 3. GLUT Thansporters Comparethe localizationof GLUT4 with that of GLUT2 and GLUT3,and explain why these localizationsare important in the responseof muscle,adiposetissue,brain, and liver to insulin. 4. Ethanol Production in Yeast Whengrown anaerobically on glucose, yeast (S. cereui'si'ae)converts plruvate to acetaldehyde,then reducesacetaldehydeto ethanol using electrons from NADH. Write the equationfor the secondreaction, and calculateits equilibriumconstantaL25'C, giventhe standard reduction potentialsin Table 13-7. 5. Energetics of the Aldolase Reaction Aldolase catreaction alyzesthe glycol5,'tic Fructose1,6-bisphosphate-----+ glyceraldehyde3-phosphate+ dihydroxyacetonephosphate The standardfree-energrcharge for this reactionin the direction written is +23 8 kJ/mol.The concentrationsof the three intermediates in the hepatoc''te of a marnrnalare: fructose 1,6-bisphos3 x 10-ou; and phate,1.4x 10 5u; $yceraldehyde3-phosphate, temperature x At body phosphate, 10-5 rra. 1.6 dihydroxyacetone (37 'C), what is the actual free-energr changefor the reaction? 6. Pathway of Atoms in Fermentation A "pulse-chase" l4olabeled carbon sourcesis carried out on experimentusing a yeast extract maintainedunder strictly anaerobicconditions to produce ethanol. The experiment consistsof incubating a taO-labeledsubstrate (the pulse) with the small amount of yeastextract just long enoughfor eachintermediatein the fermentation pathway to become labeled The label is then "chased"through the pathwayby the addition of excessunlabeled glucose.The chaseeffectively prevents any further entry of labeledglucoseinto the pathway. (glucoselabeledat C-l with toc.; is (a) If [1-14c]glucose lac in the product used as a substrate,what is the location of ethanol?Explain. (b) Where would raO have to be located in the starting 14Cactivity is liberated as r4CO2 glucoseto ensurethat all the during fermentationto ethanol?Explain. 7. Ileat from Fermentations Large-scaleindustrial fermentersgenerallyrequire constant,vigorouscooling.Why? 8. Fermentation to Produce Soy Sauce Soy sauceis prepared by fermenting a salted mixture of soybeansand wheat with severalmicroorganisms,including yeast, over a period of 8 to 12 months.The resulting sauce(after solidsare removed) is rich in lactate and ethanol. How are these two compounds produced?To prevent the soy saucefrom havinga strongvinegary taste (vinegaris dilute acetic acid), oxygenmust be kept out of the fermentationtank. WhY?

r l I 566

G l y c o l yG s i lsu, c o n e o g e naensdtihs e, p e n t o speh o s p h aptaet h w a y

9. Equivalence of Thiose Phosphates 1aC-Labeied glyceraldehyde 3-phosphatewas added to a yeast extract. A_ftera short time, fructose1,6-bisphosphate labeledwith 14Cat C-B and C-4 was isolated What was the locationof the IlC label in the starting glyceraldehyde3-phosphate?Where did the second taO label in fructose 1,O-bisphosphate come from? Explain. 10. Glycolysis Shortcut Supposeyou discovereda mutant yeast whose glycolytic pathway was shorter becauseof the presenceof a new enzyrnecatalyztngthe reaction NAD*

NADH + H+

Glyceraldehyde 3-phosphate + HrO \Z 3-phosphoglycerate Would shorteningthe glycolytic pathway in this way beneflt the cell?Explain. 11. Role of Lactate Dehydrogenase During strenuous activity, the demand for AIP in muscle tissue is vastly increased.In rabbit leg muscleor turkey flight muscle,the ATp is produced almost exclusivelyby lactic acid fermentation. ATP is formed in the payoff phase of glycolysis by two reactions,promotedby phosphoglycerate kinaseand pyruvatekinase. Suppose skeletal muscle were devoid of lactate dehydrogenaseCould it carry out strenuousphysicalactivity; that is, could it generateATP at a high rate by glycolysis? Explain. 12. Efficiency of ATP Production in Muscle The transformation of glucoseto lactatein myocytesreleasesonly about 7o/oof the free energyreleasedwhen glucoseis compietelyoxidized to CO2and H2O Doesthis mean that anaerobicglycolysis in muscieis a wasteful use of glucose?Explain. 13. Free-Energy Change for T[iose phosphate Oxidation The oxidation of glyceraldehydeB-phosphateto 1,8bisphosphoglycerate, catalyzedby glyceraldehyde3-phosphate dehydrogenase,proceeds with an unfavorable equilibrium constant(K!n : 0.08;LG'o:6.3 kJ/mol),yet the flow through this point in the glycoly'ticpathway proceedssmoothly.How doesthe cell overcomethe unfavorableequilibrium? 14. Arsenate Poisoning Arsenateis structurally and chemically similar to inorganic phosphate (p), and many enzyrnes that require phosphatewiil also use arsenate Orgamc compounds of arsenateare less stable than analogousphosphate compounds,however.For exampie, acyl arsenates decom_ poserapidly by hydrolysis:

oo ilti

R-C-O-As-O

+ H2O ------->

o

*-8-o +no-L-o-+H* I o

On the other hand,acylphosphales,suchas 1,3-bisphosphoglycerate, are more stable and undergo further enzymecatalyzedtransformationin cells (a) Predict the effect on the net reaction catalyzedby glyceraldehydeS-phosphatedehydrogenaseif phosphatewere replacedby arsenate. (b) What would be the consequenceto an organismif arsenatewere substitutedfor phosphate?Arsenateis very toxic to most organisms.Expiain why. 15. Requirement for Phosphate in Ethanol Fermentation In 1906Hardenand Young,in a seriesof classicstudies on the fermentationofglucose to ethanoland CO2by extracts of brewer'syeast, made the following observations.(1) Inorganic phosphatewas essentialto fermentation;when the supply of phosphatewas exhausted,fermentationceasedbeforea^ll the glucosewasused.(2) Duringfermentationurder theseconditions, ethanol,CO2,and a hexosebisphosphateaccumulated. (3) When arsenatewas substituted for phosphate,no hexose bisphosphateaccumulated,but the fermentationproceeded until all the glucosewas convertedto ethanoland CO2. (a) Why did fermentation ceasewhen the supply of phosphate was exhausted? (b) WhV did ethanol and CO2accumulate?Was the conversion of py'ruvateto ethanol and CO2essential?Why? Identify the hexosebisphosphatethat accumulated.Whv did it accumulate? (c) \ 4ry did the substitution of arsenatefor phosphate prevent the accumulationof the hexose bisphosphateyet allow fermentation to ethanol and CO2to go to completion? (SeeProblem14.) 16. Role of the Vitamin Niacin Adults engagedin strenuous physicalactivity require an intake of about 160g of carbohydrate daily but only about 20 mg of niacin for optimal nutrition. Given the role of niacin in glycolysis,how do you expiain the observation? 17. Synthesis of Glycerol Phosphate The glycerol 3phosphaterequired for the syrrthesisof glycerophospholipids can be synthesizedfrom a glycolytic intermediate.Proposea reactionsequencefor this conversion. 18. Severity of Clinical Symptoms Due to Enzyme Defrciency The clinical symptoms of two forms of galactosemia-deficiencyof galactokinaseor of UDp-glucose: galactosel-phosphateuridytyltransferase-show radicallydifferent severity.Atthough both t5,pesproduce gastric discomfort after milk ingestion, deficiency of the transferase also leadsto liver, kidney, spleen,and brain dysfunctionand eventual death.What products accumulatein the blood and tissues with each tlpe of enzyrnedeflciency?Estimate the relative todcities of these products from the aboveinformation. 19. Muscle Wasting in Starvation One consequenceof starvationis a reduction in musclemass.What happensto the muscleproteins? 20. Pathway of Atoms in Gluconeogenesis A liver extract capableof carrSringout all the normal metabolic reactions of

r----l

P r o b l e m1s 5 b / l

the liver is briefly incubated in separate experiments with the . 14^ lollowlng t,-la0eleo precursors.

0 (a) [14C]Bicarbonate, Ho-14C\

o (b) tt-l4Cleyruvate, CHB-p-14COO-

Explain how this reactioninhibits the transformationof lactate to pyruvate.Why doesthis lead to hruoglycemia? 26. Blood Lactate Levels during Vigorous Exerc i s e T h e c o n c e n t r a t i o n so f l a c t a t e i n b l o o d p l a s m ab e fore, during, and after a 400 m sprint are shown in the graph. Run *Before*t

o Ttace the pathwayof eachprecursorthrough gluconeogenesis Indicatethe locationof lac in all intermediatesand in the product,glucose. 21. Energy Cost of a Cycle of Glycolysis and Gluconeogenesis What is the cost (in ATP equivalents)of transforming glucoseto plruvate via glycolysisand back againto glucosevia dlrrnnnendoncsiq?

22. Relationship between Gluconeogenesisand Glycois not the exact lysis Why is it important that gluconeogenesis reversalof glycolysis? 23. Energetics of the Ppuvate Kinase Reaction Explain in bioenergetictermshow the conversionof pyruvateto phosphoenolpyruvate in gluconeogenesis overcomesthe large, negativestandardfree-energychangeof the pyruvate kinase reactionin glycolysis. 24. Glucogenic Substrates A commonprocedurefor determining the effectivenessof compoundsas precursorsof glucose in mammals is to starve the animal until the liver glycogenstores are depletedand then administerthe compoundin question A substratethat leadslo anet increasein ilver glycogenis termed glucogenic,becauseit must flrst be convertedto glucose6-phosphate.Showby meansof known enzymaticreactionswhich of the following substancesare glucogenic. -OOC-CH2-CH2-COO (a) Succinate, ( b ) G l y c e r oO l. H

OHOH

I

cH"-c-cH, -l H (c) Acetyl-CoA ? CHr-C-S-CoA (d) Pyruvate,

-

150

t

100

After--

------r

DU

0

0204060 Time(min)

(a) What causesthe rapid rise in lactate concentration? (b) What causesthe deciine in iactate concentrationafter completion of the sprint? Why does the decline occur more slowly than the increase? (c) Why is the concentrationof lactatenot zero during the resting state? 27. Relationship betrveen Fructose 1'6-Bisphosphatase and Blood Lactate Levels A congenitalderesults in fect in the liver enz;rynefructose 1,6-bisphosphatase plasma Explain. in the blood abnormallyhigh levelsof lactate 28. Effect of Phloridzin on Carbohydrate Metabolism Phioridzin, a toxic glycosidefrom the bark of the pear tree, blocks the normai reabsorptionof glucosefrom the kidney tubule,thus causingbloodglucoseto be almostcompletelyexcreted in the urine. In an experiment,rats fed phloridzin and sodiumsuccinateexcretedabout0.5 mol of glucose(madeby gluconeogenesis) for every 1 mol of sodium succinateingested How is the succinatetransfotmedto glucose?Explain the stoichiometry.

?

cH3-c-coo-

OH

(e) Butyrate, CHB-CH2-CH2-COO

C-CH

Blood Glucose Levels The consumption of alcohol (ethanol), especiallyafter peri-

25. Ethanol

Affects

ods of strenuousactivity or after not eating for severalhours, results in a deficiencyof glucosein the blood, a condition known as hypoglycemia.The flrst step in the metabolismof ethanolby the liver is oxidation to acetaldehyde,catalyzedby : Iiver alcoholdehydrogenase cH3cH2oH + NAD+ -----+CHTCHO+ NADH + H*

d OH

OH Phloridzin 29. Excess 02 Uptake during Gluconeogenesis Lactate absorbedbv the liver is convertedto glucose,with the input of

F"l

G l y c o l yG s i lsu, c o n e o g e naensdtihse, P e n t o sPeh o s p h aPtaet h w a y

6 mol of ATP for every mole of $ucose produced The extent of this process in a rat liver preparation can be monitored by administerurg [14C]lactateand measuring the amount of [1aO]$ucose produced. Because the stoichiometry of O2 consumption and AIP production is lcrou'n (about 5 ATP per O2), we can predict the extra 02 consumption above the normal rate when a given amount of lactate is administered. However, when the extra 02 used in the syrrthesisof glucose from lactate is actually measured, it is always higher than predicted by known stoichiometric relationships. Suggest a possible explanation for this observatron. 30. Role ofthe Pentose Phosphate Pathway Ifthe oxidation of glucose 6-phosphate via the pentose phosphate pathway were being used primarily to generate NADpH for bioslnthesis, the other product, ribose b-phosphate,would accumulate What problems might this cause?

DataAnalysis Problem 31. Engineering a Fermentation System Fermentationof plant matter to produce ethanol for fuel is one potential method for reducingthe use of fossilfuels and thus the CO2 emissionsthat lead to global warming Many microorganisms canbreakdown cellulosethen fermentthe glucoseto ethanol. However,many potentiaicellulosesources,includingagricuitural residues and switchgrass,also contain substantial amountsof arabinose,which is not as easilvfermented. H.O \// C

which interconvertsL-arabinoseand r,-ribulose;aro,B, r-r1buIokinase,which uses ATP to phosphorylatel-ribulose at C-5; aro,D, r-rlbttlose 5-phosphateepimerase,which lnterconverts r-ribulose 5-phosphateand l-xylulose 5-phosphate;taIB, transaldolase;and tktA. transketolase (b) For each of the three clra enzymes,briefly describe the chemicaltransformationit catalyzesand, where possible, name an enzlrne discussedin this chapter that carries out an analogousreaction The flve E. coli, genesinsertedtn Z. nnbi,lzs allowedthe entry of arabinoseinto the nonoxidativephaseof the pentose phosphatepathway (Fig. 14-22), where it was converted to glucoseO-phosphate and fermentedto ethanol. (c) The three ara enz''rneseventually converted arabinoseinto which sugar? (d) The product from part (c) feeds into the pathway shovm in Figure 14-22. Combiningthe flve E coli. enzJ.rnes iisted abovewith the enz).rnesof this pathway, describe the overall pathway for the fermentation of 6 moleculesof arabinoseto ethanol. (e) What is the stoichiometryof the fermentationof 6 moleculesof arabinoseto ethanoland CO2?How many ATP moleculeswouldyou expectthis reactionto generate? (t) Z. mobi,Li,s usesa slightly different pathwayfor ethanol fermentationfrom the one describedin this chapter.As a result, the expectedATP fleld is only I ATP per moleculeof arabinose.AJthoughthis is less beneflcialfor the bacterium,it is better for ethanolproduction.Why? Another sugarcommonlyfound in plant matter is xylose HO \//

[IO-C-H

I

c I

H-C-OH

H-C-OH

I

H-C-OH

I I H-C-OH I cH2oH

I

HO-C-H

cH2oH o-Arabinose Escheri,chi,acoli is capable of fermenting arabinoseto ethanol,but it is not naturally tolerant of high ethanol levels, thus limiting its utility for commercialethanolproduction Arother bacterium,Zymomonas mobi,li,s,is naturally tolerant of high levels of ethanol but cannot ferment arabinose.Deanda, Zhang,Eddy,and Picataggio(1996)describedtheir effortsto combine the most useful features of these two organismsby introducing the E co|i, genes for the arabinose-metabolizinA enzlnnesinlo Z. mobtLi,s. (a) Why is this a simpler strategy than the reverse:engrneering-4. coli to be more ethanol-tolerant? Deandaand coileagues insertedflveE coli,genesinto the Z. mobi,Li,sgenome:araA,, codingfor r,-arabinoseisomerase,

o-Xylose (g) What additional enzyrnes would you need to introduce into the modifled Z. mobi,Ii,s strain described above to enable it to use xylose as well as arabinose to produce ethanol? you don't need to name the enz].'rnes (they may not even exist in the real world!); just give the reactions they would need to catalyze Reference Deanda, K., Zhang, M., Eddy, C., & Picataggio, S. (19g6) Development of an arabrnose-fermenting Zgmomoncts mobzti,sstrain by metabolic pathway engineering AppL Enui,ron Mi,crobiol 62, 4465-4470

Formationof liver glycogenfrom lactic acid is thus seento establish an importantconnectionbetweenthe metabolismof the muscleand that of the liver.Muscle glycogenbecomesavailableas blood sugar throughthe interventionof the liver,and blood sugarin turn is convertedinto muscleglycogen.Thereexiststhereforea completecycle o f t h e g l u c o s em o l e c u l ei n t h e b o d y . . . E p i n e p h r i nw e asfoundto a c c e l e r a t et h i s c y c l e i n t h e d i r e c t i o no f m u s c l eg l y c o g e nt o l i v e r glycogen. . . Insulin,on the other hand, was found to acceleratethe cycle in the directionof blood glucoseto muscleglycogen. -C. F.Cori and G. T.Cori, articlein Journalof BiologicalChemistry,1929

ples0fMetabolic Regulation Princi be partially degradedto provide acetyl-CoAfor fatty acid and sterol synthesis. And the bacterium .os(ontrol 577 15.2 Analysis ofMetabolic cheri,chi,acoli, can use glucose to produce the carbon skeleton of euery one of its severalthousand types of 15.3 (oordinated Regulation ofGlycolysis and molecules.When any cell usesglucose6-phosphatefor Gluconeogenesis 582 one purpose,that "decision"affectsall the other pathwaysfor which glucose6-phosphateis a precursoror in15.4 TheMetabolism inAnimals 594 ofGlycogen termediate: any change in the allocation of glucose (oordinated 15.5 RegulationofGlyrogenSynthesis 6-phosphateto one pathway affects, directly or indirectly, the flow of metabolitesthrough all the others' andBreakdown602 Suchchangesin allocationare conunonin the life of a cell. Louis Pasteurwas the first to describethe more than l0-fold increasein glucoseconsumptionby a yeast etabolicregulation,a centraitheme in biochemculture when it was shifted from aerobic to anaerobic istry, is one of the most remarkablefeaturesof conditions.This "Pasteureffect"occurswithout a signifIiving organisms.Of the thousandsof enzymeof ATP or most of the icant changein the concentrations catalyzedreactionsthat can take place in a cell, there is hundredsof metabolichtermediatesand productsdeprobablynot one that escapessomeform of regulation. rived from glucose.A similar effect occursin the cells of This need to regulateevery aspectof cellularmetaboskeletat muscle when a sprinter Ieavesthe starting Iism becomesclear as one examinesthe complexityof blocks. The ability of a cell to carry out all these intermetabolicreactionsequences. Althoughit is convenient lockingmetabolicprocessessimultaneously-obtaining for the student of biochemistry to divide metabolic every product in the amount needed and at the right processesinto "pathways"that play discreterolesin the in the face of major perturbationsfrom outside, time, living no exists i.n the cell's economy, such separation generatingleftovers-is an astoundi'ng without and pathway is in book we discuss this cell. Rather,every accomplishment. inextricably intertwined with all the other cellular In this chapterwe use the metabolismof glucoseto pathwaysin a multidimensionalnetwork of reactions illustrate somegeneralprinciplesof metabolicregula(FiS. 15-1). For example,in Chapter14 we discussed tion. First we look at the generalroles of regulationin four possiblefatesfor glucose 6-phosphate in a hepatoachievingmetabolichomeostasisand introduce metaglycolysis production for the of AIP, cy[e: breakdownby control analysis,a system for analyzingcomplex pathway bolic for the in the pentose phosphate breakdorn"'ri interactions quantitatively.We then describe metabolic production of NADPH and pentosephosphates,use in regulatory properties of the individual enspeciflc the of the extrathe sl,nthesisof complexpolysaccharides glucose metabolism;for glycolysisand glucoof zymes cellularmatrix, or hydrolysisto glucoseand phosphate the catalyticactivitiesof the we described neogenesis, to replenishbloodglucose.In fact, glucose6-phosphate we also discussboth the Here 14. in Chapter enzyrnes has other possiblefates in hepatocytes,too; it may,for properties of the enz;.'rnesof regulatory and catalytic example,be used to synthesizeother sugars,such as one of the bestbreakdowrl, glycogen and synthesis galactose, galactosamine, glucosamine, fucose,and neuNote that in regulation. metabolic of cases studied raminic acid, for use in protein glycosylation,or it may

15.1 Regulation ofMetabolic Pathways570

T

l 569 I

ICttl

Principtes ofMetabotic Regutation

FIGURE 15-1 Metabolismasa three-dimensional meshwork.A typical eukaryoticcell has the capacityto make about 30,000 differentproteins,which catalyzethousands of differentreactions involvingmany hundredsof metabolites,most sharedby more than one ,,pathway.,, Thisoverviewimageof metabolicpathways is from the onlineKECC

(KyotoEncyclopedia of Cenesand Cenomes)PATHWAYdatabase (www genomead jplkegg/pathway/map/map0.l 100 html). Eacharea can be furtherexpandedfor increasingly detailedinformation, to the levelof specificenzymesand intermediates.

selectingcarbohydratemetabolismto illustratethe principles of metabolic regulation,we have artificially separated the metabolismof fats and carbohydrates. In fact, these two activities are very tightly integrated, as we shallseein Chapter23.

ensurethat metabolitesmove through each pathway in the correct direction and at the correct rate to match exactly the cell's or the organism'schangingcircumstances.By a variety of mechanismsoperatingon different time scales,adjustmentsare made in the rate of metaboliteflow through an entire pathway when external circumstances change. Circumstancesdo change,sometimesdramatically. For example,the demandfor ATP in insect flrght muscle increases100-foldin a few secondswhen the insect takes flight. In humans,the availability of oxygen may decreasedue to hypoxia (diminisheddelivery of oxygen to tissues)or ischemia(diminishedflow of blood to tissues).The relativeproportionsof carbohydrate, fat, and

15.1Regulation ofMetabolic Pathways The pathways of glucose metabolism provide, in the catabolic direction, the energy essential to oppose the forces of entropy and, in the anabolic direction, biosynthetic precursors and a storage form of metabolic energy. These reactions are so important to survival that very complex regulatory mechanisms have evolved to

M e t a b oP l i ca t h w a y[st t t - l 1 5 . 1R e g u l a t ioof n

protein in the diet vary from mealto meal,and the supply of fuels obtainedin the diet is intermittent, requiring metabolicadjustmentsbetweenmealsand during periods of starvation.Woundhealingrequireshuge amounts of energyand biosyntheticprecursors.

(ellsand0rganisms Maintain Steady State a Dynamic Fuels such as glucoseenter a cell, and wasteproducts suchas CO2leave,but the massand the grosscomposition of a typical cell, organ, or adult animal do not changeappreciablyover time; cells and organismsexist in a dynamicsteadystate.For eachmetabolicreaction in a pathway,the substrateis providedby the preceding reaction at the same rate at which it is convertedto product. Thus, althoughthe rate (u) of metaboliteflow, or flux, through this step of the pathway may be high and variable,the concentrationof substrate,S, remains constant.So,for the two-stepreaction u', P A ") s when ai : az, [S] is constant.For example,changesin a1 for the entry ofglucosefromvarioussourcesinto the blood are balancedby changesin u2 for the uptake of glucosefrom the blood into varioustissues,so the concentrationof glucosein the blood ([S]) is held nearly constantat 5 mnt.This is homeostasis at the molecular level. The failure of homeostaticmechanismsis often at the root of human disease.In diabetesmellitus,for example,the regulationof blood glucoseconcentrationis defective as a result of the lack of or insensitivity to insulin,with profoundmedicalconsequences. When the external perturbation is not merely transient,or when onekind of cell developsinto another,the adjustmentsin cell compositionand metabolismcan be more dramatic and may require signiflcant and lasting changesin the allocation of energy and synthetic precursorsto bring abouta new dynamicsteadystate.Consider, for example, the differentiation of stem cells in the bone marrow into erythrocytes.The precursorcell containsa nucleus,mitochondria,and little or no hemoglobin, whereasthe fully differentiatederythroclte contains prodigiousamountsof hemoglobinbut has neither nucleus nor mitochondria;the cell's compositionhas permanentlychangedin responseto externaldevelopmental signals,with accompanyingchangesin metabolism. This cellular differentiation requires precise regulationof the levelsof cellularproteins. In the courseof evolution,organismshaveacquireda remarkablecollectionof regtrlatorymechanismsfor mainat the molecular,cellular,and organistaininghomeostasis mal levels,as reflected in the proportion of genesthat encoderegulatorymachinery.In humans,about 4,000 genes(-12% of all genes)encoderegulatoryproteins,inof gene exprescludinga variety of receptors,regr.rlators sion,and morethan 500differentprotein kinases!In many is cases,the regulatorymechanismsoverlap:one erz5.'rne subjectto regulationby severaldifferentmechanisms.

Activity andtheCatalytic BoththeAmount (an Regulated Be Enzyme ofan The flux through an enzyrne-catalyzedreaction can be modulatedby changesin the number of enzymemolecules or by changesin the cataLgticacti'ui'tAof each enzyrnemoleculealreadypresent.Such changesoccur on time scalesfrom millisecondsto many hours, in responseto signalsfrom within or outside the cell' Very rapid allostericchangesin enzl'rneactivity are generally triggered locally, by changesin the local concentration of a small molecule-a substrate of the pathway in which that reaction is a step (say, glucosefor glycolysis), a product of the pathway(ATPfrom glycolysis),or a key metabolite or cofactor (such as NADH) that indicates the cell's metabolic state. Second messengers (such as cyclic AMP and Ca2*) generatedintracellularly in response to extracellular signals (hormones, cytokines, and so forth) alsomediate allostericregulation, on a slightly slower time scale set by the rate of the e-Chapter12). mec signal-transduction srgnals Extracelluiar @) maybe hormonal or neuronal (acetylfor example) (insutin or eptnephrine, The numgowth c1'tokines. factors or choline),or maybe of function given is a in a cell erzyme ber of moleculesof a enof that q,nthesis degradation and the relativerates of The rate of syrrthesiscanbe adjustedby the activa25.'rne. tion (in responseto someoutsidesrgnal)of a transcription factor (Fig. l5-2,@; descnbedin more detail in Chapter 28). Tbanscription factors are nuclear proteins that, DNA regions(response elewhenactivated,bind speci-flc ments) near a gene'spromoter (its transcriptionalstartirg point) andactivateor repressthe transcriptionofthat gene, leadingto increasedor decreasedsyrrthesisofthe encoded protein. Activationof a transcriptionfactor is sometimes the result of its binding of a specificligand and sometimes the result of its phosphorylationor dephosphorylation. Eachgeneis controlledby one or moreresponseelements that are recognizedby speciflctranscription factors. Some geneshave severalresponseelementsand are therefore controlled by severaldifferent transcription factors, respondingto severaldifferent signals.Groupsof genesencodingproteinsthat act together,suchas the enz1rnesof often share cornmonreglycolysisor gluconeogenesis, sponseelementsequences,so that a singlesignal,acting through a particulartranscriptionfactor,turns all ofthese geneson and off together.The regulationof carbohydrate metabolismby speciflctranscriptionfactorsis describedin Section15.3. The stabilityof messengerRNAs-their resistanceto degradationby cellular ribonucleases(Fig. 15-2, @)varies, and the amount of a given mRNA in the cell is a function of its rates of synthesisand degradation(Chapter 26). The rate at which an mRNA is translatedinto a protein by ribosomes(Fig. l5_2, @) is also regulated, and dependson severalfactors describedin detail in Chapter27.Note that ann-fold increasein an mRNAdoes not alwaysmeanan z-fold increasein its protein product'

fl

P r i n c i p loefsM e t a b oR l i ce g u l a t i o n

j72-

@

Protein degradation (ubiquitin; proteasome)

mRNe translation on ribosome

FIGURE15-2 Factorsaffectingtheactivityof enzymes. Thetotalactiviry of an enzyme canbe changed by alteringthenumberof itsmolecules in the cell,or iIs effective activityin a subcellular (@ compartment

Once synthesized, protein molecules have a finite lifetime, which may range from minutes to many days (Table_15-1). The rate of protein degradation (Fig. 15-2, @; Aiffers from one enz).rne to another and depends on the conditions in the cell. Some proteins are tagged by the covalent attachment of ubiquitin for degradation in proteasomes, as discussed in Chapter 2g (see, for example, the case of cyclin, in Fig. 12_46). Rapid turnover (s;,nthesis followed by degradation) is energetically expensive, but proteins with a short half_ life can reach new steady state levels much faster than those with a long half-life, and the beneflt of this quick responsiveness must balance or outweigh the cost to the cell.

fissue

Half-life (days)

Liver

0.9

Kidney

I7

Heart

4.7

Brain

4.6

Muscle

t0.7

through@), or by modulatingthe activityof existingmolecules(@ through @), asdetailedin the text.An enzymemay be influencedby a combination of suchfactors.

Yet another way to alter the elfectzueactivity of an enzymeis to sequesterthe enz}rmeand its substratein different compartments(Fig. I5-2, @).In muscle,for example,hexokinasecannot act on glucoseuntil the sugarentersthe myocytefrom the blood, and the rate at which it entersdependson the activityof glucosetransporters (see Table 11-3) in the plasma membrane. Within cells,membrane-bounded compartmentssegregate certain enzymesand enzyme systems,and the transport of substrateacrossthese intracellularmembranesmay be the limiting factor in enzlirneaction. By these severalmechanismsfor regulatingenz),Tne level, cells can dramaticallychangetheir complementof en4,.rnesin responseto changesin metabolic circumstances.In vertebrates,liver is the most adaptabletissue; a changefrom a high-carbohydrateto high-lipid diet, for example,affects the transcription of hundreds of genesand thus the levels of hundreds of proteins. Theseglobal changesin gene expressioncan be quantifled by the use of DNA microarrays (seeFig. g-22) that display the entire complement of mRNAs present in a given cell type or organ (the transcriptome) or by twodimensionalgel electrophoresis(see Fig. B-21) that displaysthe protein complementof a cell type or organ (its proteome). Both techniquesoffer great insights into metabolic regulation. The effect of changesin the

't5.1 M e t a b oP l i ca t h w a ylst t { R e g u l a t ioof n

proteome is often a change in the total ensemble of low molecular weight metabolites, the metabolome. Once the regulatory mechanisms that involve protein synthesis and degradation have produced a certain number of molecules of each enzyme in a cell, the activity of those enz).rnes can be further regulated in several other ways: by the concentration of substrate, the presence of allosteric effectors, covalent modiflcations, or binding of regulatory proteins-all of whrch can change the activity of an individual enz;rme molecule (Fig. l5-2, @ to @). All enzymes are sensitive to the concentration of their substrate(s) (Fig l5-2,@). Recall that in the simplest case (an enzyme that follows Michaelis-Menten kinetics), the initial rate of the reaction is half-maximal when the substrate is present at a concentration equal to K- (that is, when the enz;'rne is half-saturated with substrate). Activity drops off at lower [S], and when [S] 11 K^, the reaction rate is linearly dependent on [S]. This is important because intracellular concentrations of substrate are often in the same range as, or lower than, K-. The activity of hexokinase, for example, changeswith [glucose],and intracellular [glucose]varies with the concentration of glucose in the blood. As we will see, the different forms (isozymes) of hexokinase have different K- values and are therefore differently affected by changes in intracellular [glucose], in ways that make sense physiologically.

I

W0RKED EXAMPIE 15-1 Activityof aGlucose Transporter

If K, (the equivalentof K-) for the glucosetransporter in liver (GLUT2) is 40 mlt, calculate the effect on the rate of glucoseflux into a hepatocyteof increasingthe bloodglucoseconcentrationfrom 3 mu to 10 mu. Solution:We use Equation11-1 (p. 393) to find the initial velocity (flux) of glucoseuptake. "o

-

V-.*[SJo16

K, I [sL*

At 3 mu glucose Vo : V-"* (3 mu)/(40 mlr + 3 mttl) : V-., (3 mrt/43 mu) : 0.07V-.*

Hill coeffieient (nn)

Requiredchangein [S] to increase76from l0o/otog0%V^n*

0.5 1.0 2.0 3.0 4.0

x6,600 x81 X9 x4.3 X3

to sigmoidkinetics,or vice versa (see Fig. 15-14b,for example).In the steepestpart of the sigmoidcurve, a small changein the concentrationof substrate,or of allostericeffector,can have a large impact on reaction rate.Recallfrom Chapter5 (p. 164)that the cooperativity of an allostericenzymecan be expressedas a Hill coefficient,with higher coefficientsmeaning greater cooperativity.For an allosteric enzyme with a Hill V^u*to 90o/o coefflcientof 4, actiletyincreasesfrom 10%o I/.,u, with only a 3-fold increasein [S], comparedwith the 81-fold rise in [S] needed by an enzymewrth no cooperativeeffects(Hill coefflcientof l; Table15-2). Covalentmodificationsof enz;.'mesor other proteins (Fig. 15-2,@) occurwithin secondsor minutesof a regulatory si.gnal,fupicallyan extracellttlarsignal.By far the most commonmodiflcationsare phosphorylationand dephosphorylation(Fig. f 5-3); up to half the proteinsin a eukaryotic cell are phosphorylatedunder somecircumstances.Phosphorylationby a specific protein kinase may alter the electrostaticfeaturesof an enz}rme'sactive site, causemovementof an inhibitory region of the eninz;.'rneprotein out of the active site, alter the enz}.'rne's teraction wrth other proteins,or force conformational changesthat translate into changesin 7^u* or K-. For Protein substrate ' Ser/Ihr/Iyr;OH

At 10 mvt glucose Vo: V^u' (10 mn'l)/(40mn + 10 mu) : V-^* (10 mu/50 mu) : 0.20V-"* So a rise in blood glucose from 3 mlt to 10 mM increases the rate ofglucose influx into a hepatocyte by a factor of

0 . 2 0 / 0 . :037 Enzy'rneactivity canbe either ircreasedor decreased by an allostericeffector (Fig. 15-2, @; se" Fig. 6-34). Allosteric effectorstypically convert hyperbolickinetics

-P-OI oPro15-3 Protein phosphorylationand dephosphorylation. FIGURE tein kinasestransfera phosphorylgroupfrom ATPto a Ser,Thr,orTyr Proteinphosphatases residuein an enzymeor otherproteinsubstrate. removethe phosphorylgrouPas P;.

l:,1

P r i n r i p loefsM e t a b oR l i ce g u l a t i o n

covalentmodi-flcationto be useful in regulation,the cell must be able to restorethe altered enzyrneto its original activity state.A family of phosphoproteinphosphatases, at least someof which are themselvesunder regulation, catalyzesthe dephosphorylation of proteins. Finally, many enzyrnesare regulatedby association with and dissociationfrom another,regulatoryprotein (Fig. 15-2,@;. f'or example,the cyclicAMP-dependent protein kinase(PKA; seeFig. 12-6) is inactiveuntil cAMP binding separatescataly'ticfrom regulatory subunits. These several mechanismsfor altering the flux through a step in a metabolicpathway are not mutually exclusive.It is very corrunonfor a single enz;rmeto be regulatedat the level of transcriptionand by both allosteric and covalent mechanisms.The combination providesfast, smooth,effectiveregulationin response to a very wide array of perturbationsand signals. In the discussionsthat follow,it is usefirlto think of changesin enzl'rnatic activity as serving two distinct though complementaryroles.Weusethe term metabolic regulation to refer to processesthat serveto mamtarn homeostasis at the molecularlevel-to hold somecellular parameter(concentrationof a metabolite,for example)at a steadyIevel over time, even as the flow of metabolites through the pathwaychanges.The term metabolic control refers to a processthat ieadsto a changein the output of a metabolicpathwayovertime, in responseto some outside signal or changein circumstances.The distinction, althoughuseful,is not alwayseasyto make.

Reartions FarfromEquilibrium inCells Are (ommon Points ofRegulation For some steps in a metabolicpathway the reaction is closeto equilibrium,with the cell in its dynamic steady state (Fig. 154). The net flow of metabolitesthrough these steps is the small differencebetweenthe rates of the forward and reverse reactions, rates that are very similarwhen a reactionis near equilibrium.Smallchanges in substrateor product concentrationcan produce large

o@@ y: -!n-

net rate:

v : 200

y: 500

v=0.01

v=190

V=490

10

10

10

10.01

FIGURE 15-4 Near-equilibriumand nonequilibriumstepsin a metabolicpathway. Steps @ and@ of thispathwayarenearequilibriumin the cell; for each step,the rate(V) of the forwardreactionis only slightlygreaterthan the reverserate,so the netforwardrate(10) is relativelylow and the free-energy change,AC,, is closeto zero.An increasein [C] or [D] can reversethe directionof thesestepsStep@ is maintained in the cell far fromequilibrium;itsforwardrategreatlyexceedsits reverserate.The net rateof stepO ftO) is much largerthan the reverserate(0 01) and is identicalto the net ratesof steps@and@ when the pathwayis operatingin the steadystate.Step@ hasa large, neeativeAC'

changesin the net rate, and can even changethe direction of the net flow We can identify these near-equilibrium reactionsin a cell by comparingthe mass action ratio, @,with the equilibrium constantfor the reaction, K!o. Recallthat for the reaction A + B -+ C * D, Q : tcltDl/tAltBl.WhenI andKlo are within I to 2 ordersof magnitudeof eachother,the reactionis near equil_rbrium. This is the casefor 6 of the 10 stepsin the glycolS,'tic pathway (Table15-3). Other reactions are far from equilibrium in the cell. For example,K'.o for the phosphofructokinase-l (PFK-1) reactionis about 1,000,but Q ([fructose 1,6bisphosphatel[ADP]/[fructose 6-phosphate][ATP]) in a hepatocytein the steadystateis about0.1 (Table15-3). It is becausethe reaction is so far from equilibrium that the processis exergonicunder cellularconditionsand tendsto go in the forwarddirection.The reactionis held far from equilibrium because,under prevailing cellular conditionsof substrate,product, and effector concentrations,the rate of conversionof fructose6-phosphate to fructose1,6-bisphosphate is limited by the activity of PFK-1,which is itself limited by the number of PFK-1 moleculespresentand by the actionsof allostericeffectors. Thus the net forward rate of the enzyme-catalyzed reaction is equalto the net flow of glycoly-ticintermediatesthrough other stepsin the pathway,and the reverse flow throughPFK-1remainsnearzero. The cell cannot allow reactionswith large equilibrium constantsto reachequilibrium.If [fructose6-phosphatel, [ATP],and [ADP]in the cell were held at typical levels (low millimolar concentrations)and the PFK-1reaction were allowedto reach equilibrium by an increase in [fructose 1,6-bisphosphate], the concentration of fructose 1,6-bisphosphate would rise into the molar range,wreakingosmotichavocon the cell. Consideranother case:if the reaction ATP -+ ADP * P, were allowed to approachequiiibrium in the cell, the actual free-energychange (AG') for that reaction (AGo; see Worked Example I3-2, p. 503) would approachzero, and ATP would lose the high phosphorylgroup transfer potential that makesit valuableto the cell. It is therefore essentialthat enzS,.rnes catalyzingATP breakdown and other highly exergonicreactionsin a cell be sensitiveto regulation, so that when metabolic changesare forced by externalcircumstances, the flow through these enzymeswill be adjustedto ensurethat [ATP] remainsfar above its equilibrium level. When such metabolic changesoccur, the activities of enz;rmesin all interconnectedpathwaysadjustto keepthesecriticalstepsaway from equilibrium. Thus, not surprisingly,many enzlnnes (such as PFK-I) that catalyzehighly exergonic reactions are subject to a variety of subtle regulatory mechanisms.The multiplicity of theseadjustmentsis so great that we cannot predict by examiningthe properties of any one enzyrnein a pathway whether that enzymehas a strong influence on net flow through the entire pathway.This complexproblem can be approachedby metabolic controlanalysis,as describedin Section15.2.

t--J

M e t a b oP l i ca t h w a yLs ) / ) l 15 . 1R e g u l a t ioofn

Massactionratio,0

KLs

Enzyme Hexokinase

1x103 1 . 0x 1 0 3

PFK-1 Aldolase Tliose phosphateisomerase Glyceraldehyde3-phosphate dehydrogenase* phosphoglyceratekinase Phosphoglyceratemutase

1 . 0x 1 0 - 4

Liver 2 x l0-2 g x 10-2 1 . 2x 1 0 - 6

4 x 1.0*2

2x103 1 x 10-1

6x102 1 x 10-1

Enolase

2.9

Pyruvatekinase

2xr04

Phosphoglucose isomerase

4 x 10-r

Pyruvatecarboxylase + PEP carboxykinase Glucose6-phosphatase

7 8 . 5x 1 0 2

7 x 10-1 3 . 1x 1 0 - 1

IIeart

Reaction ne&r equilibrium in vivo?*

LG'" (kJ/mol)

8 x 10-2

No

_1t7

3 x 10-2 g x 10-6

No

1l

Yes

2.4 x l0*r

Yes

9.0

Yes Yes Yes No Yes

1 . 2x 1 0 - 1 1.4 40 2.4 x r}-L

No

1 x 10-3

Yes

1.2 x r02

LDA

+7.5 -13

AG' (kJ/tnol) in heart -27 -

Zt)

-6.0 +3.8

f

it.D

Ta.a

+0.6 -0.5 -17 -r.4

-D.U

-23

LAA -6.d _DI

-17

-D.U

fromthesedata. pp.97,263.AG' andAG'' werecalculated NewYork, in Metabolism,Wtley Press, Source:K:qandQ fromNewsholme, E.A.& Start,C.(1973)Regulation +Forsimplicity, nearequilibrium. forwhichtheabsolute valueof thecalculated AG' is lessthan6 is considered anyreaction

Adenine Nucleotides Play Roles in Special Metabolic Regulation After the protection of its DNA from damage,perhaps nothing is more important to a cell than maintaininga constant supply and concentrationof ATP. Many ATPusing enzyrneshave K* values between 0.1 and I mrr,t, and the ATP concentrationin a typical cell is about5 mtr,l. If [ATP]were to drop significantly,these enzyrneswould be lessthan fully saturatedby their substrate(ATP), and the rates of hundreds of reactions that involve ATP woulddecrease(FiS. f 5-5); the cellwouldprobablynot survivethis ki,netzceffect on so many reactions. There is also an important thermodynamzc effect of lowered tATPl. BecauseATP is convertedto ADP or AMP when "spent" to accomplish cellular work, the [ATP]/[ADP]ratio profoundly affects all reactions that employ these cofactors. (The same is true for other important cofactors,such as NADH/NAD* and NADPHI\TADP*.)For example,considerthe reaction catalyzedby hexokinase:

determinesthe magnitudeand sign of AG' and therefore the drivingforce,AG', of the reaction: AG':AG'"+ft?ln

IADPI[glucoseG-phosphateJ lATPllglucosel

Becausean alteration of this driving force profoundly influences every reaction that involves ATP, organisms have evolved under strong pressure to develop regulatorymechanismsresponsiveto the IATPI/tADPl ratio. AMP concentration is an even more sensitiveindicator of a cell'senergeticstate than is [ATP].Normally

!1 d

h h

ATP + glucose-----+ADP f glucoseG-phosphate 6 't

KLq

6-phosphatel IADPI "nlglucose "o : 2 x 1 0 3 [ATP]"o[glucosel "o

Note that this expressionholds true only when reactants and products are at their equi,Li,bri,umconcentrations,where LG' : 0. At any other set of concentrations, AG' is not zero.Recall(from Chapter13) that the ratio of products to substrates(the mass action ratio, Q)

510152025303540 AIP concentration [mu] FIGURE 15-5 Effectof ATPconcentrationon the initial velocityof a datayield a K- for enzyme.Theseexperimental typicalATP-dependent of ATPin animaltissuesis -5 mtt. ATPof 5 mv. Theconcentration

EZt-

P r i n c i p loefsM e t a b oR l i ce g u l a t i o n

Adenine nueleotide

Concenfoation before ATP depletion (ntu)

Concentrationafter ATP depletion (mu)

5.0 1.0 0.1

4.5 1.0 0.6

ATP ADP AMP

cells have a far higher concentration of ATP (5 to 10 mu) than of AMP (

80

N h

60

't

t40 i a

Ftn

0

0 0.050.1 0.2 0.4 0.7 1.0 2.0 (mu) lFructose6-phosphatel

lFructose 1,6-bisphosphate]krlrr) (b)

(a) Gluconeogenesis

1

I J Glycolysis (c) FIGURE l5-16 Roleof fructose2,5-bisphosphate in regulationof gly(F268P)has colysisand gluconeogenesis. Fructose 2,6-bisphosphate oppositeeffectson the enzymaticactivitiesof phosphofructokinase-1 (PFK-1, (FBPasea glycolyticenzyme)andfructose1,6-bisphosphatase (a) 1, a gluconeogenic enzyme). PFK-I activityin the absenceof F26BP(bluecurve)is half-maximal whentheconcentration of fructose is 2 mv (thatis, K6s = 2 mv). When 0.13p,uF26BPis 6-phosphate present(redcurve),the K6r for fructose6-phosphateis only 0.08 mv.

affinityfor frucitsapparent PFK-1by increasing ThusF26BPactivates (b) (see is inhibited FBPase-1 activity Fig. 15-14b\. tose6-phosphate 25 inhibited by is pu F26BP and stronSly by aslittleas1 1t'u.Intheab(blue 1,6-bisphosfructose K65 for the curve) this inhibitor senceof ol 25 p'uF26BP(redcurve)the K65 phateis 5 pu, but in the presence moresenalsomakesFBPase-1 2,6-bisphosphate is >70 1tu.Fructose of AMP (c) Summary regulator, sitiveto inhibitionby anotherallosteric by F26BP. regulation

iroul P r i n c i p loefsM e t a b oRl i ce g u l a t i o n \

\\

f-:

| I | // ------l-

:-l Ei'":;!, l;"

trzoepr Stimulates glycolysis, inhibits gh--- - - --- - --'-

| (rnacrrve, | \ glucagon

(+ tcAMPl)

J

(a)

'/

l I 1 t t (b)

FIGURIl5-17 Regulationof fructose2,6-bisphosphate (PFK-2) level.(a) The (FBPase-2). and itsbreakdownby fructose2,6-bisphosphatase cellularconcentration of the regulator (F26BP) fructose2,6-bisphosphate (b) Bothenzymeactivitiesarepartof the samepolypeptidechain,and is determined by the ratesof its synthesis by phosphofructokinase-2 theyarereciprocally regulated by insulinand glucagon.

affinity for its substrate(Fig. t5-16c), thereby slowing gluconeogenesis. The cellularconcentrationof the allostericregulator fructose2,6-bisphosphate is set by the relative rates of its formation and breakdown (F'iS. 15-l7a). It is formed by phosphorylationof fructose 6-phosphate,cata\yzedby phosphofructokinase-2 (PFK-2), and is broken down by fructose 2,G-bisphosphatase(FBPase-2). (Note that these enzymesare distinct from PFK-I and FBPase-l, which catalyze the formation and breakdown, respectively,of fructose 1,6-bisphosphate.) PFK-2 and FBPase-2are two separateenzymaticactivitiesof a single, bifunctional protein. The balance of these two activities in the liver, which determines the cellular level of fructose 2,6-bisphosphate, is regulated by glucagonand insulin (Fig. 15-17b). As we sawin Chapter12 (p. 431), glucagonstimulatesthe adenylylcyclaseof liver to synthesiz e 3' ,b'cyclic AMP (cAMP) from ATP. Cyclic AMP then activates cAMP-dependentprotein kinase, which transfers a phosphorylgroup from ATP to the bifunctional protein PFK-2/FBPase-2. Phosphorylationof this protein enhancesits FBPase-2 activity and inhibits its PFK-2 activity. Glucagonthereby lowers the cellularlevel of fructose2,6-bisphosphate, inhibiting glycolysisand stimulatinggluconeogenesis. The resulting production of more glucose enablesthe liver to replenishblood glucosein responseto glucagon. Insulin has the opposite effect, stimulating the activity of a phosphoprotein phosphatasethat catalyzes removal of the phosphoryl group from the bifunctional protein PFK-2/FBPase-2,activating its pFK-2 activity,increasingthe level of fructose2,6-bisphosphate, stimulatingglycolysis,and inhibiting gluconeogenesis.

Xylulose 5-Phosphate lsa KeyRegulator of (arbohydrate andFatMetabolism Another regulatory mechanismalso acts by controlling the level of fructose2,6-bisphosphate. In the mammalian Iiver,xylulose5-phosphate(seep. 560),a productofthe pentose phosphatepathway (hexose monophosphate pathway), mediates the increasein glycolysisthat follows ingestion of a high-carbohydratemeal. The xylulose5-phosphateconcentrationrisesasglucoseentering the liver is convertedto glucoseO-phosphate and enters both the glycolytic and pentosephosphatepathways. Xylulose 5-phosphateactivatesphosphoprotein phosphatase2A (PP2A; Fig. 15-18), which dephosphorylates the bifunctional PFK-2/FBPase-2enzyme (Fig. 15-17). DephosphorylationactivatesPFK-2 and inhibitsFBPase-2,and the resultingrise in fructose2,6bisphosphateconcentrationstimulatesglycolysisand inhibits gluconeogenesis.The increased glycolysis boosts the production of acetyl-CoA,while the increasedflow of hexosethrough the pentosephosphate pathway generatesNADPH. Acetyl-CoA and NADPH are the starting materialsfor fatty acid synthesis,which has long been known to increasedramaticallyin responseto intake of a high-carbohydrate meal.Xylulose 5-phosphatealso increasesthe syrrthesisof all the enzymes required for fatty acid synthesis,meeting the prediction from metaboliccontrol analysis.We return to this effectin our discussionof the integrationof carbohydrateand lipid metabolismin Chapter23.

TheGlycolytic Enzyrne Pyruvate Kinase ls Allosterirally Inhibited byATP At least three isozymesof pyruvate kinaseare found in vertebrates,differing in their tissue distribution and

G l y c o l yasni sdG l u c o n e o g e n leCsti {s t eedg u l a t ioofn 1 5 . 3C o o r d i n aR (a) Catalytic subunit

2Mn2*

Scaffold,/ A subunit

their responseto modulators.High concentrationsof ATP, acetyl-CoA, and long-chain fatty acids (signs of abundant energy supply) allosterically inhibit all isozymesof pyruvate kinase (FiS. 15-19). The liver isozyme (L form), but not the muscle isozyme (M form), is subjectto further regulationby phosphorylation. When low blood glucosecausesglucagonrelease, cAMP-dependentprotein kinasephosphorylatesthe L isozymeof pyruvate kinase,inactivatingit. This slows the use of glucoseas a fuel in liver, sparingit for export to the brain and other organs In muscle,the effect of increased[cAMP]is quite different.In responseto epinephrine, cAMP activates glycogen breakdown and glycolysisand providesthe fuel neededfor the flght-orflioht

roqnnnqo

FIGURI15-18 Structureand action of phosphoproteinphosphatase 2A (PP2A).(a) Thecatalyticsubunithastwo Mn2* ionsin its active recognitionsurfaceformedby the site,positionedcloseto the substrate interfacebetweenthe catalyticsubunitand the regulatorysubunit (PDBlD 2NPP).Microcystin-LR, shownherein red, is a specificinregulatorysubunitsrestin a scaffold The catalytic and of PP2A hibitor (theA subunit)that positions them relativeto eachotherand shapes the substraterecognitionsite.(b) PP2Arecognizesseveraltargetprosubunit.Eachof several providedby the regulatory teins,itsspecificity subunitsfits the scaffoldcontainingthe catalyticsubunit, regulatory site. subunitcreatesits uniquesubstrate-binding and eachregulatory All glycolytic

Liver only

Holoenzyme 2

Holoenzyme 1

tissues, including

liver

glucagon I I

F16BP------

+ A-DP

C

ATP

1l | | 6 steps It

PEP

Pyruvate

l ltransamination i ||

i

J)

A l a n i n e- - - - - / FIGURE 15-19 Regulation of pyruvatekinase. Theenzymeis allostericallyinhibitedby ATP,acetyl-CoA, and long-chain fattyacids(allsigns of an abundantenergysupply),and the accumulation of fructose1,6bisphosphate triggersits activation. Accumulation of alanine,which from pyruvatein one step,allosterically can be synthesized inhibits pyruvate kinase,slowingthe productionof pyruvate by glycolysis. The liverisozyme(Lform)isalsoregulated hormonally. Clucagonactivates

proteinkinase(PKA;seeFig.15-35),which phoscAMP-dependent it. When the phorylates the pyruvatekinaseL isozyme,inactivating (PP)dephosphorylates glucagonleveldrops,a proteinphosphatase the liverfrom prevents it. Thismechanism pyruvatekinase,activating is low; instead, glucose when blood by glycolysis glucose consuming (M not affected form) is isozyme The muscle glucose. exports liver the mechanism by thisphosphorylation

f_l

1 5 9 0]

P r i n c i p l0ef sM e t a b 0R l i ce g u l a t i o n

TheGluconeogeni( C0nversion ofPyruvate to Phosphoenol allowing conversionof excesspyruvate to oxaloacetate (and,eventually,glucose). Pyruvate lsUnder Multiple Types ofRegulation In the pathway leading from pyruvate to glucose,the fust control point determinesthe fate of pyruvate in the mitochondrion:its conversioneither to acetyl-CoA(by the pyruvate dehydrogenasecomplex) to fuel the citric acid cycle (Chapter16) or to oxaloacetate(by pyruvate carboxylase)to start the processof gluconeogenesis (Fig. f 5-20). When fatty acidsare readilyavailableas fuels, their breakdown in liver mitochondria yields acetyl-CoA,a signalthat further oxidation of glucosefor fuel is not necessary. Acetyl-CoAis a positiveallosteric modulator of pyruvate carboxylaseand a negativemodulator of pyruvate dehydrogenase,through stimulation of a protein kinasethat inactivatesthe dehydrogenase. When the cell'senergyneedsare being met, oxidative phosphorylationslows, NADH rises relative to NAD+ and inhibits the citric acid cycle,and acetyl-CoAaccumulates.The increasedconcentrationof acetyl-CoAinhibits the pyruvate dehydrogenasecomplex,slowingthe formation of acetyl-CoAfrom pyr"uvate,and stimulates gluconeogenesisby activating pyruvate carboxylase,

Glucose

tI I

S1";""""g"""ri. ]

II

Oxaloacetate

---,o

Ora.r ","I

carbox) tuse

I

Pyruvate

--'I '""'iii;il.", l.* Acetyl-CoA

II I

+ Citric acid cycle

I I

I

+

Energy FIGURE 15-20 Two alternativefates for pyruvate.pyruvatecan be converted to glucoseandglycogenvia gluconeogenesis or oxidizedto acetyl-CoAfor energyproduction.Thefirstenzymein eachpath is regulatedallosterically; acetyl-CoA,producedeitherby fatty acid oxidation or by the pyruvatedehydrogenase complex,stimulatespyruvate carboxylase and inhibitspyruvate dehydrogenase.

Oxaloacetateformed in this way is converted to phosphoenolpy'ruvate(PEP) in the reaction catalyzed by PEP carboxykinase(Fig. 15-11). In mammals,the regulation of this key enz}rmeoccurs primarily at the level of its synthesisand breakdown,in responseto dietary and hormonal signals.Fasting or high glucagon levels act through cAMP to increase the rate of transcription and to stabilizethe mRNA. Insulin, or high blood glucose,has the oppositeeffects.We discussthis transcriptional regulation in more detail below. Generally triggeredby a signalfrom outsidethe cell (diet, hormones), these changestake place on a time scale of minutesto hours.

Transcriptional Regulation and ofGlycolysis Gluconeogenesis Changes theNumber of Enzyme Molecules Most of the regulatory actions discussedthus far are mediated by fast, quickly reversiblemechanisms:allosteric effects, covalentalteration (phosphorylation) of the enz;rme,or binding of a regulatory protein. Another set of regulatoryprocessesinvolveschangesin the number of molecules of an enzyme in the cell, through changesin the balanceof enzyme synthesis and breakdovm,and our discussionnow turns to regulation of transcription through signal-activatedtranscriptionfactors. In Chapter 72 we encounterednuclear receptors and transcription factorsin the context of insulin signaling. Insulin actsthrough its receptorin the plasmamembrane to turn on at leasttwo distrnct signalingpathways, each involving activation of a protein kinase.The MAP kinaseERK, for example,phosphorylatesthe transcription factorsSRFand EIkl (seeFig. 12-15),which then stimulate the synthesis of enzymesneeded for cell growth and division. Protein kinaseB (PKB; also called Akt) phosphorylatesanother set of transcription factors (PDX1,for example),and these stimulate the syrrthesis of enz;.'rnesthat metabolizecarbohydratesand the fats formed and storedfollowing excesscarbohydrateintake in the diet. In pancreaticp cells, PDX1 also stimulates the slnthesis of insulin itself. More than 150 genesare transcriptionallyregulated by insulin; humanshave at least sevengeneraltypes of insulin responseelements,eachrecognizedbya subset of transcription factors activated by insulin under variousconditions.Insulin stimulatesthe transcriptionof the genesthat encodehexokinasesII and IV, PFK-1, pyruvate kinase,and PFK-2lFBPase-2(all involvedin glycolysis and its regulation); several enzyrnesof fatty acid synthesis;and glucose6-phosphatedehydrogenaseand 6-phosphogluconate dehydrogenase,enzpnes of the pentose phosphatepathway that generatethe NADPH required for fatty acid synthesis.Insulin also slows the

ee d g u l a t ioof n G l y c o l yasni sdG l u c o n e o g e n [etsnits l 1 5 . 3( o o r d i n a tR

Changein geneexpression

Pathway

Increased expression HexokinaseII

Glycolysis

HexokinaseIV

Glycolysis

Phosphofructokinase1 (PFK-1)

Glycolysis

Pyruvatekinase

Glycolysis

PFK-2/FBPase-2 Glucose6-phosphatedehydrogenase

Regulationof glycolysis/gluconeogenesis Pentosephosphatepathway (NADPH)

6-Phosphogluconate dehydrogenase

Pentosephosphatepathway (NADPH)

Py'ruvatedehydrogenase

Fatty acid synthesis

Acetyl-CoAcarboxylase Malic enzyme

Fatty acid synthesis Fatty acid synthesis(NADPH)

ATP-citratelyase

Fatty acid slmthesis(providesacetyl-CoA)

Fatty acid synthasecompiex

Fatty acid s5.'nthesis

Stearoyl-CoAdehydrogenase

Fatty acid desaturation

Acyl-CoA-glyceroltransferases

Tfiacylglycerolsynthesis

Decreased expression PEP carboxykinase

Giuconeogenesis

Glucose6-phosphatase(cataly'ticsubunit)

Glucosereleaseto blood

expressionof the genesfor two enz).rnes of gluconeogeglucose nesis:PEP carboxykinaseand 6-phosphatase (Table15-5) One transcription factor important to carbohydrate metabolismis ChREBP (carbohydrate response element binding protein; Fig. 15-2f ), whichis expressed primarily in liver, adiposetissue,and kidney.It servesto coordinatethe synthesisof enzl'rnesneededfor carbohydrate and fat s;,nthesis ChREBPin its iractive state is phosphorylated,and is locatedin the cy'tosol When the phosphoprotein phosphatase PP2A(Fig. I 5-18) removes a phosphorylgroup from ChREBP,the transcriptionfactor can enter the nucleus.Here,nuclearPP2Aremoves anotherphosphorylgroup,and ChREBPnow joins with a partnerprotein,Mlx, andturns on the synthesisof several enzynes: pyruvate kinase, fatty acid s;mthase, and acetyl-CoAcarboxylase,the first enzyrnein the path to (Fig. 15-21). fatty acid syarthesis

FIGURE 15-21 Mechanismof gene regulationby the transcription WhenChREBP is phosfactorChREBP. in the cytosolof a hepatocyte phorylated on a Seranda Thrresidue, it cannotenterthe nucleusDephosphorylation PP2A allows of @Ser by protein phosphatase of ChREBP to enterthe nucleus,wherea seconddephosphorylation, with its partner @Thr, activatesChREBPso that it can associate response protein,Mlx. ChREBP-Mlx now bindsto the carbohydrate PP2Ais element(ChoRE) in the promoterand stimulates transcription activated an intermediate in the allosterically by xyluloseS-phosphate, pentosephosphatepathway.

Glucose

J

GLUT2

Plasma membrane

rrl,

'll

Cytosol

I Glucose

Glucose 6-phosphate

J

.@@

_ Xylulose

b-pnospnale

dhREBF)

Nucleus

592', Principles ofMetabolic Regulation Controllingthe activity of PP2A-and thus, r.rltimately, the synthesisof this groupof metabolicen4,'rnes-is xylulose5-phosphate,an intermediatenot of glycolysisor gluconeogenesisbut of the pentose phosphate pathway. Whenblood$ucose concentrationis high, $ucose enters the liver and is phosphorylatedby hexokinaseM The gucose 6-phosphatethus formed can enter either the glycobtic pathwayor the pentosephosphatepathway.Ifthe latter,two initial oxidationsproducexylulose5-phosphate, which servesasa signalthat the glucose-utilizugpathways arewell-suppliedwith substrate.It accomplishes this by allostericallyactivatingPP2A,which then dephosphorylates ChREBRallowingthe transcriptionfactorto tum on the expressionof genesfor enz;..rnes of glycolysisand fat ryrrthesis(Fig. l5-2I) . Glycolysisyieldspyruvate,and conversion of pyuvate to acetyl-CoAprovidesthe starting material for fatty acid syrthesis:acetyl-CoAcarboxylaseconverts acetyl-CoAto malonyl-CoA, the first committedintermediate in the path to fatty acids.The fatty acid s1r-rthase complex producesfatty acidsfor export to adiposetissueand storageastriacylglycerols(Chapter21). In this way,excess dretarycarbohydrateis storedasfat. Another transcriptionfactor in the liver, SREBPlc, a member of the family of sterol response element binding proteins (see Fig. 2l-43), turns on the syrrthesisof pyruvatekinase,hexokinaseId lipoprotein lipase,acetyl-CoAcarboxylase,and the fatty acid synthase complexthat will convert acetyl-CoA(produced from pyruvate)into fatty acidsfor storagein adipocytes. The symthesis of SREBC-1cis stimulatedby insulinand depressedby glucagon.SREBP-1calso suppressesthe expressionof severalgluconeogenicenzymes:glucose 6-phosphatase, PEP carboxykinase, and FBPase1 The transcriptionfactor CREB (cyclic AMp response element binding protein) turns on the syr-rthesisof glucose6-phosphatase and PEP carboxykinase in responseto the increase in [cAMP] triggered by glucagon.In contrast,insulin-stimulatedinactiuati,on of other transcription factors turns off severalgluconeogenicenzyrnesin the liver:PEP carboxykinase, fructose 1,6-bisphosphatase, the glucose 6-phosphate transporterof the endoplasmicreticulum, and glucose 6-phosphatase. For example,FOXOf (forkhead box other) stimulatesthe synthesisof gluconeogenicenzymesand suppressesthe syrrthesisof the enzyrnesof glycolysis,the pentosephosphatepathway,and triacylglycerol synthesis(Fig. 15-22) In its unphosphorylatedform,FOXOl actsasa nucleartranscriptionfactor. In responseto insulin,FOXO1leavesthe nucieusand in the cytosolis phosphorylatedby PKB,then taggedwith ubiquitin and degradedby the proteasome.Glucagon preventsthis phosphorylationby PKB, and FOXO1remainsactivein the nucleus. Complicatedthough the processesoutlined above may seem,regulationof the genesencodingenz}rmes of carbohydrateand fat metabolismis proving far more complex and more subtle than we have shor,r,n here.

Plasma

,/,membrane Cytosol

It-i'i i i.-

J I \ PKBI

/\

Ly'n /,- 7

','**o,'" (;;;;i' o Pi_.,!"- -l,ts'P ".'--j-f o_

DNA mRNA carboxykinase Glucose6-phosphatase FIGURE 15-22 Mechanismof gene regulationby the transcription factorFOXO1.Insulinactivates the signaling cascade shownin Figure 12-.1 6, leadingto activation of proteinkinaseB (PKB).FOXOI in the cytosolis phosphorylated by PKB,and the phosphorylated transcription factoris taggedby the attachmentof ubiquitinfor degradationby proteasomes. FOXOl thatremainsunphosphorylated or isdephosphorylatedcan enterthe nucleus,bind to a response element,and trigger transcription of theassociated genes.Insulintherefore hastheeffectof turningoflthe expression of thesegenes,which includePEPcarboxykinaseand glucose6-phosphatase

Multiple transcription

factors can act on the same gene

promoter; multiple protein kinases and phosphatases can activate or inactivate these transcription factors; and a variety of protein accessory factors modulate the action of the transcription factors. This complexity is apparent, for example, in the gene encoding PEP carboxykinase, a very well-studied case of transcriptional control. Its promoter region (Fig. 15-23) has 15 or more response elements that are recognized by at least a dozen known transcription factors, with more likely to be discovered The transcription factors act in combination on this promoter region, and on hundreds of other gene promoters, to flne-tune the levels of hundreds of metabolic enzymes, coordinating their activity in the metabolism of carbohydrates and fats. The critical importance of transcription factors in metabolic regulation is made clear by observing the effects of mutations in their genes. For example, at least flve different types of maturity-onset diabetes of the young (MODYI are associated with mutations in speciflc transcription factors (Box 15-3).

andGluconeogenesis Regulation ofGlycolysis 15.3(oordinated I sol I

HNF-4cY

COUP-TF RAR H NF-36

J

Fos/Jun ATFB CREB C/EBP SREBP-1

TSR J

+**t- -t CiXBP

Nfi I

Tlanscription factors FOXO1 forkhead box other 1 PPAR72 peroxisomeproliferator-activated receptor,y2 HNF-38 hepatic nuclear factor-3B SREBP-I sterol regulatory element binding protein-l HNF-4cy hepatic nuclear factor-4o COUP-TF chicken ovalbumin upstream promoter-transcription factor retinoic acid receptor RAR glucocorticoid receptor GR TsR thyroid hormone receptor CAAT/enhancebindingprotein C/EBP HNF-I hepatic nuclear factor-l NF1 nuclear factor 1 ATFS activating transcription factor 3 CREB cAMP regulatory element binding protein NFrB nuclear factor rB TBP TATA-box binding protein Med. mediator TFIIH transcription factor IIH

mRNA

,\//.;:

Response elements and regulatory binding sites in promoter distal accessoryfactor2 dAF2 distal accessoryfactor 1 dAFl sterol regu.latory element SRE Atr'1 accessoryfactor 1 AF2 accessoryfaclor2 glucocorticoid regulatory GRE element thynoid hormone regulatory TRE element cAMP regulatory element CRE

FIGURE 15-23 The PEPcarboxykinase promoterregion,showingthe complexityof regulatoryinputto thisgene.Thisdiagramshowsthetranscriptionfactors(smallericons,boundto the DNA)knownto regulate the transcription of the PEPcarboxykinase gene.The extentto which this geneis expressed dependson the combinedinputaffectingall of these

bloodglucoselevel, of nutrients, whichcan reflecttheavailability factors, andotherfactorsthatgo intomakingup the cell'sneedfor thisenzymeat this particulartime. Pl, P2, P3l, P3ll,and P4 are proteinbindingsites (seeBox26-1).TheTATA box istheasidentifiedby DNaseI footprinting complex. ll (Polll) transcription semblypoint for the RNA polymerase

The term "diabetes"describesa variety of medicalconditionsthat havein commonan excessiveproductionof urine. In Box 11-2 we describeddiabetesinsipidus,in which defectivewater reabsorptionin the kidney results from a mutation in the gene for aquaporin. "Diabetes mellitus" refers speciflcallyto diseasein which the ability to metabolizeglucoseis defective,due either to the failureof the pancreasto produceinsulinor to tissueresistanceto the actionsof insulin. There are two common t5,pesof diabetesmellitus. Tlrpe 1, also called insulin-dependentdiabetesmellitus (IDDM),is causedby autoimmuneattackon the insulinproducing B cells of the pancreas.Individuals with IDDM must take insulin by injection or inhalation to compensatefor their missingB cells.IDDM developsin childhoodor in the teen years;an older name for the diseaseis juvenile diabetes.Tlrpe 2, also called noninsulin-dependentdiabetesmellitus (NIDDM), typically developsin adultsover 40 yearsold. It is far more common than IDDM,and its occurrenceh the populationis strongly correlated with obesity.The current epidemic

of obesityin the more developedcountriesbrings with it the promise of an epidemic of NIDDM, providing a strongincentiveto understandthe relationshipbetween obesityand the onsetof NIDDM at the geneticand biochemicallevels.After completingour look at the metabolism of fats and proteins in later chapters,we will return (in Chapter 23) to the discussionof diabetes, which has a broad effect on metabolism:of carbohydrates,fats, and proteins. Here we consideranother type of diabetesin which carbohydrateand fat metabolismis deranged:mature onset diabetesof the young (MODY), in which genetic mutation affects a transcription factor important in carrying the insulin signalinto the nucleus,or affectsan en4nne that respondsto insulin. In MODY2,a mutation in the hexokinaseIV (glucokinase)gene affects the liver and pancreas,tissuesin which this is the main isoform of hexokinase.The $ucokinase of pancreaticB cells functions as a glucosesensor.Normally,when bloodglucose (conti,nued on nert pa'ge)

rEr-l P r i n c i p loefsM e t a b oRl i ce g u l a t i o n

rises,so doesthe glucoselevel in B cells,and because glucokinasehas a relativelyhigh K* for glucose,its activity increaseswith risingbloodglucoseIevels.MetaboIism of the glucose6-phosphateformedin this reaction raisesthe ATP level in B cells,and this triggersinsulin releaseby the mechanismshown in Figure 23-28. In healthyindir,rduals, bloodglucoseconcentrationsof -b mu trigger this insulin release.But individualswrth inactivating mutations in both copies of the glucokinase gene havevery high thresholdsfor rnsulinrelease,and consequently,from birth, they have severe hyperglycemia-permanent neonataldiabetes.In individuals with one mutated and one normal copy of the glucokinasegene,the glucosethresholdfor insulinreleaserises to about 7 mu. As a result theseindividualshaveblood glucoselevels only slightly abovenormal: they generally have only mild hyperglycemiaand no syrnptoms.This

condition(MODY2)is generallydiscoveredby accident duringroutine bloodglucoseanalysis. There are at least flve other types of MODI each the result of an inactivating mutation in one or another of the transcriptionfactors essentialto the normal developmentand function of pancreaticF cells.Individuals with these mutations have varying degrees of reducedinsulin production and the associateddefects in blood glucosehomeostasis.In MODY1and MODY3, the defectsare severeenoughto producethe long-term complications associatedwith IDDM and NIDDMproblems,kidney failure,and blindness. cardiovascular MODY4,5, and 6 are lesssevereformsof the disease.Altogether,MODYdisordersrepresenta smallpercentage of NIDDM cases.Also very rare are individualswith mutations in the insulin gene itself; they have defects in insulin signalingof varying severity.

S U M M A R1Y5 . 3 C o o r d i n a t eRd e g u l a t i o n o f G l y c o l y s iasn d Gluconeogenesis

about phosphorylationof the bifunctional enzyrne PFK-2/FBPase-2. Insulinincreases[fructose 2,6-bisphosphatel by activatinga phosphoprotein phosphatasethat dephosphorylatesand thus activatesPFK-2.

r

r

r

Gluconeogenesisand glycolysis share seven enzlmes, catalyzing the freely reversible reactions of the pathways. For the other three steps, the forward and reverse reactions are catalyzed by different enzymes, and these are the points of regulation of the two pathways. Hexokinase IV (glucokinase) has kinetic properties related to its special role in the liver: releasing glucose to the blood when blood glucose is low, and taking up and metabolizing glucose when blood glucose is high. PFK-I is allosterically inhibited by ATP and citrate. In most mammalian tissues, including liver, fructose 2,6-bisphosphateis an allosteric activator of this enz).rne. Pyruvate kinase is allosterically inhibited by ATP, and the liver isozyme also is inhibited r.ry cAMP-dependent phosphorylation. Gluconeogenesisis regulated at the level of pyruvate carboxylase (which is activated by acetyl-CoA) and FBPase-l (which is inhibited by fructose 2,6-bisphosphateand AMP) To limit substrate cycling between glycolysis and gluconeogenesis,the two pathways are under reciprocal allosteric control, mainly achieved by the opposing effects of fructose 2,6-bisphosphate on PFK-1 and FBPase-1. Glucagon or epinephrine decreases[fructose 2,O-bisphosphatel,by raising [cAMP] and bringing

Xylnlose5-phosphate,an intermediateof the pentose phosphatepathway,activatesphosphoprotein phosphatasePP2A,which dephosphorylates several target proteins,includingPFK-2/FBPase-2, tilting the balancetoward glucoseuptake,glycogenslnthesis, and lipid s;,nthesisin the liver. TranscriptionfactorsincludingChREBRCREB, SREBP,and FOXO1act in the nucleusto regulate the expressionof specificgenescodingfor enz;rmes of the glycolytic and gluconeogenicpathways. Insulin and glucagonact antagonisticallyin activatingthese transcription factors, thus turning on and off largenumbersof genes.

15.4 TheMetabolism of Glycogen in Animals Our discussionof metabolic regulation, using carbohydrate metabolismas the primary example,now turns to the synthesisand breakdownofglycogen.In this section we focuson the metabolicpathways;in Section15.5we turn to the regulatorymechamsms. In organismsfrom bacteriato plants to vertebrates, excess$ucose is convertedto poly.rnericforms for storage-$ycogen in vertebratesand many microorganisms, starchin plants.In vertebrates,glycogenis for.rndprimarily in the liver andskeletalmuscle;it mayrepresentup to 10% of the weightof liver and lo/oto 2o/oof the weightof muscle. If this much glucosewere dissolvedin the cytosolof a hepatocyte,its concentrationwould be about 0.4 nl, enough

inAnimals ofGlycogen 15.4TheMetabolism | 595i

tlGURt15-24 Glycogengranulesin a hepatocyte.Clycogen,a storparticles,oftenin ageform of carbohydrate, appearsaselectron-dense glycogenis closelyassociated aggregates or rosettes.In hepatocytes reticulum. Manvmitochondria with tubulesof thesmoothendoolasmic arealsoevidentin thismicrograph.

to dominate the osmotic properties of the cell. When storedas a large pol),mer($ycogen),however,the same massof $ucosehasa concentrationof only 0.01pM.Glycogen is storedin large cltosolic granules.The elementary particleof glycogen,the B-particle,is about21 nm in diameter and consistsof up to 55,000glucoseresidueswith about2,000nonreducingends.TWentyto 40 ofthese particles clustertogetherto form a-rosettes,easilyseenwith the microscopein tissue samplesfrom well-fed animals (Fig,. 15-24) but essentiallyabsenta^ftera 24-hourfast. The glycogenin muscle is there to provide a quick sourceof energyfor either aerobicor anaerobicmetabolism.Muscleglycogencanbe exhaustedin lessthan an hour during vigorousactivity. Liver glycogenservesas a reservoirof glucosefor other tissueswhen dietaryglucoseis not available(betweenmealsor during a fast);

this is especiallyimportant for the neurons of the brain, which cannot use fatty acids as fuel. Liver glycogencan be depleted in 12 to 24 hours. In humans, the total amountof energystoredas glycogenis far lessthan the amount stored as fat (triacylglycerol) (see Table 23-5), but fats cannotbe convertedto glucosein mammalsand cannotbe catabolizedanaerobically. Glycogengranulesare complexaggregatesof glycogen and the enz5'rnes that synthesizeit and degradeit, as The well as the machineryfor regulatingthese enz5.'rnes. generalmechanismsfor storing and mobiLizingglycogen are the samein muscleand liver' but the erzSrmesdiffer in subtle yet important ways that reflect the different rolesof glycogenin the two tissues.Glycogenis alsoobtained in the diet and brokendorvnin the gut, and this involvesa separateset of hydrolytic enzymesthat convert glycogento free glucose.(Dietary starchis hydrolyzedin a similar way.) We begin our discussionwith the breakdou'n of glycogento glucose1-phosphate(glycogenolysis), then turn to synthesisofglycogen(glycogenesis)'

by lsCatalyzed Breakdown Glycogen Phosphorylase Glycogen In skeletalmuscleand liver,the glucoseunits of the outer branches of glycogen enter the glycoly'tic pathway through the action of three enzymes:glycogenphosphorylase, glycogendebranchingenzyrne,and phospho$ucomutase.Glycogenphosphorylasecatalyzesthe reaction in wluch an (a1-+4) glycosidiclinkagebetweentwo glucose residuesat a nonreducingend of $ycogen undergoes attack by inorganic phosphate (P), removing the terminal glucoseresidue as c-D-glucose l-phosphate (Fig. 15-25). This phosphorolgszsreactionis different of glycosidicbondsby amylasedurfrom the hAd,rolAsi's ing intestinaldegradationof dietary glycogenand starch. In phosphorolysis,some of the energy of the glycosidic

Nonreducing end

6cH2oH

cH2oH

Glycogen chain (glucose)"

",

--l * 'I p

J

scrr rhor vlast

Nonreducing end

6cH2oH

O-

Glucose 1-phosphate

Glycogen shortened by one residue (glucose)", t

15-25 Removalof a glucoseresidue FIGURE from the nonreducing end of a glycogen chain by glycogen phosphorylase'This processis repetitive;the enzyme removes glucoseresiduesuntil it reaches successive the fourth glucoseunit from a branch point ( s e eF i 8 . 1 5 - 2 6 ) .

ft

ofMetabolic Regulation 1596 Principles bond is preservedin the formationof the phosphateester, glucosel-phosphate(seeSection14.2). Pyridoxalphosphateis an essentialcofactorin the glycogenphosphorylasereaction;its phosphategroup actsasa generalacid catalyst,promotingattackby p1on the glycosidic bond. (This is an unusual role for pyridoxalphosphate;its more typical role is as a cofactorin aminoacid metabolism;seeFig. 18-6.) Glycogenphosphoryiaseacts repetitively on the nonreducingendsofglycogenbranchesuntil it reaches a point four glucoseresiduesaway from an (a1-+6) branch point (see Fig. 7-14), where its action stops. Further degradationby glycogenphosphorylase can occur only after the debranching enzyme, formally known as oligo (a1--+6) to (a1-+4) glucan-transferase, catalyzestwo successive reactionsthat transfer branches(Fig. l5-26). Oncethesebranchesarerransferred and the glucosylresidue at C-6 is hydrolyzed, glycogenphosphorylase activity can continue.

(anEnter Glucose I -Phosphate Glycolysis or,inLiver, Replenish Blood Glucose Glucosel-phosphate,the end product of the glycogen phosphorylase reaction,is convertedto glucose6-phosphate by phosphoglucomutase, which catalyzesthe reversiblereaction Glucose l-phosphate =+

glucose G-phosphate

Initiallyphosphorylated at a Serresidue,the enz)ryne donatesa phosphorylgroup to C-6 of the substrate,then acceptsa phosphoryigroup from C-1 (t'ig. t5-27). The glucose6-phosphateformed from glycogenin skeletalmusclecan enter glycolysisand serveas an energy source to support muscle contraction. In liver, glycogenbreakdownservesa different purpose:to reIeaseglucoseinto the blood when the blood glucose level drops,as it doesbetweenmeals.This requiresthe enzyrneglucose6-phosphatase, presentin liver and kidney but not in other tissues.The enzymeis an integral membraneprotein of the endoplasmicreticulum, predicted to containnine transmembranehelices,with its active site on the Iumenalside of the ER. Glucose6phosphateformedin the cytosolis transportedinto the ER lumen by a speciflctransporter(T1) (Fig. fE-28) and hydrolyzedat the lumenai surfaceby the glucose6phosphatase. The resultingPi and glucoseare thought to be carried back into the cytosol by two different transporters(T2 and T3), and the glucoseleavesthe hepatoc).tevia the plasmamembranetransporter,GLUT2. Notice that by havingthe activesite of glucose6-phosphataseinsidethe ER lumen,the cell separatesthis reactionfrom the processof glycolysis,which takesplace in the cy'tosoland would be aborted by the action of glucose6-phosphatase. Genetic defectsin either glucose6-phosphatase or T1 lead to seriousderangement of glycogenmetaboiism,resulting in type Ia glycogen storagedisease(Box 15-4).

Nonreducing ends

Glycogen

gLycogen lrhosphorvlase

o

o

Glucose 1-phosphate molecules tr unsler trstr irctr\ lt\. 0t c k r l r r l u r c hI n g etn zvul('

{a1l )61 glu(l0slttLlse at t il itr, ol tlebr trnchilg ctlzvllle

ffi cln.or"

u"o:3il|;i,ii:#JnT:l-*' phosphorylaseaction FIGURI15-26 Gtycogenbreakdownnear an (at-+6) branch point. Following sequential removalof terminalglucoseresidues by glycogen (seeFig.I 5-25),glucoseresidues phosphorylase neara branchareremovedin a two-stepprocess that requires a bifunctional debranching enzyme.First,the transferase activityof the enzymeshiftsa block of threeglucoseresidues from the branchto a nearbynonreducing end, to which they are reattached in (a1--+4)linkageThe singleglucose residueremainingat the branchpoint,in (a1--+6) linkage,is then releasedas freeglucoseby the debranching (a1--+6) enzyme,s glucosidaseactivity. Theglucoseresidues areshownin shorthand form,which omitsthe-H, --{H, and-{H2OH groupsfromthe pyranose rings.

Becausemuscleand adiposetissuelack glucose6phosphatase,they cannot convert the glucose6-phosphate formed by glycogenbreakdownto glucose,and thesetissuesthereforedo not contributeAlucoseto the blood.

The5ugar Nucleotide UDP-Glucose Donates Glucose forGlycogen Synthesis Many of the reactionsin which hexosesare transformed or polymerizedinvolve sugax nucleotides, compounds in which the anomericcarbon of a sugaris activatedby attachment to a nucleotide through a phosphate ester Iinkage.Sugarnucleotidesarethe substratesfor polymerizationof monosaccharide s into disaccharides,glycogen,

lt'l

G l y c o giennA n i m a l s 1 5 . 4T h eM e t a b o l i os fm

Phosphoglucomutase

'r/rt

\

\{a

HHO

Glucose 1-phosphate

FIGURE 15-27 Reactioncatalyzedby phosphoglucomutase. on a Ser The reactionbeginswith the enzymephosphorylated residue.In step @, the enzymedonatesits phosphorylgroup (green) producing glucose1,6-bisphosto glucose1-phosphate, phate.In step@, the phosphorylgroupat C-1 of glucose1,6(red)istransferred backto the enzyme,re-forming bisphosphate andproducingglucose6-phosphate. thephosphoenzyme

HHO Glucose1,6-bisphosphate

o -o_P_o_ I o-H

V

t\ HO

HHO Glucose 6-phosphate

FIGURE l5-28 Hydrolysisof glucose 6-phosphateby glucose6of the ER,Thecatalytic phosphatase faces siteof glucose6-phosphatase the lumenof the ER.A glucose6phosphate(C6P)transporter(T1)

Cytosol G6P

Glucose G-phosphatase

G6P transporter (T1)

from the cycaniesthe substrate tosolto the lumen,and the products glucoseand P; passto the (T2 cytosolon specifictransporters and T3) Clucoseleavesthe cell in the via the CLUT2 transporter plasmamembrane.

ER lumen

Increased blood glucose concentration

o-Glucosyl group

starch, cellulose, and more complex extracellular polysaccharides. They are also key intermediates in the production of the aminohexoses and deoxyhexoses found in some of these polysaccharides, and in the synthesis of vitamin C (l-ascorbic acid). The role of sugar nucleotides in the biosynthesis of glycogen and many other carbohydrate derivatives was discovered in 1953 by the Argentine Luis Leloir, 1906-1987 biochemist Luis Leloir.

Uridine

o

o

OH OH UDP-glucose (a sugar nucleotide)

[rrd

P r i n c i p loef sM e t a b oR l i ce g u l a t i o n

Much of what is written in present-daybiochemistrytextbooks about the metabolismof glycogenwas discovered betweenabout1925and 1950by the remarkablehusband and wife team of Carl F. Cori and Gerty T. Cori. Both trained in medicrneir Europe at the end of World War I (shecompletedpremedicalstudiesandmedicalschoolin oneyear!).Theyleft Europetogetherin 1922to establish researchlaboratoriesin the United States,first for rune years in Buffalo, New York, at what is now the Roswell Park MemorialInstitute, then from 1931until the end of their lives at WashingtonUniversityin St. Louis. In their early physiologicaistudies of the origin and fate of glycogen in animal muscle, the Coris

T h e C o r i s i n C e r t y C o r i ' s l a b o r a t o r y ,a r o u n d 1 9 4 7

The suitability of sugar nucleotides for biosynthetic reactions stems from several properties: 1. Their formation is metabolically irreversible, contributing to the irreversibility of the slnthetic pathways in which they are intermediates. The condensation of a nucleoside triphosphate with a hexose 1-phosphate to form a sugar nucleotide has a small positive free-energy change,but the reaction releasesPP1,which is rapidly hydrolyzed by rnorganic pytophosphatase (Fig. lE-Zg), in a reaction that is strongly exergonic (AG'' : - 19.2 kJ/mol). This keeps the cellular concentration of PP1low, ensuring that the

demonstratedthe conversionof glycogento lactate in tissues,movementof lactate in the blood to the liver, and, in the liver, reconversionof lactateto glycogena pathwaythat cameto be known asthe Cori cycle (see Fig. 23-20). Pursuing these observationsat the biochemicallevel, they showedthat glycogenwas mobiIized in a phosphorolysisreaction catalyzedby the enzyme they discovered,glycogen phosphorylase. They identified the product of this reaction (the "Cori ester") as glucose l-phosphate and showed that it could be reincorporatedinto glycogenin the reverse reaction.Althoughthis did not prove to be the reaction by which glycogenis synthesizedin cells, it was the first in vitro demonstrationof the synthesisof a macromoleculefrom simple monomericsubunits,and it inspired others to search for polymerizing enzymes. Arthur Kornberg, discoverer of the first DNA polymerase,saidof his experiencein the Coris'lab, "Glycogen phosphorylase,not basepairing, was what led me to DNA polymerase." Gerty Cori becameinterestedin human genetic ffi II diseasesin which too much glycogenis stored in the liver. She was able to identify the biochemical defect in severalof these diseasesand to show that the diseasescould be diagnosedby assaysof the enzymesof glycogenmetabolismin small samplesof tissue obtainedby biopsy.Table I summarizeswhat we now know about 13 geneticdiseasesof this sort. I Carl and Gerty Cori sharedthe Nobel Prize in Physiologyor Medicinein1947with BernardoHoussayof Argentina, who was cited for his studies of hormonal regulationof carbohydratemetabolism.The Cori laboratoriesin St. Louisbecamean internationalcenterof biochemicalresearchin the 1940sand 1950s,and at least six scientistswho trained with the CorisbecameNobel laureates:Arthur Kornberg (for DNA synthesis,1959), SeveroOchoa (for RNA synthesis,1959), Luis Leloir (for the role of sugarnucleotidesin polysaccharidesynthesis, 1970), Earl Sutherland (for the discovery of

actual free-energy change in the cell is favorable. In effect, rapid removal of the product, driven by the large, negative free-energy change of PP, hydrolysis, pulls the synthetic reaction forward, a conunon strategy in biological poll'rnerization reactions. 2. Although the chemical transformations of sugar nucleotides do not involve the atoms of the nucleotide itself, the nucleotide moiety has many groups that can undergo noncovalent interactions with enzyrnes; the additional free energy of binding can contribute signiflcantly to catalytic activity (Chapter 6; see also p.297).

G l y c o gi enA n n i m a l s[rrol 1 5 . 4T h eM e t a b o l iosfm

cAMP in the regulation of carbohydrate metabolism, 1971),Christiande Duve (for subcellular fractionation,

1974),and Edwin Krebs (for the discoveryof phosphorylasekinase,1991).

Tlpe (name)

Enrymeaffected

Primary organ affected

I}pe o

Glycogenslnthase

Liver

Low bloodglucose,high ketonebodies,earlydeath

Tlpe Ia (von Gierke's)

Glucose6-phosphatase Microsomalglucose 6-phosphate translocase

Liver

Enlargedliver, kidney failure

Liver

As in Ia; alsohigh susceptibiiityto bacterial infections

Liver

As in Ia

lVpe II (Pompe's)

MicrosomalP1 transporter Lysosomalglucosidase

Skeletaland cardiacmuscle

Infantile form: death by age2; juveniie form: muscle defects (myopathy); aduit form: as in musculardystrophy

Tlpe IIIa (Cori'sor Forbes's)

Debranchingenz,'rne

Liver, skeletal and cardiac muscle

Enlargedliver in infants; myopathy

Dpe IIIb

Liver debranching enzlme (muscle enzlme normal)

Liver

Enlargedliver in infants

Dpe IV (Andersen's)

Branchingenzyrne

Liver, skeletal muscle

Enlargedliver and spieen, myoglobinin urine

15pe V (McArdie's)

Musclephosphorylase

Skeletalmuscle

Exercise-inducedcrampsand pain; myoglobinin urine

Tpe VI (Hers's) Tlpe VII (Tarui's)

Liver phosphorylase

Liver

Enlargedliver

MusclePFK-I

Muscle,

$pe Ib

Tlpe Ic

anrfhrnn\f !r,t

!r!r

vvJ

ac

vvv

Symptoms

As in type t also hemoMic anenua

T},pe\4b, VIII, or IX

Phosphorylasekinase

Liver, Ieukocy'tes, muscle

Enlargedliver

T\rpeXI (Fanconi-Bickel)

Glucosetransporter (GLUT2)

Liver

Failure to thrive, enlarged liver, rickets, kidney dysfunction

3. Like phosphate,the nucleotidyigroup (UMP or AMP,for example)is an excellentleavinggroup, facilitating nucleophilicattack by activatingthe sugarcarbonto which it is attached.

can be derived from free glucose in a reaction catalyzedby the isozymes hexokinase I and hexokinase II in muscle and hexokinase IV (glucokinase) in liver:

4. By "tagging"somehexoseswith nucleotidylgroups, cellscan set them asidein a pool for one purpose (glycogensynthesis,for example),separatefrom hexosephosphatesdestinedfor anotherpurpose (suchasglycolysis).

However, some ingested glucose takes a more roundabout path to glycogen. It is first taken up by ery4hroc1'tes and converted to lactate glycolytically; the lactate is then taken up by the liver and converted to glucose 6-phosphateby gluconeogenesis. To initiate glycogen synthesis, the glucose 6-phosphate is converted to glucose l-phosphate in the phosphoglucomutasereaction:

Glycogensynthesistakes place in virtually all animal tissuesbut is especiallyprominentin the liver and skeletal muscies.The starting point for synthesisof glycogenis glucose6-phosphate. As we haveseen,this

o-Glucose+ ATP -----+l-glucose 6-phosphate+ ADP

Glucose6-phosphate+

glucosel-phosphate

L600_l

P r i n c i p l eo sf M e t a b o l R i ce g u l a t i o n

FIGURE 15-29 Formationof a sugarnucleotide.A condensationreactionoccursbetweena nucleosidetriphosphate (NTP) and a sugar phosphate.The negatively chargedoxygenon the sugarphosphateservesasa nucleophile, attackingthe a phosphateof the nucleoside triphosphate and displacing pyrophosphate. The reaction is pulledin the forwarddirectionby the hydrolysis of PP; by i norganicpyrophosphatase

o

@o-r

P-O- +

I o

I | \ll o-P* o f q, f-or I vl ooo-

ntu"."-[s"!q

Sugar phosphate

*o"-.'.u. I

rrorhosrhorvlas[

oo

oo o-P--o-P-o oo

llll Suear r o-f -o-f -o-lTtnoseJ s.* tl

oo-

Pyrophosphate(PP1) inors pyrophospha

"I

Sugar nucleotide (NDP-sugar)

"J

o 2

-O-P-OH

- oI Phosphate(P)

Net reaction: Sugar phosphate + NTP -----+ NDP-sugar -t 2Pi

The product of this reactionis convertedto UDP-glucose by the action of UDP-glucose pyrophosphorylase, in a key step of glycogenbiosymthesis: Glucosel-phosphate+ UTP -----+UDP-glucose * PPi Noticethat this enz),rneis namedfor the reversereaction; in the cell, the reactionproceedsh the direction of UDP6cH2oH

o-P o

glucoseformation,becausepyrophosphateis rapidly hydrolyzedby inorganicpyrophosphatase(Fig. 15-29). UDP-glucoseis the immediate donor of glucose residues in the reaction catalyzedby glycogen sJrnthase, which promotes the transfer of the glucose residue from UDP-glucoseto a nonreducingend of a branchedglycogenmolecule(Fig. 15-30). The overall A glycogenchain is elongatedby FIGURE l5-30 Glycogensynthesis. glycogensynthase. Theenzymetransfers the glucoseresidueof UDPglucoseto the nonreducingend of a glycogenbranch(seeFig.7-14) to makea new tal -+4) linkage.

I o-cH2

UDP-glucose

\./ HOH

glycogcn synthase

New nonreducing end

Elongated glycogen withz+lresidues

Nonreducing end of a glycogen chain with n residues (n> 4)

inAnimals 15.4TheMetabolism ofGlycogen latf

.iCI"JCI"f I "f ;L"f [ "fI. Nonreducing end

(aI---+4)

Nonred.ucing,[i"{I"f)-"_c)(a1--+6) Branch pornt ^ -r\, Nonreducing end

-Z\-

crycogA.Sg cofq"'t'r

FIGURE 15-31 Branchsynthesisin glycogen.The glycogen-branching enzyme(alsocalledamylo (1+4) to (1-+6) transglycosylase, or glycosyl-(4-+6){ransferase) forms a new branch point during glycogen synthesis.

equilibrium of the path from glucose 6-phosphateto glycogenlengthenedby one glucoseunit greatly favors synthesisof glycogen. Glycogensynthasecannotmakethe (a1-+6) bonds found at the branch points of glycogen; these are formed by the glycogen-branchingenzyme,also called amylo (l-+4) to (1-+6) transglyeosylase, or glycosyl-(4-+6)-transferase.The glycogen-branchingenzyme catalyzestransfer of a terminal fragment of 6 or 7 glucoseresiduesfrom the nonreducingend of a glycogen branch having at least 11 residuesto the C-6 hydroxyl group of a glucoseresidue at a more interior position of the same or another glycogenchain, thus creatinga new branch (Fig. 154f ). Further glucose residuesmay be addedto the new branch by glycogen slmthase.The biologicaleffect of branchingis to make the glycogenmoleculemore solubleand to increasethe number of nonreducingends.This increasesthe number of sites accessibleto glycogenphosphorylaseand glycogensSmthase, both of which act only at nonreducing ends.

Glycogenin Primes thelnitialSugar Residues inGlycogen Glycogen synthase cannot initiate a new glycogen chain de novo. It requires a primer, usually a preformed (a1-+4) polyglucosechain or branch havingat Ieasteight glucoseresidues.So,how is anew glycogen moleculeinitiated?The intriguing protein glycogenin (Fig. 15-32) is both the primer on which new chains are assembledand the enzy'rnethat catalyzesthef assembly.The first step in the synthesisof a new glycogen moleculeis the transfer of a glucoseresiduefrom UDP-glucoseto the hydroxyl group of TVrt" of glycogenin, catalyzed by the protein's intrinsic glucosyltransferaseactivity (Fig. 15-33). The nascentchain is extendedby the sequentialaddition of sevenmore

glucoseresidues,each derived from UDP-glucose;the reactions are catalyzedby the chain-extendingactivity of glycogenin.At this point, glycogensynthasetakes over, further extending the glycogenchain. Glycogenin remains buried within the B-particle, covalently attachedto the singlereducingend of the glycogenmolecule(Fig. 15-33b).

15-32 Glycogeninstructure.(PDB1D 1LL2)MuscleglycoFIGURE genin(M, 37,000)formsdimersin solution.Humanshavea second (shownasa UDP-glucose Thesubstrate, isoformin liver,glycogenin-2. fold near the a Rossmann is bound to red ball-and-stickstructure), residues Tyrlea from the is some distance and terminus amino (turquoise)-15 A from theTyrin the samemonomer,12 A from theTyr is boundthroughits phosin the dimericpartner.EachUDP-glucose phatesto a Mn2* ion (green)that is essentialto catalysis.Mn2* is believedto functionasan electron-pairacceptor(Lewisacid)to stabilize the leavinggroup, UDP.The glycosidicbond in the producthas the UDP-gluaboutthe C-1 of glucoseasthe substrate sameconfiguration thatthe transferof glucosefrom UDP toTyrreaoccurs cose,suggesting in two steps.The firststepis probablya nucleophilicattackby Aspr62 (orange),forming a temporaryintermediatewith invertedconfiguration. A secondnucleophilicattackbyTyrreathen restoresthe starting configuration.

Iuul

P r i n c i p loef sM e t a b o R l i ce g u l a t i 0 n

(a)

(b)

Glycogenin

UDP-glucose

-o-P-o-P-o-

llr d

o--@-lE;EIl

UDP-glucose

cH2oH

G I

Repeatssix times

SUMMAR 1Y 5 . 4 T h eM e t a b o l i somf G l y c o g ei n A n i m a l s r

Glycogenis storedin muscleand liver as large particles.Containedwithin the particlesare the enzyrnesthat metabolizeglycogen,aswell as regulatory enzyrnes.

r

Glycogenphosphorylase catalyzesphosphorol;,tic cleavageat the nonreducingends of glycogen chains,producingglucose1-phosphate. The debranchingenzyrnetransfersbranchesonto main chainsand releasesthe residueat the (a1-+6) branchas free giucose.

r

Phosphoglucomutaseinterconvertsglucose 1-phosphateand glucose6-phosphate.Glucose 6-phosphatecan enter glycolysisor, in liver, can be convertedto free glucoseby glucose 6-phosphatase in the endoplasmicreticulum, then releasedto replenishblood glucose.

glycogenin

third tier

primer

fourth tier

secono[rer

outer tier (unbrancned)

FIGURE 15-33 Glycogeninand the structureofthe glycogenparticle. (a)Clycogenin catalyzes two distinctreactions. Initialattackby the hydroxylgroupolTyrleaon C-l of the glucosylmoietyof UDP-glucose resultsin a glucosylated Tyrresidue. TheC-1 of anotherUDP-glucose moleculeis now attackedby the C-4 hydroxylgroupof the terminal glucose,and this sequencerepeatsto form a nascentglycogenmoleculeof eightglucoseresidues attached by (a1-+4)glycosidiclinkages. (b) Structureof the glycogenparticle.Startingat a centralglycogenin molecule,glycogenchains(12 to.l4 residues) extendin tiers.Inner chainshavetwo (a1--+6) branches each.Chainsin theoutertierareunbranched. Thereare 12 tiersin a matureglycogenparticle(only5 are shownhere),consisting of about55,000glucoseresidues in a moleculeof about21 nm diameterandM. -1 x 1O7.

r

The sugarnucleotideUDP-glucose donatesglucose residuesto the nonreducingend of glycogenin the reaction catalyzedby glycogensynthase.A separatebranchingenzyrneproducesthe (a1-+6) linkagesat branch points.

r

New glycogenparticles begin with the autocataly'tic formation of a glycosidicbond betweenthe glucose of UDP-glucose and a Tlr residuein the protein glycogenin,followed by addition of severalglucose residuesto form a primer that can be actedon by glycogens;.'rLthase.

15.5Coordinated Regulation ofGlycogen

Synthesis andBreakdown As we haveseen,the mobilizationof storedglycogenis brought about by glycogenphosphorylase,which degradesglycogento glucosel-phosphate (Fig. 15-25).

andBreakdown Synthesis Regulation ofGlycogen 15.5Coordinated I 60 Glycogenphosphorylaseprovides an especiallyinstructive case of erzyme regulation. It was one of the first knovm examplesof an allostericallyregulated enzyrne and the first enz;.rneshownto be controlledby reversible phosphorylation.It was alsoone of the first ailostericenzyrnesfor which the detailed three-dimensionalstructures of the active and inactive forms were revealedby x-ray crystallographicstudies. Glycogenphosphorylase is also another illustration of how isoz}.'rnesplay their tissue-speciicroles.

Glycogen Phosphorylase lsRegulated Allosterically andHormonally In the late 1930s, Carl and Gerty Cori (Box 15-4) discovered that the glycogenphosphorylase of skeletal muscle exists in two interconvertible forms: glycogen phosphorylase a, which is cataly'tically active, and glyeogen phosphorylase b, which is less active (Fig. f5+4). Subsequent studies by Earl Sutherland EarlW. Sutherland, showed that phosphorylaseb Jr., predominatesin resting mus1915-1974 cle, but during vigorousmuscular actMty epinephrinetriggers phosphorylationof a speci-flc Ser residuein phosphorylase b, converthg it to its more active form, phosphorylasea. (Note that glycogen phosphorylaseis often referred to simply as phosphorylase-so honored becauseit was the first phosphoryIaseto be discovered;the shortenedname has persistedin commonusageand in the literature.) The enzyme (phosphorylaseb kinase) responsible for activatingphosphorylaseby transferring a phosphoryl group to its Ser residue is itself activated by Ser14 OH side

OH

chain QHz

9Hz

Serl4 side chain

Phosphorylaseb (lessactive)

glucagon -. (liver)

epinephrineor glucagonthrough a seriesof stepsshovm in Figure 15-35. Sutherland discoveredthe second messengercAMP,which increasesin concentrationin responseto stimulation by epinephrine (in muscle) or glucagon(in liver). Elevated [cAMP] initiates an enzJ'rnecascade, in which a catalyst activatesa catalyst, which activatesa catalyst(seeSection12.1).Suchcascades allow for Iarge amph-flcationof the initial signal (seepink boxesin Fig. 15-35). The rise in [cAMP]actiprotein kinase,also calledprovates cAMP-dependent tein kinase A (PKA). PKA then phosphorylatesand activatesphosphorylase b kinase, which catalyzesthe phosphorylationof Ser residuesin eachof the two identical subunits of glycogen phosphorylase,activating it and thus stimulating glycogen breakdown. In muscle, this provides fuel for glycolysisto sustain muscle contraction for the flght-or-flight responsesignaledby epinephrine.In liver, glycogenbreakdowncountersthe low blood glucosesignaledby glucagon,releasingglucose. These different roles are reflected in subtle differences in the regulatory mechanismsin muscle and liver. The glycogen phosphorylasesof liver and muscle are isozl'rnes,encoded by different genes and differing in their regulatoryproperties. In muscle,superimposedon the regulationof phosphorylaseby covalentmodification are two allosteric control mechanisms(FiS. 15-35).Ca'*, the signalfor muscle contraction, binds to and activates phosphoryb Iaseb kinase,promotingconversionof phosphorylase to the active a form. Ca2+binds to phosphorylaseb kinase through its 6 subunit, which is calmodulin (see Fig. 12-11).AMP,which accumulatesin vigorouslycontracting muscle as a result of ATP breakdown,binds to and activatesphosphorylase,speedingthe releaseof glucosel-phosphatefrom glycogen.When ATP levels are adequate,ATP blocks the allostericsite to which AMP binds, inactivatingphosphorylase. Whenthe musclereturns to rest, a secondenzlirne, phosphorylase a phosphatase, also called phosphoprotein phosphatase I (PPl), removesthe phosphoryl groups from phosphorylaseo, converting it to the Iessactive form, phosphorylaseb. Like the enzyme of muscle, the glycogen phosphorylase of liver is regulated hormonally (by phosphorylation/dephosphorylation)and allosterically.The dephosphorylatedform is essentiallyinactive.Whenthe blood glucoselevel is too low, glucagon(acting through the cascademechanismshownin Fig. 15-35) activates

phosphorvlasc6 klnase

p h o s p h o rr l a s c o phosphatasc

t,t)1, epinephrine,

--f tc^'-l, ttAMPl (muscle)

o cr-Iz Phosphorylaseo (active)

/ ,o 9H,

by cova15-34 Regulationof muscleglycogenphosphorylase FIGURE lent modification.In the moreactiveform of the enzyme/phosphorylasea, Serla residues,one on each subunit,are phosphorylated' b, a is convertedto the lessactiveform, phosphorylase Phosphorylase by enzymaticlossof thesephosphorylgroups,catalyzedby phospho(alsoknown as phosphoprotein 1, phosphatase rylasea phosphatase (reactivated) phosphoryto reconverted b can be PPi). Phosphorylase b kinase,(SeealsoFig.6-36 on lasea by the actionof phosphorylase regulation.) phosphorylase glycogen

L604l

Principles 0f Metab0lic Regulation

FIGURE 15-35 Cascademechanismof epinephrineand glucagonaction. By binding to specificsurfacereceptors, either epinephrineacting on a myocyte(left)or glucagon actingon a hepatocyte(right)activatesa CTP-bindingprotein G,o (seeFig.12-4).ActiveC.o triggersa risein [cAMP], activatingPKA.Thissetsoff a cascadeof phosphorylations; PKAactivatesphosphorylase b kinase,which then activates glycogenphosphorylase. Suchcascadeseffecta largeamplificationof the initialsignal;the figuresin pink boxesare probablylow estimatesof the actualincreasein numberof moleculesat eachstageof the cascade. Theresultingbreakdown of glycogenprovidesglucose,which in the myocyte can supplyATP(via glycolysis)for musclecontractionand in the hepatocyteis releasedinto the blood to counterthe low blood glucose.

-.,. t.-. -.. - -.-- - - _ t- - _

Myocyte

Epinephrine i*moleculesl ---"-*--*-r

G

|

gon Hepatocyte

ruIl l I I tl I I I

ATP

Cyclic AMP

izii;i:t:@l Inactive PKA

Active PKA

Active phosphorylase b --- kinase

; : ^ ^ molegules , ; , iluujr I

Inactive glycogen phosphorylaseb

Active glycogen phosphorylase o f--' -^ -' ^ -"--

I

t

IIAMP]

I

Glycogen

-----l

molecules j 11,000s r. :--.-"......"....."

Glucose1-phosphate

.t

Glucose

.1,

Blood glucose

phosphorylaseb kinase, which h turn converts phosphorylaseb to its active o form, initiating the releaseof glucoseinto the blood.Whenblood glucoselevelsreturn to normal, glucose enters hepatocytesand binds to an hhibitory allosteric site on phosphorylased. This binding alsoproducesa conformationalchangethat exposes

o

I QHz

the phosphorylatedSer residues to PPl, which catalyzestheir dephosphorylationand inactivatesthe phosphorylase(Fig. f5-36). The allostericsite for glucose allows liver glycogen phosphorylaseto act as its own glucosesensorand to respondappropriatelyto changes in bloodglucose.

O Insulin Ir

c

phosphorylase o phosphatase (PP1)

sites empty FIGURE 15-35 Glycogenphosphorylase of liver as a glucosesensor. Clucosebindingto an allosteric siteof the phosphorylase a isozymeol liverinducesa conformational changethatexposesits phosphorylated Serresiduesto the actionof phosphorylase (ppl). This a phosphatase

phosphatase convertsphosphorylase a to phosphorylase b, sharplyreducingthe activityof phosphorylase and slowingglycogenbreakdown in response to highbloodglucose.lnsulinalsoactsindirectlyto stimulatePPl and slow glycogenbreakdown.

G l y c o gS e ynn t h e a s insdB r e a k d 0 w[ n 1 5 . 5C o o r d i n aR t eedg u l a t ioofn ttt]

Glycogen Synthase lsAlso Regulated byPhosphorylation which addsphosphorylgroupsto tluee Ser residuesnear stronglyinacthe carboxylterminusof glycogens),nthase, andDephnsphorylation Like glycogenphosphorylase,glycogen synthasecan exist in phosphorylatedand dephosphorylatedforms (Fig. 15-:37).Its activeform, glycogen s5,'nthasea, is unphosphorylated.Phosphorylationof the hydroxyl side chainsof severalSer residuesof both subunitsconverts giycogen synthase a to glycogen synthase b, which is iractive unless its allosteric activator,glucose 6-phosphate,is present.Glycogensyrrthaseis remarkable for its ability to be phosphorylatedon variousresiduesby at least 11 different protein kinases The most rmportant regulatorykinaseis glycogen s5mtlnse kinase 3 (GSK3),

FIGURE 15-37 Effectsof GSK3on glycogensynthase activity.C lycogen synthase a, the activeform, hasthreeSerresiduesnearitscarboxylterminus,whicharephosphorylated by glycogen synthase kinase3 (CSK3) Thisconvertsglycogensynthase to the inactive(b)form CSK3actionre(priming)by caseinkinase(CKll) lnsulin quiresprior phosphorylation triggersactivationof glycogensynthaseb by blockingthe activityof .12-1 CSK3(seethe pathwayfor this actionin Fig. 6) and activating a (PP1 phosphoprotein phosphatase in muscle,anotherphosphatase rn liver).In muscle,epinephrine activates PKA,whichphosphorylates the glycogen-targetrng proteinCv (seeFig.15-40)on a sitethatcausesdissociationof PPl from glycogen Clucose6-phosphate favorsdephosphorylationof glycogensynthase by bindingto it and promotinga conformationthat is a good substrate for PP.l. Clucosealso promotes dephosphorylation; thebindingof glucose to glycogen phosphorylase a forcesa conformational changethatfavorsdephosphorylation to glycogenphosphorylase b, thusrelieving itsinhibition of PP.l(seeFig 15-39)

tivating it. The action of GSK3is hierarchical;it carmot phosphorylateglycogensynthaseuntil anotherprotein kinase,casein kinase II (CKII), hasfirst phosphorylated on a nearbyresidue,an eventcalled the glycogens),Trthase priming (FiS. 15-38a). In liver,conversionofglycogensynthaseb to the active form is promoted by PP1, which is bound to the glycogenparticle.PPl removesthe phosphorylgroups

I I I

Phosphoserines near carboxyl terminus

3ADP (}SK3

3AIPlo"

I

lnactive

Active site

ATP H

o

o-o-P:o i

Priming site phosphorylated kinase II

o

lycogen synthase

(a)

Ser residues phosphorylatedin glycogensynthase

tIGURE 15-38 Primingof GSK3phosphorylation of glycogensynthase. (a) Clycogensynthase (glycokinase3 firstassociates with its substrate gen synthase) by interactionbetweenthreepositivelychargedresidues Arg180, Lys2os) residue anda phosphoserine at position+4 in the 1Arge6, (Fororientation, in substrate theSerorThrresidue to be phosphorylated the substrate is assigned the index0 Residues on the amino{erminal -1 , -2, andsoforth;residues sideof thisresidue arenumbered on the +1, +2, andsoforth)Thisassocicarboxyl{erminal sidearenumbered ationalignstheactivesiteof theenzymewith a Serresidue at position 0, which it phosphorylates Thiscreatesa new primingsite,and the en-

(b)

Pseudosubstrate Pseudosubstrate

the Serresidueat posizymemovesdown the proteinto phosphorylate -8. -4, (b) has a Serresiduenearits CSK3 Ser at then the and tion by PKA or PKB(seeFig. amino terminusthat can be phosphorylated regionin CSK3thatfoldsinto 15-39) Thisproducesa "pseudosubstrate" to anotherprotein theprimingsiteandmakestheactivesiteinaccessible groupof its inhibitingCSK3until the primingphosphoryl substrate, regionis removedby PP1. Otherproteinsthataresubpseudosubstrate for CSK3alsohavea primingsiteat position+4, which mustbe strates by anotherproteinkinasebeforeCSK3canacton them phosphorylated regulation.) (SeealsoFigs6-37 and12J2b on glycogensynthase

f

l

606

Principles ofMetabolic Regulation

from the three Ser residuesphosphorylatedby GSK3. Glucose6-phosphatebindsto an allostericsite on glycogen synthaseb, making the enz}.'rnea better substrate for dephosphorylation by PPI and causingits activation. By analogywith glycogenphosphorylase, which acts as a glucosesensor,glycogenslmthasecan be regardedas a glucose 6-phosphatesensor.In muscle, a different phosphatasemay have the role played by PPl in liver, activatingglycogensynthaseby dephosphorylatingit.

Glycogen Synthase Kinase 3Mediates 5ome ofthe Artions ofInsulin As we saw in Chapter 12, oneway in which insulin triggers intracellular changesis by activating a protein kinase [PKB) that in turn phosphorylatesand inactivates GSK3(Fig. 15-39; seealsoFig. 12-16).Phosphorylation of a Ser residuenear the aminoterminus of GSK3 convertsthat regionof the proteinto a pseudosubstrate, which folds into the site at which the priming phosphorylated Ser residuenormallybinds (Fig. 15-38b). This prevents GSK3 from binding the priming site of a real substrate,thereby inactivating the enzymeand tipping the balancein favor of dephosphorylationof glycogen synthaseby PPl. Glycogenphosphorylasecan also affect the phosphorylationof glycogensynthase:active glycogenphosphorylasedirectly inhibits PPl, preventing it from activatingglycogens;mthase(Fig. 15-37). Although flrst discoveredin its role in glycogenmetabolism (hence the name glycogenslmthasekinase), GSK3 clearly has a much broader role than the regulation of glycogen synthase.It mediates signalingby insulin and other growth factors and nutrients, and it acts in the specificationof cell fates during embryonicdevelopment.Amongits targetsare cytoskeletalproteinsand proteins essential for mRNA and protein synthesis. These targets, Iike glycogensynthase,must first un-

dergo a priming phosphorylationby another protein kinasebeforethey canbe phosphorylated by GSK3.

Phosphoprotein Phosphatase 1ls(entral to Glyrogen Metabolism A single enzyme,PPl, can removephosphorylgroups from all three of the enzymesphosphorylatedin responseto glucagon (liver) and epinephrrne (liver and muscle): phosphorylasekinase, glycogenphosphorylase,and glycogensyrrthase.Insulin stimulatesglycogen synthesisby activating PP1 and by inactivating GSK3. Phosphoproteinphosphatase1 doesnot exist free in the cytosol,but is tightlybourd to its targetproteinsby one of a family of glycogen-taxgeting proteins that bind $ycogen and each of the tlree enzymes,$ycogen phosphorylase,phosphorylasekinase,and $ycogen synthase (Fig. 15-40). PPI is itsel-fsubject to covalentand allostericregulation:it is inactivatedwhen phosphorylatedby PKA and is allostericallyactivatedby gucose6-phosphate.

(arbohydrate Allosteric dndll0rm0nal 5ignals Coordinate Metabolism Globally Having looked at the mechanismsthat regulate individual enz;.'rnes, we can now consider the overall shifts in carbohydratemetabolismthat occur in the well-fed state,during fasting,and in the flght-or-flightresponsesignaledby insulin, glucagon,and epinephrine,respectively. We need to contrasttwo casesin which regulation servesdifferent ends:(1) the role of hepatocytesin supplying glucoseto the blood,and (2) the selfishuse of carbohydratefuelsby nonhepatictissues,typiied by skeletal muscle(myoc1'tes),to supporttheir own activities. After ingestionof a carbohydrate-richmeal,the elevation of blood glucose triggers insulin release (FiS. 15-41, top). In a hepatocy'te, insulin hastwo imme-

Insulin

FIGURE 15-39 The path from insulinto GSK3and glycogensynthase.Insulinbindingto its receptor activatesa tyrosineprotein kinasein the receptor, which phosphorylates insulinreceptorsubstrate-1 (lRS-1). The phosphotyrosine in this proteinis then b o u n d b y p h o s p h a t i d y l i n o s i3t o- kl i n a s e( p t - 3 K ) , which convertsphosphatidylinosirol 4,S-bisphosphate(PlP2)in the membrane to phosphatidylinositol 3,4,5-trisphosphate (PlP3).A protein kinase (PDK-I)that is activatedwhen boundto plp, activatesa secondproteinkinase(pKB),which phosphorylates glycogensynthase kinase3 (CSK3)in its pseudosubstrate region,inactivating it by the mechanismsshownin Figure15-38b.The inactivation of CSK3allowsphosphoprotein phosphatase 1 (PP.l )ro dephosphorylate and thus activateglycogensynthase In thisway,insulinstimulates glycogensynthesis (SeeFig.12-16for moredetails on insulinaction.)

G l y c o g5eynn t h e sainsdB r e a k d o w[na t l 1 5 . 5C o o r d i n aR t eedg u l a t ioof n

FIGUREl5-40 Glycogen-targetingprotein G,u. The proteinCv isoneof a familyof proteins glycogentargeting that bind otherproteins(includingPP1)to glycogenpaftiat two differentsitesin recles.Cu can be phosphorylated sponseto insulinor epinephrine. @ Insulin-stimulated PPl, whichdephosphosphorylation of Cv site1 activates phorylates phosphorylase kinase,glycogenphosphorylase, and glycogensynthase(not shown). @ Epinephrinephosphorylation of Cv site 2 causesdissocistimulated ation of PP.lfrom the glycogenparticle,preventingits and glycogensynaccessto glycogenphosphorylase a protein(inhibitor1) thase.PKA also phosphorylates inhibitsPP.l By thesemeans, that,when phosphorylated, insulin inhibits glycogenbreakdownand stimulates (or glucagonin the and epinephrine glycogensynthesis, liver)hasthe oppositeeffects.

High blood glucose

GLUT2

llnsulin

,,, f Insulin-sensitive protein kinase

,IPPl

,l.GSK-3

t Glycogen synthase

JPhosphorylase kinase

'f Glycogen phosphorylase

lGlycogen breakdown

tGlycogen breakdown

+I

t Glycogen phosphorylase

tI

I I l

tPhosphorylase kinase

FIGURE 15-41 Regulationof carbohydratemetabolism in the liver.Arrowsindicatecausalrelationshios between the changesthey connect.Forexample,an arrowfrom JA to 18 meansthata decrease in A causes an increase in B. Pink arrowsconnecteventsthat resultfrom high blood glucose;blue arrowsconnecteventsthat resultfrom lol. bloodglucose.

lPKB

Synthesis of hexokinase II, PFK-I, pyruvate kinase

, ,u

'

t lclucosel6"i6"

"

fI

608

Principles ofMetabolic Regulation

diate effects:it inactivatesGSK3,actingthrough the cascadeshownin Figure 15-39,and activatesa protein phosphatase,perhaps PPl. These two actions fully activate glycogens;mthase.PPl also inactivatesglycogenphosphorylasea and phosphorylasekinaseby dephosphorylating both, effectively stopping glycogen breakdown. Glucoseentersthe hepatocy[ethrough the high-capacity transporter GLUT2,alwayspresent in the plasmamembrane,and the elevatedintracellularglucoseIeadsto drssociationof hexokinaseIV (glucokinase)from its nuclear regulatoryprotein(Fig. 15-13).Hexokinase IVentersthe c;4osoland phosphorylatesglucose,stimulatingglycolysis and supplyingthe precursor for glycogensy'nthesis. Undertheseconditions,hepatocytesuse the excessglucosein the bloodto sy'nthesize glycogen,up to the limit of about 10%of the total weight of the liver. Betweenmeals,or during an extendedfast, the drop in blood glucosetriggers the releaseof glucagon,which, acting through the cascadeshown in Figure 15-35, activatesPKA. PKA mediatesall the effects of glucagon (Fig. 15-41, bottom). It phosphorylatesphosphorylase kinase,activatingit and leadng to the activationof $ycogen phosphorylase.It phosphorylatesglycogens;mthase, inactivatingit and blocking glycogensyrrthesisIt phosphorylatesPFK-2/FBPase-2, leadingto a drop in the concen- tration of the regulatorfructose2,6-bisphosphate, which hasthe effect of inactivatingthe glycolyticenzyrne PFK-I and activatingthe gluconeogenicenzyrneFBPase1.And it phosphorylatesand inactivatesthe glycolltic enzyrnep),'ruvatekinase.Under these conditions,the liver producesglucose6-phosphateby glycogenbreakdown and by gluconeogenesis, and it stopsusing$ucose to fuel glycolysisor make $ycogen, maximizingthe amount of glucoseit canreleaseto the blood.Thisreleaseofglucose is possibleonly in liver and kidney,becauseother tissues lack glucose6-phosphatase(Fig. 15-28). The physiologyof skeletalmuscle differs from that of liver in three ways important to our discussionof metabolicregulation(Fig. 15-42): (1) muscleusesits stored glycogenonly for its own needs; (2) as it goes from rest to vigorous contraction,muscle undergoes very large changes in its demand for ATP, which is supportedby glycolysis;(3) musclelacks the enz}..rnatic machineryfor gluconeogenesis. The regulationof carbohydratemetabolismin musclereflectsthesedifferences from liver. First, myocy[eslack receptorsfor glucagon. Second,the muscleisozymeof pyruvatekinaseis not phosphorylatedby PKA, so glycolysisis not turned off when [cAMP]is high. In fact, c.\IVP,increosesthe rate of glycolysisin muscle, probably by activatingglycogen phosphorylase. When epinephrineis releasedinto the blood in a flght-or-flight situation, PKA is activated by the rise in [cAMP] and phosphorylatesand activates glycogenphosphorylase kinase.The resultingphosphorylation and activation of glycogen phosphorylaseresults in faster glycogenbreakdown.Epinephrine is not releasedunder low-stress conditions,but with each

Epinephrine Glucagon

+

Liver

II

Glycogen Blood glucose -

Muscle

Glycogen

tGlycogenolysisf I t Y Y Glucose Glucose 6-phosphate 6-phosphate A I Pyruvate

{Glycolysist lGluconeogenesis

I V Pyruvate

FIGURE 15-42 Differencein the regulationof carbohydratemetabolism in liver and muscle.In liver,eitherglucagon(indicatinglow (signaling bloodglucose) or epinephrine the needto fightor flee)has the effectof maximizingthe outputof glucoseinto the bloodstream. ln muscle,epinephrine increases glycogenbreakdown andglycolysis, which togetherprovidefuel to producethe ATPneededfor muscle contraction

neuronal [Ca'*]

stimulation of muscle contraction, cytosolic rises briefly and activates phosphorylase kinase

through its calmodulinsubunit Elevatedinsulin triggersincreasedglycogensynthesisin myocytesby activatingPPi and inactivatingGSK3. Unlike hepatocytes,myocyteshavea reserveof GLUT4 sequesteredin rntracellularvesicles.Insultntriggerstheir movement to the plasma membrane (see Fig. 12-16), wherethey allowincreasedglucoseuptake.In response to insulin, therefore, myocytes help to lower blood glucose by increasingtheir rates of glucose uptake, glycogens;mthesis,and glycolysis.

(arbohydrate and[ipidMetabolism Arelntegrated by Hormonal andAllosteric Mechanisms As complexas the reguiationof carbohydratemetabolism is, it is far from the whole story of fuel metabolism. The metabolism of fats and fatty acids is very closely tied to that of carbohydratesHormonalsignalssuch as insulin and changesin diet or exerciseare equally important in regulating fat metabolismand integrating it with that of carbohydrates.We return to this overall metabolic integration h mammalsin Chapter 23, after first consideringthe metabolicpathwaysfor fats and aminoacids(Chapters17and 18).The message we wish to convey here is that metabolic pathwaysare overlaid wrth complex regulatory controls that are exquisitely sensitiveto changesin metaboliccircumstances. These mechanismsact to adjust the flow of metabolites through various metabolic pathways,as needed by the cell and organism,and to do so without causingmajor changesin the concentrationsof intermediatesshared with other pathways

.fI

F u r t h eRre a d i n g 6 0 9 ]

SUMMAR 1Y 5 . 5 C o o r d i n a t eRde g u l a t i o n o f G l y c o g eSny n l h e s i s a n dB r e a k d o w n r

Glycogenphosphorylaseis activatedin responseto glucagonor epinephrine,which raise [cAMP]and activatePKA. PKA phosphorylatesand activates phosphorylasekinase,which convertsglycogen phosphorylaseb to its activeo form. Phosphoprotein phosphatase1 (PPl) reversesthe phosphorylation of glycogenphosphorylaseo, inactivatingit. Glucose binds to the liver isozSrme of glycogenphosphorylase a, favoringits dephosphorylationand inactivation.

r

Glycogensynthaseo is inactivatedby phosphorylationcatalyzedby GSK3.Insulin blocksGSK3.PP1,which is activatedby insulin, reversesthe inhibition by dephosphorylating glycogensynthaseb.

r

Insulinincreasesglucoseuptakeinto myocytesand adipoc;,tesby triggering movementof the glucose transporterGLUT4to the plasmamembrane.

r

Insulin stimulatesthe synthesisof hexokinases II and IV, PFK-I, pyruvate kinase,and several enzyrnesinvolvedin lipid synthesis.Insulin stimulatesglycogensynthesisin muscleand liver. In liver, glucagonstimulatesglycogenbreakdov,n and gluconeogenesis while blockingglycolysis, thereby sparingglucosefor export to the brain and other tissues.

r

r

In muscle,epinephrinestimulatesglycogen breakdownand glycolysis,providing ATP to supportcontraction.

fructose2,6-bisphosphatase UDP-glucose pyrophosphorylase 600 (FBPase-2) 588 amylo (1-+4) to (1-+6) carbohydrateresponse trans$ycosylase 601 elementbindingprotein glycogenin 601 (chREBP) 591 ctornl raqnnnqp glycogen alemont hindino phosphorylaseo 603 glycogen protein (SREBP) 592 phosphorylaseb 603 cyclic AMP response enzyme cascade 603 elementbinding phosphorylaseb kinase 603 protein (CREB) 592 phosphoprotein forkheadbox other phosphatase1 (PPI) 603 (FOXO1) 592 glycogens5mthasea 605 glycogenolysis 595 glycogenslrrthaseb 605 glycogenesis 595 glycogensynthasekinase3 glucose1-phosphate 595 (GSK3) 605 596 debranchingenzlrne (a1-+4) (a1-+6) kinaseII (CKn) 605 casein to oligo priming 605 glucantransferase 596 glycogen-targeting phosphoglucomutase596 proteins 606 sugarnucleotides 596 freeze-clamping 611

Further Reading Regulation

of Metabolic

Pathways

Desvergne, 8., Michalik, L., & Wahli, W. (2006) T[anscriptional regulationof metabolism.Physi,ol Reu. 86, 465-514 Advancedand comprehensivereview. Gibson, D. & Harris, R.A, (2001)Metabolic Regulati'on i'n MammaLs,Taylor & Francis,New York. Excellent,readableaccountof metabolicregulation.

KeyTerms

Storey, K.B, (ed.). (2004)Functinnal Metabolism: Regulati'on and Adaptati,on, Wley-Liss,Inc., Hoboken,NJ. Excelient discussionof the principles of metabolicregulation, signaltransduction,transcriptionalcontrol, and energymetabolismin health and disease.

Tenns i,n bold are defined i,n th,eglossarg

Analysis of Metabolic Control

glucose6-phosphate569 flux 57\ homeostasis 57I eellular differentiation 571 transcriptionfactor 57I response element 571 turnover 572 transcriptome 572 proteome 572 metabolome 573 metabolicregulation 574 metabolic control 574 mass action ratio, @ 574 adenylatekinase 576 AMP-activatedprotein kinase (AMPK) 576

flux control coefflcient,C 578 flux,J 578 elasticity coefflcient,e 580 responsecoefflcient,-B 581 gluconeogenesis 582 futile cycle 583 substratecycle 583 hexokinaseII 583 hexokinaseI 583 hexokinaseIV 584 GLUTz 584 glucagon 587 fructose 2,6-bisphosphate587 phosphofructokinase-2 (PFK-2) 588

Fell, D.A. (1992) Metaboliccontrol analysis:a surveyofits theoreti cal and experimentaldevelopmentBi'ochem.J 286, 313-330 Clearstatementof the principiesof metaboliccontrol analysis. Fell, D.A, (1997) Und,erstand'i'ngthe Control oJMetaboli'sm,Portland Press,Ltd , London An excellent,clear expositionof metabolicregulation,from the point of view of metaboliccontrol ana"lysis. If you read only one treatment on metaboliccontrol analysis,this shouldbe it. Heinrich, R. & Rapoport, T.A' (1974) A linear steady-statetreatment of enzjrmaticchains:generalproperties,controi and effector strength Eur J. Bi,ochem 42,89-95. Early statementof principlesof metabolc control analysis.See alsothe paperby Kacser& Burns,Iisted below Jeffrey, F.M.H., Rqiagopal, A., Maloy, C.R.' & Sherry' A.D. (1991) r3C-NMR:a simpleyet comprehensivemethod for analysisof intermediarymetabolism.Ttends Bi'ochem Sci'. 16, 5-10. Brief, intermediatelevel review. Kacse4 H. & Burns, J.A' (1973) The control of flux. Sgm'p Soc Erp 8i,o1.32,65-104. A classicpaper in the field. Seealsothe paper by Heinrich & Rapoport,listed above

10 l

P r i n c i p l eo sf M e t a b o l R i ce g u l a t i o n

Kacser, H., Burns, J.A., & Fell, D.A. (1995) The control of flux: 21 yearson.Bi,ochemSoc Tlans.z8, 341-366.

Classicdescriptionof this regulatorymolecuieand its role rn regulatingcarbohydratemetabolism

Schilling, C,H., Schuster,S., Palsson, B.O., & Heinrich, R. (1999) Metabolicpathwayanalysis:basic conceptsand scientiflcapplicationsin the post-genomicera.Biotech:nol Prog 15, 296-303. Short, advanceddiscussionof theoreticaltreatmentsthat attempt to find ways of manipulatingmetabolismto optimizethe formation of metabolicproducts.

Hue, L. & Rider, M.H. (1987)Roleof fructose2,6-bisphosphate in the control of glycolysisin mammaliantiss;rtesB'inchem.J. 245, 3t3-324

Schuster, S., Fell, D.A., & Dandekar, T. (2000) A generaldefinition of metabolicpathwaysuseful for systematicorganizationand analysisof complexmetabolicnetworks Nat Bi,otechnol.18, 326-332 An interestingand provocativeanalysisof the interplay between the pentosephosphatepathwayand glycolysis,from a theoretical standpoint.

Kahn,8.B., Alquier, T., Carling, D., & Hardie, D.G. (2005) AMP-activatedprotein kinase:ancient energygaugeprovidesclues to modern understandingof metabolism.CeLL Metab. L,15-25 Well-illustrated,intermediate-levelreview.

Westerhoff, H,V., Hofmey'r, J.-H.S., & Kholodenko, B.N. (1994) Getting to the inside of cells using metaboliccontrol analysis.Bzophgs Chem 60,273-283. Coordinated Regulation of Glycolysis and Gluconeogenesis Armoni, M., Harel, C., & Karnieli, E. (2007) T[anscriptional regulationof the GLUT4gene:from PPAR-gamma and FOXOI to FFA and inflammation.Tfends Endocrinol Metab 18, 100-107. Barthel, A,, Schmoll, D., & Unterman, T.G. (2005) FoxO proteins in insulin action and metabolism.Tiends Endocrino| Metab 16, 183-189. Intermediate-levelrevrewof the transcriptionfactor'seffectson carbohydratemetabolism. Brady, M.J., Pessin, J,8,, & Saltiel, A.B. (1999)Spatialcompartmentalizationin the regulationof giucosemetabolismby insulin. Ttends Endocri,nol Metab 10, 408-413. Intermediate-1evel review. Carling, D. (2004) The AMP-activatedprotein kinasecascade-a unifying systemfor energycontrol. Tlends Bi,ochem Sci,.29, 78-24 intermediatelevel review of AMPK and its role in energy metabolism de la Iglesia, N., Mukhtar, M., Seoane, J., Guinovart, J.J., & Agius, L. (2000) The role of the regulatoryprotein of glucokinasein the glucosesensorymechanismof the hepatocyte.J. Bi,ol Chem, 275, 10,597-10,603 Report of the experimentaldeterminationof the flux control coefflcientsfor glucokinaseand the glucokinaseregulatoryprotein in hepatocytes Dean, L. & McEntyre, J. (2004) The GenetzcLandscape oJ Dza,betes, NationalCenterfor BiotechnologyInformation, gov/books/bvfcgi?rid=diabetes. www.ncbi.nim.nih. An excellent,highly readable,downloadablebook (free) It includesan introduction to diabetes,a history of studiesof diabetes,and chapterson the geneticfactorsin IDDM, NIDDM, and MODY Desverne,8., Michalik, L., & Wahli, W (2006) Tianscriptional regulationof metabolism Phgsi,ol Reu 86,465-514 Extensive,advancedreview of transcriptionfactors,including those that regulatecarbohydrateand fat metabolism Hardie, D.G. (2007) AMP-activatedprotein kinaseas a drug target Annu Reu PhaymacoL Tori,col.47, 185-210 Advancedreview,with emphasison the possiblerole of this enz).rnein t5,peII diabetes. Herma.n, M.A. & Kahn, B.B. (2006) Glucosetransport and sensing rn the maintenanceof glucosehomeostasisand metabolicharmony. J. Clin Inuest 116,1767-1775. Beautifullyillustrated,intermediate-levelreview. Hers, H.G. & Van Schaftingen, E. (1982)Fructose2,6-bisphosphate 2 yearsa"fterits discovery.Briochem J. 2O6,1-12.

Jorgensen, S.8., Richter, E.A., & IVojtaszewski, J.F.P, (2006) Role of AMPK in skeletalmusclemetabolicregulationand adaptation in relationto exercise.J. PhEsioI 674,17-3I

Long, Y.C. & Zierath, J.R. (2006) AMP-activatedprotein kinase signalingin metabolicregulation.J. Cli,n Inuest l16, 1776-1783. Advanced,short review of AMPK role in metaboiism,including data on knockout mice Nordlie, R.C., Foste4 J.D., & Lange, A.J. (1999)Regulationof glucoseproduction by the hver.Annu Reu Nutr. f 9, 379-406. Advancedreview. Okar, D.A., Manzano, A., Navarro-Sabate, A., Riera, L., Bartrons, R., & Lange, A.J. (2001)PFK-2/TBPase-2: makerand breaker of the essentialbiofactor fructose-2,6-bisphosphaleT?ends B'inchem Scz.26, 30-35. Pilkis, S.J. & Granner, D.K. (1992) Molecularphysiologyof the regulationof hepaticgluconeogenesis and glycolysis.Annu Reu Physiol.64, 885-909. Postic, C., Dentin, R., Denechaud,P.-D.,& Girard, J. (2007) ChREBRa transcriptionalregulatorof glucoseand lipid metabolism. Annu Reu Nutr 27,179-192. Advancedreview of the role of transcriptionfactor ChREBPin carbohydratemetabolism. Schirmer, T. & Evans, P.R. (1990) Structural basisof the allosteric behaviorof phosphofructokinase. Nafzre 343, 740-l4b Towle, H.C. (2005) Glucoseas a regulator of eukaryoticgenetranscription Ttends Endocrinol Metab 16, 489494 Intermedlatelevel review. Towler, M.C. & Hardie, D.G. (2007) AMP-activatedprotein kinase in metaboliccontrol and insulin signaling.Ci,rc. Res.100, 328-341. van Shaftingen, E. & Gerin, I. (2002) The glucose-6-phosphatase sysLem. Bi,ochem J. 362, 513-532. Veech, R.L. (2003) A humble hexosemonophosphatepathway metaboliteregulatesshort- and long-term control of lipogenesis. Proc Natl Acad Sci, US,A100,5578-5580 Short review of the work from K. Uyeda'slaboratoryon the role of xylulose5-phosphatein carbohydrateand fat metabolism;Uyeda's papersare cited in this rer,rew. Yamada, K. & Noguchi, T. (i999) Nutrient and hormonairegulation of pyruvatekinasegeneexpression. Bzochem J. 337, 1-11. The Metabolism

of Glycogen in Animals

Whelan, WJ. (i976) On the origin of primer for glycogensynthesis Tlends Biochem Sci L, 13-75. Intermediatereview of the discovery,propertiesand role of glycogenin Coordinated Regulation of Glycogen Synthesis and Breakdown Aiston, S., Hampson, L., Gomez-Foix, A.M., Guinovart, J.J., & Agius, L. (2001) Hepatlcglycogensynthestsis highly sensitiveto phosphorylaseactivity: evidencefrom metaboliccontrol analysis J. Btol. Chem 276,23,858-23,866. Jope, R.S. & Johnson, G.V.W.(2004) The glamourand gloom of glycogenslrrthasekinase-3 Ttends B,iochem Sci, 29, 95-702 Intermediatelevel,well-illustratedreview

Problems [utt

Problems l. Measurement

of Intracellular Metabolite ConcentraMeasuring the concentratlons of metabolic intermedlates in a living cell presents great experimental difflculties-usually a cell must be destroyed before metabolite concentrations can be measured Yet enz5,'rnes catalyze metabolic interconversions very rapidly, so a conunon problem associated with these t5,pes of measurements is that the findings reflect not the physiological tions

concentrations of metabolites but lhe equilibrium concentrations A reliable experimental technique requires all enzr,.,rne-catalyzed reactions to be instantaneously stopped in the intact tissue so that the metabolic intermediates do not undergo change. This objective is accomplished by rapidly compressing the tlssue between large aluminum plates cooled with liquid mtrogen (-190'C), a process called freeze-clamping. After freezing, which stops enzyne action ir-rstantly,the tissue is powdered and the enzJ,lnesare inactivated by precipitation with perchloric acid. The precipitate is removed by centrifugation, and [he clear supernatant extract ls analyzed for metabo]ites. To ca1culate intracellular concentrations, the intracellular volume is determined from the total water content of the tissue and a measurement of the extracellular volume The intracellular concentratlons of the substrates and products of the phosphofructokinase-1 reaction ln isolated rat heart tissue are given in the table below.

Metabolite Fructose6-phosphate Fructose1,6-bisphosphate ATP ADP

Concenhation(prvr)87.0 22.0 11 , 4 0 0 1,320

Source:FromWilliamson, J R (1965)Glycotytic controlmechanisms l: inhibition of glycolysis byacetate perfused andpyruvate in the isolated, rat heafi.J. Biol.Chem.240, 2308-2327. *Calculated as rrmol/mLof intracellular water

3. Effect of O2 Supply on Glycolytic Rates The regulated steps ofglycolysis in intact cells can be identifled by studying the catabolism of glucose in whole tissues or organs. For example, the glucose consumption by heart muscle can be measured by artiflcially circulating blood through an isolated intact heart and measurlng the concentration of glucose before and after the blood passes through the heart If the circulating blood ls deoxygenated, heart muscle consumes glucose at a steady rate When oxygen is added to the blood, the rate of glucose consumption drops dramatically, then ls maintained at lhe new, lower rate Explain 4. Regulation of PFK-I The effect of ATP on the allosteric enzyme PFK-1 is shown below. For a given concentration of fructose 6-phosphate, the PFK-1 activity increases with increasing concentrations of ATP, but a point is reached beyond which increasing the concentration of ATP inhibits the enz;'rne.

S80

S60 .t

40

i20 U

IATP] (a) Explain how ATP can be both a substrateand an inhibltor of PFK-1 How is the enzyrneregulatedby ATP? (b) In what ways is glycolysisregulatedby ATP levels? (c) The inhibition of PFK-1by ATP is diminishedwhen the ADP concentrationis high, as shown in the illustration. How can this observationbe explained?

5, Cellular Glucose Concentration The concentrationof glucosein human blood plasmais maintainedat about 5 mu The concentrationof free glucoseinside a myocyteis much (a) CalculateQ, [fructose 1,O-bisphosphate][ADP]/[fruc- lower Why is the concentrationso low in the cell? What happens to glucoseafter entry into the cell?Glucoseis administose 6-phosphatel[ATP], for the PFK-1reactionunderphysiotered intravenously as a food source in certain clinical logicalconditions situations.Giventhat the transformationof glucoseto glucose -I4.2 (b) Givena AG'ofor the PFK-1reactionof kJ/mol, 6-phosphateconsumesATP,why not administer intravenous calculatethe equilibrium constantfor this reaction. glucose6-phosphate instead? (c) Comparethe valuesof Q andKio. Is the physiological reaction near or far from equilibrium?Explain What doesthis 6. Enz5'rneActivity and Physiological Function The Z^u* experimentsuggestabout the role of PFK-1 as a regulatory of the glycogenphosphorylasefrom skeletal muscle is much enzl'rne? greaterthan the 7-u" of the sameenzlme from [ver tissue. (a) What is the physiologicalfunction of glycogenphos2. Are All Metabolic Reactions at Equilibrium? phorylasein skeletalmuscle?In liver tissue? (a) Phosphoenolpy'ruvate (PEP) is one of the two phos(b) Whv does the 7^u* of the muscle enzyrneneed to be phoryl groupdonorsln the synthesisof ATP duringglycolysis. greater than that of the liver enzyme? In humanerythrocytes,the steady-state concentrationof ATP ts 2.24 mM, that of ADP is 0 25 mu, and that of pyruvateis 7. Glycogen Phosphorylase Equilibrium Glycogenphos0 051mn. Calculatethe concentratlonof PEP at25"C, assumphorylasecatalyzesthe removalof glucosefrom glycogen.The ing that the pyruvatekinasereaction (see Fig 13-13) is at AG'ofor this reactionis 3 1 kJ/mol equilibrium in the cell. (a) Caiculatethe ratio of [P1]to [glucose1-phosphate] (b) The physiological concentrationof PEPin humanerywhen the reaction is at equilibrium. (Hint: The removal of throcyiesis 0 023mu. Comparethis with the valueobtainedin glucoseunits from glycogendoes not changethe glycogen (a) Explain the slgniflcanceof this difference. concentration.)

f

-

l i ce g u l a t i o n 4 6 1 2 , P r i n c i p loefsM e t a b oR (b) The measuredratio [P1]/[glucose l-phosphate]in myocytes under physiologicalconditions is more than 100:1. What doesthis indicate about the direction of metaboliteflow through the glycogenphosphorylasereaction in muscle? (c) S&y are the equilibrium and physiologicalratios different?What is the possiblesigniflcanceof this difference? 8. Regulation of Glycogen Phosphorylase In muscle tissue,the rate of conversionof giycogento glucose6-phosphate is determinedby the ratio of phosphorylasea (active) to phosphorylaseb (lessactive).Determinewhat happensto the rate of glycogenbreakdou'nif a muscle preparationcontaining glycogenphosphorylaseis treated with (a) phosphorylasekinaseandATP;(b) PPl; (c) epinephrine. 9. Glycogen Breakdown in Babbit Muscle The intracellular use of glucoseand glycogenis tightly regulatedat four points To comparethe regulationof glycoiysiswhen oxygenis plentiful and when it is depleted,considerthe utilizationof glucoseand glycogenby rabbit Ieg musclein two physiological settings:a resting rabbit, with low ATP demands,and a rabbit that sights its mortal enemy,the coyote, and dashesinto its burrow. For each setting, determine the relative levels (high, intermediate,or low) of AMP,ATP,citrate, and acetyl-CoAand describe how these levels affect the flow of metabolites through glycolysisby regulating speciic enz)'rnesIn periods of stress,rabbit leg muscleproducesmuch of its ATPby anaerobic glycolysis(lactate fermentation) and very little by oxidation of acetyl-CoAderivedfrom fat breakdown 10. Glycogen Breakdown in Migrating Birds Unlike the rabbit with its short dash, migratory birds require energy for extended periods of time. For example, ducks generally fly severalthousandmiles during their annual migration. The flight musclesof migratory birds have a high oxidative capacity and obtain the necessaryATP through the oxidation of acetyl-CoA(obtainedfrom fats) via the citric acid cycle Compare the regulation of muscle glycolysisduring short-term intense activity, as in the fleeing rabbit, and during extended activity, as in the migrating duck. Why must the regulation in these two settingsbe different? 11. Enzyme Defects in Carbohydrate Metabolism Summariesof four clinical casestudiesfollow.For each casedetermhe which enzyrneis defectiveand designatethe appropriate treatment, from the lists provided at the end of the problem.Justi-fyyour choices.Answer the questionscontahed in each case study. ffou may need to refer to information in Chapter14.) Case A The patlent develops vomiting and diarrhea shortly after milk ingestion A lactosetolerancetest is administered.(The patientingestsa standardamountof lactose,and the glucoseand galactoseconcentrationsof blood plasmaare measuredat intervals In individualswith normal carbohydrate metabolism,the levelsincreaseto a maximum in about t hour, then decline.)The patient'sblood glucoseand galactoseconcentrationsdo not increaseduringthe test.Why do bloodglucoseand galactoseincreaseand then decreaseduringthe test in healthy individuals?Why do they fail to rise in the patient?

CaseB The patient developsvomiting and diarrhea after ingestion of milk. His blood is found to have a low concentration of glucosebut a much higher than normal concentration of reducing sugars.The urine tests positivefor galactose.Why is the concentrationofreducing sugarin the blood high?Why doesgalactoseappearin the urine? Case C The patient complainsof painful muscle cramps when performing strenuousphysicalexercisebut hasno other symptoms A muscle biopsy indicates a muscle glycogen concentrationmuch higher than normal. Why does glycogen accumulate? CaseD The patient is lethargic,her liver is enlarged,and a biopsy of the liver showslarge amounts of excessglycogen. She also has a Iower than normal blood glucoselevel. What is the reasonfor the low blood glucosein this patient? DefectiueEnzyme (a) MusclePFK-I (b) Phosphomannose isomerase (c) Galactose1-phosphateuridylyltransferase (d) Liver glycogenphosphorylase (e) Ttiose kinase (f ) Lactasein intestinal mucosa (g) Maltasein intestinai mucosa (h) Muscledebranchingenzy'rne Tteatmnnt 1. Jogging5 km eachday 2 Fat-free diet 3. Low-lactosediet 4. Avoidingstrenuousexercise 5. Largedosesof niacin (the precursorof NAD') 6. Frequent feedings (smaller portions) of a normal diet 12. Effects of Insufficient Insulin in a Person with Diabetes A man with insulin-dependent diabetes is brought to the emergencyroom in a near-comatose state.While vacationingin an isolatedplace,he lost his insulin medicationand has not taken any insulin for two days. (a) For each tissue listed below, is each pathway faster, slower,or unchangedin this patient, comparedwith the normal level when he is getting appropriate amounts of insulin? (b) For eachpathway,describeat least one control mechanismresponsiblefor the changeyou predict ?issue and Pathways 1. Adipose:fatty acid sSmthesis 2. Muscle: glycolysis;fatty acid synthesis;glycogen slmthesis glycogen synthesis; 3 Liver: glycolysis;gluconeogenesis; fatty acid synthesis;pentosephosphatepathway 13. Blood Metabolites in Insulin Insufficiency For the patient describedin Problem 12, predict the levels of the following metaboiites in his blood bejore ireatment in the emergency room, relative to levels maintained during adequate insulin treatment: (a) glucose; (b) ketone bodies; (c) free fatty acids. 14. Metabolic Effects of Mutant Enz5rrnes Predict and explain the effect on glycogen metabolism of each of the foliowing

Data A n a l y sPi sr o b l e mlsa t f defectscausedby mutation: (a) loss of the cAMP-bindingsite on the regulatorysubunitof protein kinaseA (PKA); (b) lossof the proteh phosphataseinhibitor (inhibitor 1 in Fig. 1b-40); (c) overexpressionof phosphorylaseb kinasein iiver; (d) defective glucagonreceptorsin liver 15. Hormonal Control of Metabolic Fuel Between your eveningmeal and breakfast,your blood glucosedrops and your liver becomesa net producerrather than consumerof glucose.Describethe hormonalbasisfor this switch,and explain how the hormonalchangetriggersglucoseproduction by the liver. 16. Altered Metabolism in Genetically Manipulated Mice Researcherscan manipulate the genes of a mouse so that a singlegenein a singletissueeither producesan inactive proteln (a "knockout"mouse) or producesa protein that is always (constitutively) active. What effects on metabolism would you predict for mice with the following geneticchanges: (a) knockout of glycogen debranching enzyrnein the liver; (b) knockout of hexokinaseIV in liver; (c) knockout of FBPase-2in liver; (d) constitutivelyactiveFBPase-2in liver; (e) constitutively active AMPK in muscle; (f) constitutively active ChREBPin liver?

DataAnalysis Problem 17. Optimal Glycogen Structure Musclecells need rapid accessto large amounts of glucoseduring hear,yexercise This glucoseis storedin liver and skeletalmusclein pollrneric form as particles of glycogen. The typical glycogen particle contains about 55,000glucoseresidues (see Fig. 15-33b) Mel6ndez-Hevia, Waddell,and Shelton(1993) exploredsome theoreticalaspectsof the structureof glycogen,as described in this problem. (a) The cellular concentrationofglycogenin liver is about 0.01pu What cellularconcentrationof free glucosewouldbe required to store an equivalentamount of glucose?Why would this concentrationof free giucosepresenta problemfor the cell? Glucoseis releasedftom $ycogen by glycogenphosphorylase,an enzl'rnethat canremove$ucosemolecules,oneat a time, from one end of a $ycogenchain.Glycogenchainsare branched (seeFigs 15-26and 15-33b),and the degreeof branchmg-the number of branchesper chain-has a powerful influence on the rate at which $ycogenphosphorylase canrelease$ucose. (b) WhV would a degree of branching that was too low (i.e.,below an optimumlevel) reducethe rate of glucoserelease?(Hint: Considerthe extremecaseof no branchesin a chainof 55,000glucoseresidues.)

(c) \VhVwould a degree of branching that was too high also reduce the rate of glucose release?(Hint: Think of the physicalconstraints) Mel6ndez-Heviaand colleaguesdid a seriesof calculations and found that two branchesper chain (see Fig. 15-33b) was optimai for the constraints described above. This is what is found in glycogenstored in muscleand liver. To determine the optimum number of glucose residues per chain,Mel6ndez-Hevia and coauthorsconsideredtwo key parametersthat define the structure of a glycogenparticle: I : the number of tiers of glucosechainsin a particle (the moleculein Fig. 15-33bhas five tiers);9" : the number of glucoseresiduesin eachchain.They set out to find the valuesof t and g. that would maximize three quantities: (1) the amount of glucosestored in the particle (Gr) per unit volume; (2) the numberofunbranchedglucosechains(Cj per unit volume (i.e., number of chains in the outermost tier, readily accessibleto glycogenphosphorylase);and (3) the amount of glucose available to phosphorylasein these unbranchedchains(Gp1). (d) Show thaLCo: 2t 1. This is the number of chains availableto glycogenphosphorylasebefore the action of the debranchingenzyme. (e) Show that C1, the total number of chainsin the partic l e , i s g i v e n b y C r : 2 ' - 1 T h u sG r : Q " ( C " i : g . ( 2 t- 1 ) , the total number of glucoseresiduesin the particle. (f) Glycogenphosphorylasecannot remove glucosefrom glycogen chains that are shorter than flve glucose residues. Showthat Gpr : (Q. - 4)(2' t). This is the amountof glucosereadily availableto glycogenphosphorylase. (g) Basedon the sizeofa $ucose residueand the location of branches,the thicknessof onetier of $ycogenis 0.129"nm * 0.35nm Showthat the volume of a particle, 7", is givenby the ,| equationV": |nf(O.I2g" + 0.35)3nm3 o

Mel6ndez-Heviaand coauthorsthen determined the optimum valuesof I andg"-those that gavethe maximumvalueof a quality function, f, Ihat maximizes Gr, Co, and Gp1,while GrC oGo. minimizingVs:J : -;-.They

found that the optimum

value ofg" is independeniof I (h) Choosea value of f between 5 and 15 and find the optimum value of 9". How doesthis comparewith the g" found in liver glycogen(seeFig. 15-33bX (Hint: Youmay find it useful program.) to usea spreadsheet Reference E., Waddell,T.G., & Shelton, D.D. (1993) Mel6ndez-Hevia, Optimization of molecular design in the evolution of metabolism: the glycogen molecrle. Biochem J. 295,477483.

lf citrateis addedthe rateof respirationis often increased. . . the extra oxygen uptakeis by far greaterthan can be accountedfor by the completeoxidationof citrate. . . Sincecitric acid reactscatalytically in the tissueit is probablethat it is removedby a primaryreactionbut regenerated by a subsequentreaction. -H. A. Krebsand W. A. Johnson,articlein Enzymologia,1937

TheCitric AcidCycle (Activated 16.1 Production ofAcetyl-(oA Acetate)616 16.2 Reactions oftheCitric Acid(ycle 620 16.3 Regulation ofthe(itricAcid(yde 635 (ycle 638 16.4 lheGlyoxylate s we saw in Chapter 14, some cells obtain energy (ATP) by fermentation, breaking dov,n glucose in the absence of oxygen. For most eukaryotic cells and many bacteria, which live under aerobic conditions and oxidize their organic fuels to carbon dioxide and water, glycolysis is but the first stage in the complete oxidation of glucose. Rather than being reduced to lactate, ethanol, or some other fermentation product, the pyruvate produced by glycolysis is further oxidized to H2O and CO2 This aerobic phase of catabolism is called respiration. In the broader physiological or macroscopic sense, respiration refers to a multicellular organism's uptake of 02 and releaseof CO2.Biochemists and cell biologists, however, use the term in a narrower sense to refer to the molecular processesby which cells consume 02 and produce CO2-processes more precisely termed cellular respiration.

Cellular respiration occurs in three major stages (Fig. 16-1). In the first, organic fuel moleculesglucose,fatty acids, and some amino acids-are oxidized to yield two-carbonfragmentsin the form of the acetyl group of acetyl-coenzyme A (acetyl-CoA).In the second stage,the acetyl groups are fed into the citric acid cycle,which enz;'rnaticallyoxidizesthem to CO2;the energyreleasedis conservedin the reduced electron carriersNADH and FADH2.In the third stage of respiration,these reduced coenzymesare themselvesoxidized,givingup protons(H*) and electrons.

The electrons are transferred to O2-the final electron acceptor-via a chain of electron-carrying molecules known as the respiratory chain. In the course of electron transfer, the large amount of energy released is conserved in the form of ATP, by a process cailed oxidative phosphorylation (Chapter 19). Respiration is more complex than glycolysis and is believed to have evolved much later, after the appearance of cyanobacteria. The metabolic activities of cyanobacteria account for the rise of oxygen levels in the earth's atmosphere, a dramatic turning point in evolutionary history. We consider first the conversion of pyruvate to acetyl groups, then the entry of those groups into the acid citric acid cycle, also called the tricarboxylic (TCA) cycle or the Krebs cycle (after its discoverer, Hans Krebs). We next examine the cycle reactions and the enzymes that catalyze them. Because intermediates of the citric acid cycle are also siphoned off as biosynthetic precursors, we go on to consider some ways in which these intermediates are replenished. The citric acid cycle is a hub in metabolism, with degradative pathways leading in and ana-

bolic pathways leading out, and it is closely regulated in coordinationwith other pathways. The chapter ends with a description of the glyoxylate pathway,a metabolic sequence in some organisms that employs several of the same enzymesand reactions used in the citric acid cycle, bringing about the net synthesisof glucosefrom stored triacylglycerols.

H a n sK r e b s1, 9 0 0 - 1 9 8 1

t?tt]

616

I h e( i t r i cA c i d( y c l e

Amino acids

Fatty

Stage 2 Acetyl-CoA oxidation

(reduced e- carriers)

Stage 3 Electron transfer and oxidative phosphorylation

zn++ $o2 Hzo +' _Pl .

FIGURE 16-l Catabolismof proteins,fats, and carbohydrates in the 'l three stagesof cellular respiration.Stage : oxidationof fatty acids, glucose, andsomeaminoacidsyieldsacetyl-CoAStage 2: oxidationof acetylgroupsin the citricacidcycleincludes fourstepsin whichelectronsare abstractedStage3: electronscarriedby NADH and FADH2 (or, in bacteria,plasma are funneledinto a chain of mitochondrial membrane-bound) electroncarriers-therespiratory chain-ultr mately reducing02 to H2O.Thiselectronflow dnvesthe productionof ATP.

respiratory chain. Before entering the citric acid cycle, the carbon skeletons of sugars and fatty acids are degraded to the acetyl group of acetyl-CoA, the form in which the cycle accepts most of its fuel input. Many amino acid carbons also enter the cycle this way, although several amino acids are degraded to other cycle intermediates. Here we focus on how pyruvate, derived from glucose and other sugars by glycolysis, is oxidized to acetyl-CoA and CO2 by the pyruvate dehydrogenase (PDH) complex, a cluster of enzymes-multiple copies of each of three enzymes-located in the mitochondria of eukaryotic cells and in the cytosol of bacteria. A careful examination of this enz)rynecomplex is rewarding in several respects. The PDH complex is a classic, much-studied example of a multienz)..rnecomplex in which a series of chemical intermediates remain bound to the enzyrne molecules as a substrate is transformed into the final product. Five cofactors, four derived from vitamins, participate in the reaction mechanism. The regulation of this enzyrne complex also illustrates how a combination of covalent modification and allosteric reg-ulation results in precisely regulated flux through a metabolic step. Finally, the PDH complex is the prototype for two other important enzyme complexes: a-ketoglutarate dehydrogenase,of the citric acid cycle, and the branched-chain a-keto acid dehydrogenase, of the oxidative pathways of several amino acids (see Fig 18-28). The remarkable similarity in the protein structure, cofactor requirements, and reaction mechanisms of these three complexes doubtless reflects a common evolutionaryorigin.

Pyruvate ls0xidized taAcetyl-(oA and{02 The overall reaction catalyzed by the pyruvate dehydrogenase complex is an oxidative decarboxylation, an irreversible oxidation process in which the carboxyl group is removed from pyruvate as a molecule of CO2 and the two remaining carbons become the acetyl group of acetyl-CoA ( Fig. I tt-2 ). The NADH formed in this reaction gives up a hydride ion (:H ) to the respiratory chain (Fig. 16-1), which carries the two electrons to

orr

,ro-

C

C:O CH,

16,1Production ofAcetyl-(oA (Activated Acetate) In aerobic organisms, glucose and other sugars, fatty acids, and most amino acids are ultimately oxidized to CO2 and HzO via the citric acid cycle and the

Pyruvate

COz +

CoA-SH NAD* \

-.rPPl Ipoale, FAD

NADH 4

O*

,S-CoA C

I

CHe Acetyl-CoA AG'' - -33.4 kJ/mol

FIGU REI 6-2 Overallreactioncatalyzedby the pyruvatedehydrogenase complex.Thefivecoenzymes participating in thisreaction,andthethree enzymesthat makeup the enzymecomplex,are discussed in the text.

e cde t a t e ) 6 1 1 6 . 1P r o d u c t ioofnA c e t y l - ( (0AAc t i v a tA

Reactive thiol group

H

, HS-CH2-CHz-N B-Mercaptoethylamine

(tlI,r

I

Pantothenic acid

,\)

('\

S-CoA AcetyI-CoA

Ribose3'-phosphate

ooH I o:P-oI o

3'-Phosphoadenosinediphosphate

Coenzyme A

FIGURE 16-3 CoenzymeA (CoA).A hydroxylgroupof pantothenic acid is joinedto a modifiedADP moietyby a phosphate esterbond, and itscarboxylgroupis attached to B-mercaptoethylamine in amide linkage. The hydroxylgroupat the 3' positionof the ADP moietyhas

a phosphorylgroup not presentin free ADP.The -SH group of the moietyforms a thioesterwith acetatein acetylmercaptoethylamine (lowerleft). coenzymeA (acetyl-CoA)

oxygen or, in anaerobicmicroorganisms,to an alternative electron acceptor such as nitrate or sulfate The transfer of electrons from NADH to oxygen ultimately generates2.5 moleculesof ATP per pair of electrons. The irreversibility of the PDH complex reaction has been demonstratedby isotopic labeling experiments: the complexcannotreattachradioactivelylabeledCO2 to acetyl-CoAto yield carboxyllabeledpyruvate.

group attached to coenz;,'rne A may thus be thought of as "activated"for group transfer. The fifth cofactor of the PDH complex, lipoate (FiS. 16-4), has two thiol groups that can undergo reversibleoxidation to a disulfide bond (-S-S-), similar to that between two Cys residuesin a protein. Becauseof its capacityto undergooxidation-reduction reactions,lipoate can serveboth as an electron (hydrogen) carrier and as an acyl carrier, as we shall see.

ThePyruvate Dehydrogenase {0mplex Requires (oenzymes Five The combineddehydrogenationand decarboxylationof pyruvateto the acetylgroup of acetyl-CoA(Fig. 16-2) requires the sequentialaction of three different enzymes and five different coenzlrnes or prosthetic groups-thiamine pyrophosphate(TPP), flavin adenine dinucleotide(FAD), coenz),rne A (CoA, sometimesdenoted CoA-SH, to emphasizethe role of the -SH group), nicotinamideadeninedinucleotide(NAD), and lipoate.Four different vitaminsrequired in humannutrition are vital componentsof this system: thiamine (in TPP), riboflavin (in FAD), niacin (in NAD), and pantothenate(in CoA).Wehavealreadydescribedthe roles of FAD and NAD as electroncarriers(Chapter13), and we have encounteredTPP as the coenzS,'rne of pyruvate (seeFig. 14-14). decarboxylase Coenz;.'rne A (FiS. 16-8) hasa reactivethiol (-SH) group that is critical to the role of CoA as an acyl carrier in a numberof metabolicreactions.Acyl groupsare covalently linked to the thiol group, forming thioesters. Becauseof their relatively high standardfree energiesof hydrolysis (see Figs 13-16, 13-17), thioestershave a high acyl group transfer potential and can donate their acyl groupsto a variety of acceptormolecules.The acyl

Reduced form

Ils-cH2

O

'"""

il cHa-c-s-c{r,

.CHo

TIS-CH 9H"

Lipoic acid

Acetylated

, .CHq /-

HS-CH

CH,

Lys residue of E,

$N"c

H

Polvpeptidechainof

]t ()

n, idiihydronpoyr tr'ansacetylaiei

tIGURE16-4 Lipoicacid (lipoate)in amidelinkagewith a Lysresidue. The lipoyllysylmoiety is the prostheticgroup of dihydrolipoyl (E,of the PDHcomplex). Thelipoylgroupoccursin oxtransacetylase (dithiol) (disulfide) forms reduced and actsas a carrierof and idized both hydrogenand an acetyl(or otheracyl)group

L61B__jTheCitricAcidCycle

(onsists ThePyruvate Dehydrogenase [0mplex Distinct ofThree Enzymes The PDH complex containsthree en4rnes-pyruvate dehydrogenase (E i), dihydrolipoyl transacetylase (E2), and dihydrolipoyl dehydrogenase (E3)-each presentin multiplecopies.The numberof copiesof each enzymeand therefore the size of the complex varies amongspecies.The PDH complexisolatedfrom mammals is about 50 nm in diameter-more than five times the sizeof an entireribosomeand big enoughto be visualizedwith the electron microscope(Fig. 16-5a). In the bovineenzyrne,60 identicalcopiesof E2form a pentagonal dodecahedron(the core) with a diameter of about25 nm (Fig. 16-5b). (The coreof theEscheri,chi,a col'i enzymecontains24 copiesof E2.) E2is the point of connectionfor the prosthetic group lipoate, attached through an amide bond to the e-amino group of a Lys residue (Fig. 16-4). E2 has three functionallydistinct domains (Fig. 16-5c): the amino-terminalli,pogl domai.n, containingthe lipoyl-Lysresidue(s);the central E1- and E3-bi,ndi,ngdoma'in; and the innercore acyltransJerase domai,n, which contains the acyltransferase active site. The yeast PDH complex has a single lipoyl domain with a Iipoate attached,but the mammalian complex has two, and E. coli, has three (Fig. 16-5c). The domainsof E2 are separatedby linkers,sequencesof 20 to 30 amrnoacid residues,rich in Ala and Pro and interspersedwith chargedresidues;theselinkers tend to assumetheir extendedforms, holding the three domainsapart. The active site of E1 has bound TPP, and that of E3 has bound FAD. Also part of the complex are two

FIGURE 16-5 The pyruvatedehydrogenase complex.(a) Cryoelectron micrograph of PDHcomplexesisolatedfrom bovinekidney In cryoelectron microscopy, biologicalsamplesare viewedat extremelylow temperatures; this avoidspotentialartifactsintroducedby the usualprocess of dehydrating, fixing,and staining.(b) Three-dimensional imageof PDH complex,showingthe subunitstructure:E1,pyruvatedehydrogenase;E2,dihydrolipoyltransacetylase; and Er,dihydrolipoyldehydrogenase.Thisimageisreconstructed by analysis of a largenumberof images suchasthosein (a),combinedwith crystallographic studiesof individual subunits. Thecore(green)consists of 60 moleculesof Er,arrangedin 20 trimersto form a pentagonal dodecahedron. Thelipoyldomainof E2 (blue)reaches outwardto touchthe activesitesof E.,molecules(yellow) arrangedon the E2core SeveralE3subunits(red)arealsoboundto the core,wherethe swingingarm on E2can reachtheir activesitesAn asteriskmarksthe sitewherea lipoyl group is attachedto the lipoyl domainof E2.To makethe structure clearer,abouthalfof the complexhas beencut awayfrom the front Thismodelwas preparedby Z. H. Zhou (2001);in anothermodel,proposed andcolleagues by J. L. S.Milneand (2002),the E, subunitsare locatedmoretowardthe periphcolleagues (c) E2consists ery (seeFurtherReading). ofthreetypesofdomainslinked by shortpolypeptidelinkers:a catalyticacyltransferase domain;a bindingdomain,involvedin thebindingof E, to E''andE3;andoneor more (depending on the species) lipoyldomains.

, 50nm ,

Number of lipoyl domains varies by species. E. coli

(3)

Mammals

(2)

Yeast

\\ L

N-i

FIexibIe polypeptide Iinker

r-

\

Binding domain (involvedin E2-E1 and E2-Eg Lipoyl domain (c)

Acyltransferase domain (inner core)

(Activated Acetate) 0fA(etyl-coA l6.l Production [or s l regulatory proteins, a protein kinase and a phosphoprotein phosphatase, discussed below. This basic E1E2-E3 structure has been conserved during evolution and used in a number of similar metabolic reactions, including the oxidation of a-ketoglutarate in the citric acid cycle (described below) and the oxidation of aketo acids derived from the breakdown of the branched-chain amino acids valine, isoleucine, and leucine (see F ig. 1B-28). Within a given species,E3 of PDH is identical to E3 of the other two enzyme complexes. The attachment of Iipoate to the end of a Lys side chain in E2 produces a long, flexible arm that can move from the active site of E1 to the active sites of E2 and E3, a distance of perhaps 5 nm or more.

(hanneling, In5ubstrate Intermediates Never leave theEnzyme Surface Figure 16-6 shows schematicallyhow the pyruvate dehydrogenasecomplex carries out the five consecutive reactions in the decarboxylationand dehydrogenationofpyruvate. StepO is essentiallyidenticalto the reaction catalyzedby pyruvatedecarboxylase(see Fig. 14-14c);C-1 of pyruvateis releasedas CO2,and C-2, which in pyruvate has the oxidation state of an aldehyde,is attachedto TPP as a hydroxyethylgroup. This first step is the slowest and therefore limits the rate of the overall reaction.It is also the point at which the PDH complexexercisesits substratespeciflcity. In step @ the hydroxyethyl group is oxidized to the level of a carboxylicacid (acetate).The two electrons

? //o cH3-c-c\^

dl*

of a Iipoyl removedin this reactionreducethe -S-Sgroup on E2 to two thiol (-SH) groups. The acetyl moiety produced in this oxidation-reductionreaction is first esterifiedto one of the lipoyl -SH groups,then transesterifiedto CoA to form acetyl-CoA(step @). Thus the energyof oxidationdrivesthe formation of a high-energythioester of acetate.The remainingreactions catalyzedby the PDH complex(by E3,in steps@ and @) are electrontransfersnecessaryto regenerate the oxidized (disulfide)form of the lipoyl group of E2 to prepare the enzymecomplex for another round of oxidation. The electrons removed from the hydroxyethyl group derived from pyruvate pass through FAD to NAD+. Centralto the mechanismof the PDH complexare the swinginglipoyllysyl arms of E2, which accept from E1 the two electronsand the acetylgroup derivedfrom pyruvate,passingthem to E3. All these enzymesand coenz).rnesare clustered,allowing the intermediatesto react quickly without diffusing awayfrom the surfaceof the enzymecomplex.The flve-reactionsequenceshoum in Figure 16-6 is thus an example of substrate channeling. The intermediatesof the multistep sequence neverleavethe complex,and the local concentrationof the substrate of E2 is kept very high. Channelingalso preventstheft of the activatedacetyl group by other enz).rnesthat use this group as substrate.As we shall see,a similar tethering mechanismfor the charmelingof substratebetweenactivesitesis usedin someother enzymes,with lipoate,biotin, or a CoA-likemoiety serving as cofactors,

^ CoA-SH

o I

CH3-C-S-CoA

()

)^t;; NADH + H+

TPP

NAD+ Pyruvate dehydrogenase, Er

Dihydrolipoyl transacetylase, E2

tIGURE 16-6 Oxidativedecarboxylation of pyruvateto acetyl-CoAby the PDH complex.Thefateof pyruvateistracedin red.ln step@ pyru(TPP)of pyruvate vate reactswith the bound thiaminepyrophosphate (E1),undergoing to the hydroxyethyl dehydrogenase decarboxylation (seeFig. 14-14) Pyruvate derivative dehydrogenase alsocarriesout step@, the transferof two electronsand the acetylgroupfromTPPto the oxidizedform of the lipoyllysylgroupof the core enzyme,dihy(Er),to form the acetylthioesterof the reduced drolipoyltransacetylase in whichth" -SH qrottn Iipoylgroup Step@ is a transesterification

Dihydrolipoyl dehydrogenase, E3

of CoA replacesthe -SH groupof E2to yield acetyl-CoAand the fully reduced(dithiol)form of the lipoyl group.In step @ dihydrolipoyl (E3)promotestransferof two hydrogenatomsfrom the dehydrogenase groups of E, to the FADprostheticgroupof E3,restoring lipoyl reduced groupof E2.In step@ the reduced the oxidizedformof the lipoyllysyl a hydrideion to NAD*, formingNADH. The FADH2of E3transfers enzymecomplexis now readyfor anothercatalyticcycle.(Subunit to thosein Fi8.16-5b.) colorscorrespond

l%rtl

IheCitric Acid Cycle

As one might predict, mutationsin the genesfor the subunits of the PDH complex, or a dietary thiaminedeficiency,canhavesevereconsequences. Thiamine-deflcientanimals are unable to oxidize pytuvate normally. This is of particular importance to the brain, which usually obtainsall its energyfrom the aerobicoxidation of glucosein a pathwaythat necessarilyincludes the oxidation of pyruvate.Beriberi, a diseasethat results from thiamine deflciency,is characterizedby loss of neuralfunction.This diseaseoccursprimarilyin populations that rely on a diet consistingmainly of white (polished) rice, which lacks the hulls in which most of the thiamine of rice is found. Peoplewho habitually consume large amounts of alcohol can also develop thiamine deflciency,becausemuch of their dietary intake consistsof the vitamin-free "empty calories"of distilled spirits. An elevatedlevel of pyruvate in the blood is often an indicator of defects in pytuvate oxidation due to oneofthesecauses. r

S U M M A R1Y6 . 1 P r o d u c t i oonf A c e t y l - C o A ( A c t i v a t eAdc e t a t e ) r

Pyruvate,the product ofglycolysis,is converted to acetyl-CoA,the starting material for the citric acid cycle,by the pyruvate dehydrogenase complex.

r

The PDH complexis composedof multiple copies pyruvate dehydrogenase,E 1 of three enz),Tnes: (with its bound cofactorTPP); dihydrolipoyl transacetylase,E2 (with its covalentlybound lipoyl group); and dihydrolipoyldehydrogenase, E3 (with its cofactorsFAD and NAD),

r

Er catalyzesflrst the decarboxylationof pyruvate, producing hydroxyethyl-TPP,and then the oxidation of the hydroxyethyl group to an acetyl group.The electronsfrom this oxidationreduce the disulfideof lipoateboundto E2,and the acetyl group is transferredinto thioester linkagewith one -SH group of reducedlipoate.

r

Ez catalyzesthe transfer of the acetyl group to coenzpe A, formingacetyl-CoA.

r

Ea catalyzesthe regenerationofthe disulflde (oxidized)form oflipoate; electronspassfirst to FAD,then to NAD+.

r

The long lipoyllysyl arm swingsfrom the active site of E1to E2to E3,tetheringthe intermediates to the enzlme complexto allow substrate channeling.

r

The organizationof the PDH complexis very similar to that of the enzymecomplexesthat catalyzethe oxidation of a-ketoglutarateand the branched-chain a-keto acids.

16.2Reactions oftheCitric Acid(ycle We are now readyto trace the processby which acetylCoA undergoesoxidation. This chemical transformation is carried out by the citric acid cycle, the first cgcli,cpathwaywe have encountered(FiS. 16-7). To begin a turn of the cycle,acetyl-CoAdonatesits acetyl group to the four-carbon compound oxaloacetateto form the six-carboncitrate.Citrateis then transformed into isocitrate,alsoa six-carbonmolecule,which is dehydrogenatedwith loss of CO2to yield the five-carbon compounda-ketoglutarate(also called oxoglutarate). a-Ketoglutarateundergoesloss of a secondmolecule of CO2and ultimatelyyieldsthe four-carboncompound succinate.Succinateis then enzymaticallyconverted in three steps into the four-carbon oxaloacetatewhich is then ready to react with another moleculeof acetyl-CoA.In eachturn of the cycle,one acetyl group (two carbons)entersas acetyl-CoAand two molecules of CO2leave;one moleculeof oxaloacetateis used to form citrate and one molecule of oxaloacetateis regenerated.No net removalof oxaloacetateoccurs;one moleculeof oxaloacetatecan theoreticallybring about oxidation of an infinite number of acetyl groups,and, in fact, oxaloacetateis presentin cellsin very low concentrations.Four of the eight stepsin this processare oxidations,in which the energyof oxidationis very efficiently conservedin the form of the reduced coenzymesNADH and FADH2. As noted earlier, although the citric acid cycle is central to energy-yreldingmetabolismits role is not limited to energyconservation.Four- and flve-carbonintermediatesof the cycle serve as precursorsfor a wide variety of products.To replaceintermediatesremoved for this purpose,cells employanaplerotic(replenishing) reactions,which are describedbelow. Eugene Kennedy and Albert Lehninger showed in 1948that, in eukaryotes,the entire set of reactionsof the citric acid cycletakesplacein mitochondria.Isolatedmitochondria were found to contain not only all the enz),'rnesand coenz;rmesrequired for the citric acid cycle, but also all the enz).rnesand proteins necessaryfor the last stageof respiration-electron transferand ATP synthesis by oxidative phosphorylation.As we shall see in later chapters,mitochondriaalsocontainthe enz;rmesfor the oxidation of fatty acids and some amino acids to acetyl-CoA,and the oxidativedegradationof other amino acids to a-ketoglutarate,succinyl-CoA,or oxaloacetate. Thus, in nonphotos;.'ntheticeukaryotes,the mitochondrion is the siteof mostenergy-yielding oxidativereactions and of the coupledsy-rthesisof AIP. In photosyntheticeukaryotes,mitochondriaare the major site of ATP production in the dark,but in dayffit chloroplastsproducemost of the organism's ATP.In mostbacteria,the erzS..rnes of the citric acid cycle are in the cy'tosol,and the plasmamembraneplaysa role analogousto that of the inner mitochondrial membranein AIP slmthesis(Chapter19).

1 6 . 2R e a c t i oonftsh e( i t r i cA c i dC t l c l e

[rrl

AcetyI-CoA

e

o tl

Cendensation

CH3+S*g-9o6

HO-

O:C-COO-

I

fiIxusCIoCI'ri

cH2-cooOxaloacetate nalate dehydrogcnase

cooI

HO-CH

I

Malate

CHz I

coo

coo I

CH

H-C-COO-

Fumarate

I I coo-

HC

Isocitrate

HO-C-H

I coo succinatc dch,ydlogcnascr

rsocrtrate dehydrogenase

@

@ Oxidative decarboxylation

Dehydrogenation

C

I

tr-ketoglutarate dc.hydrogenase compk:x

succinll-(loA synthetase

C C Succinate

Coz

CHO

tc:o I coo a-Ketoglutarate

CoA-SH GTP (ATP)

l-

C-S-CoA

GDP (ADP)

o Succinyl-CoA

tPi lo, Substrate-level phosphorylation

FIGURE 15-7 Reactionsof the citric acid cvcle.The carbon atoms shadedin pink arethosederivedfrom the acetateof acetyl-CoAin the firstturn of the cycle;thesearenot the carbonsreleasedas CO2 in the first turn. Note that in succinateand fumarate,the two-carbongroup derivedfrom acetatecan no longerbe specificallydenoted;because succinateand fumarateare symmetricmolecules,C-1 and C-2 are indistinguishable from C-4 and C-3. The numberbesideeach reaction

(ycleHasEight TheCtricAcid Steps In examiningthe eightsuccessive reactionstepsof the citric acid cycle,we placespecialemphasison the chemical

Coz

@

.'... ..&i.darivb 4th*'bd*Yti,iislr to a numberedheadingon pages622-628.The red stepcorresponds arrowsshowwhereenergyis conservedby electrontransferto FADor NAD+, formingFADH2or NADH + H+. Stepse, @, and @ are The in the cell; all otherstepsare reversible. irreversible essentially productof step @ may be eitherATPor CTP,dependingon which isozymeis the catalyst. succinyl-CoAsynthetase

transformations taking place as citrute formed from acetyl-CoAand oxaloacetateis oxidizedto yield CO2and the energyof this oxidationis conservedin the reduced coenz).rnesNADH and FADH2.

The(itricAcid cycte

3rl

@ Formation of Citrate The flrst reaction of the cycle is the condensationof acetyl-CoAwith oxaloacetate to form citrate, catalyzedby citrate synthase:

CH.-C -\

(a) Acetyl-CoA analog Oxaloacetate

//,o

//,o

S-CoA Acetyl-CoA

+

o:c-coo I

fH'-cr

HrO

Io

HO-C-COO I cH2-coo Citrate

cH2-coo-

Oxaloacetate

AG'' -

32.2kJ/rnol

In this reaction the methyl carbon of the acetyl group is joined to the carbonyl group (C-2) of oxaloacetate.CitroylCoA is a transient intermediate formed on the active site of the enz;'rne (see F1g.16-9) It rapidly undergoeshydrolysis to free CoA and citrate, which are released from the active site. The hydrolysis of this tugh-energ/ thioester intermediate makes the forward reaction highly exergonic. The large, negative standard free-energy change of the citrate symthase reaction is essentialto the operation of the cycle because,as noted earlier, the concentration of oxaloacetateis normally very low The CoA liberated in this reaction is recycled to participate in the oxidative decarboxylation of another molecule of pyruvate by the PDH complex. Citrate synthase from mitochondria has been crystallrzed and visualized by x-ray diffraction in tlie presence and absence of its substrates and ilrhibitors (FiS. 16-3). Each subunit of the homodimeric enzyrne is a single polypeptide with two domains, one large and rigid, the other smaller and more flexible, with the active site between them. Oxaloacetate, the first substrate to bind to the erzy.rne,induces a large conformational change in the flexible domain, creating a binding site for the second substrate, acetyl-CoA. When citroyl-CoA has formed ur the enz),rre active site, another conformational change br4gs about thioester hydrolysis, releasing CoA-SH. This induced fit of the enz;.rnefirst to its substrate and then to its reaction intermediate decreases the likelihood of premature and unproductive cleavage of the thioester bond of acetyl-CoA. Kinetic studies of the erz;.'rne are consistent with this ordered bisubstrate mechanism (see Fig. 6-lg). The reaction catalyzed by citrate s;mthase is essentially a Claisen condensation (p. 497), involving a thioester (acetyl-CoA) and a ketone (oxaloacetate) (Fig. 16-9). @ Formation of Isocitrate via cis-Aconitate The enzyme aconitase (more formally, aconitate hydratase) catalyzes the reversible transformation of citrate to isocitrate, through the intermediary formation of the tricarboxylic acid cis-aconitate, which normally does not dissociate from the active site. Aconitase can promote the reversible addition of H2O to the double bond of enzyme-bound ces-aconitate in two different ways, one leading to citrate and the other to isocitrate:

E $ $ t $ q. E E

E (b)

F F E E

€'

tIGURE16-8 Structureof citratesynthase. The flexibledomainof each subunitundergoes a largeconformational changeon binding oxaloacetate, creatinga bindingsitefor acetyl-CoA.(a) Open form of the enzymealone(PDBlD 5CSC);(b) closedform with boundoxaloacetate and a stableanalogof acetyl-CoA(carboxymethyl-CoA) (derivedfrom PDB lD SCTS).In theserepresentations one subunitis coloredtan and one qreen

cis-Aconitate CHO-COO

t-

H-C-COO-

I I coo-

HO-C-H

Isocitrate AG'" = 13.3 kJ/mol

Acid Cycle[tr-l 0ftheCitric 16.2Reactions Although the equilibrium mixture at pH 7.4 and 25'C containslessthan 10%isocitrate,in the cell the reaction is pulled to the right because isocitrate is rapidly consumed in the next step of the cycle, Iowering its steady-stateconcentration.Aconitasecontainsan ironsulfur eenter (Fig. f6-f0), which acts both in the binding of the substrateat the active site and in the catalfiic addition or removalof H2O.In iron-depletedcells, aconitaselosesits iron-sulfur center and acquiresa new role in the regulation of iron homeostasis.Aconitaseis one of many enzlmes known to "moonlight" in a second role (Box 16-1). @ Oxiaation of Isocitrate to a-Ketoglutarate and COz In the next step, isoeitrate dehydrogenase on ofisocitrate to form catalyzesoxidativ Mnz* in the active site c-ketogluta,rate group of the intermediate carbonyl with the interacts but doesnot transiently which is formed oxalosuccinate, converts it until decarboxylation site binding Ieavethe formed the enol stabilizes Mn2* also to a-ketoglutarate. transiently by decarboxYlation. There are two djfferent forms of isocitrate dehydrogenasein all cells,one requiringNAD+ as electronacceptor

citrate In themammalian 16-9 Citratesynthase' FIGURE MEffAtllSM binds first, in a strictlyorderedreacsynthasereaction,oxaloacetate a conformationchangethat opens triggers This binding tion sequence. is specificallyoriOxaloacetetate for acetyl-CoA. site up the binding entedin the activesiteof citratesynthaseby interactionof its two carboxylateswith two positivelychargedArg residues(not shown here). Citrate SynthaseMechanism

H

H

Citrate

The iron-sulfurcenteris 16-10 lron-sulfurcenterin aconitase. FIGURE in red,the citratemoleculein blue.ThreeCys residuesof the enzyme bind threeiron atoms;the fourth iron is boundto one of the carboxyl groupsof citrate and also interactsnoncovalentlywith a hydroxyl group of citrate (dashedbond)' A basic residue(:B) in the enzyme helpsto positionthe citratein the activesite.The iron-sulfurcenteracts Thegeneralpropertiesof ironbindingand catalysis. in both substrate 19 (seeFi8.19-5)' in Chapter discussed sulfurproteinsare

I h eC i t r iA c c i dC y c l e

[.'{

The "one gene-one enzyrne"dictum, put forward by GeorgeBeadleand Edward Tatum in 1940 (see Chapter 24), went unchallengedfor much of the twentieth century as did the associated assumptionthat eachprotein had only one role. But in recent years,many striking exceptions to this simple formula have been discovered-casesin which a singleprotein encodedby a singlegeneclearlydoesmore than onejob in the cell. Aconitaseis one such protein: it acts both as an enzyrne and as a regulator of protein sy'nthesis. Eukaryoticcellshavetwo isozymesof aconitase.The mitochondrialisoz;'rneconvertscitrate to isocitratein the citric acid cycle. The cytosolicisozSrme has two distinct ftmctions.It catalyzesthe conversionof citrate to isocitrate, providingthe substratefor a cytosolicisocitratedehydrogenasethat generatesNADPH as reducing power for fatty acid sy-rthesisand other anabolicprocessesin the cytosol.It alsohas a role in cellulariron homeostasis. All cellsmust obtainiron for the activity of the many proteins that requrreit as a cofactor.In humans,severe iron deflciencyresults in anemia,an insufflcient supply of efihrocytes and a reduced oxygen-carryingcapacity that can be life-threatening.Too much iron is alsoharmful: it accumulatesin and damagesthe liver in hemochromatosisand other diseases.Iron obtainedin the diet is carried in the blood by the protein transferrin and enters cells via endocy'tosismediated by the transferrin receptor. Onceinside cells,iron is usedin the synthesis of hemes,cytochromes,Fe-S proteins, and other I.e-

dependentproteins,and excessiron is storedbound to the protein ferritin. The levelsof transferrin,transferrin receptor,and ferritin are thereforecrucialto cellulariron homeostasis.The synthesisof thesethree proteinsis regulated in responseto iron availability-and aconitase,in its "moonlighting"job, plays a key regulatoryrole. Aconitasehas an essentialFe-S cluster at its active site (seeFrg. 16-10). When a cell is depletedof iron, this Fe-S cluster is disassembledand the erz;'rne loses its aconitaseactivity. But the apoenz],'me(apo-aconitase, lacking its Fe-S cluster) so formed has now acquiredits secondactivity-the ability to bind to specificsequences in the mRNAs for the transferrin receptor and ferritin, thus regulatingprotein synthesisat the translationallevel. TWoiron regulatory proteins, IRPI and IRP2, wereindependentlydiscoveredas regulatorsof iron metabolism. As it turned out, IRP1 is identicalto cytosolicapo-aconitase,and IRP2is very closelyrelatedto IRPI in structure andfunction,but unlikeIRP1it cannotbe convertedto en4nnaticallyactiveaconitase.Both IRP1 and IRP2bind to regionsin the mRNAsencodingferrith and the transferrin receptor,with effectson iron mobiljzationandiron uptake. These mRNA sequencesare part of hairpin structures (p. 285) callediron response elements (IREs), located at the 5' and 3' endsof the mRNAs(Flg. 1). Whenbound Low [iron]

High [iron]

@:

@t;)

IRP bound to iron response element (IRE)?

f]

Ferritin mRNA Ferritin mRNA translation Ferritin synthesis

*;*-----\^r4u

FIGURE 1 Effectof tRpt and tRp2on the mRNAsfor ferritinand the transferrin receptor.

coo I CHr- o H-C-C

lo-

HO-C-H i C

/\

ooIsocitrate

Repressed Decreased

Activated Increased

Transferrin receptor

,

''-*

'*'"fiil.*lH:g llf|$l1l-_AAA(A,zs, iilffi::: B::ffi::$ coo-

ls(x llt ttc (teD!a1 i ogrrnlts(,

CHz

coz

I

H-

H-C-H

I

\t/

Isocitrate is oxidized by hydride transfer to NAD+ or NADP+ (depending on the isocitrate dehydrogenase isozyme).

cooI

I

NAD(P)*NAD(P)H+H+ \,,

{

Jto-'M,,,*

Oxalosuccinate

MICHANISM tlGURt16*11 lsocitratedehydrogenase. In this reaction, the substrate,isocitrate,losesone carbon by oxidativedecarboxyla_

\e)

QJI

c :o (1

Decarboxylation

is facilitated by electron withdrawal by bound Mn2+.

Rearrangement of the enol intermediate generaf,eS o-ketoglutarate.

//"'r O Oa_Ketoglutarate

tion. See Fig. 14-13 for more information on hydride transfer reactions involving NAD* and NADP+.

c c i dC y c l e[trt] 16 . 2R e a c t i oonftsh eC i t r iA

to the 5'-untranslatedIRE sequencein the ferritin mRNA, IRPs block ferritin synthesis;when bound to the 3'untranslatedIRE sequencesin the transferrin receptor mRNA, they stabilize the mRNA, preventing its degradation and thus allowingthe synthesisof more copiesof the receptorprotein per mRNAmolecule.So,in iron-deflcient cells,iron uptakebecomesmore efflcientand iron storage (boundto ferritin) is reduced.Whencellulariron concentrations return to normal levels, IRPI is converted to aconitase,and IRP2 undergoesproteolytic degradation, endingthe low-ironresponse. The enzS.rnatically active aconitaseand the moonlighting, regulatory apo-aconitasehave different structures.As the activeaconitase,the protein hastwo lobes that close around the Fe-S cluster; as IRPI, the two Iobesopen, exposrngthe mRNA-bindingsite (Fig. 2). Aconitase is just one of a growing list of enzyrnes known (or believed)to moonlightin a secondrole. Many of the glycolytic enzyrnesare included in this group. Pyruvatekinaseacts in the nucleusto regulatethe transcription of genes that respond to thyroid hormone. Glyceraldehyde3-phosphatedehydrogenasemoonlights both as uracil DNA glycosylase,effecting the repair of damagedDNA, and as a regulator of histone H2B transcription. The crystallins in the lens of the vertebrate eye are severalmoonlightingglycolytic enzymes,including phosphoglyceratekinase, triose phosphate isomerase,and lactatedehydrogenase. Until recently,the discoverythat a protein hasmore than one function was largely a matter of serendipity: two groups of investigatorsstudying two unrelated questions discoveredthat "their" proteins had sirrrilar properties,comparedthem carefully,and found them to be identical. With the growth of annotated protein and DNA sequencedatabases, researcherscan now deliberately look for moonlighting proteins by searching the databasesfor any other protein with the samesequence as the one under study, but with a different function. This also meansthat in the databases.a orotein amo-

tated ashavinga given function doesn'tnecessarilyhave only that function. Protein moonlighting may also explain some pvzling findings: experiments in which a protein with a known function is madeinactive by a mutation, and the resulting mutant organismsshow a phenotype with no obviousrelation to that function.

and the other requiringNADP+.The overallreactionsare otherwiseidentical.In eukaryoticcells,the NAD-dependent eru;.'rneoccursin the mitochondrial matrix and serves in the citric acid cycle.The mainfunctionof the NADP-dependent enz)rrne,found in both the mitochondnalmatrix and the cytosol,may be the generationof NADPH,which is essentialfor reductiveanabolicreactions.

acceptorand CoA as the carrier of the succinylgroup. The energyof oxidation of a-ketoglutarateis conserved in the formation of the thioester bond of succinyl-CoA:

@ Oxiaafion of c-Ketoglutarate to Succinyl-CoA and CO2 The next stepis anotheroxidativedecarboxyIation,in which o-ketoglutarateis convertedto succinylCoA and CO2 by the action of the c-ketoglutarate dehydrogenase complex; NAD+ servesas electron

with two distinct 2 Two forms of cytosolicaconitase/lRP1 FIGURE are closedand the (a) major lobes two the In aconitase, functions. hereto Fe-Sclusteris buried;the proteinhas been madetransparent show the Fe-Scluster(PDB lD 2B3Y).(b) In lRP1,the lobesopen up, exposinga bindingsite for the mRNA hairpinof the substrate ( P D BI D 2 I P N .

cH2-cooI

NA-DH

CHr

tc- cooo a-Ketoglutarate

a-ketoglutarate dehydrogenase complex

cH2-cooQH" I C-S-CoA

+ ,CS,E1,

o Succinyl-CoA LG'o: -33.5 kJ/mol

I

626

I h e( i t r i cA c i d( y c l e

This reaction is virtually identical to the pyruvate dehydrogenasereaction discussedabove,and the aketoglutaratedehydrogenasecomplex closely resemblesthe PDH complexin both structureand function.It includesthree enzymes,homologousto E1,E2,and E3of the PDH complex,aswell as enzyme-bound TPP,bound lipoate, f'AD, NAD, and coenz;.rneA. Both complexes are certainlyderivedfrom a corrrmonevolutionaryancestor. Althoughthe E, componentsof the two complexes are structurallysimilar,their aminoacid sequencesdiffer and, of course,they have different binding speciflcities: E1 of the PDH complexbinds pyruvate,and E, of the o-ketoglutaratedehydrogenasecomplex binds aketoglutarate.The E2componentsof the two complexes are alsovery similar,both havingcovalentlybound lipoyl moieties.The subunitsof E* are identicalin the two enzymecomplexes @ Conversion of Succinyl-CoA to Succinate Succinyl-CoA, like acetyl-CoA,hasa thioesterbondwith a strongly negative standard free energy of hydrolysis (AG'' : -36 kJ/mol).In the next step of the citric acid cycle,energyreleasedrn the breakageofthis bondis used to drive the synthesisof a phosphoanhydridebond in either GTP or ATP,with a net AG'oof only -2.9 kJ/mol. Succinate is formedin the orocess:

cH"-coo-

tCH9 t-

GDP + Pi

C-S-CoA

o Succinyl-CoA

GTP CoA-SH

-/

'/

snccinr / ('rr.\ svutilcLls(,

cooI

CHo

Succinvl(lo;\ srnthctlsc

CoA-SH

Enzyme-bound succinyl phosphate

o c-cH2

cH2-c O-

Succinate

Phosphohistidyl enzyme

tlcoo CH,

Succinate

AG'' : -2.9 kJ/mol The enzyme that catalyzes this reversible reaction is called succinyl-CoA synthetase or succinic thiokinase; both names indicate the participation of a nucleoside triphosphate in the reaction (Box 16-2). This energy-conserving reaction involves an intermediate step in which the enzyme molecule itself becomes phosphorylated at a His residue in the active site (Fig. l6-12a). This phosphoryt group, which has

FIGURE 16-12Thesuccinyl-CoA (a)Instep@ a synthetase reaction. phosphoryl groupreplaces theCoAof succinyl-CoA boundto theenzyme,forming a high-energy acylphosphate In step@ thesuccinyl phosphate donates itsphosphoryl groupto a Hisresidue of theenzyme,forming a high-energy phosphohistidyl enzymeIn step@ the phosphoryl groupis transferred fromthe Hisresidue to theterminal phosphate of CDP(orADP),forming CTp(orATp).(b)Activesiteof succinyl-CoA synthetase ol E.coli(derived frompDBlD ISCU).The

0 subunit

Coenzyme A

te group

active site includes part of both the a (blue)and the g (brown) subunits The power helices (blue, brown) place the partial positive c h a r g e so f t h e h e l i x d i p o l e n e a r t h e p h o s p h a t eg r o u p o f @His216 in t h e a c h a i n , s t a b i l i z i n gt h e p h o s p h o h i s t i d y e l n z y m e .T h e b a c t e r i a la n d m a m m a l i a n e n z y m e s h a v e s i m i l a r a m i n o a c i d s e q u e n c e sa n d t h r e e _ d i m e n s i o n a ls t r u c t u r e s .

c subunit ver helix

c subunit

3r1

c c i dC y c l e 1 6 . 2R e a c t i oonftsh eC i t r iA

Citrate synthaseis one of many enzJ,.rnes that catalyze yielding product condensationreactions, a more chemically complexthan its precursors.Synthases catalyze condensationreactionsin which no nucleosidetriphosphate (ATP,GTP,and so forth) is required as an energy source. Synthetases catalyzecondensationsthat do useATP or anothernucleosidetriphosphateas a source of energyfor the slnthetic reaction.Succinyl-CoAsynthetaseis such an enz).rne.Ligases (from the Latin Li,gare, "to tie together") are enzymes f,hat,catalyze condensationreactionsin which two atomsare joined, usingATP or anotherenergysource.(Thus synthetases are ligases.)DNA ligase,for example,closesbreaksin DNA molecules,usingenergysuppliedby eitherATP or NAD-; it is widely used in joining DNA pieces for geneticengineedng.Ligasesarenot to be confusedwith lyases, enzymes that catalyze cleavages (or, in the reverse direction, additions) in which electronic rearrangementsoccur. The PDH complex,which oxidatively cleavesCO2from pyruvate, is a member of the Iargeclassof lyases. The namekinase is appliedto enzymesthat transfer a phosphorylgroup from a nucleosidetriphosphate such as ATP to an acceptormolecule-a sugar (as in hexokinaseand glucokinase),a protein (as in glycogen phosphorylase kinase),anothernucleotide(as in nucleosidediphosphatekinase),or a metabolicintermediate suchas oxaloacetate(asin PEPcarboxykinase)The reaction catalyzedby a kinase is a phosphorylat'i,on. On the other hand,phosphorolys'isis a displacementreaction in which phosphateis the attacking speciesand becomescovalentlyattachedat the point ofbond breakage. Such reactions are catalyzedby phosphorylases. for example,catalyzesthe phosGlycogenphosphorylase, phorolysisof glycogen,producurg$ucose l-phosphate. DephosphorgLati,on,the removalof a phosphorylgroup from a phosphateester,is cata\yzedby phosphatases,

with water as the attackingspecies.Fructosebisphosto frucphatase-l convertsfructose 1,6-bisphosphate phosphorylase gluconeogenesis, and in 6-phosphate tose a phosphataseremovesphosphoryl groups from phosphoserinein phosphorylatedglycogenphosphorylase. Whew! Unfortunately,these descriptions of enz;.'rnetyBes overlap,and many enzymesare commonlycalledby two or more names.Succinyl-CoAsynthetase,for example, is alsocalledsuccinatethiokinase;the enzyrneis both a synthetasein the citric acid cycle and a kinase when acting in the direction of succinyl-CoAsynthesis.This raises another source of confusion in the naming of enzyrnes.An enzymemay have been discoveredby the use of an assayin which, say,A is convertedto B. The enzymeis then namedfor that reaction.Later work may show, however, that in the cell, the enzyme functions primarily in convertingB to A. Commonly,the first name continuesto be used,althoughthe metabolicrole of the enzyrnewould be better describedby naming it for the reversereaction.The glycolytic enzymepyruvate kinase illustratesthis situation(p. 538). To a beginnerin biochemistry,this duplication in nomenclaturecan be bewildering. International committees have made heroic efforts to systematizethe nomenclatureof enzymes (seeTable6-3 for a brief summaryof the system),but some systematicnameshave proved too long and cumbersomeand are not frequently used in biochemical conversation. We have tried throughout this book to use the enzyme name most commonly used by working biochemistsand to point out casesin which an enzyrne hasmorethan onewidely usedname.For current information on enzymenomenclature,refer to the recommendations of the Nomenclature Committee of the InternationalUnion of Biochemistryand MolecularBiology (wrnrv.chem.qmw.ac.uViubmb/nomenclature/)'

a high group transfer potential, is transferred to ADP (or GDP) to form ATP (or GTP). Animal cells have two isozymesof succinyl-CoAsynthetase,one specific for ADP and the other for GDP The enzymehas two subunits,a (M,32,000), which has the €)His residue (His2ao)and the binding site for CoA, and B (M, 42,000),which confers specificity for either ADP or GDP The active site is at the interfacebetween subunits.The crystal structure of succinyl-CoAsynthetaserevealstwo "power helices" (one from each subunit), oriented so that their electric dipolessituate partial positive chargesclose to the negatively chargedQlHis (Fig. 16-12b), stabilizingthe phosphoenzymeintermediate. (Recall the similar role of helix dipolesin stabilizingK* ions in the K* channel; seeFig. 11-48.)

The formation of ATP (or GTP) at the expenseof the energy releasedby the oxidative decarboxylationof aketoglutarate is a substrate-levelphosphorylation,like the syrrthesisof ATP in the glycolt'ticreactionscatalyzed by glyceraldehyde3-phosphate dehydrogenaseand pyruvatekinase(seeFig. 14-2).The GTPfotmedby succinyl-CoAsynthetasecan donateits termrnalphosphoryl group to ADP to form ATP,in a reversiblereaction catalyzedbynucleoside diphosphate kinase (p. 510): GTP+ ADP -----+GDP+ ATP

AG'o: 0 kJ/mol

of Thus the net result of the activity of either isoz5,'rne energy as succinyl-CoAsynthetaseis the conservationof ATP Thereis no changein free energyfor the nucleoside diphosphatekinasereaction;ATP and GTP are energetically equivalent.

!'*l

T h eC i t r iA c c i dC y c l e

@ Oxidation of Succinate to Fumarate The succinate formed from succinyl-CoAis oxidizedto fumarate by the flavoproteinsuccinate dehydrogenase:

coo I

FAD

FADHz

CHO

t-

H._a-.COO

-/

CHo

This enzymeis highly stereospecific;it catalyzeshydration of the trans doublebond of fumaratebut not the cis double bond of maleate (the cis isomer of fumarate). In the reversedirection (from l-malate to fumarate), fumaraseis equally stereospecific:t-malate is not a substrate.

-ooc-c-H

l-

H\

coo

H\

,/coo

c

Succinate

Fumarate C

\H

oocl

AG'" : 0 kJ/mol

H

Fumarate

In eukaryotes,succinatedehydrogenase is tightly bound to the mitochondrialinner membrane;in bacteria,to the plasmamembrane.The enz).ryne containsthree different iron-sulfur clusters and one molecule of covalently bound FAD (see Fig. 19-10). Electronspassfrom succinate through the FAD and iron-sulfur centers before entering the chain of electron carriers in the mitochondrial inner membrane (the plasmamembranein bacteria). Electronflow from succinatethroughthesecarriers to the final electronacceptor,02, is coupledto the synthesisof about 1.5ATP moleculesper pair of electrons (respiration-linked phosphorylation).Malonate,an analog of succinatenot normallypresentin cells,is a strong competitiveinhibitor of succinatedehydrogenase, and its addition to mitochondriablocks the activity of the citric acid cycle.

/coo-

c c

coo I

COOMaleate

cooI

HO_C-H

H-C-OH

CHr

cH2

I

I

1coo

tcoo-

l-Malate

o-Malate

@ Oxiaation of Malate to Oxaloacetate In the last reaction of the citric acid cycle, NADlinked r,-malate dehydrogenase catalyzesthe oxidation of r,-malateto oxaloacetate:

cooI

HO-C-H

I

CH"

NAD+

NADH + H+

\\/.0/ l : 6

\/.cHo

t- Tt--" coo rlelrrrlrogcnusc l-Malate

COO-

COO-

Oxaloacetate

AG'' :29.7 kJ/mol

zzc\ ooo'o-

,c.

Malonate

Succinate

@ Hydration of Fumarate to Malate The reversible hydration of fumarate to r,-malate is catalyzedby fumarase (formally, fumarate hydratase). The transition state in this reaction is a carbanion: H\^..coo-

oT{ "i-

u

il\

c ooc/'\H

t r r ,r, r r . , .

H\^--COO U

I -ooc--C'- oH \ H

Fumarate

Carbanion transition state

H'2 H--C-

-COO-

I oH -ooc--c'," \

H l-MaIate AG'' : -3.8 kJ/mol

The equilibrium of this reaction lies far to the left under standard thermodynamic conditions, but in intact cells oxaloacetate is continually removed by the highly exergonic citrate synthase reaction (step e of Fig. 16-T). This keeps the concentration of oxaloacetate in the cell extremely low (rylatecycle serves as a mechanism for converting acetate to carbohydrate.

(ycleProduces TheGlyoxylate Four-Carbon Compounds fromA(etate In plants, certain invertebrates,and some microorganisms (including E co|i, and yeast) acetate can serve both as an energy-richfuel and as a sourceof phosphoenolpyruvate for carbohydrate synthesis. In these organisms,enz).rnesof the glyoxylate cycle catalyze the net conversionof acetateto succinateor other fourcarbonintermediatesof the citric acid cycle: 2Acetyl-CoA+ NAD+ + 2HrO -----+ + 2CoA+ NADH + H+ succinate In the glyoxylate cycle, acetyl-CoAcondenseswith oxaloacetateto form citrate,and citrate is convertedto isocitrate, exactly as in the citric acid cycle. The next step, however,is not the breakdownof isocitrateby isocitrate dehydrogenasebut the cleavageof isocitrate by isocitrate lyase, forming succinateand glyoxylate. The $yoxylate then condenseswith a second molecule of acetyl-CoAto yield malate,in a reaction catalyzedby malate synthase. The malate is subsequentlyoxidized to oxaloacetate,which can condensewith anothermolecule of acetyl-CoAto start anotherturn of the cycle (Fig. 16-20). Eachtum of the glyoxylatecycle consumestwo moleculesof acetyl-CoAand producesone molecule of succinate,which is then availablefor biosynthetic purposes.The succinatemaybe convertedthrough fumarate and malate into oxaloacetate,which can then be converted to phosphoenolpytuvateby PEP carboxykhase, Vertebratesdo and thus to glucoseby gluconeogenesis. not have the enzyrnesspeciic to the glyoxylate cycle (isocitratelyaseand malatesyrrthase)and thereforecannot bring about the net synthesisof glucosefrom Jipids. In plants, the enz;,'rnesof the glyoxylate cycle are sequesteredin membrane-boundedorganellescalled glyoxysomes,which are specializedperoxisomes(Fig. 16-21). Those enzyrnescommonto the citric acid and glyoxylate cycleshave two isoz;rmes,one speciflcto mitochondria,the other to glyoxysomes. Glyoxysomes are not presentin all plant tissuesat all times.They develop in lipid-rich seedsduring germination,before the developing plant acquiresthe ability to makeglucoseby photosynthesis.In addition to glyoxylate cycle enzymes, glyoxysomescontain all the enzymesneeded for the degradationof the fatty acids stored in seed oils (see Fig. 17-13). Acetyl-CoAformed from lipid breakdown is converted to succinatevia the glyoxylate cycle, and the succinateis exported to mitochondria,where citric acid cycle enz).rnestransform it to malate. A cy'tosolic isozyrneof malatedehydrogenaseoxidizesmalateto oxaloacetate,a precursorfor gluconeogenesis. Germinating seedscan therefore converf the carbon of stored lipids into glucose.

Frrl

1 6 . 4T h eG l y o x y l aCtyec l e

(ycles Are The(itricAcid and6lyoxylate (oordinately Regulated

o II

CHg*C-S-CoA Acetyl-CoA

o:C-COocH2-coo Oxaloacetate

cH2-coo HO-C-COO-

I cH2-coo

Glyoxylate cycle

coo HO-CH I

In germrnatingseeds,the enzymatictransformationsof dicarboxylic and tricarboxylic acids occur in three intracellular compartments:mitochondria,glyoxysomes, and the cytosol. There is a continuous interchange of metabolitesamongthese compartments(Fig. 16-22). The carbon skeletonof oxaloacetatefrom the citric acid cycle (in the mitochondrion) is carried to the glyoxysomein the form of aspartate.Aspartate is converted to oxaloacetate,which condenseswith acetyl-CoA derived from fatty acid breakdown. The citrate thus formed is convertedto isocitrateby aconitase,then spJit into glyoxylateand succinateby isocitratelyase.The succinate returns to the mitochondrion,where it reenters

CHO

tcoo-

CH"-COO

l-

CH_COO_

Malate

I

HO-CH-COO Isocitrate

oI c:o I

Lipid body Triacylglycerols

lso(rlt itle l\ asc

Fatty acids

C:O tl

I

CH3-C-S-CoA H Acetyl-CoA Glyoxylate

?H,-coocH2-coo Succinate

FIGURE 16-20 Glyoxylatecycle.The citratesynthase,aconitase,and malatedehydrogenase of the glyoxylatecycleare isozymes of the citric acidcycleenzymes; isocitrate lyaseandmalatesynthase areuniqueto the glyoxylate cycle.Noticethattwo acetylgroups(pink)enterthe cycleand four carbonsleaveas succinate(blue).The glyoxylatecyclewas elucidatedby HansKornberg andNeilMadsenin the laboratory of HansKrebs.

le

Citra # s

/ ,8{ Isocitrate

Lipid body

,;."/

Glyorysome

i/

Oxaloacetate

f

Cytosol

Malate

.* Glyoxysome

Mitochondria

tIGURE 16-21 Electronmicrograph of a germinating cucumberseed, showinga glyoxysome, mitochondria, and surrounding lipid bodies.

7

.trumarate

alate

Citric acid cycle FIGUREI6-22Relationshipbetweentheglyoxylateandcitricacidcycles.Thereactionsofthe glyoxylate cycle(inglyoxysomes) proceedsimultaneously with,and meshwith,thoseof thecitric acid cycle (in mitochondria), The conas intermediates passbetweenthesecompartments. versionof succinate to oxaloacetate is catalyzed Theoxidationof by citricacidcycleenzymes. fattyacidsto acetyl-CoAis describedin Chapter17; the synthesis of hexosesfrom oxaloacetate is described in Chaoter20.

+

Oxaloacetate

| 0+0I

Ihe(itricAcid Cycle

the citric acid cycleand is transformedinto malate,wtuch entersthe cltosol andis oxrdized(by cytosolicmalatedehydrogenase)to oxaloacetate.Oxaloacetateis converted via gluconeogenesis into hexosesand sucrose,which can be transportedto the growingroots and shoot.Four distinct pathwaysparticipatein theseconversions:fatty acid breakdownto acetyl-CoA(in glyoxysomes),the gyoxyIate cycle (in glyoxysomes),the citric acid cycle (in mito(in the cytosol). chondria),and gluconeogenesis The shanng of commonintermediatesrequiresthat these pathwaysbe coordinatelyregulated.Isocitrateis a crucial intermediate,at the branch point betweenthe $yoxylateandcitric acidcycles(Fig. f 6-23). Isocitratedehydrogenaseis regulatedby covalentmodification:a specific Acetyl-CoA intermediates of citric acid cycle and glycolysis, AMP, ADP

Isocitrate

intemediates of citric acid cycle and glycolysis, AMP, ADP

!

O "fr* Succinate, Ketoglutarate

protein kinasephosphorylates and therebyinactivatesthe dehydrogenase. This inactivationshuntsisocitrateto the $yoxylate cycle, where it beginsthe s;,ntheticroute toward $ucose.A phosphoproteinphosphatase removesthe phosphorylgroup from isocitratedehydrogenase, reactivating the enz),rneand sendingmore isocitratethrough the energy-yieldingcitric acid cycle.The regulatoryprotein kinaseand phosphoproteinphosphataseare separateerzymatic activitiesof a singlepolypeptide. Somebacteria,includmg.O.col'i,havethe full complement of erz;.'rnesfor the g$oxylate and citric acid cyclesin the cy'tosoland canthereforegrow on acetateastheir sole sourceof carbonand energr.The phosphoproteinphosphatasethat activatesisocitratedehydrogenase is stimulatedby intermediatesof the citric acidcycleandglycolysis and by indicatorsof reducedcellularenergysupply (Fig. 16-23). The samemetabolitesinlyibit the protein kinase activityof the bifunctionalpolypeptide.Thus,the accumulation of rntermediatesof the central energy-yieldingpathways-indicating energydepletion-results in the activation of isocitratedehydrogenase. Whenthe concentration ofthese regulatorsfalls, srgnajrnga sufficient flux through the energy-yielding citric acid cycle,isocitratedehydrogenaseis inactivatedby the protein kinase. The sameintermediatesof glycolysisand the citric acid cycle that activate isocitrate dehydrogenaseare allosteric inhibitors of isocitrate lyase.When energyyielding metabolism is sufficiently fast to keep the concentrationsof glycolytic and citric acid cycle intermediateslow, isocitrate dehydrogenase is inactivated, the inhibition of isocitrateIyaseis relieved,and isocitrate flows into the glyoxylatepathway,to be usedin the biosl'nthesisof carbohydrates,amino acids,and other cellularcomponents.

S U M M A R1Y6 . 4 T h eG l y o x y l a t(ey c l e r

The glyoxylate cycle is active in the germinating seedsof someplants and in certain microorganisms that canlive on acetateas the solecarbonsource. In plants,the pathwaytakesplacein glyoxysomes in seedlings.It involvesseveralcitric acid cycle enzyrnesand two additional enzymes:isocitrate lyaseand malate slmthase.

r

In the glyoxylatecycle, the bypassingof the two decarboxylationstepsofthe citric acid cycle makespossiblethe net formationof succinate, oxaloacetate, and other cycleintermediatesfrom acetyl-CoA.Oxaloacetate thus formed canbe used to synthesizeglucosevia gluconeogenesis.

Oxaloacetate

FIGURE 16-23 Coordinatedregulationof glyoxylateand citric acid cycles.Regulation of isocitrate dehydrogenase activitydetermines the partitioningof isocitrate betweentheglyoxylate andcitricacidcyclesWhen the enzymeis inactivated (by a specificproteinkiby phosphorylation nase),isocitrate is directedinto biosynthetic reactions via the glyoxylate cycle Whentheenzymeisactivated (bya specific by dephosphorylation phosphatase), isocitrate entersthe citricacidcycleandATPis produced.

Vertebrates lack the glyoxylate cycle and cannot synthesize glucose from acetate or the fatty acids that give rise to acetyl-CoA. The partitioning of isocitrate between the citric acid cycle and the glyoxylate cycle is controlled at the level of isocitrate dehydrogenase,which is regulated by reversible phosphorylation.

F-l

F u r t h eRre a d i n g

KeyTerms Tenns'in bold are defi,nedin the glossarg respiration 615 ceilularrespiration 615 citric acid cycle 615 tricarboxylic acid (TCA) cycle 615 Krebs cycle 615 pyruvate dehydrogenase (PDH) complex 616 oxidative decarboxylation 616 thioester 617 lipoate 6\7 substrate channeling 619 iron-sulfur center 623 moonlighting enzJrrnes 624 a-ketoglutarate dchrrdrndpneqa

nucleoside diphosphate kinase 627 synthases 627 synthetases 627 ligases 627 lyases 627 kinases 627 phosphorylases 627 phosphatases 627 prochiral molecule 629 amphibolic pathway 631 anaplerotic reaction 631 biotin 633 avidin 633 metabolon 637 glyoxylate cycle 638

complex 625

Further Reading General Holmes, F.L. (1990, 1993)Hans Krebs,YoI l: Fonnation of a Scientzj,cLi,fe, 1900-1933;YoI 2: Archi,tect oJIntermedi,arry Metabo|istn, I 933- I 937, Oxford UniversityPress,Oxford. A scienti-ficand personalbiographyof Krebsby an eminent historian of science,with a thorough descriptionof the work that revealedthe urea and citric acid cycles Kay, J. & Weitzman, P.D.J. (eds). (1987) Krebs' Citri,cAcid Cycle: HaIJ a Centutg and Sti,LlTfutning, BiochemrcalSocietyS;..rnposium 54, The BiochemicalSociety,London A mu-ltiauthor bookon the citric acidcycle,includingmoleculargenetics,regulatorymechanisms, variationson the cyclein microorganismsfrom unusualecologicalniches,and evolution of the pathway. Especiailyrelevantarethe chaptersby H Gest(EvolutionaryRootsof the Citric Acid Cyclein Prokaryotes),W.H Holms(Controlof Flux througft the Citric Acid Cycleand the GlyoxylateBypasstnEscheri,chia colz),and R. N. Perhamet al. (a-KetoAcid Dehydrogenase Complexes). Pyruvate Dehydrogenase Complex Harris, R.A., Bowker-Kinley, M.M., Huang,8., & Wu, P. (2002) Regulationof the activity of the pyruvate dehydrogenasecomplex Adu Enzyme Regul 42,249-259. Milne, J.L.S., Shi, D., Rosenthal, P.B.,Sunshine, J.S., Domingo, G.J., Wu, X., Brooks,8.R., Perham, R.N., Henderson, R., & Subramaniam, S. (2002) Moleculararchitectureand mechanism of an icosahedralpyruvate dehydrogenasecomplex:a multifunctional catalltic machine EMBO J 21, 5587-5598 Beautiful illustration of the power of imagereconstruction methodologywith cryoelectronmicroscopy,here usedto developa plausiblemodel for the structure of the PDH complex Comparethis model with that in the paper by Zhou et al (below) Perham, R.N. (2000) Swingingarms and swingingdomainsin multifunctronalenzyrnes:catal)'ticmachinesfor multistep reactionsAnnu Reu Bzochem 69,961-1004 Reviewof the roles of sw'rngrng arms containinglipoate,biotin, and pantothenatein substratecharurelingthrough multienz5rme comolexes

Zhort,Z,H,, McCarthy, D.8., O'Conner, C.M., Reed, L.J., & Stoops, J,K. (2001) The remarkablestructural and ftrnctionalorganizationof the eukaryoticpyruvate dehydrogenasecomplexes Proc NatI Acad Sci. USA 98, 14,802-14,807. Another striking paper in which imagereconstructionwith cryoelectronmicroscopyflelds a model of the PDH complex Comparethis model with that in the paper by Milne et al (above). Citric Acid Cycle Enzymes Fraser, M.D., James, M.N., Bridger, \M.A.,& Wolodko, W.T. (1999) A detailedstruclural descriptionof Escherichi,a co\'i synthetaseJ. MoI Bi,oI 285, 1633-1653.(Seealso succinyl-CoA the erratuminJ. MoI Bi,oI 288,501 (1999)) Goward, C.R. & Nicholls, D.J. (1994) Malatedehydrogenase: a model for structure, evolution,and catalysisProtein Sci. 3, 1883-1888. A good,short review. Hagerhall, C. (1997) Succinate:quinoneoxidoreductases:variations on a conservedlheme Bi,ochi,mBi,ophus Acta 132O,107-141. A review of the stmcture and function of succinate dehydrogenases Jitrapakdee, S. & Wallace, J.C. (1999) Structure,function, and regulationof pymvate carboxylaseBi,ochem J 340, 1-16 Knowles, J. (1989) The mechanismof biotin-dependentenz5.'rnes. Annu Reu Bi,ochem,58, 195-221. Matte, A,, Thri, L.Iry.,Goldie, H., & Delbaere, L.T.J. (1997) Structure and mechanismof phosphoenolpyruvatecarboxykinase. J. Bi,oI Chem 272,8105-8108. Ovadi, J. & Srere, P. (2000) Macromolecuiarcompafimentation and channelingInt Reu, Cgtol 192,255-280. Advancedreview of the evidencefor channelingand metabolons Remington, S.J. (1992) Structure and mechanismof citrate synthase.Curr Top CeII Regul.33,209-229. A thorough reviewof [his enz],rne. Singer, T.P. & Johnson, M.K. (1985) The prosthetic groupsof 30 yearsfrom discoveryto identiflcation. succinatedehydrogenase: FEBSLett 190,189-198. A descriptionof the structure and role of the iron-sulfur centers ln this enz5,'rne Weigand, G. & Remington, S.J. (1986) Citrate s}'nthase:structure, control, and mechanismAnnu Reu.Bi,ophqs Bi,ophEs.Chem 16, 97-117. Wolodko, W.T., Fraser, M.E., James, M.N.G., & Bridger' W.A. (1994) The crystal structure of succinyl-CoAsynthetasefrom E scheri,chiacoti,at 2.5-A resolution.J. Bi'ot. Chem 269, 10,883-10,890 Moonlighting

Enzymes

Eisenstein, R,S. (2000) Iron regulatoryproteins and the moiecular control of mammalianiron metabolism.Annu Reu Nutr.2O, 627 662 Kim, J.-W. & Dang, C.V. (2006) Multifacetedroles of glycoly'tic enzyrnesTlends Biochem Sci, 3O, 142-150. Intermediate-levelreview of moonlightingenzlrnes Rouault, T.A. (2006) The role of iron regulatoryproteins in mammalianiron homeostasisand disease.Nat Chem Bi'oL 2, 406-414. An advancedreview. Regulation of the Citric Acid Cycle Briere, J.-J., Favier, J., Gimenez-Roqueplo, A.-P.' & Rustin, P, (2006) Tlicarboxylic acid cycle dysfunctionas a causeofhuman diseasesand tumor formation.Am J. Physio| CeIIPhgsi,ol 291, 1114-1120

(itric (ycle 1642_)The Acid Intermediatelevel review of clinical effects of mutations in succinate dehydrogenase, fumarase, and a-ketoglutarate dehydrogenase Hansford, R.G. (1980) Control ofmitochondrial substrate oxidation Curr Top Bi,oenerget 1O,217-278 A detailed review of the regulation of the citric acid cycle. Kaplan, N.O. (1985) The role of pyridine nucleotides in regulating cellularmetabolism Czrrr Top CeLl Regul 26,371-381 An excellent general discussion of the rmportance of the

FAD dehydrogenate/hydrogenateorganic moleculesin the presenceof the proper enzlrnes.

o

o.- Hl

8r-ti

reduction

+H-H-

cH3-c-H

oxtdanon

Hj

Acetaldehyde

reduction .

[NADH]/[NAD+] ratio in cellular regulation

.-

King, A., Selak, M.A., & Gottlieb, E. (2006) Succinate dehydrogenase and fumarate hydralase: linking mitochondrial dysfunction

oxrdatlon

o-H I

CH"-C-H H Ethanol

and cancer Oncogene 25, 46754682 Reed, L.J., Damuni, 2., & Merryfield, M.L. (1985) Regulation of mamrnalian p).r'uvate and branched-chain a-keto acid dehydrogenase complexesbyphosphorylation-dephosphorylation Cum Top Cell Regul 27,4749

(1)

|

o cH3-c\

+H-+H-H

o

Glyoxylate

Acetate

Cycle

Eastmond, P.J. & Graham, I.A. (2001) Re-examrning the role ofthe glyoxylate cycle in oilseeds Trends Plant Sci 6,72-77 intermediatelevel revrew of studies of the glyoxylate cycle in Aro,bidopsis Holms, W.H. (1986) The central metabolic pa[hways of Escheri,chia colz: relationship between flux and control at a branch point, efficiency of conversion to biomass, and excretion of acetate Cum Top CeII Regul 28,69-106

Problems 1. Balance Sheet for the Citric Acid Cycle The citric acid cycle has eight enz),mes: citrate s)'nthase, aconitase, isocitrate dehydrogenase, a-ketoglutarate dehydrogenase, succinyl-CoA s5,nthetase, succinate dehydrogenase, fumarase, and malate dehydrogenase. (a) Write a baianced equation for the reaction calalyzed by each enzlrrne (b) Name the cofactor(s) required by each eruyrne reaction. (c) For each enzJ,.rnedetermine which of the following describes the type of reaction(s) calalyzed: condensation (carbon-carbon bond formation) ; dehydration (loss of water); hydration (addition of water); decarboxylation (loss of CO);

(2)

|

H

For each of the metabolictransformationsin (a) through (h), determine whether oxidation or reduction has occurred. Balanceeachtransformationbyinserting H-H and,where necessary H2O.

o -----) H-C-H Formaldehyde

(a) CH3-OH Methanol

oo ------ H-Cy

H-C-H

ft)

+ H*

oFormaldehyde

Formate

o (c)

-----+

O:C:O

+ H+ "-C..

?" ?^ ,.o + H*

(d) cH2-c-c\

fro-

OH OH tt//O ------)CH"-C-C -t

l

OHOH

tt

(e) CH2-C-CH2 I H

OH +

Glycerol H 'l' (f)

C-H_-(

_t_ H Toluene

\

HH Glyceraldehyde

Glycerate

OH

o-

Formate

Carbon dioxide

including all cofactors.

form of ATP. It is important to be able to recognize oxidationreduction processes in metabolism. Reduction of an organic molecule results from the hydrogenation of a double bond (Eqn 1, below) or of a single bond with accompanying cleavage (Eqn 2). Conversely, oxidation results from dehydrogenation. In biochemical redox reactions, the coenzyrnes NAD and

,/

Acetaldehyde

acetyl-CoA to CO2.

3. Recognizing Oxidation and Reduction Reactions One biochemicai strategy of many living organisms is the stepwise oxidation of organic compounds to CO2 and H2O and the conservation of a major part of the energy thus produced in the

ll

+O CH"-C-H -

: oxrdatron

oxidation-reduction; substrate-levei phosphoryiation; isomerization. (d) Write a balanced net equation for the catabolism of

2. Net Equation for Glycolysis and the Citric Acid Cycle Write the net biochemical equation for the metabolism of a molecuie of glucose by glycolysis and the citric acid cycle,

o

reduction .

t

O OH

ill

CH2-C-CH2

Dihydroxyacetone

,^-\

\:/

\)-C

,r/o

.o_ + H +

Benzoate

Problems F-t

o

o

o* (g)

-o/C-CII2-CH2-Cx

/o'o

HC

\ ,,,\ o------) ,rc:c.. o-co H o

Succinate

\

+ co2

Acetate

Substrate + NADH + H+ i-

product + NAD*

Reduced

Reduced

Oxidized

For each of the reactions in (a) through (f), determine whetherthe substratehasbeen oxidizedor reducedor is unchangedin oxidation state (see Problem3) If a redox change has occurred,balancethe reactionwith the necessaryamount of NAD-, NADH, H-, and H2O.The objectiveis to recognize when a redox coenzl'rneis necessaryin a metabolicreaction.

(a) CH3CH2OH -----+ Ctr-{:

H Acetaldehyde

oPoSgPoS oH o | | .//?, _____+ (b) CH2-C-C\ cH2_g_a\ 'oPo'zfiH 'l 1,3-Bisphosphoglycerate

+ CO2

Acetone

?. Thiamine Deficiency Individuals with a thiaminedeflcient diet have relatively high levels of pyruvate ir their blood. Explain this in biochemicalterms 8. Isocitrate Dehydrogenase Reaction What type of chemicalreaction is involvedin the conversionof isocitrate to a-ketoglutarate?Nameand describethe role of any cofactors. What other reaction(s) of the citric acid cycle are of this same type? 9. Stimulation of Oxygen Consumption by Oxaloaeetate and Matate In the early 1930s,Albert Szent-Gyorgyi reported the interesting obseryationthat the addition of small amounts of oxaloacetateor malate to suspensionsof minced pigeon breast muscle stimulated the oxygen consumptionof the preparation.Surprisingly,the amount of oxygenconsumed was about seventimes more than the amount necessaryfor completeoxidation(to CO2and H2O) of the added oxaloacetate or malate.Why did the addition of oxaloacetateor malate stimulate oxygen consumption?Why was the amount of oxygen consumedso much greaterthan the amountnecessaryto or malate? completelyoxidizethe addedoxaloacetate 10. Formation of Oxaloacetate in a Mitochondrion In the last reaction of the citric acid cycle, malate is dehydrogenatedto regeneratethe oxaloacetatenecessaryfor the entry of acetyl-CoAinto the cYcle: l-Malate + NAD+ -----+oxaloacetate+ NADH + HA'G'"= 30.0kJ/mol

,o

Glyceraldehyde 3-phosphate

+ HPo?

(a) Calculatethe equilibrium constantfor this reaction at 25'C (b) BecauseAG'oassumesa standardpH of 7, the equilibrium constantcalculatedin (a) correspondsto KLq:

cH,-

C6

_>

Ca

------) -CoA

l7-8 The p-oxidation pathway.(a) In each passthrough this FIGURE one acetylresidue(shadedin pink) is removedin four-stepsequence, the form of acetyl-CoAfrom the carboxylend of the fatty acyl chain(b) Six in this examplepalmitate(Cro),which entersas palmitoyl-CoA. more passesthrough the pathwayyield seven more moleculesof acetyl-CoA,the seventharisingfrom the lasttwo carbonatomsof the 16-carbonchain.Eightmoleculesof acetyl-CoAareformedin all.

(atabotism los+_l FattyAcid

This first step is catalyzed by three isozymes of acyl-CoA dehydrogenase, each speciflc for a range of fatty-acyl chain lengths: veryJong-chain acyl-CoA clehydrogenase (VLCAD), acting on fatty acids of 12 to 18 carbons; medium-chain (MCAD), acting on fatty acids of 4to 14 carbons; and short-chain (SCAD), acting on fatty acids of 4 to 8 carbons. All three isozymes are flavoproteins with FAD (see Fig. 13-27) as a prosthetic group. The electrons removed from the fatty acyl-CoA are transferred to FAD, and the reduced form of the dehydrogenase immediately donates its electrons to an electron carrier of the mitochondrial respiratory chain, the electron-transferring flavoprotein (ETF) (see Fig. 19-8). The oxidation catalyzed by an acyl-CoA dehydrogenaseis analogousto succinate dehydrogenation in the citric acid cycle (p. 628); in both reactions the enzyme rs bound to the inner membrane, a double bond is introduced into a carboxylic acid between the a and p carbons, FAD is the electron acceptor, and electrons from the reaction ultimateiy enter the respiratory chain and pass to 02, with the concomitant syrrthesisof about 1.5 ATP molecules per electron pair. In the second step of the B-oddation cycle (Fig. 17-8a), water is added to the double bond of the trans-Lzenoyl-CoA to form the I stereoisomer of B-hydroxyacylCoA (3-hydroxyacyl-CoA). This reaction, catalyzed by enoyl-CoA hydratase, is formally analogousto the fumarase reaction in the citric acid cycle, in which H2O adds across an a-B double bond (p. 628). In the third step, l-B-hydroxyacyl-CoA is dehydrogenated to form p-ketoacyl-CoA, by the action of dehydrogenase; NAD+ is the B-hydroxyacyl-CoA electron acceptor. This enzlrne is absolutely speciflc for the I stereoisomer of hydroxyacyl-CoA. The NADH formed in the reaction donates its electrons to NADH dehydrogenase, an electron carrier of the respiratory chain, and ATP is formed from ADp as the electrons pass to 02. The reaction catalyzed. by B-hydroxyacylCoA dehydrogenase is closely analogous to the malate dehydrogenasereaction ofthe citric acid cycle (p. 628). The fourth and last step of the B-oxidation cycle is catalyzed by acyl-CoA acetyltransferase, more commonly called thiolase, which promotes reaction of 6ketoacyl-CoA with a molecule of free coenzyme A to split off the carboxyl-terminal two-carbon fragment of the original fatty acid as acetyl-CoA. The other product is the coenzyme A thioester of the fatty acid, now shortened by two carbon atoms (Fig. 17-8a) . This reaction is called thiolysis, by analogy with the process of hydrolysis, because the B-ketoacyt-CoA is cleaved by reaction with the thiol group of coenzyme A. The Iast three steps of this four-step sequence are catalyzed by either of two sets of enzymes, with the enzymes employed depending on the length of the fatty acyl chain F or fatty acyl chains of 12 or more carbons, the reactions are catalyzed by a multienzyme complex associatedwith the inner mitochondrial membrane. the

trifunctional protein (TFP). TFP is a heterooctamer of aaBa subunits. Each a subunit contains two activities, the enoyl-CoA hydratase and the B-hydroxyacylCoA dehydrogenase;the p subunits contain the thiolase activity. This tight association of three enz).rnes may allow efficient substrate channeling from one active site to the next, without diffusion of the intermediates away from the enzyme surface. When TFP has shortened the fatty acyl chain to 12 or fewer carbons, further oxidations are cala\yzed by a set of four soluble enzymes in the matrix. As noted earlier, the single bond between methylgroups in fatty acids is relatively stable. ene (-CH2-) The B-oxidation sequence is an elegant mechanism for destabilizing and breaking these bonds. The first three reactions of B oxidation create a much less stable C-C bond, in which the a carbon (C-2) is bonded to two carbonyl carbons (the B-ketoacyl-CoA intermediate). The ketone function on the B carbon (C-3) makes it a good target for nucleophilic attack by the-SH of coenzyme A, catalyzed by thiolase The acidity of the c hydrogen and the resonance stabilization of the carbanion generated by the departure of this hydrogen make the terminal-CH2-CO-S-CoA a good leaving group, facilitating breakage of the a-B bond.

p-Oxidation TheFour Steps AreRepeated toYield Acetyl-CoA andATP In onepassthroughthe B-oxidationsequence,one moleculeof acetyl-CoA,two pairsof electrons,and four protons (H-) are removed from the long-chain fatty acyl-CoA,shorteningit by two carbonatoms.The equation for one pass,beginningwith the coenzyrneA ester of our example,palmitate,is Palmitoyl-CoA + CoA + FAD + NAD+ + H2O -> myristoyl-CoA + acetyl-CoA + FADH2 + NADH + H+ (17-2)

Following removal of one acetyl-CoAunit from palmitoyl-CoA,the coen4,'rneA thioester of the shortened fatty acid (now the l4-carbonmyristate)remains.The myristoyl-CoAcan now go through anotherset of four Boxidation reactions,exactly analogousto the first, to yield a secondmoleculeof acetyl-CoAand lauroyl-CoA, the coenzymeA thioesterof the l2-carbonlaurate.Altogether,sevenpassesthrough the B-oxidationsequence arerequiredto oxidizeonemoleculeof palmitoyl-CoAto eight moleculesof acetyl-CoA(Fig. 17-8b).The overall equationis Palmitoyl-CoA + TCoA+ TFAD+ TNAD++ 7H2O-----> 8 acetyl-CoA + ZFADH2+ TNADH+ 7H+ (12_g) Each moleculeof MDH2 formed during oxrdationof the fatty acid donatesa pair of electronsto ETF of the respiratory chain,and about 1.5moleculesof ATp are generated during the ensuingtransfer of each electron pair

1 7 . 20 x i d a t i o n o f F a t t y|A6c5i5d| s

to 02. Similarly,eachmoleculeof NADH formed delivers a pair of electronsto the mitochondrialNADH dehydrogenase,and the subsequenttransfer of each pair of electronsto 02 resultsin formationof about 2.5 moleculesof ATP.Thus four moleculesof ATP are formed for each two-carbonunit removedin one passthrough the sequence.Note that water is also produced in this process.T?ansferof electronsfrom NADH or FADH2to 02 yieldsone H2Oper electronpair.Reductionof 02 by NADH also consumesone H* per NADH molecule: NAD+ + H2o. In hibernatNADH + H+ + iO, ing animals, fatty acid oxidation provides metabolic energy, heat, and water-all essential for survival of an animal that neither eats nor drinks for long periods (Box 17-1). Camelsobtain water to supplementthe meagersupply availablein their natural environmentby oxidation of fats stored in their hump.

The overall equationfor the oxidation of palmitoylCoA to eight moleculesof acetyl-CoA,including the electron transfersand oxidativephosphorylations,is + TCoA+ 7O2+ 28Pi+ 28ADP-) Palmitoyl-CoA + 28ATP+ 7H2O (77-4) 8 acetyl-CoA

Many animals depend on fat stores for energy during hibernation,dudng migratoryperiods,and in other situations involvingradicalmetabolicadjustments.One of the most pronouncedadjustmentsof fat metabolismoccurs in hibernating grizzly bears. These animalsremain in a continuousstateof dormancyfor periodsaslong asseven months.Unlike most hibernatingspecies,the bear mainoC,closeto tainsa bodytemperatureof between32 and35 the normal (nonlLibernating) level. Although expending about 25,000kJ/day (6,000kcaVday),the bear doesnot eat, drink, utinate, or defecatefor monthsat a time. Experimental studies have shov,nthat hibernating grizzlybearsusebodyfat astheir solefuel. Fat oxidation yields sufficient energy for maintenanceof body temperature, active slrrthesis of amino acids and proteins, and other energy-requiringactivities,suchasmembrane transport. Fat oxidation also releaseslarge amounts of

water, as describedin the text, which replenisheswater lost in breathing. The glycerol releasedby degradation of triacylglycerolsis convertedinto blood glucoseby Urea formed during breakdown of gluconeogenesis. amino acids is reabsorbedin the kidneys and recycled, the amino groups reused to make new amino acids for maintainingbody Proteins. Bears store an enormous amount of body fat in preparation for their long sleep. An adult gtizzly consumes about 38,000 kJ/day during the late spring and surruner,but aswinter approachesit feeds20 hoursa day, consumingup to 84,000kJ daily This changein feedingis a responseto a seasonalchangein hormone secretion' Large amounts of triacylglycerolsare formed from the huge intake of carbohydratesduring the fattening-up period. Other hibernating species,including the tiny dormouse,alsoaccumulatelarge amountsof body fat.

0xidized CanBeFurther Acetyl-(oA (itric AcidCycle inthe The acetyl-CoAproducedfrom the oxidation of fatff acids canbe oxidizedto CO2and H2Oby the citric acid cycle.The followingequationrepresentsthe balancesheetfor the second stagein the oxidation of palmitoyl-CoA,together with the coupledphosphorylationsof the third stage: 8 Acetyl-CoA+ 1602 + 80Pi + 80ADP ---+ 8CoA + 80ATP + 16CO2+ 16H2O

nest,nearthe McNeilRiverin Canada its hibernation Agrizzlybearprepares

(17-5)

i.l

(arabotism Acid L656I Fatty

Numberof NADII or FADH' formed

Enzymecatalyzingthe oxidation step Acyl-CoAdehydrogenase

7FADHz

B-Hydroxyacyl-CoAdehydrogenase Isocitrate dehydrogenase

8 NADH

NumberofATP ultimately formed* 10.5 17.5 20 20

7 NADH

a-Ketoglutaratedehydrogenase Succinyl-CoAsy'nthetase

8 NADH

Succinatedehydrogenase

8 FADHz

Malatedehydrogenase

8 NADH

8T

72 20 108

Total

*These calculations assume thatmitochondrial phosphorylation oxidative produces 1.5ATPperFADH2 oxidized and2 5 ATpperNADHoxidizeo. 1GTP produced directly in thisstepyieldsATP in the reaction catalyzed by nucleoside diphosphate kinase(p.510).

Combining Equations l7-4 and 1Z-5, we obtain the overall equation for the complete oxidation of palmitoylCoA to carbon dioxide and water: Palmitoyl-Co| + 2BO2+ 108pi + 108ADp -----+ coA + 108ATP+ 16CO2+ 23H2O

(17_6)

Table 17-1 summarizesthe yields of NADH, FADH2, and ATP in the successivesteps of palmitoyl-CoA oxidation. Note that because the activation of palmitate to palmitoylCoA breaks both phosphoanhydride bonds in ATp (Fig. 17-5), the energetic cost of activating a fatty acid is equivalent to two ATP, and the net gain per molecule of palmitate is i06 ATP. The standard free-energy change for the oxidation of palmitate to CO2 and H2O is about 9,800 kJ/mo]. Under standard conditions, the energy recovered as the phosphate bond energy of ATp is 106 X 30.5 kJ/mol : 3,230 kJ/mol, about 33% of the theoretical maximum. However, when the free_energy changes are calculated from actual concentrations of reactants and products under intracellular conditions (see Worked Example I3_2, p.508), the free-energy recovery is more than 60%; the energy conservation is remarkably efflcient.

Oleate is an abundant 18-carbonmonounsaturated fatty acid with a cis doublebond between C-9 and C-10 (denotedAe). In the first step of oxidation,oleateis converted to oleoyl-CoAand, Iike the saturatedfatty acids, entersthe mitochondrialmatrix via the carnitine shuttle (Fig. 17-6). Oleoyl-CoAthen undergoesthree passes through the fatty acid oxidationcycle to yield three moleculesof acetyl-CoAand the coerzlrne A ester of a A3, 12-carbonunsaturatedfatty acid,cris-A3-dodecenoyl-CoA (FiS. f7-9). This product carLnotserveasa substratefor

o

,// _c-s-coA oleoyl-CoA

I

B oxidationI Ithreecvclesr I

B Acetvl_coA

J HFo ,, \ J/ coA + 16CO2 + 108ATP + 23H2O

Oleoyl-CoA + mitochondria

Stearoyl-CoA + mitochondria

Elaidoyl-CoA + mitochondria

Water is also produced in the reactlon C1s-CoA

ADP+Pi+ATP+H2O but is not lncluded as a product rn the overall equation. Why? 27. Biological Importance of Cobalt In cattle, deer, sheep, and other ruminant animals, large amounts of propionate are produced in the rumen through the bacterial fermentation of ingested plant matter. Proplonate ls the princlpal source of glucose for these animals, via the route propionate -+ oxaloacetate -+ glucose In some areas of the world, notably Australia, ruminant animals sometimes show s5.'rnptomsof anemia with concomitant loss of appetite and retarded growth, resulting from an inability to transform propionate to oxaloacetate This condition is due to a cobalt deficiency caused by very low cobalt levels in the soil and thus in plant matter Explain. 28. Fat Loss during Hibernation Bears expend about 25 x 106 J/day during periods of hibernation, which may last as long as seven months The energy required to sustain life is obtained from fatty acid oxidatlon How much welght loss (in

n N

o 6

21

30 L2

2t Time (min)

30 12

27

In the figure, IS indicates an internal standard (pentadecanoyl-CoA) added to the mixture, after the reaction, as a molecular marker. The researchers abbreviated the CoA derivatives as follows: stearoyl-CoA, C1s-CoA;cas-A5-tetrade-

i672)

F a t tA y c i d( a t a b o l i s m

cenoyl-CoA,cAsCrn-CoA; oieoyl-CoA,cAeCrr-CoA;trans-L5tetradecenoyl-CoA,tAsCr4-CoA;and elaidoyl-CoA,tAeC18CoA.

cis-A5-Tetradecenoyl-CoA

o '-"oO lrans-A5-Tetradecenoyl-CoA (a) Why did Yu and colleagues need to use CoA derivatives rather than the free fatty acids in these experiments? (b) WhV were no lower molecular weight CoA derivatives found in the reaction with stearoyl-CoA? (c) How many rounds of p oxidation would be required to convert the oleoyl-CoA and the elaidoyl-CoA to cib-A5-tetradecenoyl-CoA and.trans- L5 -Ietradecenoyl-CoA,respectively? There are two forms of the enz)'rne acyl-CoA dehydrogenase (see Frg I 7-8a) : Iong-chain acyl-CoA dehydrogenase (LCAD) and very-long-chain acyl-CoA dehydrogenase fl{,CAD) Yu and coworkers measured the kinetic parameters of both enzgnes. They used the CoA derivatives of [Lree fatty acids: tetradecanoylCoA (C1a-CoA),czs-A5-tetradecenoyl-CoA(cA5Crn-CoA),and trans- L5 -telradecenoyl-CoA(tA5Ci4-CoA). The resr-r-lts are shown below. (See Chapter 6 for definitions of the kinetic parameters )

LCAI) CrnCoA

v^u* K^ kcat k"dlK^

3.3 0.41 9.9 24

cAo0laCoA

VI,CAI) tAocr4CoA

3.0 0 40

,cl

g.g

8.5 5

22

10

CuCoA

r.4 0.57 2.0 4I

cAoCla-

tAoC14-

CoA

CoA

0 32 0 44 0.42

0.88 0.97 L.L2 I

(d) For LCAD, the K^ differs dramaticallyfor the cis and trans substrates.Provide a plausible explanationfor this observationin terms of the structuresof the substratemolecules. (Hint: Youmay want to refer to Fig. 10-2.) (e) The kinetic parametersof the two enz)'rnesare relevant to the differential processingof these fatty acids only lf the LCAD or WCAD reaction (or both) is the rate-limiting step in the pathway.What evidenceis there to support this assumption? (f) How do these different kinetic parametersexplain the different levels of the CoA derivativesfound after incubation of rat liver mitochondria with stearoyl-CoA,oieoyl-CoA,and elaidoyl-CoA(shovmin the three-panelflgure)? Yu and coworkers measuredthe substrate speciflcity of rat liver mitochondrial thioesterase,which hydrolyzes acylCoA to CoA and free fatty acid (see Chapter21). This enz).rne was approximatelytwice as active with C1a-CoAthioestersas thioesters. with C1s-CoA (g) Other researchhas suggestedthat free fatty acids can pass through membranes.In their experiments,Yu and colleaguesfound trans - L5 -Letradecenoic acid outside mitochondria (i.e., in the medium) that had been incubated with elaidoyl-CoADescribethe pathwaythat led to acid. Be this extramitochondrialtrans-L5-tetradecenoic sure to indicate where in the cell the various transformations take place,as well as the enzymesthat cataiyzethe transformations. (h) It is often saidin the popular pressthat "trans fats are not broken down by your cells and insteadaccumulatein your body." In what senseis this statement correct and in what senseis it an oversimpliflcation? Reference Yu, W., Liang, X., Ensenauer, R., Vockley, J., Sweetman, L., & Schultz, H. (2004) Leaky B-oxidation of a trans-fatty acid.J. Bi,oL Chem 279. 52.160-52,167

I chosethe study of the synthesisof urea in the liver becauseit appearedto be a relativelysimpleproblem. -Hans Krebs,articlein Perspectives in Biologyand Medicine,1970

Amino AcidOxidation andthe Production ofUrea 1 8 . 1 Metabolic Fates ofAmino Groups 674 1 8 . 2 Nitrogen Excretion andtheUrea(ycle 682

breakdownand are not neededfor new protein synthesisundergooxidative degradation.

2. When a diet is rich in protein and the ingested

1 8 . 3 Pathways ofAminoAcidDegradation687

aminoacidsexceedthe body'sneedsfor protein synthesis,the surplusis catabolized;aminoacids cannotbe stored.

e now turn our attentionto the aminoacids,the final class of biomoleculesthat, through their oxidative degradation,make a signiflcantcontribution to the generationof metabolic energy.The fraction of metabolic energy obtained from amino acids, whether they are derivedfrom dietary protein or from tissue protein, varies greatly with the type of organism and with metabolic conditions.Carnivorescan obtain (immediately following a meal) up to 90% of their energy requirementsfrom amino acid oxidation, whereas herbivoresmay flll only a smail fraction of their energy needsby this route. Most microorganisms can scavenge amino acids from their environment and use them as fuel when required by metabolic conditions. Plants, however, rarely if ever oddize amino acids to provide energy;the carbohydrateproducedfrom CO2and H2O in photosynthesisis generallytheir sole energysource. Amino acid concentrationsin plant tissuesare carefully regulatedto just meet the requirementsfor biosynthesis of proteins,nucleic acids,and other moleculesneeded to supportgrowth.Amino acid catabolismdoesoccurin plants, but its purpose is to produce metabolitesfor other bioslmtheticpathways. In animals,amino acidsundergo oxidative degradation in three differentmetaboliccircumstances:

During starvationor in uncontrolleddiabetes mellitus,when carbohydratesare either unavailable or not properlyutilized,cellularproteinsare used as fuel.

1

During the normal synthesisand degradationof cellular proteins (protein turnover; Chapter27), someaminoacidsthat are releasedfrom nrotein

Under all these metabolicconditions,amino acidslose their amino groups to form a-keto acids,the "carbon skeletons"of aminoacids.The o-keto acidsundergooxidation to CO2and H2Oor, often more importantly, provide three- and four-carbonunits that can be converted by gluconeogenesisinto glucose,the fuel for brain, skeletalmuscle,and other tissues. The pathwaysof amino acid catabolismare quite similar in most organisms.The focus of this chapteris on the pathwaysin vertebrates,becausethesehavereceivedthe most researchattention.As in carbohydrate and fatty acid catabolism,the processesof amino acid degradationconverge on the central catabolic pathways, with the carbon skeletonsof most amino acids finding their way to the citric acid cycle. In some casesthe reactionpathwaysof amino acid breakdown closely parallel steps in the catabolismof fatty acids fChapter17). One important feature distinguishesamino acid degradationfrom other catabolicprocessesdescribedto this point: every amino acid contains an amino group, and the pathwaysfor amino acid degradationtherefore include a key step in which the a-amino group is separated from the carbon skeletonand shunted into the

Ftr]

f

l

I 6 7 4)

A m i nA o c i d0 x i d a t i oann dt h eP r o d u c t ioofnU r e a

Intracellular protein

ll

ll

Dietarv proteii €

- Amino acids

NHil

ft

\+,f&:t31.

Biosynthesis of amino acids, nucleotides, and biological amines

+

a-Keto acids

Carbamoyl phosphate

Aspartateargininosuccinate shunt of citric acid cycle

Urea (nitrogen excretion product)

Citric acid cycle

COz+ H2O + ATP

Oxaloacetate

II

v Glucose (synthesized in gluconeogenesis) FIGURE 18-l Overviewof aminoacid catabolismin mammals.Theaminogroupsandthe pathways. carbonskeletontakeseparate but interconnected

pathwaysof aminogroup metabolism(Fig. 18-1). We deal first with amino group metabolism and nitrogen excretion,then with the fate of the carbon skeletons derivedfrom the aminoacids;alongthe way we seehow the pathwaysare interconnected.

18.1Metabolic Fates Groups ofAmino Nitrogen,N2,is abundantin the atmospherebut is too inert for use in most biochemicalprocesses.Because only a few microorganismscan convert N2to biologically useful forms such as NH3 (Chapter 22), amino groups are carefully husbandedin biologicalsystems. Figure l8-2a providesan overviewof the catabolic pathwaysof ammoniaand amino groups in vertebrates. Amino acidsderived from dietary protein are the source of most aminogroups.Most aminoacidsare metabolized in the liver. Some of the ammoniageneratedin this processis recycledand usedin a variety of biosynthetic pathways;the excessis either excreteddirectly or converted to urea or uric acid for excretion,dependingon the organism(Fig. 18-2b). Excessammoniagenerated in other (extrahepatic) tissuestravelsto the liver (in the form of amino groups,as describedbelow) for conversion to the excretory form.

Glutamate and glutamine play especially critical roles in nitrogen metabolism,acting as a kind of general collectionpoint for aminogroups.In the cytosolof hepatocfies, amino groups from most amino acids are transferred to a-ketoglutarateto form glutamate,which enters mitochondria and gives up its amino group to form NHf . Excess ammoniageneratedin most other tissuesis convertedto the amidenitrogenof glutamine, which passesto the liver, then into liver mitochondria. Glutamine or glutamate or both are present in higher concentrationsthan other amino acidsin most tissues. In skeletalmuscle,excessamrnogroupsare generally transferred to pyruvate to form alanine,another important moleculein the transportof aminogroupsto the liver. We begin with a discussionof the breakdownof dietary proteins, then give a generaldescriptionof the metabolicfates of amino groups.

ls[nzymatically Degraded to Dietary Protein Amino Acids In humans, the degradation of ingested proteins to their constituent amino acids occurs in the gastrointestinaltract. Entry of dietaryprotein into the stomach stimulatesthe gastric mucosato secretethe hormone

o r o u o s,-*r) 1 8 . 1M e t a b oF lia c t eosfA m i n G

Amino acids from ingested protein

Cellular protern

Liver

1l il, cooNHf

Hrll-c-H

iln

Ammonia (as ammonium ion)

Alanine from muscle

Ammonotelic animalsl most aquatic vertebrirtes, cuch aE bony fishes end the larvae ofremphibia

HoN-C-NHo -ll

o Urea Ureotelic animals: many terrestrial vertebrates; also sharks

Glutamine from muscle and other tissues

o

Uric acid

NHf,, urea, or uric acid

Uricotelic animals: birds, reptiles

(b)

(a) (a) Overviewof catabolism FIGURI 18*2 Aminogroupcatabolism. of aminogroups(shaded)in vertebrateIiver.(b) Excretory formsof nitrogen Excess NHf, is excreted asammonia(microbes, bonyfishes), urea (mostterrestrial vertebrates), or uric acid(birdsandterrestrial reptiles)

Noticethatthecarbonatomsof ureaand uricacidarehighlyoxidized; mostof itsavailable carbononly afterextracting discards theorganism energyof oxidation.

gastrin, which in turn stimulatesthe secretionof hydrochloricacid by the parietal cellsand pepsinogenby the chiefcellsof the gastricglands(Fig" 18-ila). The acidicgastricjuice (pH 1.0to 2.5)is both an antiseptic, killing most bacteriaand other foreign cells,and a denaturing agent, unfolding globular proteins and rendering their internal peptide bondsmore accessibleto enzymatichydrolysis.Pepsinogen (M, 40,554),an tnactiveprecursor,or zymogen(p 227), is convertedto activepepsin (M,34,614) by an autocatalyticcleavage (a cleavagemediatedby the pepsinogenitself) that occurs only at low pH. In the stomach,pepsinhydrolyzes ingested proteins at peptide bonds on the aminoterminai side of the aromaticamino acid residuesPhe,

Ttp, and T}rr(seeTable3-7), cleavinglong polypeptide chainsinto a mixture of smallerpeptides. As the acidicstomachcontentspassinto the smallintestine,the low pH triggerssecretionof the hormonesecretin into the blood.Secretinstimulatesthe pancreasto secretebicarbonateinto the smallintestine to neutralize the gastnc HCl, abruptly increasingthe pH to about 7. (All pancreatic secretionspass into the small intestine through the pancreaticduct.) The digestionof proteins now continues in the small intestine. Arrival of amino acids in the upper part of the intestine (duodenum) causesreleaseinto the blood of the hormone cholecystokinin, which stimulatessecretionof severalpancreatic with activity optimaat pH 7 to B.Thypsinogen, enz)rrnes

T--

1676

A m i nA o c i d0 x i d a t i oann dt h eP r o d u c t ioofn Urea

(a) Gastric glands in lining

Parietal cells (secreteHCI)

Chief cells (secrete pepsinogen) Gastric mucosa (secretesgastrin) (b) Exocrine cells of pancreas

Rough ER

Collecting duct

FIGURE 18-3 Part of the human digestive (gastrointestinal) tract. (a) The parietalcells and chiefcellsof the gastricglandssecrete their productsin responseto the hormone gastrin.Pepsinbeginsthe processof protein degradationin the stomach.(b) The cytoplasmof exocrinecells is completelyfilled with roughendoplasmic reticulum,the site of synthesis of the zymogensof manydigestive enzymes.The zymogensare concentrated in membrane-enclosed transDort particles calledzymogengranules. When an exocrinecell is stimulated, its plasmamembrane fuses with the zymogen granule membraneand zymogensare releasedinto the lumenofthe collectingductby exocytosis.The collectingducts ultimatelylead to the pancreatic duct and thenceto the small intestine.(c) Amino acids are absorbed throughthe epithelialcell layer(intestinal mucosa) of the vi|| i andenterthe capiIlaries. Recallthatthe products of lipid hydrolysis in the smallintestineenterthe lymphaticsystem aftertheirabsorptionby the intestinalmucosa (seeFig..l7-1).

Villus Intestinal mucosa (absorbs amino acids)

chymotrypsinogen, and procarboxypeptidases A and B-the z),'mogensof tr5rysin, chymotrypsin, and carboxypeptidases A and B-are syr.rthesized and secretedby the exocrinecellsofthe pancreas(Fig. 18-3b). T\psinogen is convertedto its active form, trypsin, by enteropeptidase, a proteolltic en4..rnesecretedby intestinal cells.Free trypsin then catalyzesthe conversion of additional trypsinogento trypsin (see Fig. 6-38). Trypsin also activates chlmotrypsinogen, the procarboxypeptidases,and proelastase. Why this elaboratemechanismfor getting active digestiveenz).rnesinto the gastrointestinaltract? Sfrthesis of the enzlrnes as inactive precursors protects the exocrine cells from destructive proteol),tic attack. The pancreasfurther protects itself againstself-digestionby making a specif,cinhibitor, a protein called pancreatic trypsin inhibitor (p.227), that effectivelyprevents premature production of active proteolytic enzyrnes within the pancreaticcells.

I$psin and chymotrypsin further hydrolyze the peptides that were producedby pepsinin the stomach.This stageof protein digestionis accomplishedvery efflciently, becausepepsin,trypsin, and chymotrypsinhavedifferent amino acid speciflcities(see Table 3-7). Degradationof the short peptidesin the smallintestineis then completed by other intestinalpeptidases.Theseincludecarbox;peptidases A and B (both of which are zinc-containing enz),Tnes),which remove successivecarboxyl-terminal residues from peptides, and an aminopeptidase that hydrolyzes successive amino-terminal residues from short peptides.The resultingmixture of free aminoacids is transported into the epithelial cells lining the small intestine (Fig. 18-3c), through which the amino acids enter the blood capillariesin the villi and travel to the liver. In humans, most globular proteins from animal sourcesare almostcompletelyhydrolyzedto amino acids in the gastrointestinaltract, but some fibrous proteins, such as keratin, are only partly digested.In addition,the

Fates ofAmino Groups 18.1Metab0lic furl protein contentof someplant foodsis protectedagainst breakdownby indigestiblecellulosehusks. Acute pancreatitis is a diseasecausedby obffi struction of the normal pathway by which panI! creaticsecretionsenter the intestine The zymogensof the proteoly'ticenzymesare convertedto their catalltically active forms prematurely, i,nside the pancreatic cells,and attackthe pancreatictissueitself.This causes excruciatingpain and damageto the organ that can prove fatal. I

Pyridoxal Phosphate Participates intheTransfer of a-Amino Groups toa-Ketoglutarate The first step in the catabolismof most r,-aminoacids, once they have reached the liver, is removal of the aaminogroups,promotedby en4'rnescalledaminotransferases or transaminases. In these transamination reactions,the a-amino group is transferred to the ocarbon atom of a-ketoglutarate,Ieavingbehind the corresponding a-keto acid analog of the amino acid (Fig. f 8-4). Thereis no net deamination(lossof amino groups) in these reactions,becausethe a-ketoglutarate becomesaminatedas the a-aminoacid is deaminated. The effect of transaminationreactionsis to collect the amino groups from many different amino acids in the form of l-glutamate. The glutamatethen functions as an amino group donor for biosynthetic pathwaysor for excretion pathways that lead to the elimination of nitrogenouswasteproducts. Cells contain different types of aminotransferases. Manyare speciflcfor a-ketoglutarateasthe aminogroup acceptor but differ in their speciflcity for the l-amino acid. The en4..rnesare namedfor the aminogroup donor

cooI C:O

I

HsN-C-H -l CHo

C}lq

CHo

tcooc-Ketoglutarate

coo

H.N-C-H "l R l-Amino

coo

CHq

t-

*l

*l

acid

1-

tcoor,-Glutamate

cooI C:O

I

(alanine aminotransferase,aspartateaminotransferase, for example). The reactions catalyzedby aminotransferasesare freely reversible,having an equilibrium constant of about 1.0 (LG' : 0 kJ/mol). AII aminotransferaseshave the same prosthetic group and the samereactionmechanism.The prosthetic group is pyridoxal phosphate (PLP), the coenzl'rne form of pyridoxine, or vitamin 86. We encounteredpyridoxal phosphatein Chapter 15, as a coenzymein the glycogenphosphorylase reaction,but its role in that reaction is not representativeof its usual coenzymefunction. Its primary role in cells is in the metabolismof moleculeswith aminogroups. Pyridoxal phosphate functions as an intermediate carrier of amino groups at the active site of aminotransferases.It undergoesreversible transformationsbetween its aldehydeform, pyridoxal phosphate,which can acceptan amino group, and its aminatedform, pyridoxaminephosphate,which can donateits amino group phosphateis to an a-keto acid (Fig. 18-5a). PS,ridoxal generally covalently bound to the enz;rme'sactive site through an aldimine (Schiff base)linkageto the e-amino groupofa Lysresidue(Fig. 18-5b, d). Pyridoxal phosphateparticipatesin a variety of reactionsat the a, B, and 7 carbons(C-2to C-4) of amino acids.Reactionsat the a carbon (Fig. 18-6) include racemizations(interconvertingl- and l-amino acids) and decarboxylations,as well as transaminations.Pyridoxal phosphateplaysthe samechemicalrole in eachof thesereactions.A bond to the a carbonof the substrate is broken, removingeither a proton or a carboxylgroup. The electron pair left behind on the a carbon would form a highly unstable carbanion,but plryidoxalphosphate providesresonancestabilizationof this intermediate (Fig. 18-6 inset). The highly conjugatedstructure of PLP (an electron sink) permits delocalizationof the negativecharge. (Fig. 1B-5) are classicexamples Aminotransferases of enzymescatalyzingbimolecularPing-Pongreactions (seeFig. 6-13b), in which the first substratereactsand the productmust Ieavethe activesitebeforethe second substratecan bind. Thus the incoming amino acid binds to the activesite, donatesits aminogroup to pyridoxal phosphate,and departsin the form of an a-keto acid. The incominga-keto acid then binds, acceptsthe amino group from pyridoxaminephosphate,and departsin the form of an amino acid. As describedin Box l8-1 on page678,measurementof the alanineaminotransferase levelsin blood serumis and aspartateaminotransferase importantin somemedicaldiagnoses.

R a-Keto acid

FIGURE 18-4 Enzyme-catalyzed transaminations. In many aminoreactions,a-ketoglutarate transferase is the amino groupacceptor.All aminotransferases have pyridoxalphosphate(PLP)as cofactor.Althoughthe reactionis shownhere in the directionof transfer of the aminogroupto a-ketoglutarate, it is readilyreversible

Group AsArnmonia Releases ltsAmino 6lutamate intheLiver As we have seen,the amino groups from many of the a-amino acidsare collectedin the liver in the form of the amino group of L-glutamatemolecules.These amino

o c i d0 x i d a t i oann dt h eP r o d u c t ioofnU r e a | 6 7 8 | A m i nA

o-

I -O-P:O

o-

I o

I O-P:O

I o I

Pyridoxal phosphate (PLP)

o o-P:o o +

CH. \" lJ*

H |

HsN-9-< t\/ H

o I -o-P:o I o

NH

OH CHS Pyridoxamine phosphate (a)

H H

CHo \-

lll__\*

Lys-N:C{

Schiffbase

NH

\/ /

oH

\

cHa

ft) FIGURE 18-5 Pyridoxalphosphate,the prostheticgroup of amino(a) Pyridoxalphosphate(PLP)and its aminatedform, transferases. pyridoxamine phosphate, arethe tightlyboundcoenzymes of aminotransferases. The functionalgroupsare shaded.(b) Pyridoxalphosphateis boundto the enzymethroughnoncovalentinteractions and a (aldimine)linkageto a Lysresidueat the activesite.The Schiff-base stepsin the formationof a Schiffbasefrom a orimarvamineand a car-

bonylgrouparedetailedin Figure14-5. (c) PLP(red)boundto oneof the two activesitesof the dimericenzymeaspartate aminotransferase, (d) close-upview of the activesite,with a typical aminotransferase; PLP(red,with yellow phosphorus) in aldiminelinkagewith the side (purple);(e) anotherclose-upview of the activesite, chain of Lys2s8 (green)via a with PLPlinkedto the substrate analog2-methylaspartate Schiffbase(PDBlD l AJS).

Analysesof certain enzymeactivities in blood serum give valuablediagnosticinformation for severaldisease conditions. Alanine aminotransferase(ALT; also called glutamate-pyruvate transaminase, GPT) and aspartate aminotransferase(AST; also called glutamate-oxaloacetate transaminase,GOT) are impoftant in the diagnosis of heart and liver damagecausedby heart attack, drug toxicity, or infection. A-fter a heart attack, a variety of enzyrnes,including these aminotransferases,leak from injured heart cellsinto the bloodstream.Measurements of the blood serum concentrationsof the two aminotransferasesby the SGPT and SGOT tests (S for serum)-and of another enzyme,creatine kinase, by the SCK test-can provide information about the severity of the damage.Creatine kinase is the first

heart enzy'rneto appearin the blood after a heart attack; it alsodisappearsquickly from the blood.GOTis the next to appear,and GPTfollows later.Lactate dehydrogenase alsoleaksfrom rnjured or anaerobicheart muscle. The SGOTand SGPT tests are also important in occupationalmedicine, to determine whether people exposedto carbontetrachloride,chloroform,or other industrial solventshave sufferedliver damage.Liver degenerationcausedby thesesolventsis accompaniedby leakageof various enzymesfrom injured hepatocytes into the blood. Aminotransferasesare most usefirlin the monitoring of people exposedto these chemicals,becausethese enzyrneactivities are high in liver and thus are likely to be among the proteins leaked from damaged hepatocytes;also, they can be detected in the bloodstreamin very small amounts.

Groups Fates ofAmino 18.1Metabolic furnl

Hydrolysis of Schiff base to form a-keto acid and py'ridoxamine HrO

\ =^ (9

Pyridoxal phosphate (internal aldimine form, on enzyme)

lvs-fru"

I

R _

cOO ,-:----r (Enz,)

=-a -S

cH Hor.\|

, ,,"

(P.)

-

R

R

I

'NH i CH

H-C

i,:

I

R R

I

*NH

H-C-H I *NH3

Coz

CH

H

_), .^ (q) Schiff base intermediate (external

Resonance structures for stabili zation of a carbanion bv PLP

regenerated enzyme)

H-

I *NH e

*NH, o-Amino acid ., Pyridoxal phosphate (internal aldimine

\ ) cH.'H

Quinonoid intermediate

H-C:

H

R-(-coo

s

H

Quinonoid intermediate

+

Amine

Pyridoxal phosphate (internal aldimine form, on regenerated enzyme)

aldimine)

ME(HANlSM FIGURE l8-6 Someaminoacidtransformations at the c carbonthat are facilitatedby pyridoxalphosphate. Pyridoxalphosphateis generallybondedto the enzymethrougha Schiffbase,also calledan internalaldimine.Thisactivated formof PLPreadilyundergoestransimination to forma new Schiffbase(external aldimine)with the a-aminogroupof the substrate aminoacid (seeFig. 1B-5b,d) Threealternative fatesfor the externalaldimineareshown:@ transamination,@racemization, and@decarboxylation ThePLP-amino acid with thepyridinering,an electron Schiffbaseis in conjugation sinkthat permitsdelocalization of an electronpair to avoidformationof an

is A quinonoidintermediate on thea carbon(inset). carbanion unstable route@is Thetransamination involvedin all threetypesof reactions. The especiallyimportantin the pathwaysdescribedin this chapter. only partof the pathwayhighlightedhere(shownleftto right)represents To completethe overallreactioncatalyzedby aminotransferases. and the one that is released, process, a seconda-ketoacid replaces of the reactionsteps to an aminoacid in a reversal this is converted reactions (rightto left).Pyridoxalphosphateis also involvedin cer (not Pyridoxal acids shown). of someamino at theF and7 carbons ReactionMechanisms Phosphate

groups must next be removedfrom glutamateto prepare them for excretion.In hepatocytes,glutamateis transportedfrom the cltosol into mitochondria,whereit undergoesoxidative deamination cataiyzedby Lglutamate dehydrogenase(Mr 330,000).In mammals, this en4,me is present in the mitochondrialmatrix. It is the only enzyrnethat canuseeitherNAD+ or NADP+as the acceptorof reducingequivalents(Irig. 18-7). The combinedaction of an aminotransferaseand glutamate dehydrogenaseis referred to as trans-

deamination. deamination

A few amino acids bypass the transpathway and undergo direct oxidative

deamination.The fate of the NHi producedby any of these deaminationprocessesis discussedin detail in Section 18.2. The a-ketoglutarateformed from glutamate deaminationcan be used in the citric acid cycle and for glucosesynthesis. operatesat an rmporGlutamatedehydrogenase tant intersectionof carbonand nitrogenmetabolism. An allostericenzymewith six identical subunits,its

.t

A m i nA o c i d0 x i d a t i oann dt h eP r o d u c t ioofnU r e a

-680-

coo *l Hsbr-c-H

frn"

I -ooc-cH2-cH2-cH-coo-

I

*l

coo-

H2N :C CH"

t-

CHo

+

oo NHg iltl I o-P-o-c-cH2-cH2-cH-coot-

o

tcoo

- NH;

l;:l;iHJ'

gl utirf sVllth('

.1pi

+

NH* q. \\l ,/.c-cH2-cH2-cH-coo HzN

coo a-Ketoglutarate

r,-Glutamine

FIGURE 18-7 Reactioncatalyzedby glutamatedehydrogenase. Theglutamatedehydrogenase of mammalianliverhasthe unusualcapacityto useeitherNAD* or NADP+ascofactor. Theglutamatedehydrogenases of plantsand microorganisms aregenerallyspecificfor one or the other Themammalian enzymeisallosterically regulated by CTPandADP.

activity

is influenced

by a complicated

array of al-

Iosteric modulators.The best-studiedof these are the positivemodulatorADP and the negativemodulator GTP.The metabolicrationalefor this regulatory pattern has not been elucidated in detail. Mutations that alter the allostericbinding site for GTP or otherwise cause permanent activation of glutamate dehydrogenaselead to a human genetic disorder called hyperinsulinism-hyperammonemia syndrome,characterized by elevated levels of ammonia in the bloodstreamand hypoglycemla.

oo o

l-Glutamate FIGURE 18-8 Ammonia transportin the form of glutamine.Excess ammoniain tissues is addedto glutamate to formglutamine, a process catalyzedby glutaminesynthetaseAftertransportin the bloodstream, theglutamine entersthe liverand NHi is liberated in mitochondria by theenzymeglutaminase.

produce glutamine

Glutamine Transports Ammonia inth*Sloodstrearn Ammonia is quite toxic to animal tissues (we examine some possible reasons for this toxicity later), and the levels present in blood are regulated. In many tissues, including the brain, some processes such as nucleotide degradation generate free ammonia. In most animals much of the free ammonia is converted to a nontoxic compound before export from the extrahepatic tissues into the blood and transport to the liver or kidneys. F or this transport function, glutamate, critical to 'intracelluLar amino group metabolism, is supplanted by t -glutamine. The free ammonia produced in tissues is combined with glutamate to yreld glutamine by the action of glutamine synthetase. This reactiorr requrres ATP and occurs in two steps (Fig. lS-S). First, glutamate and ATP react to form ADP and a 7-glutamyl phosphate intermediate, which then reacts with ammonia to

T"'

/c-c}l2-cH2-cH-coo-

and inorganic

phosphate.

Glutamine

is a nontoxic transport form of ammonia;it is normally present in blood in much higher concentrationsthan other amino acids.Glutaminealso servesas a sourceof amino groupsin a variety of biosyntheticreactions.Glutamine synthetaseis found in all organisms,alwaysplaying a central metabolic role. In microorganisms,the enzyme serves as an essential portal for the entry of fixed nitrogen into biological systems.(The roles of glutamineand glutaminesynthetasein metabolismare further discussedin Chapter22.) In most terrestrial animals,glutamine in excessof that requiredfor biosynthesisis transportedin the blood to the intestine, liver, and kidneys for processing.In thesetissues,the amidenitrogenis releasedas atrrmonium ion in the mitochondria,where the enzymeglutaminase converts glutamine to glutamate and NHf, (Fig. 18-8). The NHf from intestine and kidney is transported in the blood to the liver. In the liver, the

o rouos 681 1 8 . 1M e t a b 0 F l i ca t eosfA m i n G

anlmoniafrom all sourcesis disposedof by urea synthesis.Someof the glutamateproducedin the glutaminase reacti.onmay be further processedin the liver by glutamate dehydrogenase, releasingmore ammoniaand producing carbon skeletonsfor metabolicfuel. However, most glutamateentersthe transaminationreactionsrequired for aminoacid biosynthesisand other processes (Chapter22). lG

Tn motehnlin ani6l6si5

(p. 667) there is an increase w:"":":-"":'"-.in glutamineprocessingby the kidneys.Not all N

the excess NHi thus produced is releasedinto the bloodstreamor convertedto urea; some is excreted directiy into the urine. In the kidney, the NHi forms salts with metabolic acids, facilitating their removal in the urine.Bicarbonateproducedby the decarboxylation of a-ketoglutaratein the citric acid cyclecan alsoserve asa bufferin bloodplasma.Takentogether,theseeffects of glutaminemetabolismin the kidney tend to counteract acidosis. I

":1.-

a-Ketoglutarate Alanine. ,,

I '=dl

i Blood alanine

Blood glucose

Alanine Transports Ammonia fromSkeletal Muscles to theLiver Alanine also plays a specialrole in transportingamino groups to the liver in a nontoxic form, via a pathway called the glucose-alanine cycle (Fig. 18-9). In muscle and certain other tissuesthat degradeamino acids for fuel, amino groups are collectedin the form of glutamateby transamination(Fig. 18-2a). Glutamate can be converted to glutamine for transport to the liver, as describedabove,or it can transferits aaminogroup to pyruvate,a readily availableproduct of muscle glycolysis,by the action of alanine aminotransferase (Fig. 18-9). The alanine so formed passesinto the blood and travelsto the liver. In the cytosol of hepatocytes,alanine aminotransferasetransfers the amino group from alanineto a-ketoglutarate, forming pyruvate and glutamate.Glutamatecan then enter mitochondria,where the glutamatedehydrogenase reactionreleasesNHi Gig. 18-7), or can undergo transamination with oxaloacetateto form aspartate,anothernitrogen donor in urea synthesis,as we shallsee. The use of alanine to transport ammonia from skeletalmusclesto the liver is another exampleof the intrinsic economyof living organisms.Vigorouslycontracting skeletal muscles operate anaerobically,producing pyruvate and lactate from glycolysisas well as ammonia from protein breakdov,'n.These products must find their way to the iiver, where pyruvate and lactate are incorporatedinto glucose,which is returnedto the muscles,and ammoniais convertedto urea for excretion. The glucose-aianine cycle,in concertwith the Cori cycle (see Box l4-2 and Fig. 23-20), accomplishesthis transaction.The energeticburden ofgluconeogenesisis thus imposedon the liver rather than the muscle,and all availableATP in muscleis devotedto musclecontraction.

Glucose + gl,t"n,a"ul gcDesls

Liver

.ri

r1;r cycle.Alanineservesas a carrierof l8-9 Glucose-alanine FIGURE from skeletalmuscle of pyruvate ammoniaand of the carbonskeleton and the pyruvateis usedto produce to liver.Theammoniais excreted which is returnedto the muscle. glucose,

lsToxit toAnimals Arnmonia The catabolic production of ammonia poses a serious biochemical problem, because ammonia

is very toxic. The molecular basis for this toxicity is not entirely understood.The terminal stagesof ammonia intoxication in humansare characterizedby onset of a comatosestate accompaniedby cerebral edema (an increase in the brain's water content) and increasedcranialpressure,so researchand speculation on ammoniatoxicity havefocusedon this tissue.Speculation centers on a potential depletion of ATP in brain cells. Riddingthe cy'tosolof excessammoniarequiresreductive amination of a-ketoglutarate to glutamate by glutamatedehydrogenase(the reverseof the reaction

t

l

682

Amino Acid 0xidation andtheProduction ofUrea

describedearlier; Fig. 18-7) and conversionof glutamate to glutamineby glutamine synthetase.Both enz).rynes are present at high levels in the brain, although glutamine the synthetasereaction is almost certainly the more important pathway for removal of ammonia. High levelsof NHf lead to increasedlevelsof glutamine, which acts as an osmoticallyactive solute (osmolyte) in brain astrocytes,star-shaped cellsofthe nervoussystem that provide nutrients, support, and insulation for neurons. This triggersan uptake of water into the astrocl'tes to maintain osmotic balance,leading to swelling of the cellsand the brain,leadingto coma. Depletionof glutamatein the glutaminesynthetase reaction may have additional effects on the brain. Glutamate and its derivative y-aminobutyrate (GABA; see Fig.22-29) are importantneurotransmitters;the sensitivity of the brain to ammoniamay reflect a depletion of neurotransmittersaswell as changesin cellularosmotic balance.I As we closethis discussionof amino group metabolism, note that we have describedseveralprocessesthat deposit excessammoniain the mitochondriaof hepatocy[es(Fig. 18-2).Wenow]ook at the fate of that ammonia.

S U M M A R1Y8 . 1 M e t a b o lFi ca t eosf A m i n o Groups r

Humansderive a small fraction of their oxidative energyfrom the catabolismof amino acids.Amino acidsare derived from the normal breakdown (recycling)of cellularproteins,degradationof ingestedproteins,and breakdownof body proteins in lieu of other fuel sourcesduring starvationor in uncontrolleddiabetesmellitus.

r

Proteasesdegradeingestedproteinsin the stomach and smallintestine.Most proteasesare initially synthesizedas inactivezymogens.

r

An early step in the catabolismof amino acids is the separationof the amino group from the carbonskeleton.In most cases,the aminogroupis transferred to a-ketoglutarateto form glutamate. This transaminationreaction requiresthe coenz;,me pyridoxal phosphate.

r

Glutamateis transportedto liver mitochondria, where glutamatedehydrogenaseliberatesthe amino group as ammoniumion (NHf). Ammonia formedin other tissuesis transportedto the liver as the amide nitrogen of glutamine or, in transport from skeletalmuscle,as the aminogroup of alanine.

r

The pyruvateproducedby deaminationof alanine in the liver is convertedto glucose,which is transportedbackto muscleaspaft of the glucosealaninecycle.

18.2Nitrogen Excretion andtheUrea Cycle If not reused for the syrrthesisof new amino acids or other nitrogenousproducts,aminogroupsare charmeled into a singleexcretoryend product (Fig. f 8-10). Most aquatic species, such as the bony fishes, are ammonotelic, excreting amino rutrogen as ammonia.The toxic ammoniais simply diluted in the surroundingwater. Terrestrial animals require pathways for nitrogen excretionthat minimizetoxicity and water loss.Mostterrestrial animalsare ureotelic, excretingamino nitrogen in the form of urea;birds and reptiles are uricotelic, excretrngaminonitrogen asuric acid. (The pathwayof uric acid synthesisis describedin Fig. 22-45.) Plantsrecycle virtually all amrnogroups,and nitrogen excretion occurs orLlyunder very unusualcircumstances. In ureotelic organisms,the ammonia deposited in the mitochondriaof hepatocytesis convertedto urea in the urea cycle. This pathwaywas discoveredin l932by HansKrebs (who later alsodiscoveredthe citric acid cycle) and a medical student associate,Kurt Henseleit. Urea production occurs almost exclusivelyin the liver and is the fate of most of the ammoniachanneledthere. The urea passesinto the bloodstreamand thus to the kidneysand is excretedinto the urine. The production of urea now becomesthe focusof our discussion.

UrealsProduced fromAmmonia in FiveEnzymatic Steps The urea cycle begins inside liver mitochondria,but three of the subsequentsteps take place in the cytosol; the cycle thus spanstwo cellular compartments (Fig. 18-10). The first amino group to enter the urea cycle is derived from ammonia in the mitochondrial matrix-NHf, arisingby the pathwaysdescribedabove. The liver also receivessome ammoniavia the portal vein from the intestine,from the bacterialoxidationof amino acids.Whateverits source,the NHf generated in liver mitochondriais immediatelyused,togetherwith CO2(asHCOI) producedby mrtochondrialrespiration,to

FIGURE l8-1 0 Ureacycleand reactionsthat feedaminogroupsinto the cycle. The enzymescatalyzingthesereactions(namedin the text)are distributedbetweenthe mitochondrialmatrix and the cytosol.One amino groupentersthe ureacycle as carbamoylphosphate, formedin the matrix;theotherentersasaspartate, formedin the matrixby transaminationof oxaloacetate and glutamate,catalyzedby aspartate aminotransferase. The urea cycle consistsof four steps @ Formationof citrullinefrom ornithineand carbamoylphosphate(entryof the first amino group);the citrullinepassesinto the cytosol.@ Formationof argininosuccinate (entryof the secthrougha citrullyl-AMPintermediate ond aminogroup).@ Formation of argininefrom argininosuccinate; this reactionreleasesfumarate,which entersthe citric acid cycle of urea;thisreactionalsoregenerates ornithine.Thepath@ Formation waysby which NHf arrivesin the mitochondrialmatrixof hepatocvtes werediscussed in Section18.1.

Cycle[ttt] andtheUrea Excretion 18.2Nitrogen

frr,

I cH3-cH-cooAlanine (from muscle)

ox

Tt'

,C-CH2-CH2-CH-COOfi2N

Glutamine (from extrahepatic tissues)

f

NH-(CH2)s-CH-COO-

l)IHe I

o I

H3N-(CH2)3-CH-COO Ornithine Urea cycle

T"t

(CH2)3-CH-COO-

qCIo-

**u

lll -OOC:-CHz-Cll-NH-C-NH-

Fumarate

*"t (CH2)3- CH-COO-

Argininosuccinate

ft

Acid 0xidation andtheProduction ofUrea | 6841 Amino form carbamoylphosphatein the matrix @ig. l8-tla; see also Fig. 18-10). This ATP-dependentreaction is catalyzedby carbamoyl phosphate synthetase I, a regulatory enzlrne (seebelow). The mitochondrialform of the enzyrneis distinct from the c;tosolic (II) form, which hasa separatefunction in pyrimidine biosyrrthesis (Chapter22). The carbamoylphosphate,which functions as an activated carbamoylgroup donor, now enters the urea cycle. The cycle has four enzymaticsteps. First, carbamoyl phosphate donates its carbamoyl group to ornithine to form citrulline, with the release of P1 (Fig. 18-10, step @). Ornithineplaysa role resembling that of oxaloacetatein the citric acid cycle,acceptingmaterial at eachturn of the cycle.The reactionis catalyzed by ornithine transearbamoylase, and the citrulline passesfrom the mrtochondrionto the cy'tosol The secondaminogroup now entersfrom aspartate (generated in mitochondria by transamination and transportedinto the cytosol)by a condensation reaction between the amino group of aspartate and the ureido (carbonyl) group of citrulline, forming argininosuccinate (step@ in fg. 18-10). This cytosolicreaction,catalyzed by argininosuecinate synthetase, requires ATP and proceedsthrough a citrullyl-AMP intermediate (Fig. 18-1lb). The argininosuccinate is then cleavedby argininosuccinase (step@ in Fig. 18-10) to form free

oo ll -o-PI

arginine and fumarate,the latter entering mitochondria to join the pool of citric acid cycleintermediates.This is the only reversiblestepin the urea cycle.In the last reactionof the urea cycle (step @;, ttre cytosolicenzyme arginase cleavesarginine to yield urea and ornithine. Ornithine is transportedinto the mitochondrionto initiate anotherround of the urea cycie. As we noted in Chapter 16, the enzyrnesof many metabolic pathwaysare clustered (p. 619), with the product of one enzymereaction being channeleddirectly to the next enz).rnein the pathway.In the urea cycle,the mrtochondrialand cytosolicenzyrnesseemto be clustered in this way. The citrulline transported out of the mitochondrionis not dilutedinto the generalpool of metabolitesin the cytosolbut is passeddirectly to the active site of argininosuccinatesynthetase.This channeling between enzymescontinuesfor argininosuccinate, arginine,and ornithine.Only urea is releasedinto the generalcytosolicpool of metabolites.

(yeles The(itricAcid andUrea CanBeLinked Becausethe fumarateproducedin the argininosuccinase reaction is also an intermediateof the citric acid cycle, the cyclesare,in principle,interconnected-in a process dubbedthe "Ituebsbicycle"(Fig. 18-12). However,each cycle can operate independently, and communication

Pi

il c-oH

oH3

Bicarbonate is phosphory- Carbonic-phosphoric acid anhydride Iated by ATP

(a)

) @ Ammonia displaces phosphoryl group to generate carbamate.

o tl

H2N-C-OCarbamate

oo il

@ Carbamate is phosphorylated to yield carbamoyl phosphate.

*

NH I

t?rr'j H-C-NH3

cooCitrulline

PPr

AMP

,/

o

Rearrangementleads to addition ofAMP, activating the carbonyl oxygenofcitrulline.

oCarbamoyl phosphate

lf', ?ooC_N-C-H

U I

tl

H -e lN - C - O - P - O -

@ Aspartate addition is facilitated by displacement ofAMP

lHl

T' ?',

(?Hr)3cooH-C-NH3

cooArgininosuccinate

ATP il4E(l{ANlsM FIGURE 18-11 Nitrogen-acquiring reactionsin the synthesisof urea.The ureanitrogensare acquiredin two reactions,each requiringATP.(a) In the reactioncatalyzedby carbamoylphosphate synthetase l, thefirstnitrogenentersfromammonia. Theterminalphosphategroupsof two moleculesof ATp are usedto form one molecule of carbamoylphosphate.In otherwords,this reactionhastwo activa-

tion steps(@and Ol O CarbamoylPhosphate Synthetase I Mechanism(b) In the reactioncatalyzedby argininosuccinate synthetase, the secondnitrogenentersfrom aspartate. Activationof the ureido oxygeno itrullinein step@ setsup the additionof aspartatein step@. ArgininosuccinateSynthetaseMechanism

f

l

a y c l e| 6 8 5 I 1 8 . 2N i t r o g eEnx c r e t iaonndt h eU r e C

FumaraterL

-> Arsinine -

rea

Aspartate

e"purtut"/ a-Ketoglutarate Glutamate

CarbamoYl phosphate

NADH NAD* Malate A \ Fumarate

Citric acid cycle

Mitochondrial matrix

18-12 Links betweenthe urea cycle and citric acid cycle. FIGURE The interconnected cycleshavebeen calledthe "Krebsbicycle" The pathwayslinkingthe citric acid and urea cyclesare known as the aspartate-argininosuccinate shunt;theseeffectivelylink the fatesof the aminogroupsand the carbonskeletons of aminoacids.The interconnectionsareevenmoreelaborate thanthe arrowssuggestForexample,

somecitric acid cycleenzymes,suchasfumaraseand malatedehydroisozymes.Fumarate genase,haveboth cytosolicand mitochondrial producedin the cytosol-whetherby the ureacycle,purinebiosynthesis,or other processes-canbe convertedto cytosolicmalate,which (viathe malateintomitochondria is usedin thecytosolor transported acid cycle' enter the citric Fig 19-29) to see shuttle; aspartate

betweenthem dependson the transportof key intermediatesbetweenthe mitochondrionand cytosol.Several enzyrnesof the citric acid cycle,including fumarase(fu(p. 628), maratehydratase)and malatedehydrogenase are also present as isozymesin the cytosol. The fumarate generatedin cytosolic arginine synthesiscan therefore be convertedto malate in the cvtosol. and these intermediates can be further metabolizedin the cytosolor transportedinto mitochondriafor use in the citric acid cycle. Aspartate formed in mitochondria by transaminationbetween oxaloacetateand glutamate can be transportedto the cytosol,where it servesas nitrogen donor in the urea cycle reaction catalyzedby argininosuccinate s;nthetase.Thesereactions,making up the aspartate-argininosuccinate shunt, provide metabolic links between the separate pathways by which the amino groups and carbon skeletonsof amino acidsare processed.

amino groups. During prolonged starvation, when breakdownof muscle protein beginsto supply much of the organism'smetabolic energy,urea production also increasessubstantially. These changesin demand for urea cycle activity are met over the long term by regulationof the rates of synthesis of the four urea cycle enzymes and carbamoyl phosphate synthetaseI in the liver. All five enzymesare synthesizedat higher rates in starving animals and in animals on very-high-protein diets than in well-fed animals eating primarily carbohydratesand fats.Animalson protein-freedietsproducelower levels of urea cycle enzymes. On a shortertime scale,allostericreguiationof at least one key enzlrne adjuststhe flux through the urea cycle' The first enz).rnein the pathway,carbamoylphosphate synthetaseI, is allostericallyactivatedby N-acetylglutamate, which is rynthesizedfrom acetyl-CoAand glutamatebyN-acetylglutamate synthase (Fig. 18-f3)' In this enzlrne catalyzesthe flrst plantsandmicroorganisms of argininefrom glutamate slmthesis novo step in the de (see Fig. 22-10), but in mammalsl/-acetylglutamate synthase activity in the liver has a purely regulatory function (mammalslack the other enz;.'rnesneeded to convert glutamateto arginine). The steady-statelevels

(yclelsRegulated TheActivity oftheUrea atTwoLevels The flux of nitrogen through the urea cycle in an individual animalvarieswith diet. Whenthe dietary intake is primarily protein, the carbon skeletonsof amino acids are usedfor fuel, producingmuch urea from the excess

-

686

l

A m i nA o c i d0 x i d a t i oann dt h eP r o d u c t ioofnU r e a

//o -c\

cH3

+ S-CoA

*l

(ycle(anBe Genetic intheUrea Defects Life-Threatening

coo-

H3N-C-H 'l

CH,

Acetyl-CoA

I CHr

Glutamate

tcoo

o ill

coo-

cH3-C-NH-C-H I CHo

ttcoo CH, N-Acetylglutamate

Carbamoylphosphate FIGURE 18-13 Synthesis of N-acetylglutamate and its activationof carbamoyl phosphate synthetase l.

of N-acetylglutamate are determined by the concentrations of glutamate and acetyl-CoA (the substrates for l/-acetylglutamate synthase) and arginine (an activator of N-acetylglutamate synthase, and thus an activator of the urea cycle).

(ostof Pathway Interconnections Reduce theEnergetic Urea Synthesis If we consider the urea cycle in isolation, we see that the synthesis of one molecule of urea requires four highenergy phosphate groups (FiS. lB-10). T\ryoATP molecules are required to make carbamoyl phosphate, and one AIP to make argininosuccinate-the latter ATp undergoing a pyrophosphate cleavage to AMp and pp,, which is hydrolyzed to two P,. The overall equation of the urea cycle is

2NHi+ HCot+ tTi,..,x;?J+ +p?-+AMp2+ 2H+ However, the urea cycle also causes a net conversion of oxaloacetate to fumarate (via aspartate), and the regeneration of oxaloacetate (Fig. 18-12) produces NADH in the malate dehydrogenase reaction. Each NADH molecule can generate up to 2.5 ATP during mitochondrial respiration (Chapter 19), greatly reducing the overall energetic cost of urea synthesis.

People wrth genetic defects in any enzyme inE volved in urea formation cannot tolerate proE tein-rich diets. Amino acids ingested in excess of the minimum daily requirements for protein synthesis are deaminated in the liver, producing free ammonia that cannot be converted to urea and exported into the bloodstream, and, as we have seen, ammonia is highly toxic. The absence of a urea cycle enzyme can result in hyperammonemia or in the build-up of one or more urea cycle intermediates, depending on the enzyme that is missing. Given that most urea cycle steps are irreversible, the absent enzyme activity can often be identified by determining which cycle intermediate is present in especially elevated concentration in the blood and/or urine. Although the breakdown of amino acids can have serious health consequences in individuals with urea cycle deficiencies, a protein-free diet is not a treatment option. Humans are incapable of synthesizing half of the 20 common amino acids, and these essential amino acids (Table 18-l) must be provided in the diet. A variety of treatments are available for individuals with urea cycle defects. Careful administration of the aromatic acids benzoate or phenylbutyrate in the diet can help lower the level of ammonia in the blood. Benzoate is converted to benzoyl-CoA, which combines with glycine to form hippurate (Fig. 18-14, Ieft). The glycine used up in this reaction must be regenerated, and ammonia is thus taken up in the glycine synthase reaction. Phenylbutyrate is converted to phenylacetate by B oxidation. The phenylacetate is then converted to phenylacetyl-CoA, which combines with glutamine to form phenylacetylglutamine (Fig. 18-14, right). The resulting removal of glutamine triggers its further synthesis by glutamine synthetase (see Eqn 22-I) in a

Nonessential

Conditionally essential*

Essential

Alanine

Arginine

Histidine

Asparagine

Cysteine

Isoleucine

Aspartate

Glutamine

Leucine

Glutamate

Glycine

Lysine

Serine

Proline

Methionine

Tlrosine

Phenylalanine Threonine Tf1ptophan Valine

+Required growing to somedegree in young, animals, and/orsometimes duringillness

Acid Degradation ofAmino 18.3Pathways Ioaz-]

cH,-cH"-cH,-coo t-



AMP + PPt J> O S-CoA \\/ C

o CH2-C-S-CoA

Benzoyl-CoA

Phenylacetyl-CoA

H,-Coo

V

CH.

Phenylacetate

2*, L

l-

ttcoo

_sH

ll *

I

Benzoate

?ooCHo

/\

ll + coA-sH

1

H2N-C-NH-C-H

t

Supplementingthe diet with arginine is useful in treating deficienciesof ornithine transcarbamoylase,arginiMany nosuccinatesynthetase,and argininosuccinase. district by be accompanied must of thesetreatments amino acids. of essential etary controland supplements In the rare casesof arginasedeflciency,arginine,the substrateof the defectiveenzJrme,must be excluded from the diet. r

1Y 8 . 2 N i t r o g eEnx c r e t iaonnd SUMMAR Cycle theUrea

I' H-CH2-COO

r

Hippurate (benzoylglycine)

o 1l

coo

I CH2-C-NH-CH I

z-\ lrr

\l

gH,

?', C:O

r

NH, Phenylacetylglutamine FIGURE 18-14 Treatmentfor deficienciesin urea cycle enadand phenylbutyrate, zymes.Thearomaticacidsbenzoate ministered in the diet,aremetabolized andcombinewith glycineand respectively. Theproductsareexcretedin the urine.Subseglutamine, quentsynthesis to replenish thepoolof these of glycineandglutamine removes ammoniafromthe bloodstream. intermediates

reaction that takes up ammonia.Both hippurate and phenylacetylglutamineare nontoxic compoundsthat are excretedin the urine. The pathwaysshownin Figure 18-14 make only minor contributions to normal metabolism,but they become prominent when aromatic acidsare ingested. Other therapiesare more specific to a particular enzyme deficiency. Deficiency of N-acetylglutamate

r

Ammoniais highly toxic to animaltissues.In the urea cycle, ornithine combineswith ammonia, in the form of carbamoylphosphate,to form citrulline. A secondamino group is transferredto citrulline from aspartateto form arginine-the immediateprecursor of urea. Arginase catalyzes hydrolysisof arginineto urea and ornithine; thus ornithine is regeneratedin eachturn of the cycle. The urea cycleresultsin a net conversionof oxaloacetateto fumarate,both of which are intermediatesin the citric acid cycle. The two cyclesare thus interconnected. The activity of the urea cycle is regulatedat the Ievel of enzyrnesy'nthesisand by allosteric regulationof the enzymethat catalyzesthe formation of carbamoylphosphate.

AcidDegradation ofAmino 18.3Pathways The pathways of amino acid catabolism,taken toto l5% of the gether,normallyaccountfor only 10%o production; these pathwaysare human body'senergy glycolysis and fatty acid oxinot nearly as active as dation. Flux through these catabolic routes also variesgreatly,dependingon the balancebetweenrequirements for biosynthetic processesand the availability of a particular amino acid. The 20 catabolic pathways convergeto form only six major products,

t

[

l

6BB

A m i nA o c i d0 x i d a t i oann dt h eP r o d u c t ioofnU r e a

Some Amino Acids Are(onverted toGlucose,Others to Ketone Bodies

all of which enter the citric acid cycle (Fig. lS-f b). From here the carbon skeletons are diverted to gluconeogenesis or ketogenesis or are completely oxidized to CO2 and H2O. AII or part of the carbon skeletons of seven amino acids are ultimately broken down to acetyl-CoA. Five amino acids are converted to a-ketoglutarate, four to succinyl-CoA, two to fumarate, and two to oxaloacetate. Parts or all of six amino acids are converted to pyruvate, which can be converted to either acetyl-CoA or oxaloacetate. We later summarize the individual pathways for the 20 amino acids in flow diagrams, each Ieading to a specific point of entry into the citric acid cycle. In these diagrams the carbon atoms that enter the citric acid cycle are shown in color. Note that some amino acids appear more than once, reflecting different fates for different parts oftheir carbon skeletons. Rather than examining every step of every pathway in amino acid catabolism, we single out for special discussion some enzymatic reactions that are particularly noteworthy for their mechanisms or their medical significance.

Leucine Lysine Phenylalanine Tryptophan Tyrosine

The seven amino acids that are degraded entirely or in part to acetoacetyl-CoA and,/or acetyl-CoA-phenylalanine, tyrosine, isoleucine, leucine, tryptophan, threonine, and lysine-can yield ketone bodies in the liver, where acetoacetyl-CoAis converted to acetoacetateand then to acetone and B-hydroxybutyrate (see Fig. 17-l B) . These are the ketogenic amino acids (Fig. 18-15). Their ability to form ketone bodies is particularly evident in uncontrolled diabetes mellitus, in which the liver produces large amounts of ketone bodies from both fatty acids and the ketogenic amino acids. The amino acids that are degraded to pyruvate, a-ketoglutarate, succinyl-CoA, fumarate, and,/oroxaloacetate can be converted to glucose and glycogen by pathways described in Chapters 14 and 15. They are the glucogenic amino acids. The division between ketogenic and glucogenic amino acids is not sharp; flve amino acids-tryptophan, phenylalanine, ty'osine, threonine, and isoleucine-are both ketogenic and glucogenic.

Arginine Glutamine Histidine Proline

Ketone bodies

Isocitr

1

I Citrate

Isoleucine Methionine Threonine Valine

Citric acid cycle

*-'

PhenYlalanine 'fyrosine

Malate

Glucose

Isoleucine Leucine Threonine Tryptophan

Alanine Cysteine Glycine Serine Threonine Tryptophan

Glucogenic Asparagine Aspartate

FIGURE 18-15 Summaryof aminoacid catabolism.Aminoacidsare groupedaccordingto their major degradative end product Some aminoacidsare Iistedmorethanoncebecause differentpartsof their carbon skeletonsare degradedto differentend products The figure showsthe mostimportantcatabolicpathwaysin vertebrates, but there areminorvariations amongvertebrate species. Threonine, for instance, is degradedvia at leasttwo differentpathways(seeFigslB_19, 1B_27),

Ketogenic

a n d t h e i m p o r t a n c eo f a g i v e n p a t h w a yc a n v a r y w i t h t h e o r g a n i s ma n d i t s m e t a b o l i c c o n d i t i o n s T h e g l u c o g e n i c a n d k e t o g e n i ca m i n o a c i d s a r e a l s o d e l i n e a t e di n t h e f i g u r e , b y c o l o r s h a d i n g . N o t i c e t h a t f i v e o f t h e a m i n o a c i d s a r e b o t h g l u c o g e n i c a n d k e t o g e n i c .T h e a m i n o a c i d s d e g r a d e dt o p y r u v a t ea r e a l s o p o t e n t i a l l y k e t o g e n i c O n l y t w o a m i n o a c i d s , l e u c i n e a n d l y s i n e ,a r e e x c l u s i v e l yk e t o g e n i c .

Acid Degradatiot ofAmino 18.3Pathways [Ltr l

o rr HN'"tNH HC-CH H.C. - \ ^ / -CH \ s\ "

CH.-CH.-CHO-CH9-COO_ methionine valerate

Biotin p-aminobenzoate

6-methylpterin

Tetrahydrofolate (Ha folate) tIGURE18-16 Someenzymecofactorsimportant in one-carbontransferreactions.The nitrogen are shownin blue atomsto which one-carbongroupsareattachedin tetrahydrofolate

Catabolismof amino acids is particularly critical to the survival of animals with high-protein diets or during starvation.Leucine is an exclusivelyketogenicamino acid that is very common in proteins. Its degradation makes a substantialcontribution to ketosis under starvation conditions.

(ofactors in lmportant Roles Enzyme Play Several (atabolism Acid Amino occur A variety of interestingchemicalrearrangements in the catabolicpathwaysof amino acids. It is useful to begin our study of these pathwaysby noting the classes of reactionsthat recur and introducing their enzyme cofactors.We have already consideredone important class: transamination reactions requiring pyridoxal phosphate.Another colnmon type of reaction in amino acid catabolismis one-carbontransfers, which usually involveone of three cofactors:biotin, tetrahydrofolate, (Fig. 18-f 6). These cofacor S-adenosylmethionine tors transfer one-carbongroups in different oxidation states:biotin transferscarbonrn its most oxidizedstate, CO2 (see Fig. 14-18); tetrahydrofolatetransfersonecarbon groups in intermediate oxidation states and sometimesasmethyl groups;and S-adenosylmethionine transfersmethyl groups,the most reducedstate of carbon. The latter two cofactorsare especiallyimportant in amino acid and nucleotidemetabolism. Tetrahydrofolate (Ha folate), synthesizedin bacteria, consistsof substitutedpterin (6-methylpterin), p-aminobenzoate, and glutamatemoieties(Fig. 18-16).

o Pterin

The oxidizedform, folate, is a vitamin for mammals;it is converted in two steps to tetrahydrofolate by the enzyme dihydrofolatereductase.The one-carbongroup undergoingtransfer, in any of three oxidation states,is bondedto N-5 or N-10 or both. The most reducedform of the cofactor carries a methyl group, a more oxidized form carries a methylene group, and the most oxidized forms carry a methenyl, formyl, or formimino group (Fig. 18-f 7). Mostforms of tetrahydrofolateare interconvertibleand serveas donors of one-carbonunits in a variety of metabolic reactions. The primary source of one-carbonunits for tetrahydrofolate is the carbon removed in the conversionof serine to glycine,producing M, Nr 0-methylenetetrahydrofolate. Although tetrahydrofolate can carry a methyl group at N-5, the transfer potential of this methyl group is insufficientfor most biosyntheticreactions.SAdenosylmethionine (adoMet) is the preferred cofactor for biological methyl group transfers. It is synthesizedfrom ATP and methionineby the action of methionine adenosyl transferase (Fig. 18-18, step @). This reaction is unusual in that the nucleophilic sulfur atom of methionineattacksthe 5' carbon of the ribose moiety of ATP rather than one of the phosphorusatoms. Ttiphosphateis releasedand is cleavedto P1and PP1on the enzyme,and the PP1is cleaved by inorganic pyrophosphatase;thus three bonds,including two bonds of high-energyphosphate groups, are broken in this reaction. The only other known reaction in which triphosphate is displaced from ATP occursin the synthesisof coenzyrneB12(see Box l7-2, Fig. 3). S-Adenosylmethionineis a potent alkylating agent by virtue of its destabilizingsulfonium ion. The methyl group is subject to attack by nucleophilesand is about 1,000times more reactive than the methyl group of AF-methylt etrahydrofolate.

Acid 0xidation andtheproduction | 590 i Amino ofUrea O:ridation state trangferred)

Serine

coo-

*l H3N-q-H

I CH2OH

Glycine

cooHrfr-g-x

M,Nro-methylenetetrahydrofolate reductase

+H+

I

H

HrO

/ serine hydroxymethyl transferase

Tetrahydrofolate

N5,Nlo-methylenetetrahydrofolate dehydrogenase

+H+

FIGURE l8-17 Conversions of one-carbonunits on tetrahydrofolate. The differentmolecularspeciesare groupedaccordingto oxidation state,with the mostreducedat the top and mostoxidizedat the bot_ tom. All specieswithin a singleshadedbox are at the sameoxidation state.The conversionof Ns,Nlo-methylenetetrahydrofolate to Ns_ methyltetrahydrofolate is effectivelyi rreversible. The enzymatictransfer offormyl groups,as in purinesynthesis (seeFig.22-33) and in the formationof formylmethionine in bacteria(Chapter27),generallyuses

Nt o-formyltetrahydrofolate ratherthan Ns-formyltetrahydrofolate. The lafterspeciesis significantlymorestableand thereforea weakerdonor of formyl groups.Ns-Formyltetrahydrofolate is a minor byproductof the cyclohydrolase reaction,and can also form spontaneously. Conversionof Ns-formyltetrahydrofolate to N5,Nlo-methenyltetrahydrofolate requiresATP,becauseof an otherwiseunfavorableequilibrium. Note that Ns-formiminotetrahydrofolate is derivedfrom histidinein a pathwayshownin Figure18-26.

Tlansfer of the methyl goup from S-adenosylmethionine to an acceptor yields ,S-adenosylhomocysteine (Fig. 18-18,step @), which is subsequentlybrokendown to homocysteineand adenosine(step @). Methionineis regenerated by transfer of a methyl group to homocysteine in a reaction catalqed by methionine synthase (step @), and metlLionineis reconvertedto S-adenosylmethionine to completean activated-methylcycle.

One form of methionine sytrthasecorrrmonin bacteria uses M-methyltetrahydrofolate as a methyl donor. Another form of the enzyme present in some bacteria and mammalsuseslF-methyltetrahydrofolate, but the methyl group is first transferred to cobalamin,derived from coenzyfil€ 812, to form methylcobalamin as the methyl donor in methionine formation. This reaction and the rearrangement of l-methylmalonyl-CoA to

o c i dD e g r a d a t i o["'] n 1 8 . 3P a t h w aoyfsA m i nA

N

+

HaNN

I H

CHo

N

cH" t-

N

methionine adenosyl

*At NH

c-H

t-

PR+Pi

*-a",

coo I

o

lNH

Tetrahydrofolate coenzFne

CHs

Iranslerase

-CH,

S-Adenosylmethionine

(A nethionine syntha-.e

*l

coo-

H3N-C-H

CH"

t-

cHo

t-

,/@\ Adenosine

Homocysteine

R-cH3

I

-____Fr N5-Methyltetrahydrofolate

a variety of methyJ transrerases

HrO

S-Adenosine S-Adenosylhomocysteine

to cobalaminto form methylcobalamin, which in turn is the methyl

which S-Adenosylmethionine, donorin the formationof methionine. chargedsulfur(andis thusa sulfoniumion),is a powhasa positively erful methylatingagentin severalbiosyntheticreactions.The methyl groupacceptor(step@ is designatedR.

succinyl-CoA(seeBox |7-z,Fig.la) arethe only knou'n coenz),rneB12-dependentreactionsin mammals. The vitamins B12and folate are closelylinked in fr E these metabolic pathways.The B12 deficiency E diseasepernicious anemia is rare, seen only in individuals who have a defect in the intestinal absorption pathwaysfor this vitamin (see Box 17-2) or in strict vegetarians(B12is not presentin plants). The disease progressesslowly, becauseonly small amounts of vitamin B12are requiredandnormalstoresof 812in the liver can last three to five years. S5rmptoms include not only anemiabut a variety of neurologicaldisorders. The anemiacan be traced to the methioninesynthase reaction. As noted above,the methyl group of methylcobalaminis derived from N5-methyltetrahydrofolate, and this is the only reaction in mammals that uses N5-methyltetrahydrofolate. The reaction converting the Ns,Nl0-methylene form to the l/5methyl form of tetrahydrofolate is irreversible (Fig. 18-17). Thus, if coenzymeB12is not availablefor the synthesis of methylcobalamin,metabolic folates become trapped in the N5-methyl form. The anemia associatedwith vitamin B12deficiencyis calledmegaloblastic anemia. It manifestsas a declinein the production of mature erythrocytes (red blood cells) and the appearancein the bone marrow of immature precursor cells,or megaloblasts. Erythrocytesare gradually replaced in the blood by smaller numbers of

abnormallylarge erythrocytes called macrocytes. The defect in erythroclte developmentis a direct consequence of the depletion of the N5,Nr0-methylenetetrahydrofolate,which is required for synthesisof the thymidine nucleotides needed for DNA synthesis (see Chapter 22). Folate deficiency, in which all forms of tetrahydrofolateare depleted, also produces anemia, for much the samereasons.The anemiasymptomsof B12deflciencycan be alleviatedby administeringeither vitamin B12or folate. However,it is dangerousto treat perniciousanemia by folate supplementationalone,becausethe neurological symptoms of B12 deficiency will progress.These s1'rnptomsdo not arise from the defect in the methionine synthasereaction. Instead, the impaired methylmalonyl-CoA mutase causesaccumulation of unusual, odd-numberfatty acids in neuronal membranes.The anemia associatedwith folate deficiency is thus often treated by administeringboth folate and vitamin Bp, at least until the metabolic source of the anemiais unambiguously defined. Early diagnosisof 812 deflciency is important becausesome of its associatedneurological conditionsmay be irreversible. Folate deflciencyalsoreducesthe availabilityof the AF-methyltetrahydrofolaterequired for methionines],'nthase function. This leadsto a rise in homocysteinelevels in blood, a condition linked to heart disease, hlpertension, and stroke. High levels of homocysteine

FIGURE 18-18 Synthesis of methionineand S-adenosylmethionine in an activated-methyl cycle.The stepsare describedin the text. In the methioninesynthasereaction(step@), the methylgroupis transferred

Iosr

A m i nA o c i d0 x i d a t i oann dt h eP r o d u c t ioofnU r e a

may be responsiblefor 10%of all casesof heart disease. The condition is treated with folate supplements.r Tetrahydrobiopterin, another cofactor of amino acid catabolism,is similar to the pterin moiety of tetrahydrofolate,but it is not involved in one-carbon transfers;insteadit participatesin oxidationreactions. We considerits modeof actionwhen we discussohenvlalaninedegradation(seeFig. 18-24).

SixAmino Acids AreDegraded toPyruvate The carbonskeletonsof six amino acidsare converted in whole or in part to pyruvate. The pyruvate can then be convertedto acetyl-CoAand eventuallyoxidizedvia the citric acid cycle, or to oxaloacetateand shunted into gluconeogenesis. The six amino acidsare alanine, tryptophan, cysteine, serine, glycine, and threonine (Fig. 18-19). Alanine yields pyruvate directly on transaminationwith a-ketoglutarate,and the side chain of tryptophan is cleavedto yield alanineand thus p5,r'uvate.Cysteine is convertedto py'uvate in two steps;one removesthe sulfur atom, the other is a transamination. FIGURE 18-19 Catabolicpathwaysfor alanine,glycine,serine, cysteine,tryptophan,and threonine.Thefateof the i ndolegroup of tryptophanis shownin Figure18-2.1 Detailsof mostof the reactionsinvolvingserineand glycineareshownin Figure'l8-20. The pathwayfor threoninedegradationshownhereaccountsfor only abouta third of threoninecatabolism(for the alternatrve pathway,seeFig.1B-27).Severalpathwaysfor cysteinedegradation leadto pyruvate.Thesulfurof cysteinehasseveralalternative fates,one of which is shownin Figure22-15. Carbonatomshere and in subsequent figuresare color-codedas necessary to trace theirfates

Serine is convertedto pyruvate by serine dehydratase. Both the B-hydroxyland the a-aminogroupsof serineare removed in this single pytidoxal phosphate-dependent reaction(Fig. 18-20a). Glycine is degradedvia three pathways,only one of which leads to pyruvate. Glycine is convertedto serine by enzymaticaddition of a hydroxSrmethylgroup (Figs 18-19, 18-20b). This reaction,catdyzedby serine hydroxymethyl transferase, requires the coenzyrnes tetrahydrofolateand pl'ridoxal phosphate.The serine is convertedto py'ruvateas describedabove.In the second pathway,which predominatesin animals,glycine undergoes oxidative cleavageto CO2,NHf , and a methylene group (-CHz-) Grgs 18-19, i8-20c). This readilyreversible reaction, catalyzedby glycine cleavage enzyme (also called glycine sy'nthase), also requires tetrahydrofolate,which acceptsthe methylenegroup. In this oxidativecleavagepathway,the two carbonatomsof glycine do not enter the citric acid cycle. One carbonis lost as CO2and the other becomesthe methylenegroup (Fig. 1S- 17), a oneof AF,ly'1O-methylenetetrahydrofolate carbongroup donor in certainbiosyntheticpathways.

f"'

CH3-CH-CHOH

tittt"

t" cH3-c-cH-coo tl O

2-Amino-3-ketobutyrate

CoA Acetyl'CoA NAD'NADH

CO2+ NH;

NHs

I HO- CH2-CH-COO-l Serine I

H

In.r"o, T

frH. t"

cH3-cH-ctoo-

f a-Ketoglutarate

er.r \ Glutamate

o cH3-c-cooPyruvate

SH _2 steps

ll

+

NH'

cH2-cH-coo-

o c i dD e g r a d a t i o[urr] n 1 8 . 3P a t h w aoyfsA m i n A (a) Serrne dehydratase reaction

(b) Serine hydroxymethyltransferase reaction

(c) Glycine cleavage enzyme reaction

Hi o-H \_ '-\:B-

Pppl-cH:rtH-

I

Covalently bound serine proton "OOabstraction at I o carbon leads CDl. -l-HrO toBelimination of OH. il-

HBFr,p-l-crr:rtrn-i I coorhe link ^lf @ (?[ to PLP isreversed. }@D H2 H2

coo

LYs plp

The carbanion .. attacKs tne methvlene of

l-Ilr

@r I *l

CH.

H,N:? coo Hydrolytic deamination yields pymvate.

t,-H,O

HB-

OO-

PlP-stabilized caroanlon

f,f,,o;methvlene ^.:/ ll,il##etnvrene fi4 lotate to lL yield serine.

l['Hn folate

HO

*l IPLPI-CH:NH

CHz

C-H I

The aldimine

reversed.and

theproduct is released.

Lys

lf-"@

pr,p

. ?"'o" H3N-C-H

F;E

CH"

;..-b;;i carbanion

@ The carbanion attacks the oxidizedlipoic acid component of enzymeH.

1 @ll lf C-S HS

Thelink ^ Ia\ry (9, to PLP - L is reversed. l\@

@ LYS pl.p

+

H3N-CH2-S\ HS Methylene is transferred to Hn folate with elimination of ammonta.

I'NHs

PLP-stabilized

H+

coo-

@[

O:C

I

n

1r@

fD - lf

H

lFiFl-crr:trn-^C: Hi

+H I P L PI _ C H - N H

coo-

ffi;;er,;;*

Rearrangement of the enamine forms arr imine.

carbanion.

-H

Flfl-cH:frH-

CHo

Iene

1I

cooPyruvate Lipoic acid is reoxidized.

MECHANISM tl6URE18-20 Interplayof the pyridoxalphosphate and tetrahydrofolate cofactorsin serineand glycinemetabolism.The first stepin eachof thesereactions(notshown)involvesthe formationof a covalentiminelinkagebetweenenzyme-bound PLPandthe substrate aminoacid-serinein (a),glycinein (b) and (c) (a)A PlP-catalyzed eliminationof waterin the serinedehydratase reaction(step@) begins the pathwayto pyruvate(b) In the serinehydroxymethyltransferase reaction,a PLP-stabilized carbanion(productof step@) is a key intermediate in the reversibletransferof the methylenegroup (as

to form serine. f rom Ns,Nro-methylenetetrahydrofolate with comcomplex, (c)Theglycinecleavage is multienzyme a enzyme conwhich is reversible, ponentsB H, I and L. Theoverallreaction, vertsglycineto CO2and NHf , with the secondglycinecarbontaken Pyrito form N5,N10-methylenetetrahydrofolate. up by tetrahydrofolate thea carbonof aminoacidsat criticalstages activates doxalphosphate carriesone-carbonunits in in all thesereactions,and tetrahydrofolate two of them (seeFigs18-6, 18-17). -CH2-OH)

o c i d0 x i d a t i oann dt h eP r o d u c t ioofnU r e a L 6 9 4 _ l A m i nA

This second pathway for glycine degradation seemsto be critical in mammals.Humanswith serious defects in glycine cleavageenzlrne activity suffer from a condition known as nonketotic hyperglycinemia. The condition is characterizedby elevatedserum levels of glycine, leading to severemental deflcienciesand death in very early childhood.At high levels,glycine is an inhibitory neurotransmitter,perhaps explaining the neurologicaleffectsof the disease.Many geneticdefects of amino acid metabolism have been identified in humans (Table 18-2). We will encounterseveralmore in this chapter.r In the third and final pathway of glycine degradation, the achiral glycine molecule is a substratefor the enzyrneo-amino acid oxidase.The glycine is converted to glyoxylate,an alternativesubstratefor hepaticlactate dehydrogenase(p. 547). Glyoxylateis oxidized in an NAD--dependentreactionto oxalate:

frn, I

9H, |

Qz I^{zo NH:

:#;#";;;;'"

coo

o I

iH

N4D* NADH

I coo Glyoxylate

Medicaleondition

Approximate incidenee (per 100,000 births)

Albinism

cooI coo-

Oxalate

The primary function of o-amino acid oxidase, present at high levelsin the kidney,is thought to E be the detoxi-flcationof ineestedl-amino acids derived from bacterial cell walls and from grilled foodstuffs (high heat causessomespontaneousracemizationof the l-amino acidsin proteins). Oxalate,whether obtainedin foods or produced enzymaticallyin the kidneys, has medical significance.Crystals of calcium oxalate account for tp to 75o/oof all kidney stones.t There are two significant pathways for threonine degradation.One pathway leadsto pyruvate via glycine (Fig. 18-19). The conversionto glycine occurs in two steps, with threonine first converted to 2-amino3-ketobutyrateby the action of threonine dehydrogenase. This is a relatively minor pathway in humans,accounting for 10%to 30% of threonine catabolism,but is more important in someother mammals.The major pathway in humans leads to succinyl-CoAand is described later. In the Iaboratory serine hydroxymethyltransferase will catalyzethe conversionof threonine to glycine and acetaldehydein one step, but this is not a signiflcant pathwayfor threonine degradationin mammals.

Defectiveprocess

Defectiveenzyme

Symptomsand effects

H2o

Eqn 13-5, p. 515). A second method for determining the sequence of electron carriers involves reducing the entire chain of carriers experimentally by providing an electron source but no electron acceptor (no 02). When 02 is suddenly introduced into the system, the rate at which each electron carrier becomes oxidized (measured spectroscopically) reveals the order in which the carriers function. The carrier nearest 02 (at the end of the chain) gives up its electrons first, the second carrier from the end is oxidized next, and so on. Such experiments have confirmed the sequence deduced from standard reduction potentials In a final conirmation, agents that inhibit the flow of electrons through the chain have been used in combination with measurements of the degree of oxidation of each carrier. In the presence of 02 and an electron donor, carriers that function before the inhibited step become fully reduced, and those that function after this step are completely oxidized (FiS. f9-6). By using several inhibitors that block different steps in the chain, investigators have determined the entire sequence;it is the same as deduced in the first two approaches.

(arriers Electron Function inMultienzyme Complexes The electron carriers of the respiratory chain are organizedinto membrane-embeddedsupramolecular complexesthat can be physically separated.Gentle treatment of the inner mitochondrial membrane with detergentsallowsthe resolutionof four unique electroncarrier complexes,each capableof catalyzingelectron transfer through a portion of the chain (Table 19-3; Fig. 19-7). ComplexesI and II catalyzeelectrontransfer to ubiquinone from two different electron donors: NADH (ComplexI) and succinate(ComplexII). Complex III carrieselectronsfrom reducedubiquinoneto cytochromec, and ComplexIV completesthe sequenceby transferring electronsfrom cytochromec to 02. We now look in more detail at the structure and function of each complex of the mitochondrial respiratory chain. Complex I: NADH to Ubiquinone Figure l9-8 illustrates the relationshipbetween ComplexesI and II and ubiqurnone.Complex I, alsocalledNADH:ubiquinone

F"il;l NADH

Q -

+ Cytb -------+ Cytct -----+ Cytc -"---+ Cyt(a+as)

+ O,

Ett'"d;l NADH ---------+Q ----+

a", U @'

Cyt c, --------+Cyt c ------+ Cyt (o + aB)

> 92

reN"'col 19 NADH ---------+Q -------+Cyt b ------+ Cyt c1 FIGURE l9-6 Methodfor determiningthe sequenceof electroncarriers.Thismethodmeasures the effectsof inhibitorsof electrontransfer on the oxidationstateof each carrier.ln the oresenceof an electron

> Clt c ------->Cyt @ +or)

€/-

O,

donor and 02, each inhibitor causesa characteristicpattern of oxidized/ reduced carriers: those before the block become reduced (blue), and t h o s e a f t e rt h e b l o c k b e c o m e o x i d i z e d ( p i n k )

Reactions inMitochondria 19.1Electron-Iransfer tDt t

EnzJmeeomplex/protein

Mass(kDa)

I NADH dehydrogenase T Succinatedehydrogenase ilI tlbiquinone:cytochromec oxidoreductasr:

850

43 (14\

r40 250 13 160

CS,tochrome ct

ry

Numberof subunits*

Cytochromeoxidase

4

hosthetic gfoup(s) FMN, Fe-S FAD, Fe-S

tI

Hemes,Fe-S

1 13 13--41

Heme Hemes;Cua,Cug

*Numbers 0f subunits in thebacterial equivalents in parentheses. tCytochrome c is notpartof an enzyme complex; it movesbetween Complexes lll andlVas a freelysolubleprotein.

Interrnembrane space (e side)

Trea with digitonin

s ."1R

,' - Osn

r.

\

'4fl I

...,.

ic ruoture

'rS

1

i,'i

ATP synthase

Solubilization with detergent followed by ion-exchangechromatography

IilIIIIVATP synthase

)H Q NADH

Suc- a crnate

ETF:Q

z^r Q Cyt c Cyt c 02

ATP ADP

Reactions catalyzed.byisolated fractions in vitro

Pi

FIGURt 19-7 Separationof functionalcomplexesof the respiratorychain. Theoutermitochondrial membrane is firstremovedby treatment with the detergent digitonin.Fragments of innermembrane arethenobtainedby osmoticruptureof the mitochondria, andthefragments aregentlydissolved in a seconddetergent. Theresulting mixtureof innermernbrane proteinsis resolvedby ion-exchange chromatography into differernt complexes(l throughlV)of the respiratory chain,eachwith is uniqueproteincomposi(sometimes tion (seeTable19-3),and the enzymeATPsynthase called ComplexV). The isolatedComplexes I throughlV catalyzetransfers betweendonors(NADH and succinate), intermediate carriers(Q and cytochromec), and O:, as shown.In vitro,isolated ATPsynthase hasonly (AThse),notATP-synthesizing, ATP-hydrolyzing activity.

19-8 Pathof electronsfrom NADH, succinate, fatty acyl-CoA, FIGURE from NADH pass to ubiquinone.Electrons and glycerol3-phosphate througha flavoproteinto a seriesof iron-sulfurproteins(in Complexl) and then to Q. Electronsfrom succinatepassthrougha flavoprotein and severalFe-Scenters(in Complexll) on the way to Q. Clycerol3phosphatedonateselectronsto a flavoprotein(glycerol3-phosphate membrane, on the outerfaceof the innermitochondrial dehydrogenase) (thefirstenzymeof fromwhichtheypassto Q. Acyl-CoAdehydrogenase flavoprotein B oxidation)transferselectronsto electron-transferring (ETF),from which they passto Q via ETF:ubiquinone oxidoreductase

oxidoreductase or NADH dehydrogenase, is a large enzymecomposedof 42 different polypeptidechains, includingan FMN-containingflavoproteinand at leastsix iron-sulfur centers.High-resolutionelectron microscopy showsComplexI to be L-shaped,with one arm of the L in the membraneand the other extendinginto the matrix. As shovmin Figure 19-9, ComplexI catalyzestwo simultaneousand obligatelycoupledprocesses:(1) the exergonictransferto ubiquinoneof a hydride ion from NADH and a proton from the matrix, expressedby NADH+ H* + Q ---> NAD- + QH2

(19-1)

and (2) the endergonictransfer offour protons from the matrix to the intermembranespace.ComplexI is therefore a proton pump driven by the energy of electron transfer, and the reaction it catalyzesis vectorial: it movesprotons in a speciflcdirection from one location (the matrix, which becomesnegativelychargedwith the

Vro)

O x i d a t iP v eh o s p h o r y l aatni odPn h o t o p h o s p h o r y l a t i o n

Intermembrane space (e side)

(Complexl). ComFIGURE 19-9 NADH:ubiquinoneoxidoreductase plex I catalyzesthe transferof a hydrideion from NADH to FMN,from which two electronspassthrougha seriesof Fe-Scentersto the ironsulfurproteinN-2 in the matrixarm of the complex.Thedomainthat extendsinto the matrix has beencrystallizedand its structuresolved (PDBlD 2FUC);the structure domainof ComplexI of the membrane is not yet known. Electrontransferfrom N-2 to ubiquinoneon the membranearm formsQH2, which diffusesinto the lipid bilayer.This fromthe matrixof four proelectrontransfer alsodrivesthe expulsion Thedetailedmechanism thatcoupleselectonsper pair of electrons. tron and protontransferin ComplexI is not yet known, but probably involvesa Q cyclesimilarto thatin Complexlll in whichQH2 participatestwice per electronpair (seeFig.19-12).Protonflux producesan potential (N membrane electrochemical across the innermitochondrial

Complex I

Matrix (ruside)

someof the energyresidenegative,e sidepositive),which conserves potenleasedby the electron-transfer reactions.This electrochemical tial drivesATPsynthesis. NADH + H*

NAD+

Series of Fe-S centers

location of the protons: p for the positive side of the inner membrane(the intermembranespace),N for the negativeside (the matrix):

FMN

NADH + 5H* + Q -----+NAD* + QHz+ 4H; (19-2) Amytal (a barbiturate drug), rotenone (a plant product cornrnonlyused as an insecticide), and piericidin A (an antibiotic) inhibit electron flow from the Fe-Scentersof Complex I to ubiquinone (Table 19-4) and therefore block the overall processof oxidative phosphorylation. ubiquinol (QH2,the fully reduced form; Fig. 19-2) diffuses in the inner mitochondrial membrane from ComplexI to ComplexIII, where it is oxidizedto Q in a processthat alsoinvolvesthe outward movementof H+.

departure of protons) to another (the intermembrane space,which becomespositivelycharged).To emphasizethe vectorialnatureofthe process,the overallreaction is often written with subscriotsthat indicate the

Tlpe of interference

Compound*

TargeUmode of action

Inhibition of electron transfer

Cyanide ] Carbonmonoxide J

Inhibit cytochromeoxidase

Antimycin A

Blockselectron transfer from cytochromeb to cltochrome c1

Myxothiazol Rotenone Amytal Piericidin A

I I I )

DCMU Inhibition of AIP slnthase

Prevent electrontransfer from Fe-Scenter to ubiquhone Competeswith QBfor binding site in PSII Inhibits F1

Aurovertin .t

urgomycm Venturicidin

t )

Blocksproton flow through Fo and CFo

DCCD Uncouplingof phosphorylation from electron transfer

Inhibition of ATP-ADPexchange

FCCP DNP

Inhibit Fo and CFo

] )

Hydrophobicproton carriers

Valinomycin

K

Thermogenin

In brown adiposetissue,forms proton-conductingpores in inner mitochondrialmembrane

Atractyloside

Inhibits adeninenucleotidetranslocase

lOnOpnore

* DCI\4U is 3-(3,4-dichlorophenyl)-1,1-dimethylurea; DCCD, dicyclohexylcarbodiimide; FCCB cyanide-p{rifluoromethoxyphenylhydrazone; DNB2,4-dinitrophenol.

inMitochondria Reactions 19.1Electron-Transfer [ttt] Intermembrane space (e side)

Fe-S centers

B

-{. -l

l*

Substratebinding site

4 A

FIGURE 19-10 Structureof Complex ll (succinatedehydrogenase). (PDBlD lZOY) This complex(shownhere is the porcineheartenzyme)hastwo transmembrane subunits, C and D; the cytoplasmic exA and B.Justbehindthe FADin subunitA is tensions containsubunits the bindingsitefor succinate.SubunitB hasthreesetsof Fe-Scenters; between ubiquinoneis boundto subunitB; and hemeb is sandwiched moleculesare so subunitsC and D. Two phosphatidylethanolamine tightlyboundto subunitD thattheyshowup in the crystalstructure. Electronsmove(bluearrows)from succinateto FAD,then throughthe Thehemeb is not on the mainoath threeFe-Scentersto ubiouinone. of electrontransferbut protectsagainstthe formationof reactiveoxygen species{ROStby electronsthat go astray.

Complex II: Succinate to Ubiquinone We encountered Complex II in Chapter 16 as succinate dehydrogenase, the only membrane-boundenzyme in the citric acid cycle (p. 628). Although smaller and simpler than ComplexI, it containsflve prosthetic groups of two typesand four differentprotein subunits(FiS. f 9-10). SubunitsC and D are integral membraneproteins, each with three transmembranehelices.They contain a heme group, heme b, and a binding site for ubiquinone,the flnal electronacceptorin the reactioncatalyzedby Complex II. Subunits A and B extend into the matrix; they containthree 2Fe-2Scenters,boundFAD,and a binding site for the substrate,succinate.The path of electron transfer from the succinate-bindingsite to FAD, then through the Fe-Scentersto the Q-bindingsite, is more than 40 A long, but none of the individual electrontransfer distancesexceedsabout 11 A-a reasonable distancefor rapid electrontransfer(Fig. 19-10). The heme b of ComplexII is apparentlynot in the ffi direct path of electron transfer; it may serve inE steadto reducethe frequencywith which electrons"leak" out of the system,moving from succinateto molecular

oxygento producethe reactive oxygen speeies (ROS) hydrogenperoxrde(HzOz)and the superoxide radical ('Ot), as describedbelow.Humanswith point mutations n ComplexII subunitsnear heme b or the quinone-bindThis hhering site sufferfrom hereditaryparaganglioma. ited condition is characterizedby benign tumors of the head and neck, co[tmonly in the carotid body, an organ that senses02 levelsin the blood.Thesemutationsresult in greaterproduction of ROSand perhapsgreatertissue damageduring succinateoxidation.r Other substratesfor mitochondrial dehydrogenases pass electronsinto the respiratory chain at the level of ubiquinone,but not through Complex II. The flrst step in the B oxidation of fatty acyl-CoA, catalyzedby the flavoproteinacyl-CoA dehydrogenase (seeFig. t7-8), involvestransfer of electronsfrom the substrateto the FAD of the dehydrogenase,then to electron-transferring flavoprotein (ETF), which in turn passesits electrons to ETF:ubiquinone oxidoreductase (FiS. 19-8). This enzyme transfers electrons into the respiratory chain by reducing ubiquinone.Glycerol 3-phosphate, formed either from glycerol releasedby triacylglycerol breakdown or by the reduction of dihydroxyacetone phosphate from glycolysis,is oxidized by glycerol 3phosphate dehydrogenase (see Fig. 17-4). This enzyme is a flavoprotein located on the outer face of the inner mitochondrial membrane,and like succinate dehydrogenaseand acyl-CoA dehydrogenaseit channels electrons into the respiratory chain by reducing ubiquinone (FiS. 19-8). The important role of glycerol 3-phosphate dehydrogenasein shuttling reducing equivalentsfrom cytosolicNADH into the mitochondrial matrix is describedin Section19.2(seeFig. 19-30)' The is effect of each of these electron-transferringenz5.'rnes to contribute to the pool of reduced ubiquinone' QHz from all thesereactionsis reoxidizedbyComplexIII' Complex III: Ubiquinone to Cytochrome c The next respiratory complex, Complex III, also called cytochrome bcq complex or ubiquinone:c5rtochrome c oxidoreductase, couplesthe transfer of electronsfrom ubiquinol (QH2) to cytochrome c with the vectorial transport of protons from the matrix to the intermembrane space.The determinationsof the completestructure of this hugecomplex(Fig. 19-f f ) and of Complex IV (below) by x-ray crystallography,achievedbetween 1995 and 1998,were landmarksin the study of mitochondrial electron transfer, providing the structural framework to integrate the many biochemicalobservations on the functions of the respiratory complexes. The functional unit of Complex III is a dimer, with the two monomericunits of cytochromeb surroundinga "cavern" in the middle of the membrane, in which ubiquinoneis free to move from the matrix side of the membrane(site QNon one monomer) to the intermembrane space(site Qeof the other monomer)asit shuttles electronsand protons acrossthe inner mitochondrial membrane(FiC.19-11b).

jto,

O x i d a t iP ve h o s p h o r y l aatni odP nh o t o p h o s p h o r y l a t i o n

(a)

(b)

Interrnembrane space (n side) Heme c,

Intermembrane space (e side)

Rieske iron2Fe-2S

Cytochrome c1

Heme 61

Heme b1

Heme bs

Cytochrome

Heme

Heme b1 Cavern

Heme 611

Matrix (N side)

Rieske ironprotems

Heme c,

Cytochromec,

Heme b1

Cyt c

Matrix (N side)

:.*t,

FIGURE 19-11 Cytochrome bc1complex(Complexlll). Thecomplex is a dimer of identicalmonomers,each with 11 differentsubunits. (a)Thefunctional coreof eachmonomeristhreesubunits: cytochrome b (green) with itstwo hemes(bp andbL);the Rieskeiron-sulfur protein (purple)with its 2Fe-2Scenters;and cytochromec, (blue)with its heme(PDBlD l BCY) (b) Thiscartoonview of the complexshows how cytochrome c1andthe Rieskeiron-sulfur proteinprojectfromthe p surface andcan interact with cytochrome c (notpartof thefunctional complex)in the intermembrane space.Thecomplexhastwo distinct bindingsitesfor ubiquinone, and to thesites Q* ep, whichcorrespond of inhibitionby two drugsthat block oxidativephosphorylation. AntimycinA, which blockselectronflow from hemeb;1to e, bindsat sideof the membraneMvxothQ*, closeto hemebp1on the N (matrix)

iazol,whichprevents electronflow fromQH, to the Rieskeiron-sulfur protein,bindsat Qp,nearthe2Fe-2S centerandhemeb1on the e side Thedimericstructure isessential to thefunctionof Complexlll.Theinterfacebetweenmonomersformstwo caverns/each containinga Qp sitefrom one monomerand a Q, sitefrom the otherTheubiquinone intermediates movewithinthesesheltered caverns. Complexlll crystallizes (notshown). in two distinctconformations In one,the RieskeFe-Scenteris closeto itselectronacceptor.the heme of cytochromec1, but relativelydistantfrom cytochromeb and the electrons. In QHr-bindingsiteat whichthe RieskeFe-Scenterreceives the other,the Fe-Scenterhasmovedawayfrom cytochromec1 and toward cytochromeb. The Rieskeproteinis thoughtto oscillatebetween thesetwo conformations as it is firstreduced,then oxidized.

Based on the structure of Complex III and detailed biochemical studies ofthe redox reactions, a reasonable model, the Q cycle, has been proposed for the passage of electrons and protons through the complex. The net equation for the redox reactions of the O cvcie (Fig. r9-r2) is

from ComplexIII, c1'tochrome c movesto ComplexIV to donatethe electronto a binuclearcoppercenter.

QHz + 2 cy,tc1(oxidized)+ 2Hfi -+ Q + 2 clt cr (reduced)+ 4Hi

(19_S)

The Q cycle accommodates the switch between the twoelectron carrier ubiquinone and the one-electron carners-c),tochomes br.62,bs66, c1,atd c-and explains the measured stoichiometry of four protons translocated per pair of electrons passing through Complex III to cytochrome c Although the path of electrons through this segment of the respiratory chain is complicated, the net effect of the transfer is simple: QHz is oxidized to e and two molecules of c1tochrome c are reduced. Cy'tochrome c is a soluble protein of the intermembrane space. After its single heme accepts an electron

Complex IV Cytochrome c to Oz In the final step of the respiratory chain, Complex IV, also called cytochrome oxidase, carries electronsfrom c;,'tochrome c to molecularoxygen,reducingit to H2O.ComplexIV is a large enzyme(13 subunits;M,204,000) of the inner mitochondrial membrane.Bacteria contain a form that is much simpler,with only three or four subunits,but still capableof catalyzingboth electrontransferand proton pumping.Comparisonof the mitochondrialand bacterial complexessuggeststhat three subunitsare critical to the function(Fig. 19-13). MitochondrialsubunitII containstwo Cu ions complexed with the -SH groups of two Cys residuesin a binuclearcenter (Cua;Fig. 19-13b) that resemblesthe 2Fe-2Scenters of iron-sulfur proteins. Subunit I contains two heme groups,designateda and a3, and another copperion (Cus).Hemea3 and Cusform a second

inMitochondria Reacti0ns 19.1Electron-Transfer 714 Intermembrane space (r "tU.) ,

. ar,

Cyt c

"

Heme c1

Cyt c,

QHz Heme bt. Heme6s

a Matrix (N side) 2H+

Cytochrome b --------> QHz + Q + cyt c1 (oxidized) 'Q+ Q + 2Hi + cytcl(reduced)

QHz +'O

Net equation: qH2+ 2 cyt c1(oxidized) + 2H* FIGURE 19-12 TheQ cycle,shownin two stages.The pathof electrons throughComplexIll is shownby bluearrows.In the firststage(left),Q on the N sideis reducedto the semiouinone radical.which in thesecond stage(right)is converted to QH2.Meanwhile, on the p sideof the membrane, two moleculesof QH2 are oxidizedto Q, releasing two

-)

+ 2Hft + cytcr(oxidized) -> QH, + 2Hi + Q + cytcl(reduced) Q + 2 cyt ct (reduced) + 4HF

protonsper Q molecule(fourprotonsin all) into the intermembrane space.EachQH2 donatesone electron(via the RieskeFe-Scenter)to cytochromecl, and one electron(via cytochromeb) to a moleculeof Q nearthe I side,reducingit in two stepsto QH2.Thisreductionalso usestwo protonsper Q, which aretakenup from the matrix

(a) Interrnembrane space (p side)

Matrix (N side) l9-13 Structureof cytochromeoxidase(ComplexlV). This tIGURE .l has 3 subunits, butonlyfourcore complexfrombovinemitochondria proteinsare shownhere(PDBlD lOCC). (a) ComplexlV with four subunits in eachof two identicalunitsof a dimer Subunit| (yellow)has two heme groups,a and a3, near a singlecopper ion, Cus (green Hemea3 and Cus form a binuclearFe-CucenterSubunitll sphere). (purple) with the-SH groupsof two containstwo Cu ionscomplexed the2Fe-2S cenin a binuclear center,Cua,thatresembles Cysresidues proteins. Thisbinuclearcenterand the cytochrome tersof iron-sulfur

from c-bindingsitearelocatedin a domainof subunitll thatprotrudes Subp (into space) the intermembrane membrane inner the sideof the submovementthrough proton (light for rapid is essential blue) lll unit is notyetknown.(b)Thebinuclear unit ll.Theroleof subunitlV (green) Whenthecenteris equally. centerof Cuo TheCu ionsshareelectrons Cul*Cu1*;whenoxidized, the ionshavetheformalcharges reduced, s*. areligandsaroundtheCu ions: Cul 5*Cu1 Sixaminoacidsresidues two His,two Cys,Clu, and Met

f

--t

l|1Bl

OxidativePhosphorylP a thi o n t oapnhdo s p h o r y l a t i o n

binuclear center that accepts electrons from heme a and transfers them to 02 bound to heme a3. Electron transfer through Complex IV is from cytochrome c to the Cua center, to heme a, to the heme a3-Cus center, and finally to 02 (Fig. 19-f 4). For every four electrons passing through this complex, the enzyrne consumes four "substrate" H* from the matrix (N side) in converting O2to 2H2O.It also uses the energy of this redox reaction to pump one proton outward into the intermembrane space (e side) for each electron that passesthrough, adding to the electrochemical potential produced by redox-driven proton transport through Complexes I and III. The overall reaction catalyzed by Complex IV is 4 Cyt c (reduced)+ 8H1d* 02 ------+ cytc,oxidized)+ 4Hi + 2}J2O (19-4) This four-electron reduction of 02 involves redox centers that carry only one electron at a time, and it must occur

4 Cytc Au+

+c

Interrnembrane space (p side)

o2

without the releaseof incompletelyreduced intermediates such as hydrogen peroxide or hydroxyl free radicals-very reactivespeciesthat would damagecelluIar components.The intermediatesremaintightly bound to the complexuntil completelyconvertedto water.

(omplexes Mitochondrial MayAssociate inRespirasomes There is growingexperimentalevidencethat in the intact mitochondrion,the respiratorycomplexestightly associatewith each other in the inner membraneto form respirasomes, functional combinationsof two or more electron-transfercomplexes.For example, when complex III is gently extracted from mitochondrial membranes,it is found to be associatedwith ComplexI and remainsassociatedduring gentle electrophoresis.Supercomplexesof ComplexIII and IV can also be isolated,and when viewed with the electron microscopeare of the right size and shapeto accommodatethe crystal structures of both complexes (Fig. 19-15). The kinetics of electronflow through the seriesof respiratorycomplexeswould be very different in the two extreme casesof tight versusno association:(1) if complexeswere tightly associated, electron transfers would essentiallyoccur through a solid state; and (2) if the complexesfunctioned separately, electrons would be carried between them by ubiquinone and cytochrome c. The kinetic evidence supports electron transfer through a solid state, and thus the respirasomemodel. Cardiolipin,the Iipid that is especiallyabundant in the inner mitochondrial membrane (see Figs 10-9 and Il-2), may be critical to the integrity of respirasomes; its removalwith detergents,or its absencein certain yeast mutants, results in defective mitochondrial electron transfer and a Ioss of affinity between the respiratory complexes.

(onserved ThelnergyofElectron Transfer lsEfficiently in a Proton Gradient

Matrix (N side) 4IJ+ (substrate) 4H* 2H2O (pumped) FIGURE 19-14 Pathof electronsthroughComplextV.The three proteinscriticalto electronflow aresubunitsl, ll, and lll Thelargergreen structure includes theother10 proteinsin thecomplex.Electron transfer throughComplexIV beginswith cytochrome c (top).Two molecules of reducedcytochromec each donate an electronto the binuclearcenterCuA.Fromhereelectrons passthroughhemea to the Fe-Cucenter(cytochrome a3and Cus) Oxygennow bindsto hemea3 and is reducedto its peroxyderivative(O3 ; not shown here)by two electronsfrom the Fe-Cucenter.Deliveryof two more electronsfrom cytochrome c (top,makingfour electronsin all) convertsthe O22-to two moleculesof water,with consumptionof four ,,substrate,, protons from the matrix.At the sametime, four protonsare pumpedfrom the matrixby an asyet unknownmechanism

The transfer of two electrons from NADH through the respiratory chain to molecular oxygen can be written as NADH + H+ + iO, ----- NAD+ + H2O

(19-5)

This net reaction is highly exergonic. For the redox pair NAD+AJADH,.U'o is -0.320 V, and for the pair O2/H2O, E'" is 0.816 V. The LE'o for this reaction is therefore 1.14 V, and the standard free-energy change (see Eqn 13-7, p. 5i5) is AG'' : -n,7LE''

(19-6)

: -2(96.5 kJ/V . molX1.14V) : -220 kJ/mol (of NADH) This standard free-energy change is based on the assumption of equal concentrations (1 vr) of NADH and

inMitochondria Reactions 19.1Electron-Transfer Ft tl

(a)

(b)

FIGURE 19-15 A putative respirasomecomposedof Complexeslll lll and lV and lV. (a) Purifiedsupercomplexes containing Complexes from yeast,visualizedby electronmicroscopyafter stainingwith uranylacetateTheelectrondensities of hundreds of imageswereaveragedto yield this compositeview (b) The x-ray structuresof one

NAD-.

In actively respiring mitochondria,

the actions of

many dehydrogenases keep the actual [NADHI/[NAD*] ratio well aboveunity, and the real free-energychangefor the reactionsho\&nin Equation19-5 is thereforesubstantially greater (more negative)than -220 kJ/moLA simiIar calculationfor the oxidation of succirate showsthat electrontransfer from succinate(fl'" for fumarate/succinate : 0.031\D to 02 has a smaller,but still negative, standardfree-energychangeof about - 150kJ/mol.

moleculeof Complexlll (red;from yeast)and two of ComplexlV map (green;from bovineheart)could be fittedto the electron-density to suggestone possiblemode of interactionof thesecomplexesin a Thisview is in the planeof the bilayer(yellow)' respirasome.

Much of this energy is used to pump protons out of the matrix. I'or each pair of electrons transferred to 02, four protons are pumped out by Complex I, four by Complex III, and two by Complex IV (Fig. 19-16). The uectori,cllequation for the process is therefore NADH + 11H* + ;O2-----+NAD"+ toHfr + H2O (19-7) The electrochemical energy inherent in this difference in proton concentration and separation of charge

Cyt c Intermembrane space (e side)

4IJ+ 4H+

zn++ |o2 4"r'

NADH + H+ Matrix (N side) 19-16 Summaryofthe flow ofelectronsand protonsthrough FIGURE four complexesof the respiratorychain. Electronsreach Q the as a mobile I and ll. ThereducedQ (QHz)serves throughComplexes carrierof electronsand protons.lt passeselectronsto Complexlll, c link,cytochrome which passes themto anothermobileconnecting electronsfrom reducedcytochromec to 02. ComplexlV thentransfers by proflow throughComplexes I, lll, and lV is accompanied Electron

space.Recallthat electon flow from the matrixto the intermembrane trons from p oxidationof fatty acids can also enter the respiratory shownhereare from chainthroughQ (seeFig.19-B).The structures (PDB lD 2FUC); thermophilus severalsources:Complex l, Thermus .lZOY); lll, bovineheart (PDB Complex lD heart Complexll, porcine c, equineheart(PDBlD lHRC); Com(PDBlD lBCY); cytochrome olexlV.bovineheart(PDBlD lOCC).

J2\

0 x i d a t i vPeh o s p h o r y l aatni odP nh o t o p h o s p h o r y l a t i o n

p side lH+l"

N side

I

H-

OH

H-

oHoH-

HH-

OH_

AG-R?ln(C2/C)+ZJLqr :2.3RT Lp}l + J^4r FIGURE 19-17 Proton-motive force,The inner mitochondrial memlrraneseparates two compartments of different[H*], resultingin differencesin chemicalconcentration (ApH)and chargedistribution (Ary') acrossthe membraneThe net effectis the proton-motiveforce (AC), whichcanbe calculated asshownhere.Thisisexplained morefully in the text

representsa temporary conservationof much of the energyof electrontransfer.The energystoredin such a gradient,termed the proton-motive force, has two components:(1) the chemica| potent?,alenergg due to the difference in concentration of a chemical species (H+) in the two regions separatedby the membrane,and (2) the electri,caLpotenti,al energy that results from the separationof chargewhen a proton movesacrossthe membranewithout a counterion (Fig. 19-17). As we showed in Chapter 11, the free-energy changefor the creation of an electrochemicalsradient by an ion pump is

LG: Rru (*) + zr^tr

Energeticsof Electron Transfer

Calculatethe amountof energyconservedin the proton gradientacrossthe inner mitochondrialmembraneper pair of electronstransferred through the respiratory chain from NADH to oxygen.AssumeArl is 0.15V and the pH differenceis 0.75units.

oHoHoH-

n

(19-8)

where C2 and C 1 are the concentrations of an ion in two regions, and C2 > Cl Z is the absolute value of its electrical charge (1 for a proton); and Ary'is the transmembrane difference in electrical potential, measured in volts. F o r p r o t o n sa t 2 5 " C ,

t(fr):

W0RKED EXAMPTE 19-l

lH+I.o: cr

: 6,

- loslH+l*) 2.BooelH*lp : 2.3(pHN - pHp) - 2.3 ApH

and Equation 19-8 reduces to LG : 2.3RTApH + JArT (19-9) : (5.70kJ/mol)ApH + (96.5kJ/V.mol)Arl In actively respiring mitochondria, the measured Ary'is 0 15 to 0.20 V and the pH of the matrix is about O.Zb units more alkaline than that of the intermembrane space.

Solution:Equation 19-9 gives the free-energychange when one mole of protons movesacrossthe inner membrane. Substituting the measured values for ApH (0.75units) and Ary'(0.15\) in this equation€fvesAG : 19kJ/mol(of protons).Becausethe transferof two electrons from NADH to 02 is accompaniedby the outward pumping of 10 protons (Eqn I9-7), rougruy 200 kJ (of Ihe 220 kJ releasedby oxidation of 1 mol of NADH) is conservedin the proton gradient. When protons flow spontaneouslydown their electrochemicalgradient, energy is made availableto do work. In mitochondria,chloroplasts,and aerobicbacteria, the electrochemicalenergyin the proton gradient drives the synthesisof ATP from ADP and P1.We return to the energeticsand stoichiometryof ATP synthesis driven by the electrochemicalpotential of the proton gradientin Section19.2.

Reactive 0xygen Species AreGenerated during 0xidative Phosphorylation Several steps in the path of oxygen reduction in mitochondria have the potential to produce highly reactive free radicals that can damage cells. The passageof electrons from QHz to Complex III, and passageof electrons from Complex I to QH2, involve the radical 'Q as an in'Qtermediate. The can, with a low probability, pass an electron to 02 in the reaction O, + e- --+

'Ot

The superoxide free radical thus generated is highly reactive; its formation also leads to production of the even more reactive hydroxyl free radical, 'OH (FiS. 19-f 8). These reactive oxygen species can wreak havoc, reacting with and damaging enzymes, membrane lipids, and nucleic acids. In actively respiring mitochondria, 0 lo/o to as much as 4o/oof the 02 used in respiration 'Of -more forms than enough to have lethal effects unless the free radical is quickly disposed of Factors that slow the flow of electrons through the respiratory chain increase the formation of superoxide, perhaps by prolonging the lifetime of 'Of generated in the Q cycle. 'Oi, To prevent oxidative damage by cells have several forms of the enzyme superoxide dismutase, which catalyzes the reaction 2',O; + 2H*

---> }l2O2 + 02

?,r

s itochondria 1 9 . 1 E l e c t r o n - T r aR ne s faecrt i 0i n M

Nicotinamide nucleotide transhydrogenase

tI "

Inner mitochondrial membrane

Cyt c

produced by a processknown as photorespiration,is convertedto serine (seeFig. 20-21): 2 Glycine+ NAD* ----->serine+ CO2+ NHB+ NADH + H*

I

III

I

rt\

IV

'oH 'loi

NADH

NADPH inactive

f'or reasonsdiscussedin Chapter 20, plants must carry out this reaction evenwhen they do not need NADH for ATP production.To regenerateNAD+ from unneeded NADH,plant mitochondriatransferelectronsfrom NADH directly to ubiquinoneand from ubiquinone directly to 02, bypassingComplexesIII and IV and their proton pumps.In this processthe energyin NADH is dissipated as heat, which can sometimesbe of value to the plant (Box 19-1). Unlike q,'tochromeoxidase (Complex I\|, the alternativeQH2oxidaseis not inhibited by cyanide. Cyanide-resistantNADH oxidation is therefore the hallmark of this uniqueplant electron-transferpathway.

1 9Y. 1 E l e c t r o n - T r a n s f e r SUMMAR R e a c t i oinnsM i t o c h o n d r i a tIGURE 19-18 ROSformationin mitochondria andmitochondrial defenses.When the rateof electronentry into the respiratorychain and the rateof electrontransfer throughthe chainare mismatchedi superoxideradical('Oi) productionincreases at Complexes I and lll asthe partiallyreducedubiquinoneradical('Q-) donatesan electronto 02. Superoxide actson aconitase, protein,to release a 4Fe-4S Fe2+In the presence of Fe2*,the Fentonreactionleadsto formationof the highly reactivehydroxylfreeradical('OH).The reactions shownin blue defendthecell againstthedamaging effects of superoxide. Reduced glutathione(CSH;seeFig 22-27)donateselectrons for the reductionof H2O2and of the oxidized Cys residues(-S-S-) of enzymesand otherproteins, andCSH is regenerated fromtheoxidizedform(CSSC) by reductionwith NADPH.

The hydrogenperoxide (HzOz)thus generatedis rendered harmlessby the action of glutathione peroxidase (Fig. 19-18). Glutathionereductaserecyclesthe oxidizedglutathioneto its reducedform,usingelectrons from the NADPHgeneratedby nicotinamidenucleotide (in the mitochondrion)or by the pentranshydrogenase tose phosphatepathway (in the c1'tosol;seeFig. 14-20). Reduced glutathione also serves to keep protein sulfhydryl groups in their reduced state, preventing some of the deleterious effects of oxidative stress (Fig. 19-18), Nicotinamidenucleotide transhydrogenaseis critical in this process:it producesthe NADPH essentialfor glutathionereductaseactivity.

r

theoryprovidesthe intellectual Chemiosmotic framework for understandingmany biological energytransductions,including oxidative phosphorylationand photophosphorylation. The mechanismof energycouplingis similar in both cases:the energyof electronflow is conservedby the concomitantpumpingof protonsacrossthe gradient, membrane,producingan electrochemical the proton-motiveforce.

r

In mitochondria,hydride ions removedfrom substratesby NAD-linked dehydrogenasesdonate electronsto the respiratory(electron-transfer) chain,which transfersthe electronsto molecular 02, reducingit to H2O.

r

Shuttle systemsconveyreducing equivalentsfrom cltosolic NADH to mitochondrialNADH. Reducing equivalentsfrom all NAD-linked dehydrogenations are transferredto mitochondrial NADH (ComplexI). dehydrogenase

r

Reducingequivalentsare then passedthrough a seriesof Fe-Scentersto ubiquinone,which transfersthe electronsto cy'tochromeb, the first carrierin ComplexIII. In this complex,electrons take two separatepaths through two b-type c1to an Fe-Scenter. and cy'tochrofiI€ c1'tochromes passes one at a time, Fe-S center electrons, The IV, into Complex c and through c1'tochrome This copper-containing cltochromeoxidase. enzyme,which also containscy'tochromeso and a3, accumulateselectrons,then passesthem to 02, reducingit to H2O.

r

Someelectronsenter this chainof carriersthrough alternativepaths. Succinateis oxidizedby (ComplexII), which succinatedehydrogenase containsa flavoproteinthat passeselectrons

Plant Mitcchondria Have Alternative Meehanisms f0r Oxidizing ttlADh{ Plant mitochondria supply the cell with ATP during periods of low illumination or darknessby mechanisms entirely analogousto those used by nonphotosynthetic organisms.In the light, the principal source of mitochondrialNADH is a reaction in which glycine,

f

722

l

OxidativePhosphorylationandPhotophosphorylation

through several Fe-S centers to ubiquinone. Electrons derived from the oxidation of fatty acids pass to ubiquinone vra the electron-transferrino flavoprotein r

of protectiveenzymes,includingsuperoxide dismutaseand glutathioneperoxidase. r

Potentially harmful reactive oxygen species produced in mitochondria are inactivated by a set

Many flowering plants attract insect pollinators by reIeasingodorantmoleculesthat mimic an insect'snatural food sourcesor potential egg-layingsites.Plantspollinated by flies or beetlesthat normally feed on or lay their eggs in dung or carrion sometimes use foulsmellingcompoundsto attract theseinsects. Onefamrlyof stinkingplantsis the Araceae,which includes philodendrons,arutn lilies, and skunk cabbages. These plants have tiny flowers denselypacked on an erect structure,the spadix,surroundedby a modifled leaf,the spathe.The spadixreleasesodorsofrotting flesh or dung. Before pollination the spadix also heats up, in somespeciesto asmuchas20 to 40 oCabovethe ambient temperature.Heat production (thermogenesis)helps evaporateodorantmoleculesfor better dispersal,and becauserotting flesh and dr-mgare usually warm from the hyperactivemetabolismof scavengingmicrobes,the heat itself might alsoattract insects.In the caseof the eastem skunk cabbage(Fig. 1), which flowers rn late winter or early spring when snow still coversthe ground, thermogenesisallows the spadix to grow up through the snow How doesa skunkcabbageheat its spadix?The mitochondriaof plants,fungr,and unicellulareukaryoteshave electron-transfersystemsthat are essentiallythe sameas thosein animals,but they alsohavean alternativerespiratory pathway.A cyanide-resistantQH2 oxidasetransfers electronsfrom the ubiquinonepool directlyto oxygen,bypassi4gthe two proton-translocatingstepsof Complexes III and IV (Fig. 2) Energr that might havebeenconserved asATPis insteadreleasedasheat.Plantmitochondriaalso

FIGURE 1 Eastern skunk cabbage. have an altemative NADH dehydrogenase,insensitive to the ComplexI inhibitor rotenone (see Table 19-4), that transfers electrons from NADH in the matrix directly to proton ubiquinone,bypassingComplexI andits associated pumpmg.And plant mitochondriahaveyet another NADH dehydrogenase, on the extemal face of the inner membrane,that transferselectronsfrom NADPHor NADH in the intermembranespaceto ubiquinone,againbypassmg ComplexI. Thus when electronsenter the alternativerespiratory pathwayttrough the rotenone-insensitive NADH dehydrogenase,the external NADH dehydrogenase,or (ComplexII), and passto 02 via succinatedehydrogenase the cyanide-resistantalternative oxidase,energy is not conservedasATPbut is releasedasheat.A skurk cabbage canusethe heatto melt snow,producea foul stench,or attract beetlesor flies.

External NAD(P)H dehydrogenase

Interrnembrane space (r side)

III Alternative NADH

NADH

dehydrogenase

(N side)

A.vt

?

NAD(P)H

I

Matrix

Plants,fungi, and unicellulareukaryoteshave,in path for additionto the typical cyanide-sensitive electrontransfer,an alternative,cyanide-resistant NADH oxidation pathway.

NAD+

,Lo,

ry

a ,l':,

HrO

Alternative oxidase

f IGURE 2 Electron carriersof the innermembrane of plantmitochondria. Electrons can flow throughComplexes l, lll, and lV, as in animalmitochondria, or throughplanfspecificalternative carriersby the pathsshownwith blue arrows.

synthesis 19.2ATP ?rt

19.2ATP Synthesis How is a concentration gradient of protons transformed into ATP? We have seen that electron transfer releases, and the proton-motive force conserves, more than enough free energy (about 200 kJ) per "mole" of electron pairs to drive the formation of a mole of ATP, which requires about 50 kJ (p. 503). Mitochondrial oxidative phosphorylation therefore poses no thermodynamic problem. But what is the chemrcal mechanism that couples proton flux with phosphorylation? The chemiosmotic model, proposed by Peter Mitchell, is the paradigm for this mechanism. According to the model (l-ig. 19-19), the electrochemical energy inherent in the difference in proton concentration and the separation of charge across the inner mrtochondrial membrane-the proton-motive force-drives the synthesis of ATP as protons flow passively back into the matrix through a proton pore associated with ATP synthase. To emphasize this crucial role of the proton-motive force, the equation for ATP synthesis is sometimes written ADP + P, + nH$ ------+ATP + H2O + zHfi (19-10) Mitchell used "chemiosmotic" to describe erz;rmatic reactions that involve, simultaneously, a chemrcal reaction and a transport process. The operational definition of "coupling" is shown in Figure f9-20. When isolated mitochondria are suspended in a buffer containing ADP, P1,and an oxidizable substrate such as succinate, three easily measured processesoccur: (1) the substrate is oxidized

(succinateyields fumarate), (2) 02 is consumed,and (3) ATP is slmthesized.Oxygen consumption and ATP sy'nthesisdepend on the presenceof an oxidizable substrate(succinatein this case) aswell asADP and Pi. Becausethe energyof substrate oxidation drives ATP synthesisin mitochondria,we would expect inhibitors of the PeterMitchell, passageof electronsto 02 (such 1920-1992 as cyanide, carbon monoxide, and antimycin A) to block ATP synthesis(Fig. 19-20a). More surprising is the finding that the converseis also true: inhibition of ATP synthesisblockselectrontransfer in rntact mitochondria This obligatory couplirLgcan be demonstratedin isolated mitochondria by providing Oz and oxidizablesubstrates,but not ADP (Fig. 19-20b). Under these conditions,no ATP synthesiscan occur and electrontransferto O2doesnot proceed.Couplingof oxidation and phosphorylationcan also be demonstrated using oligomycin or venturicidin, toxic antibiotics that bind to the ATP synthasein mitochondria.These compounds are potent inhibitors of both ATP synthesisand the transfer of electronsthrough the chain of carriersto 02 @ig. 19-20b). Becauseoligomycinis knov,rrto interact not directly with the electron carriersbut with ATP it follows that electron transfer and ATP synsSmthase, thesis are obligatelycoupled:neither reaction occurs without the other.

AIf +

+++++

zu*+ |o2

Matrix (N side)

Hzo ADP+ Pi

Succinate

Fumarate

ATP NADH + H+

Chemical potential ApH (inside alkaline)

ATP synthesis driven by

proton-motive force

of FIGURE 19-19 Chemiosmotic model.In this simplerepresentation the chemiosmotic theory appliedto mitochondria,electronsfrom passthrougha chainof carriers NADH andotheroxidizablesubstrates Electron flow is acarranged asymmetrically in the innermembrane. producing both a by proton transfer across the membrane, companied

Electrical potential NL (inside negatrve,

Theinnermitochemicalgradient(ApH)and an electricalgradient(Ary'). can reenterthe protons protons; to impermeable chondrialmembraneis (Fo). proton-motive The channels proton-specific through matrix only forcethatdrivesprotonsbackintothe matrixprovidesthe energyfor ATP with Fo' catalyzedby the Fr complexassociated synthesis,

l:r1

Oxidative Phosphorylation andPhotophosphorytation

Add venturicidin

Add DNP

E o N

N a

a o

a

Pi

a N

N

-

3

(a)

Time

(b)

Time

FIGURE 19-20 Couplingof electrontransferand ATp synthesisin mitochondria. In experiments to demonstrate coupling,mitochondria are suspended in a bufferedmediumand an 02 electrodemonrtors02 consumption. At intervals, samplesare removedand assayed for the presence of ATP.(a)Additionof ADPand P;aloneresultsin littleor no increase in eitherrespiration (O2consumption; black)orATpsynthesis (red).When succinateis added,respiration beginsimmediately and

ATPis synthesized. Additionof cyanide(CN-),which blockselectron transfer betweencytochrome oxidase(ComplexlV)andOz, inhibitsboth (b) Mitochondriaprovidedwith succinate respiration andATPsynthesis. respireand synthesize ATPonly when ADP and P; are added Subsequentadditionof venturicidin or oligomycin, inhibitors of ATPsynthase, (DNP)isan unblocksbothATPsynthesis andrespiration. Dinitrophenol coupler,allowingrespiration to continuewithoutATPsynthesis.

Chemiosmotic theory readily explains the dependence of electron transfer on ATP s],'nthesis in mitochondria.

tiflcially createdproton gradient shouldbe able to replace electron transfer in driving ATP synthesis.This hasbeenexperimentallyconflrmed(Fig. f 9-22). Mitochondria manipulated so as to impose a difference of proton concentrationand a separationof chargeacross the inner membranesynthesizeNIP i,n the absenceoJ an onidi,zablesubstrate;the proton-motiveforce alone sufflcesto drive ATP srmthesis.

Whenthe flow of protonsinto the matrix throughthe proton charurelof AIP slnthaseis blocked(with oligomycin, for example), no path existsfor the return of protonsto the matrix, and the continuede\trusion of protons driven by the activity ofthe respiratorychaingeneratesa largeproton gradient.The proton-motiveforce builds up until the cost (free energ/) of purnpingprotons out of the matnx againstthis gradientequalsor exceedsthe energyreleased by the transferof electronsfrom NADHto 02.At this point electron flow must stop; the free energy for the overall processof electronflow coupledto proton pumpmgbecomeszero,and the systemis at equilibrium. Certainconditionsand reagents,however.,can uncoupleoxidationfrom phosphorylation. Whenintact mitochondriaare disruptedby treatmentwith detergentor by physical shear,the resulting membranefragments can still catalyzeelectron transfer from succinateor NADHto 02, but no ATP synthesisis coupledto this respiration.Certainchemicalcompoundscauseuncoupling without disrupting mitochondrialstructure. Chemical uncouplersinclude 2,4-dinitrophenot(DNp) and carb onylcyanide-p -trifluoromethoxyphenylhydrazone (FCCP) (Table19-4; Fig. l9-21), weak acidswfth hydrophobicpropertiesthat permit them to diffusereadily across mitochondrial membranes.After entering the matrix in the protonatedform, they can releasea proton, thus dissipatingthe proton gradient. Ionophores such as valinomycin (see Fig. 11-4b) allow inorganic ions to pass easilythrouglr membranes.Ionophoresuncoupleelectrontransferfrom oxidativephosphorylation by dissipatingthe electricalcontributionto the electrochemical gradientacrossthe mitochondrialmembrane. A predictionof the chemiosmotictheoryis that, becausethe role of electron transfer in mitochondrialATp synthesisis simply to pump protonsto createthe eiectrochemicalpotentialof the proton-motiveforce,an ar-

Noz +H-

2,4-Dinitrophenol (DNP)

I\T

"\-

\T

- c- ,cy'"" ll

N

NH

T\T

"\c-

T\T

-c- ,cr';"' ll N

N_

I z'\ tI \-/

o I

F-C-F I

F

+H-

I

o I

F-C-F

$

Carbonylcyanide-ptrifl uoromethoxyphenylhydrazone (FCCP) FIGURE 19-21 Two chemicaluncouplersof oxidativephosphorylation. BothDNP and FCCPhavea dissociable protonand arevery hydrophobic.Theycarryprotonsacross the innermitochondrial membrane, dissipatingthe protongradient Both also uncouplephotophosphorylation (seeFie.19-63).

19.2ATP S y n t h e s i s725iL

:-

g*1-

1ut*"':

:

t K + l: t c l - l = 0 . 1 M

&w

(a)

pH lowered from 9 to 7; , valinomycin present; no K-

$

(b) FIGURE 19-22 Evidencefor the role of a proton gradientin ATPsynthesis.An artificiallyimposedelectrochemical gradientcan driveATP synthesis in the absence of an oxidizablesubstrate as electrondonor. (a) isolatedmitochondria In this two-stepexperiment, are first incubatedin a pH 9 buffercontaining0.1 v KCl.Slow leakageof buffer and KCIinto the mitochondria eventually bringsthe matrixinto equilibriumwith the surrounding medium.No oxidizablesubstrates are present(b) Mitochondriaare now separated from the pH 9 bufferand resuspended in pH 7 buffercontainingvalinomycinbut no KCl.The changein buffercreatesa difference of two pH unitsacrossthe inner membrane. mitochondrial Theoutwardflow of K*, carried(byvalinomycin)down its concentration gradientwithouta counterion, creates (matrixnegative) Thesumof a chargeimbalance across the membrane and the electrithe chemicalpotentialprovidedby the pH difference cal potentialprovidedby the separation of chargesis a proton-motive forcelargeenoughto supportATPsynthesis in the absence of an oxidizablesubstrate.

Flas ATF 5ynthase TwoFunctional ilomains, F,,anci tu Mitochondrial ATP synthase is an F-g,pe MPase (see Fig. 11-39) similar in structure and mechanism to the ATP slmthases of cNoroplasts and bacteria. This large en4.'rne complex of the imer mitochondrial membrane catalyzes the formation of ATP from ADP and Pi, accompanied by the flow of protons from the p to the N side of the membrane (Eqn 19-10). ATP slrrthase, also called Complex V, has two distinct components: F1, a peripheral membrane protein, and Fo (o denoting ohgomycin-sensitive), which is integral to the membrs.n€.F1, the first factor recognized

as essentialfor oxidativephosphorylation,was identi-fiedand purified by Efraim Rackerand in the early1960s. his colleagues hr the Iaboratorysmallmembrane vesiclesformed from inner mitochondrial membranes carryout ATPsynthesiscoupled to electrontransfer.When F1 is gently extracted,the "stripped" vesiclesstill containintact respiratory chainsand the Foportion of ATP srmthase.The vesicles

Efraim

can catalyze electron transfer from NADH to 02 but cannot produce a proton gradient: f'o has a proton pore ttuouglt which protons leak as fast as they are pumped by electron transfer, and without a proton gradient the F1-depleted vesicles cannot make ATP. Isolated F1 catalyzesATP hydrolysis (the reversal of synthesis) and was therefore originally called FlATPase. When purifled F1 is added back to the depleted vesicles, it reassociates with Fu, plugging its proton pore and restoring the membrane's capacity to couple electron transfer and ATP synthesis.

t0 ADP0nthe Relative ATFlsStabilized Surface of F,' Isotope exchange experiments with purifled F1 reveal a remarkable fact about the enzyme's catalytic mechanism: on the enzyme surface, the reaction ADP + Pi 9070 efficiency. Within 3 ps of the excitation of P870, pheoph;,tin has received an electron and become a negatively charged radical; less than 200 ps later, the electron has reached the quinone Qe (Ftg 19-55b) The electron-transfer reactions not only are fast but are thermodynamically "downhill"; the excited special pair (Chl)} is a very good electron donor (E'o : - I \D, and each successiveelectron transfer rs to an acceptor of substantially less negative fl'o. The standard free-energy change for the process is therefore negative and large; recali from Chapter 13 that LG'" = -tzJLE'o; here, A.E'ois the drfference between the standard reduction potentials of the two half-reactions (1) (Chl)t ----+ '(Chl)i + e(2) Q + 2Ht + 2e- ---+ QH,

E ' , ': - 1 . 0 V E ' " : - 0 . 0 4 5V

Thus LE',": -0.045V - (-1.0 V) - 0.95V

and AG'" : -2(96.5 kJ/V . mol)(0.95V) : -180 kJ/mol The combination of fast kinetics and favorable thermodynamics makes the process virtually irreversible and higily efflcient. The overall energy yield (the percentage ofthe photon's energy conservedin QHz) is >30%, with the remainder of the energy dissipated as heat and entropy.

(enters InPlants, TwoReaction ArtinTandem The photosynthetic apparatus of modern cyanobacteria, algae, and vascular plants is more complex than the one-center bacterial systems, and it seems to have evolved through the combination of two simpler bacterial photocenters. The thylakoid membranes of chloroplasts have two different kinds of photosystems, each with its own type of photochemical reaction center and set of antenna molecules. The two systems have distinct and complementary functions (I,'ig. 19-l-r{i). Photosystem II (PSII) is a pheophy-tin-quinone type of

Photosystem I

Photosystem II

\ A1

- 1 . 0-

\

ffil

-s

Fe

\ Fd

\ Fd:NADP+ oxidoreductase

q

NADPN NA-DPH

0-

f Light

PQa = plastoquinone PQB = secondquinone At = electron acceptorchlorophyll A, = phylloquinone

FIGURE 19-56 Integrationof photosystems I and il in chloroplasts. This "Zscheme"showsthe pathwayof electrontransfer from HrO (lowerleft) to NADP- (far right)in noncyclicphotosynthesis The positionon the verticalscaleof eachelectroncarrierreflectsits standardreductionpotential.To raisethe energyof electronsderivedfrom H2O to the energy level requiredto reduceNADP+ to NADPH, each electronmust be "lifted"twice (heavyarrows)by phoronsabsorbedin pSlland pSl.One photon is requiredper electronin each photosystem. After excitation,

t h e h i g h - e n e r g ye l e c t r o n sf l o w " d o w n h i l l " t h r o u g h t h e c a r r i e r c h a i n s shown. Protons move acrossthe thylakoid membrane during the watersplitting reaction and during electron transferthrough the cytochrome b6f complex, producing the proton gradient that is essentialto ATp formation. An alternative path of electrons is cyclic electron transfer, in which electrons move from ferredoxin back to the cytochrome buf complex, instead of reducing NADP* to NADPH. The cyclic pathway prod u c e s m o r e A T P a n d l e s sN A D P H t h a n t h e n o n c y c l i c .

e c t r oFnl o w [ t U Ul 19 . 8 T h e( e n t r aPl h o t o c h e mEi cvaelnLt :i g h t - D r i vEel n

system (like the single photosystem of purple bacteria) containing roughly equal amounts of chlorophylls a and b Excitation of its reaction-center P680 drives electrons through the cytochrome b61fcomplex with concomitant movement of protons across the thylakoid membrane. Photosystem I (PSI) is structurally and functionally related to the type I reaction center ofgreen sulfur bacteria. It has a reaction center designated P700 and a high ratio of chlorophyll n to chlorophyll b. Excited P700 passes electrons to the Fe-S protein ferredoxin, then to NADP-, producing NADPH. The thylakoid membranes of a single spinach chloroplast have many hundreds of each kind of photosystem. These two reaction centers in piants act ln tandem to catalyze the light-driven movement of electrons from H2O to NADP- (FiC. 19-56). Electrons are carried between the two photosystems by the soluble protein plastocyanin, a one-electron carrier functionally simiiar to cytochrome c of mitochondria. To replace the electrons that move from PSII through PSI to NADP-, cyanobacteria and plants oxidize H2O (as green sulfur bacteria oxidize H2S), producing 02 (Fig. 19-56, bottom left). This process is called oxygenic photosynthesis to distinguish it from the anoxygenic photosynthesis of purple and green sulfur bacteria. All O2-evolvingphotosynthetic cells-those of piants, algae, and cyanobacteria-contain both PSI and PSII; organismswith only one photosystem do not evolve 02. The diagram in Figure 19-56, often called the Z scheme because of its overall form, outlines the pathway of electron flow between the two photosystems and the energy relationships in the light reactions The Z scheme thus describes the complete route by which electrons flow from H2O to NADP-, according to the equation 2H2O+ 2NADP+ * Sphotons

> 02 + 2NADPH +2l{*

For every two photons absorbed (one by each photosystem) , one eiectron is transferred from H2Oto NADP-. To form one molecule of 02, which requires transfer of four electrons from two HrO to two NADP+, a total of eight photons must be absorbed, four by each photosystem The mechanistic details of the photochemical reactions in PSII and PSI are essentially similar to those of the two bacterial photosystems, with several important additions In PSII, two very similar proteins, Dl and D2, form an almost symmetric dimer, to which all the electron-carrying cofactors are bound (Fig. 19-57). Excitation of P680 in PSII produces P680'k,an excellent electron donor that, within picoseconds, transfers an electron to pheophytin, giving it a negative charge ('Pheo-). With the loss of its electron, P680* is trans'Pheo very rapidly formed into a radical cation, P680+ passes its extra electron to a protein-bound plastoquinone, PQa (or QJ, which in turn passesits electron to another, more loosely bound plastoquinone, PQs (or Qs). When PQs has acquired two electrons in two such transfers from PQa and two protons from the solvent

Tyrz /.R\ ( ,1/

P680 ,1,

| \+/

Pheo

\ \_

(Chl ptr"o

/6\

,. ).. .). Stroma (N side) ll of the cyanobacteriumSynechococcus 19-57 Photosystem FfGURE form of the complexshown here hastwo The monomeric elongates. proteins, Dl and D2, eachwith itssetof cofacmajortransmembrane electronflow ocarenearlysymmetric, tors Althoughthetwo subunits curs throughonly one of the two branchesof cofactors,that on the right (on D-l) The arrowsshow the path of electronflow from the Mn enzymeto the quinonePQs ion cluster(Mno)of the water-splitting by the step indicated eventsoccurin the sequence Thephotochemical of cofactors numbersNoticetheclosesimilaritybetweenthepositions centershownin hereand the positionsin the bacterialphotoreaction laterin the text is discussed residues The role oftheTyr 19-55. Figure water, it is in its fully reduced quinol form, PQ3H2 The overall reaction initiated by light in PSII is 4P680 + 4}l* + 2 PQB * 4photons -+ 4 P680* + 2 PQBH2 (19-12) Eventually, the electrons in PQgH2 pass through the cytochrome b6;f complex (Fig. 19-56). The electron initially removed from P680 is replaced with an electron obtained from the oxidation of water, as described below.

The binding site for plastoquinone is the point of action of many commercial herbicides that kill plants by blocking electron transfer through the cy'tochrome b6;f complex and preventing photosynthetic ATP production. The photochemical events that follow excitation of PSI at the reaction-center P700 are formally similar to those in PSII. The excited reaction-center P700* loses an electron to an acceptor, A0 (believed to be a special form of chlorophyll, functionally homologous to the pheophytin of PSII), creating As and P700* (Fig. 19-56, right side); again, excitation results in charge separation at the photochemical reaction center. P700- is a strong oxidizing agent, which quickly acquires an electron from plastocyanin, a soluble Cu-containing electron-transfer protein. Ae is an exceptionally strong reducing agent that passesits electron through a chain of carriers that leaclsto NADP+ First, phylloquinone (A1) accepts an electron and passesit to an iron-sulfur protein (through three Fe-S centers in PSI). From here, the electron moves to ferredoxin (Fd), another iron-sulfur protein loosely associated with the thylakoid membrane. Spinach ferredoxin (M,I0,700) contains a 2Fe-2S center (trig. 19-5) that undergoes one-electron oxidation and reduction reactions. The fourth electron carrier in

f-t

1754

0xidativePhosphorylationandPhotophosphorylation

the chain is the flavoprotein ferredoxin:NAl)P+ oxidoreductase, which transfers electrons from reduced ferredoxin (fd."J to NADP*:

Antenna Chlorophylls AreTightly Integrated with ileetrsn {arriers The electron-carryingcofactorsof PSI and the lightharvestingcomplexesare part of a supramolecularcomplex (Fig. 19-58a), the structure of which has been solvedcrystallographically.The protein consistsof three

2Fd..d + 2H+ + NADP* ------+2Fdu*+ NADPH + H+ This enz;.rne is homologous to the ferredoxin:NAD reductase ofgreen sulfur bacteria (Fig. 19-54b). (a)

Lumen (p side) Subunit B )2 p ^

,/ chl

too

I

chr

(chl)A __0

/rrl.l\

ex

wK PSI Subunit C

r' A

\

Stroma (w side)

Feor

FIGURE 19-58 Thesupramolecular complexof pSl and its associated antennachlorophylls. (a) Schematic drawingof the essential proteins andcofactors in a singleunitof PSl.A largenumberof antennachlorophyllssurroundthe reactioncenterand conveyto it (redarrows)the energyof absorbedphotons.The resultis excitationof the pair of p700,greatlydecreasing chlorophyllmolecules that constitute its reductionpotential;P700then passes an electronthroughtwo nearby chlorophylls (Q*; alsocalledA1).Reducedphylloto phylloquinone quinoneis reoxidized as it passes two electrons, one at a time (blue arrows),to an Fe-Sprotein(Fy)nearthe N sideof the membrane.From Fx, electronsmove throughtwo more Fe-Scenters(FAand FB)to the Fe-Sproteinferredoxinin the stroma.Ferredoxin thendonateselectrons

to NADP- (not shown),reducingit to NADPH,one of the formsin whichthe energyof photonsis trappedin chloroplasts. (b) Thetrimericstructure(derivedfrom PDB lD lJBO),viewed from the thylakoidlumenperpendicular to the membrane,showing all proteinsubunits(gray)and cofactors.(c) A monomerof pSlwith all the proteinsomitted,revealingthe antennaand reaction-center c h l o r o p h y l l s( g r e e nw i t h d a r k g r e e n M g 2 * i o n s i n t h e c e n t e r ) , (yellow),and Fe-Scentersof the reactioncenter(spacecarotenoids filling red and orangestructures). The proteinsin the complexhold the componentsrigidlyin orientations that maximizeefficientexciton transfers betweenexcitedantennamoleculesand the reaction center.

Electron FlowFttl Event:Light-Driven Photchemical 19.8TheCentral identical complexes,each composedof l1 different proteins (Fig. 19-58b).In this remarkablestructurethe many antenna chlorophyll and carotenoidmoleculesare preciselyarrayedaround the reaction center (Fig. 19-58c). The reaction center's electron-carryingcofactorsare therefore tightly integrated with antenna chlorophylls. This arrangementallowsvery rapid and efflcient exciton transfer from antenna chlorophylls to the reaction center.In contrastto the singlepath of electronsin PSII, the electronflow initiated by absorptionof a photonis believedto occur through both branchesof carriersin PSL

llandI Photosystems b6f(0mplex Links The(ytochrome Electronstemporarilystoredin plastoquinolas a result of the excitation of P680in PSII are carried to P700 of PSI via the cytochromeb6lcomplex and the solubleprotein plastocyanin(Frg.19-56,center). Like ComplexIII of mitochondria,the cytochromeb6J complex(FiS. 19-59) contains a b-type cy[ochrome with two heme groups (designated bs and br), a Rieske iron-sulfur protein (lr[, 20,000), and cy'tochrome/ (named for the Latin '). frons, "leaf Electronsflow through the cytoctrome b6f

Heme/

Heme b"

Lumen (p side)

ata'

Heme bq

Heme r Stroma (w side) (a)

4H* Rieske ironsulfur protein

Thylakoid (p side)

2Ho

lumen

Strorna (N side)

FIGURE19-59 Electron and proton flow through the cytochromeb6f complex.(a)The crystalstructureof the complex (PDBlD .lUM3) revealsthe positionsof the cofactorsinvolved In additionto the hemesof cytochromeb in electrontransfers. (hemebp and b1;alsocalledhemeb^ and 6p,respectively, bep and of the bilayefl proximity N and sides to the of their cause cytochromef (hemef), thereis a fourth(hemex) nearhemebn; of unknownfunction.Two sitesbind thereis alsoa B-carotene plastoquinone: the PQH2sitenearthe p sideof the bilayer,and the PQ sitenearthe N side.The Fe-Scenterof the Rieskeprotein liesjustoutsidethe bilayeron the p side,and the heme/site is well intothethylakoidlumen. on a proteindomainthatextends (b) The complex is a homodimerarrangedto createa cavern thiswith the structhe PQH, and PQ sites(compare connecting .l 1).Thiscavernallll in Fig 9-1 Complex tureof mitochondrial to move betweenthe sitesof its oxidation lows plastoquinone and reduction. (PQHr)formedin PSllis oxidizedby the (c) Plastoquinol cytochromeb6f complex in a seriesof stepslike thoseof the bcl complex(Complexlll) of mitoQ cyclein the cytochrome to chondria(seeFig. I9-12). One electronfrom PQH2passes the Fe-Scenterof the Rieskeprotein,the other to hemeb1 of of electronsfrom PQH2 cytochromeb6.Theneteffectis passage which carriesthemto PSl. plastocyanin, to the solubleprotein

F"l

0 x i d a t i vPeh o s p h o r y l aatni odP nh o t o p h o s p h o r y l a t i o n

complex from PQ3H2 to cytochrome /, then to plastocyanin, and finally to P700, thereby reducing it. Like Complex III of mitochondria, cytochrome b61[ conveys electrons from a reduced quinone-a mobile, Iipid-soluble carrier of two electrons (Q in mitochondria, PQs in chloroplasts)-to a water-soluble protein that carries one electron (cytochrome c in mitochondria, plastocyanin in cNoroplasts). As in mitochondria, the function of this complex involves a Q cycle (Fig. 19-12) in which electrons pass, one at a time, from PQ3H2to cytochrome b6. This cycle results in the pumping of protons across the membrane; in chloroplasts, the direction of proton movement is from the stromal compartment to the thylakoid lumen, up to four protons moving for each pair of electrons. The result is production of a proton gradient across the thylakoid membrane as electrons pass from PSII to PSI. Because the volume of the flattened thylakoid Iumen is small, the influx of a small number of protons has a relatively large effect on lumenal pH. The measured difference in pH between the stroma (pH 8) and the thylakoid lumen (pH 5) represents a 1,000-fold difference in proton concentration-a powerful driving force for ATP syrrthesis.

(ycficElectron Flow between PSI andtheCytochrome b6f (omplex Increases theProduction 0fATP Relative to NADPH Electron flow from PSII through the cytochrome b6l complex, then through PSI to NADP+, is sometimes called noncyclic electron flow, to distinguish it from cyclic electron flow, which occurs to varying degrees depending primarily on the light conditions. The noncyclic path produces a proton gradient, which is used to drive ATP synthesis, and NADPH, which is used in reductive biosynthetic processes. Cyclic electron flow involves only PSI, not PSII (Fig. 19-56). Electrons passing from P700 to ferredoxin do not continue to NADP+, but move back through the cytochrome b6;fcomplex to plastocyanin. (This electron path parallels that in green sulfur bacteria, shown in Fig. 19-54b.) Plastocyanin then donates electrons to P700, which transfers them to ferredoxin. In this way, electrons are repeatedly recycled through the cy'tochromeb6;f complex and the reaction center of PSI, each electron propelled around the cycle by the energy of one photon. Cyclic electron flow is not accompanied by net formation of NADPH or evolution of 02. However, it es accompanied by proton pumping by the cytochrome b6l complex and by phosphorylation of ADP to ATP, referred to as cyclic photophosphorylation. The overatl equation for cyclic electron flow and photophosphorytation is simply ADP + P,4

ATP + H2O

By regulating the partitioning of electrons between NADP+ reduction and cyclic photophosphoryiation, a plant adjusts the ratio of ATP to NADPH produced in the light-dependent reactions to match its needs for these products in the carbon-assimilationreactions and other

biosyntheticprocesses.As we shall see in Chapter20, the carbon-assimilationreactions require ATP and NADPHin the ratio 3:2. This regulationof electron-transferpathwaysis parl of a short-termadaptationto changesin Iight color (wavelength) and quantity (intensity), asfilther describedbelow.

StateTransitions Change theDistribution of LHCII between theTwoPhotosystems The energy required to excite PSI (P700) is less (light of longer wavelength, lower energy) than that needed to excite PSII (P680). If PSI and PSII were physically contiguous, excitons originating in the antenna system of PSII would migrate to the reaction center of PSI, leaving PSII chronically underexcited and interfering with the operation of the two-center system. This imbalance in the supply of excitons is prevented by separation of the two photosystems in the thylakoid membrane (Fig. 19-60) PSII is located almost exclusively in the tightly appressed membrane stacks of thylakoid grana; its associated lightharuesting complex (LHCII) mediates the tight association of adjacent membranes in the grana. PSI and the ATP symthase complex are located almost exclusively in the nonappressed thylakoid membranes (the stromal lamellae), where they have access to the contents of the stroma, including ADP and NADP+. The cytochrome b6l complex is present primarily in the grana. The associationof LHCII with PSI and PSII depends on light intensity and wavelength, which can change in the short term, leading to state transitions in the chloroplast. In state 1, a critical Ser residue in LCHII is not phosphorylated, and LHCII associates with PSII. Under conditions of intense or blue light, which favor absorption by PSII, that photosystem reduces plastoquinone to plastoquinol (PQHz) faster than PSI can oxidize it The resulting accumulation of PQH2 activates a protein kinase that triggers the transition to state 2 by phosphorylating a Thr residue on LHCII (Fig. f 9-6f ). Phosphorylation weakens the interaction of LHCII with PSII, and some LHCII dissociatesand moves to the stromal lamellae; here it captures photons (excitons) for PSI, speeding the oxidation of PQH2 and reversing the imbalance between electron flow in PSI and PSII. In Iess intense light (in the shade, with more red light), PSI oxidizes PQHz faster than PSII can make it, and the resulting increase in [PQ] triggers dephosphorylation of LHCII, reversing the effect of phosphorylation. The state transition in LCHII localization is mutually regulated with the transition from cyclic to noncyclic photophosphorylation, described above; the path of electrons is primarily noncyclic in state 1 and primarily cyclic in state 2.

Water lsSplitbythe0xygen-Evolving Complex The ultimate source of the electrons passed to NADPH in plant (o:ygenic) photos5,nthesisis water. Having given up an electron to pheophy[ir:r, P680+ (of PSII) must acquire

n c t r oFnl o w I z S z ah l o t o c h e mEi cvaelnLt :i g h t - D r i vEel e f 9 . 8 T h eC e n t rP

ATP synthase i Ferredoxin: NADPT

Photosystem II

Light-haruesting Stroma ATP synthase

Nonhppressed membranes (stromal lamellae)

(b)

rlt

FIGURE 19-60 Localization of PSIand PSllin thylakoidmembranes. (a)Thestructures of the complexesand solubleproteinsof the photosynthetic apparatus of a vascularplantor alga,drawnto the same 'l scale.ThePDBlD for the structure of PSllis 2AXT;for PSl, QZV;for .1A70; cytochrome bof, 2E74;for plastocyanin , 1AC6;for ferredoxin, for ferredoxin:NADP* reductase, 1QCO TheATPsynthase structure shown is a compositeof that from yeastmitochondria(PDB lD (PDBlD l BMF).(b) Lightharvest1QO1)and bovinemitochondria

are locatedboth in appressed ing complexLHCIIand ATPsynthase (granal lamellae,in whichseveral membrane regionsof the thylakoid regions(stromal membranesare in contact)and in nonappressed and havereadyaccessto ADP and NADP+in the stroma. lamellae), regions,and PSI PSllis presentalmostexclusivelyin the appressed regions,exposedto the stroma. almostexclusivelyin nonappressed lamellaetogether(see LHCIIis the "adhesive"that holdsappressed .l Fie. 9-61).

Thylakoid membrane FIGURE 19-61 Balancingof electron flow in PSI and PSll by state transition.A hydrophobic domainof LHCIIin one thylakoidlamella insertsinto the neighboringlamellaand closelyappresses the two (state1).Accumulation (notshown)stimumembranes of plastoquinol latesa proteinkinasethat phosphorylates a Thr residuein the hydrophobic domain of LHC|l, which reducesits affinity for the neighboringthylakoidmembraneand convertsappressedregionsto nonappressed regions(state2).A specificproteinphosphatase reverses phosphorylation thisregulatory whenthe tPQl/tPQHrlratioincreases

an electronto retum to its groundstatein preparationfor captureof anotherphoton.In principle,the requiredelectron might comefrom any mrmberof organicor inorganic compounds.Photosymthetic bacteriausea variety of electron donorsfor this purpose-acetate, succinate,malate,

protern kinase ATP

ADP

\t,

*T p.

,"

protein tase

Appressed (state 1)

Nonappressed (state 2)

or sulflde-depending on what is available in a particular ecological niche. About 3 billion years ago, evolution of primitive photosynthetic bacteria (the progenitors of the modern cyanobacteria) produced a photosystem capable of taking electrons from a donor that is always available-

D"l

0 x i d a t i vPeh o s p h o r y l aatni odP nh o t o p h o s p h o r y l a t i o n

2nd Exciton

4th Exciton

t_

Ttl el

.1,_l---o, [9.M"d ', "' L

[q-.], L M"+l H*

H*

FIGURE 19-62Water-splitting activityof the oxygen-evolving complex. Shownhereis the process that produces a four-electron oxidizing agent-amultinuclear center withseveral Mn ions-inthewater-splitting complexof PSllThesequential (excitons), absorption of fourphotons

eachabsorption causingthe lossof oneelectronfromthe Mn center,producesan oxidizingagentthatcan removefourelectrons from two moleculesof water,producing02 Theelectronslostfromthe Mn centerpass one at a time to an oxidizedTyrresiduein a PSllprotein,thento P680*.

water. T\arowater molecules are split, flelding four electrons, four protons, and molecular oxygen:

cluster are not fully understood,but this chemistryis essentialto life on Earth and of great interest both for its biologicalsignificanceand as a challengein bioinorganic chemistry.Manganesecan exist in stable oxidation statesfrom 2 + to 7+ , so a cluster of Mn ions can certainly donate or accept four electrons.Determination of the structure of the polymetalliccenter has inspired severalreasonableand testable hypotheses. Stay tuned.

zHzO ------+4IJ* + 4e + Oz A single photon of visible light does not have enough energy to break the bonds in water; four photons are required in ttus photolytic cleavage reaction. The four electrons abstracted from water do not pass directly to P680+, which can accept only one electron at a time. Instead, a remarkable molecular device, the oxygen-evolving complex (also called the watersplitting complex), passes four electrons one at a ti,me to P680+ (FiS. f 9-62). The immediate electron donor to P680* is a T5rrresidue (often designated Z or t'.r'z) in subunit Dl of the PSII reaction center. The Tlr residue loses both a proton and an electron, generating 'Tlr: the electrically neutral T).r free radical, 4P680+ + ATyr -> 4P680+ 4 'Tyr. (19-13) The Tirr radical regains its missing electron and proton by oxidizing a cluster of four manganese ions in the watersplitti4g complex. With each sirgle-electron transfer, the Mn cluster becomes more oxidized; four single-electron transfers, each corresponding to the absorption of one photon, produce a charge of 4* on the Mn complex (Fig. 19-62): 'Tyr+ 4 4 Tyr + [Mncomplex]a*(19-14) [Mn complex]o------+

SUMMAR 1Y 9 . 8 T h eC e n t r P a lh o t o c h e m i c a l E v e nLt :i g h t - D r i v e n E l e c t r oFnl o w r

Bacteriahave a singlereaction center; in purple bacteria,it is of the pheophytin-quinonetype, and in greensulfurbacteria,the Fe-Stype.

r

Structuralstudiesof the reactioncenterof a purple bacterium have provided information about light-driven electron flow from an excited special pair of chlorophyll molecules,through pheophytin, to quinones.Electronsthen passfrom quinones throughthe cytochromebc1complex,and backto the photoreactioncenter. An alternativepath, in green sulfur bacteria,sends electronsfrom reducedquinonesto NAD+ Cyanobacteriaand plants have two different photoreactioncenters,arrangedin tandem. Plant photosystemI passeselectronsfrom its excitedreactioncenter,P700,througha seriesof carriersto ferredoxin,which then reducesNADP+ to NADPH.

r

In this state, the Mn complex can take four electrons from a pair of water molecules, releasing 4 H+ and 02:

r

lMn complexln* + 2H"O ----[Mn complex]o+ 4H+ + O, (19-15)

r

Because the four protons produced in this reaction are released into the thylakoid lumen, the oxygen-evolving complex acts as a proton pump, driven by electron transfer. The sum of Equations 19-12 through 19-15 is 2}J2O+ 2PQB+ 4photons

r

> 02 + 2PQBH2 (19-16)

The oxygen-evolving complex is associated with a peripheral membrane protein (M,33,000) on the lumenal side of the thylakoid membrane that stabilizes the cluster of four Mn ions (in various oxidation states), one Caz* ion, five O atoms, and a Cl ion, with precise geometry. The chemical changes that take place in this

r

The reactioncenterofplant photosystemII, P680, passeselectronsto plastoquinone, and the electronslost from P680are replacedby electrons from H2O(electrondonorsother than H2Oare used in other organisms). Flow of electronsthroughthe photosystems producesNADPH and ATP,in the ratio of about 2:3.A secondtype of electronflow (cyclicflow) producesATP only and allowsvariability in the proportions of NADPH and ATP formed.

Tsgl 19.9ATP Synthesis byPhotophosphorylation

The localization of PSI and PSII between the granal and stromal lamellae can change and is indirectly controlled by Iight intensity, optimizing the distribution of excitons between PSI and PSII for efflcient energy capture.

structurescalledchromatophores, derivedfrom photosyntheticbacteria. Investrgatorsconcluded that someof the light energycapturedby the photosyntheticsystemsof these organismsis transformedinto the phosphatebond energzofAIP. This processrs calledphotophosphorylation, to distinguishit from ondative phosphorylationin respiring mitochondria.

The light-driven splitting of H2O is catalyzed by a Mn-containing protein complex; 02 is produced. The reduced plastoquinone carries electrons to the cytochrome b6lcomplex; from here they pass to plastocyanin, and then to P700 to replace those lost during its photoexcitation Electron flow through the cytochrome b6;f complex drives protons across the plasma membrane, creating a proton-motive force that provides the energy for ATP synthesis by an ATP synthase.

DanielArnon, 1910-1994

(ouples Electron Flow and AProton Gradient Phosphorylation Severalpropertiesof photosyntheticelectron transfer in chloroplastsindicatethat and photophosphorylation a proton gradientplaysthe samerole as in mitochondrial oxidativephosphorylation.(1) The reactioncenters, electroncarriets,and MP-forming enz1rnesare located in a proton-impermeablemembrane-the thylakoid membrane-which must be intact to support photophosphorylation.(2) Photophosphorylationcan be uncoupledfrom electronflow by reagentsthat promote the passageof protons through the thylakoid memcanbe blockedby venbrane.(3) Photophosphorylation turicidin and similar agentsthat inhibit the formation of ATP from ADP and P1by the mitochondrial ATP synthase (Table 19-4). (4) ATP synthesisis catalyzedby FoFl complexes,locatedon the outer surfaceof the thyIakoidmembranes,that are very similar in structure and functionto the F.F1 complexesof mitochondria. moleculesrn the chainof carriers Electron-transferring in the connectingPSII and PSIare oriented as}.'rnmetrically thylakoidmembrane,sophotoinducedelectronflow results inthe net movementof protonsacrossthe membrane,from the stromalside to the thylakoidlumen (Fig. f9-63). In 1966An&6 Jagendorfshowedthat a pH gradientacrossthe thylakoid membrane (alkaline outside) cottld fumtsh the ddving force to generateAIP. His ear$ observationsprovidedsomeof the mostimportantexperimentalevidencein supportof Mitchell'schemiosmotichypothesis.

19.9ATP Synthesis byPhotophosphorylation The combrnedactivities of the two plant photosystems moveelectronsfrom water to NADP+,conservingsomeof the energyof absorbedlight as NADPH (Frg. 19-56). Simultaneously,protons are pumped acrossthe thylakoid membraneand energyis conservedasan electrochemical potential.Weturn now to the processby which this proton gradient drives the synthesisof ATP,the other energvconseruingproduct of the light-dependentreactions. In 1954DanielArnon and his colleaguesdiscovered that ATP is generatedfrom ADP and P1during photosynthetic electron transfer in illuminated spinach chloroplasts.Supportfor thesefindingscamefrom the work of Albert Frenkel,who detectedlight-dependent ATP production in pigment-containingmembranous

NADP++H+ NADPH .:.\,.=..,.e, bt/'

Fd

19-63 Proton and electron circuits in thyFIGURE lakoids.Electrons(blue arrows)move from H2O chainof carrithroughPSll,throughthe intermediate I (red ers,throughPSl,and finallyto NADP . Protons arrows)arepumpedintothe thylakoidlumenby the throughthecarrierslinkingPSlland flow of electrons PSl,and reenterthe stromathroughprotonchannels CF")of ATP synthase. formed by the Fo (designated (CF1) ofATP. catalyzes synthesis TheF1subunit

D"l

O x i d a t iP ve h o s p h o r y l aatni odPn h o t o p h o s p h o r y l a t i o n

Jagendorf incubated cNoroplasts, in the dark, in a pH 4 buffer; the buffer slowly penetrated urto the irLner compartment of the thylakoids, lowerirg their internal pH. He added ADP and P1to the dark suspension of cNoroplasts and then suddenly raised the pH of the outer medium to 8, momentarily creatAnd16 Jagendorf ing a large pH gradient across the membrane. As protons moved out of the thylakoids into the medium, ATP was generated from ADP and P1.Because the formation of ATP occurred in the dark (with no input of energr from light), this experiment showed that a pH gradient across the membrane rs a high-energr state that, as in mrtochondrial oxidative phosphorylation, can mediate the transduction of energz from electron transfer into the chemical eners/ of AIP.

energy-enough energyto drive the synthesisof several molesof ATP (AG'" = 30.5kJ/mol).Experimentalmeasurementsyield valuesof about3 ATP per 02 produced. At least eight photons must be absorbedto drive four electronsfrom H2O to NADPH (one photon per electronat each reactioncenter). The energyin eight photons of visible light is more than enoughfor the sy'nthesisof three moleculesof ATP. ATP synthesisis not the only energy-conservingreaction of photosynthesisin plants;the NADPHformed in the final electron transfer is also energeticallyrich. The overall equationfor noncyclic photophosphorylation (a term explainedbelow) is 2Il2O+ Sphotons+ 2NADP++ -3ADP -l- -3P, -----+ 02 + -3ATP + 2NADPH (19-17)

TheATP Synthase of[hloroplasts lt l-ike ThatofMitochondria

TheApproximate Stoichi0metry 0f Photophosph0rylati0fi The enzyme responsible for ATP synthesis in chloroplasts is a Iarge complex with two functional compoHasBeen Established As electrons move from water to NADP* in plant cNoroplasts, about 12 H* move from the stroma to the thylakoid Iumen per four electrons passed (that is, per 02 formed). Four of these protons are moved by the oxygenevolving complex, and up to eight by the cy'tochrome b6l complex. The measurable result is a 1,000-fold difference in proton concentration across the thylakoid membrane (ApH : 3). Recall that the energy stored in a proton gradient (the electrochemical potential) has two components: a proton concentration difference (ApH) and an electrical potential (Arl) due to charge separation In chloroplasts, ApH is the dominant component; counterion movement apparently dissipates most of the electrical potential. In illuminated chloroplasts, the energy stored in the proton gradient per mole of protons is LG :2.1RTLpH + ZJArlt: -17 kJ/mol so the movement of 12 mol of protons across the thylakoid membrane represents conservation of about 200 kJ of Mitochondrion

nents, CFo and CF 1 (C denoting its location in chloroplasts). CFo is a transmembrane proton pore composed of several integral membrane proteins and is homologous to mitochondrial Fo. CF 1 is a peripheral membrane protein complex very similar in subunit composition, structure, and function to mitochondrial F 1. Electron microscopy of sectioned chloroplasts shows ATP synthase complexes as knoblike projections on the outside (stromal, or N) surface of thylakoid membranes; these complexes correspond to the ATP synthase complexes seen to project on the i,nside (matrix, or N) surface of the inner mitochondrial membrane. Thus the relationship between the orientation of the ATP slnthase and the direction of proton pumping is the same in chloroplasts and mitochondria. In both cases, the F1 portion of ATP slmthaseis located on the more alkaline (u) side of the membrane through which protons flow down their concentration gradient; the direction of proton flow relative to F1 is the same in both cases:p to N (Fis. 19-64). Bacterium (E.coli)

Chloroplast

tgr

Matrix

(N side)

Cytosol (x side)

Thylakoid lumen (p side)

FIGURE 1 9 - 6 4 C o m p a r i s o no f t h e t o p o l o g y o f p r o t o n m o v e m e n t a n d A T P s y n t h a s eo r i e n t a t i o ni n t h e m e m b r a n e so f m i t o c h o n d r i a ,c h l o r o p l a s t s a , nd the bacterium E coli ln each case, orientation of the proton gradient relative to ATP synthase activity is the same

qr" Stroma (N side)

Photosynthesis 19.10 TheEvolution ofOxygenic [ttl and imported (Chapter27). Whenplant cellsgrow and divide,chloroplastsgive rise to new chloroplastsby division, during which their DNA is replicated and divided between daughter chloroplasts.The machinery and mechanismfor light capture, electron flow, and ATP synthesisin modern cyanobacteriaare similar in many respects to those in plant chloroplasts.These observationsled to the now widely acceptedhypothesis that the evolutionaryprogenitorsof modern plant cells were primitive eukaryotesthat engulfedphotoSUMMAR 1Y 9 . 9 A T PS y n t h e sbi sy synthetic cyanobacteriaand established stable enP h o t o p h o s p h o r y l a t i o ndosymbioticrelationshipswith them (see Fig. 1-36). At least half of the photosyntheticactivity on Earth r In plants,both the water-splittingreactionand now occurs in microorganisms-algae, other photosynelectron flow through the cytochromeb61fcomplex thetic eukaryotes, and photosynthetic bacteria. are accompaniedby proton pumpingacrossthe Cyanobacteriahave PSII and PSI in tandem, and the thylakoid membrane.The proton-motiveforce thus PSII has an associatedwater-splittingactivity resemcreateddrivesATP synthesisby a CF"CFi complex bling that of plants. However,the other groups of photosimilar to the mitochondrialFoFl complex. synthetic bacteria have single reaction centers and do r The ATP synthaseof chloroplasts(CFoCFl)is very not split H2O or produce 02. Many are obligate anaersimilar in both structure and catalytic mechanism obesand cannottolerate02; they must use somecomto the ATP s;mthasesof mitochondriaand bacteria. pound other than H2O as electron donor. Some Physicalrotation driven by the proton gradient is photosyntheticbacteria use inorganic compoundsas accompaniedby ATP synthesisat sites that cycle electron (and hydrogen) donors. For example,green through three conformations,one with high affinity sulfur bacteriause hydrogen sulflde: for ATP,one with high affinity for ADP * P,, and j's5 (CH2O)+ H2O+ 25 one with low affinity for both nucleotides. 2H2S+ CO' The mechanismof chloroplastATP synthaseis also believed to be essentiallyidentical to that of its mitochondrial analog;ADP and P, readily condenseto form ATP on the enzyme surface,and the release of this enzyme-boundATP requires a proton-motive force. Rotational catalysissequentiallyengageseach of the three B subunits of the ATP slmthasein ATP synthesis, ATP release,and ADP + P,binding(Figs 19-26,19-27).

19.10 TheEvolution of0xygenic Photosynthesis The appearance of oxygenic photosynthesis on Earth about 2.5 billion years ago was a crucial event in the evolution of the biosphere. Until then, the earth had been essentially devoid of molecular oxygen and lacked the ozone layer that protects living organisms from solar IIV radiation. Oxygenic photosynthesis made available a nearly limitless supply of reducrng agent to drive the production of organic compounds by reductive biosyerthetic reactions. And mechanisms evolved that allowed organisms to use 02 as a terminal electron acceptor in highly energetic electron transfers from organic substrates, employing the energy of oxidation to support their metabolism. The complex photosynthetic apparatus of a modern vascular plant is the culmination of a series of evolutionary events, the most recent of which was the acquisition by eukaryotic cells of a cyanobacterial endosS.'rnbiont.

(hloroplasts Evolved fromAncient Photosynthetic Bacteria Chloroplastsin modern organismsresemble mitochondria in severalproperties,and are believedto have originatedby the samemechanismthat gaverise to mitochondria:endosymbiosisLike mitochondria, chloroplastscontain their own DNA and protein-synthesizing machinery.Some of the polypeptidesof chloroplastproteins are encodedby chloroplastgenes and synthesizedin the chloroplast;othersare encoded by nucleargenes,synthesizedoutsidethe chloroplast,

Thesebacteria,insteadof producingmolecular02, form elementalsulfur as the oxida,tionproduct of H2S.(They further oxidize the S to SOi- ) Other photosynthetic bacteriause organiccompoundssuchas lactateas electron donors: 2Lactate+ CO, -l's5 (CH2O)+ H2O+ 2 pyruvate The fundamental similarity of photosymthesisin plants and bacteria, despite the differencesin the electron donors they employ,becomesmore obvious when the equation of photosynthesisis written in the more general form + co, -tisht>(cH2o) + H2o + 2D 2]F'2D in which H2Dis an electron (and hydrogen) donor and D is its oxidizedform. H2Dmay be water,hydrogensulfide, lactate,or someother organiccompound,dependingon the species.Most likely, the bacteriathat first developed photosymthetic abilityusedH2Sastheir electronsource. The ancient relatives of modern cyanobacteria probably arose by the combination of genetic material from two types of photoslrrthetic bacteria,with systems of the tlpe seenin modern purple bacteria (with a PSIIlike electron path) and green sulfur bacteria (with an electron path resemblingthat in PSI). The bacterium with two independentphotosystemsmay haveused one in one set of conditions, the other in different conditions. Over time, a mechanismto connectthe two photosystems for simultaneous use evolved, and the PSII-like system acquired the water-splitting capacity found in modern cyanobacteria.

D'1

0 x i d a t i vPeh o s p h o r y l aatni odP nh o t o p h o s p h o r y l a t i o n

Modern cyanobacteria can slmthesize ATP by oxidative phosphorylation or by photophosphorylation, although they have neither mitochondria nor chloroplasts. The enzymatic machinery for both processes is in a highly convoluted plasma membrane (see Fig 1-6). Three protein components function in both processes, giving evidence that the processes have a corunon evolutionary origin (Fig. 19-65). First, the proton-pumping cytochrome b6l complex carries electrons from plastoquinone to cytochrome c6 in photosynthesis, and also carries electrons from ubiqutnone to cy'tochrome c6 in oxidative phosphorylation-the role played by cytochrome bc1 in mitochondria. Second, cytochrome c6, homologousto mitochondrial cytochrome c, carries electrons from Complex III to Complex IV in cyanobacteria; it can also carry electrons from the cytochrome b61fcom-

HzO Light

iO,

NADH+H+

NAD*

e

\ PSII one

Cyt baf Complex il Lig ht

\

'i{

PSI

\

Cyta + a, Complex

\*

Hzo

Photophosphorylation (a)

io"

Oxidative phosphorylation (b)

FIGURE 19-65 Dual roles of cytochromeb"f and cytochromecu in cyanobacteriareflect evolutionaryorigins. Cyanobacteriause cytochromeb6{, cytochrom€c6, and plastoquinonefor both oxidative phosphorylation (a) In photophosphorylaand photophosphorylation. tion, electronsflow (topto bottom)from waterto NADP+.(b) In oxidative phosphorylation, electronsflow from NADH to 02. Both processes are accompanied by proton movementacrossthe membrane,accomplished by a Q cycle.

plex to PSI-a role performed in plants by plastocyanin. We therefore see the functional homology between the cyanobacterial cytochrome b6l complex and the mitochondrial cytochrome bct complex, and between cyanobacterial cy'tochron€ c6 and plant plastocyanin. The third conserved component is the ATP synthase, which functions in oxidative phosphorylation and photophosphorylation in cyanobacteria, and in the mitochondria and chloroplasts of photosynthetic eukaryotes. The structure and remarkable mechanism of this enz).rne have been strongly conserved throughout evolution.

lnHalobscte, a Single Protein Absorbs Lightand Pumps Protons toDrive ATP Synthesis In some modern archaea,a quite different mechanism for convertingthe energyof light into an electrochemical gradient has evolved.The halophilic ("salt-loving") archaeanHalobacteri,um sali,narunz is descendedfrom ancient evolutionary progenitors.This archaean (commonly referred to as a halobacterium)Iives only in brine ponds and salt lakes (Great Salt Lake and the Dead Sea, for example),where the high salt concentration-which can exceed4 u-results from waterlossby evaporation; indeed, halobacteriacannot live in NaCl concentrations lower than 3 u. Theseorganismsare aerobesand normally use 02 to oxidize organicfuel molecules.However, the solubility of 02 is so low in brine ponds that sometimes oxidative metabolism must be supplementedby sunlight as an alternativesourceof energy. The plasma membrane of H. sal'inan-um contains patches of the light-absorbing pigment bacteriorhodopsin, which containsretinal (the aldehydederivative of vitamin A; see FiC. 10-21) as a light-harvesting prosthetic group. When the cells are illuminated,alltrans-retinal bound to the bacteriorhodopsinabsorbs a photon and undergoesphotoisomerization to 13-czsretinal, forcing a conformationalchangein the protein. The restorationof all-trans+etinalis accompaniedby the outward movement of protons through the plasma membrane.Bacteriorhodopsin,with olly 247 aminoacid residues, is the simplest light-driven proton pump known. The difference in the three-dimensionalstructure of bacteriorhodopsinin the dark and after illumination (F'ig. l9-66a) suggestsa pathway by which a concertedseriesof proton "hops"couldeffectivelymove a proton acrossthe membrane.The chromophoreretinal is bound through a Schiff-baselinkage to the e-amino group of a Lys residue.In the dark, the nitrogen of this Schiffbaseis protonated;in the light, photoisomerization of retinal lowers the pK" of this group and it releasesits protonto a nearbyAsp residue,triggeringa seriesofproton hops that ultimately result in the releaseof a proton at the outer surfaceof the membrane(Fig. 19-66b). The electrochemicalpotential acrossthe membrane drives protons back into the cell through a membrane ATP synthasecomplexvery similar to that of mitochon-

Photosynthesis 19.10 TheEvolution of0xygenic [tot]

Cytosol

Extemal medium (a)

Dark

FIGURE 19-65 Evotutionof a secondmechanismfor light-drivenproton (M.26,000)of pumpingin a halophilicarchaean.(a) Bacteriorhodopsin Halobacteriumhalobiumhassevenmembrane-spanning a helices(PDB (purple)is covalentlyar lD lCBR).The chromophoreall-trans-retinal tachedvia a Schiffbaseto the a-aminogroupof a Lysresiduedeepin the membraneinterior.Runningthroughthe proteinarea seriesof Asp and Clu residues and a seriesof closelyassociated watermoleculesthattogetherprovidethe transmembrane pathfor protons(redarrows).Steps@ through@ indicateproton movements,describedbelow. (b) In the dark (leftpanel),the Schiffbaseis protonated.lllumination (rightpanel)photoisomerizes the retinal,forcingsubtleconforma-

Light

the Schiff tional changesin the proteinthat alterthe distancebetvveen baseand its neighboringamino acid residues.Interactionwith these neighborslowers the pK" of the protonatedSchiff base,and the base givesup its protonto a nearbycarboxylgroupon Aspsslstep@ in {a;;. Thisinitiatesa seriesof concertedproton hopsbetweenwatermolecules (seeFig.2-13) in the interiorof the protein,which endswith @ the releaseof a proton that was sharedby Clulea and Clu2oanear the extracellularsurface.@ fhe Schiff base reacquiresa proton from Aspe6,which @ takesup a protonfrom the cytosol.@ Finally,Aspss pair. of the Glu2oa-Clursa givesup its proton,leadingto reprotonation Thesystemis now readyfor anotherroundof protonPUmpinS.

t-' I J6L)

0xidative Phosphorylation andPhotophosphorylation

dria and chloroplasts. Thus,when 02 is limited,halobacteria can use light to supplementthe ATP synthesized by oxidativephosphorylation.Halobacteriado not evolve 02, Ilor do they carry out photoreductionof NADP+; their phototransducingmachinery is therefore much simpler than that of cyanobacteriaor plants. Never-theless, its proton-pumpingmechanismmay prove to be prototypical for the many other, more complex, ion pumps. I Bacteriorhodopsin

malate-aspartate shuttle 73I glycerol 3-phosphate shuttle 732 acceptor control 733 mass-action ratio 733 brown adipose tissue (BAT) 736 thermogenin 736 cytochrome P-450 736 xenobiotics 736 S U M M A R1Y9 . 1 0 T h eE v o l u t i o no f apoptosis 737 0 x y g e n iPch o t o s y n t h e s i sapoptosome 738 caspase 738 r Modern cyanobacteriaare derived from an ancient heteroplasmy 740 photosystems, organismthat acquiredtwo one of homoplasmy 740 purple the type now found in bacteria,the other light-dependent of the type found in green sulfur bacteria. reactions 742 r Many photosyntheticmicroorganismsobtarn light reactions 742 electronsfor photosynthesis not from water but carbon-assimilation from donorssuchas H2S. reactions 742 r Cyanobacteriawith the tandem photosystemsand a carbon-fixation water-splittingactivity that releasedoxygeninto reaction 742 the atmosphereappearedon Earth about2.5 billion thylakoid 743 yearsago. grane 743 stroma 743 r Chloroplasts,like mitochondria,evolvedfrom Ilill reaction 743 bacterialiving endosy'rnbiotically in early photon 744 eukaryoticcells.The ATP synthasesof bacteria, quantum 744 cyanobacteria,mitochondria,and chloroplasts excited state 744 sharea commonevolutionaryprecursor and a ground state 744 colnmon enz;rmaticmechanism. fluorescence 745

r

An entirely different mechanismfor converting Iight energyto a proton gradient has evolvedin the modern archaea,in which the light-harvesting pigment is retinal.

exciton 745 exciton transfer 745 chlorophylls 745 Iight-harvestingcomplexes (LHCs) 746 accessory pigments 747 carotenoids 747 actionspectrum 747 photosystem 747 photochemical reaction center 747 light-harvesting(antenna) molecules 747 photosystemII (PSil) 752 photosystemI (PSI) 753 plastocyanin 753 oxygenicphotoslnthesis / D.J

plastoquinone(PQf 753 ferredoxin 753 cyclic electron flow 756 noncyclic electron flow 756 cyclic photophosphorylation 756 oxygen-evolving complex 758 water-splittingcomplex 758 photophosphorylation 759

Further Reading History and General Background

KeyTerms

Arnon, D.I. (1984) The discoveryof photosyrrtheticphosphorylation Ttends Bt ochem Sci, 9, 258-262.

Tertns i,n bo\d are defined i,n the glossary chemiosmotic theory 707 nicotinamide nucleotide-linked dehydrogenases 709 flavoproteins 709 reducingequivalent 710 ubiquinone (coenzyrneQ, Q) 7I0 cytochromes 710 iron-sulfurproteins 71,I Rieske iron-sulfirr proteins 7II ComplexI 7I2 vectorial 713 ComplexII 715 succinate dehydrogenase 715

reactive oxygen species (ROS) 715 superoxide radical('O) 715 ComplexIII 715 cy'tochrome bc1 complex 7I5 Q cycle 716 ComplexIV 716 c5,'tochrome oxidase 716 respirasomes 718 proton-motive force 720 ATP synthase 723 FlATPase 725 rotationalcatalysis 728 P/O ratio 729 P/Ze-ralio 729

Beinert, H. (1995) Theseare the momentswhen we live! From Thunbergtubes and manometryto phone,fax and FedEx. In Selected Topi,csi,n the Hzstory oJBi,ochem'i,strg: Personal Recollectzons,ComprehensiveBiochemistry Vol 38, ElsevierScrence PublishingCo , Inc , New York An engagingpersonalaccountof the exciting period when the biochemistryof respiratoryelectrontransfer wasworked out Blankenship, R.E. (2002)Molecular Mechani,smsoJPhotosynthesis, BlackwellScienceInc., London An intermediate-leveldiscussionof all aspectsof photosynthesis. Govin{iee, Beatfi J.T., Gest, H., & Allen, J.F. (eds). (2006) Discoueries in Photosynthesris,Advancesin Photosyrrthesis and Respiration,Vol 20, SpringerVerlag,Dordrecht,The Netherlands. A wonderful descriptionof the historicalbackgroundto discoveries in photosynthesis,told by the peoplewho madethat history. Harold, F.M. (1986) The Vr,talForce: A Studg in Bi,oenergeti,cs, W. H. Freemanand Company,New York A very readablesynthesisof the principles of bioenergeticsand their applicationto energytransductions. Heldt, H.-W. (1997)Plont Bi,ochemi,strAand Mo\ecuLarBioLogg, Oxford UniversltyPress,Oxford

Further Reading I zo{ A textbook of plant biochemistrv wrth exceilent discussions of photophosphorylation Keilin, D. (1966) The H.istory oJ CeLl Respr,ration and, Cgtochrome, Cambridge University Press, London. An authoritative and absorbing account of the discovery of c5,tochromes and their roles in respiration, written by the man who discovered cytochromes Kresge, N., Simoni, R.D., & Hill, R.L. (2004) Britton Chance: Ol5'rnpian and developer of stop-flow methods I Bi,oL Chem 279, e10, www.jbc org A JBC Classic (on the website under "Classic Articles,') describing the technology used to determine the sequence of electron carriers Kresge, N., Simoni, R.D., & Hill, R.L. (2006) Forty years of superoxide dismulase research: the work of hwin Fridovich I Briol Chem 281, e17, www.jbc.org A JBC Classic Article

Heinemeyer, J., Braun, H.-P., Boekema, E.J., & Kouril, R. (2007) A structural model ofthe cytochromec reductase/oxidase supercomplexfrom yeastmitochondria.J. Bi,ol Chem.282, 12.240-12.248 Primary researchsupportingthe existenceof supercomplexesin mitochondria. Hosler, J.P., Ferguson-Miller, S., & Mills, D.A. (2006) Energy transduction:proton transfer through the respiratorycomplexes. Annu Reu Bi,ochem.75, 165-187 Advanceddescriptionof electrontransfer Lenaz, G., Fato, R., Genova, M.L., Bergamini, C., Bianchi, C., & Biondi, A. (2006) MitochondrialComplexI: structura.land functional aspects.B'inchi,m Bi.ophys Acta 1757, 1406-1420. Intermediatelevel reviervof ComDlexI stmcture and function.

Lane, N. (2005) Powe4 Ser, Sui,cide: Mi,tochond,ri,a and the Meaning oJ LiJe, Oxford University Press, Oxford An entryJevel description of the roles of mitochondria in energy conservation and in apoptosis

Lenaz, G. & Genova, M.L. (2007) Kinetics of integratedelectron transfer in the mitochondrialrespiratorychain:random collisionsvs solid state electron channeling.,4Tiz J. Phgs'iol CelLPhysi,ol 292, r22t-t239 Test of the hlpothesis that supercomplexesexist in mitochondria

Mitchell, P. (1979) Keiiin's respiratory chain concept and its chemiosmotic consequences.Sci,ence 206, 1148-1 1bg Mitchell's Nobel lecture, outlining the evolution of the chemiosmotic hypothesis.

Michel, H., Behr, J., Harrenga, A., & Kannt, A. (1998) Cy'tochromec oxidase:structure and spectroscopy.Anrru Reo B'iophgs Bzomol Stnrct 27,329-356 Advancedreview of ComplexIV structure and function.

Nicholls, D.G. & Ferguson, S.J. (2002) B,i,oenergettcs S, Academic Press, Amsterdam. Up-to-date, comprehensive, well-illustrated treatment of all aspects of mitochondrial and chloroplast energy transductions.

Osyczka, A., Moser, C.C., & Dutton, P.L. (2005) Fixing the Q cycle.Tlends Bi,ochem ,Sca30, 176-182. Intermediatelevel review of the Q cycle

Scheffi er, I.E. ( I 999) M i,tochond,ria, Wiley-Liss, New York An excellent survey of mitochondrial structure and function.

Rottenberg, H. (1998) The generationofproton electrochemical potential gradientby cytochromec oxidase.Biochi,m Bi,ophys Acta 1364,1-16.

Slater, E.C. (1987) The mechanism of the conservatron of energr of biological oxidations Eur J. Bi,ochem 166,489-504 A clear and critical account of the evoiution of the chemiosmotic model.

Sazanov, L.A. (2007) RespiratorycomplexI: mechanistic and stmcturai insightsprovidedby the crystal structure of the hydrophilic domain.Bi,ochemistrg 46, 2275-2286. Primary researchpaper on structure of ComplexI.

OXIDATIVE PHOSPHORYLATION

Smith, J.L. (1998) Secretlife of cytochromebc1 Sci,ence28t, 58-59

Electron-Thansfer

Reactions in Mitochondria

Adam-Vizi, V. & Chinopoulos, C. (2006) Bioenergeticsand the formation of mitochondrialreactiveoxygenspecies. Tlends Pharmacol 27,639-645 Babcock, G.T. & Wikstriim, M. (1992) Oxygenactivationand the conseryationof energyin cell respiration. Nature 356, 30i-309. Advanceddiscussronof the reduction of \4/aterand pumping of protons by cytochromeoxidase Boekema, E.J. & Braun, H.-P. (2007) Supramolecularstructure of the mitochondrialoxidativephosphorylationsystem..l Bzol Chem 282,\-4 Brandt, U. (1997) Proton-translocationby membrane-bound NADH:ubiquinone-oxidoreduclase (complexI) through redox-gated ligand conductton Bi,ochi,mBiophgs Acta 1318, 79-91. Advanceddiscussionof modelsfor electronmovementthrouqh ComplexI Brandt, U. (2006) Energy convertingNADH:quinoneoxidoreductase(complexI) Annu Reu Biochem 76,69-92. Advanceddiscussionof the structure of ComplexI and the rmplicationsfor function. Brandt, U. & Thumpowea B. (1994) The protonmotiveQ cycle inmitochondriaandbacteria Crit Reu Bi,ochem.Mot Bi,oL 29, 165-197 Advanceddescriptionof the possiblemechanismsof the Q cycle Crofts, A.R. & Berry, E.A. (1998) Structure and function of the cltochrome bcr complexof mitochondriaand photosyrrthetic bacteria Curr. Opi,n Struct BioI 8, 501-509

Sun, F., Huo, X., Zhai,Y.r lVang, A,, Xu, J., Su, D., Ba.rtlam, M., & Rao, Z. (2005) Crystalstructure of mitochondriairespiratory protein complexll. CeLLl2l, 1043-1057 Tlelens, A.G.M., Rotte, C., van Hellemond, J.J., & Martin, W. (2002) Mitochondriaas we don't know them Ttends Bi,ochem.Sci, 27,564-572. Intermediatelevel discussionof the many organismsin which mitochondriado not dependon oxygenas the final electron donor Tbukihara, T., Aoyama, H., Yamashita, E., Tomizaki, T,, Yamaguchi, H., Shinzawa-Itoh, K., Nakashima, R., Yaono, R., & Yoshikawa, S. (1996) The whole structure ofthe l3-subunit oxidized cy'tochromec oxidaseat 2.8 A. Science 272, 1136-1144. The solutionby x-ray crystallographyof the structure of this hugemembraneprotein Wikstriim, M. & Verkhovsky, M.I. (2007) Mechanismand energetrcs of proton translocationby the respiratoryheme-copperoxidases. Biochim Bi,ophys Acta 1767, 1200-1214. Xia, D,, Yu, C.-A., Kim, H., Xia, J.-2., Kachurin, A.M., Zhang, L., Yu, L., & Deisenhofer, J. (1997) Crystalstructure of the cy4ochromebc1complexfrom bovineheart mitochondia. Sci,ence277, 60-66. Report revealingthe crystallographicstructure of ComplexIIL Yankovskaya, V., Horsefield, R., Tiirnroth, S., Luna-Chavez, C., Myoshi, H., L6ger, C., B5irne, B., Cecchini, G., & Iwata, S. (2003) Architecture of succinatedehydrogenaseand reactiveoxygen speciesgeneration.Science 299, 700-704. Advancedreview of this classof electron-transferprocesses.

J66)

O x i d a t i vPeh o s p h o r y l a tai onndP h o t o p h o s p h o r y l a t i o n

Zhang, M., Mileykovskaya, E., & Dowhan, W, (2005) Cardiolipin is essentialfor organizationof complexesIII and IV into a supercomplex in intact yeastmitochondria.J. Bi,ol Chem 28O,29,403-29,408. Primaryresearchpaper ATP Sgtthesis Abraframs, J.P., Leslie, A.G.IV., Lutter, R., & Walker, J.E. (1994) The structure of F1-ATPasefrom bovineheart mitochondria determinedat 2 8 A resolution Nature 870, 621-628. Bianchet, M.A., Hullihen, J., Pedersen,P.L.,& Amzel, L.M. (1998)The 2 80 A structureof rat liver F1-ATPase: of a configuration critical intermediatein ATP synthesis-hydrolysis. Proc Natl Acad Sci US,A95. 11.065-11,070. Researchpaper that providedimportant structurai detail in support of lhe cataly ic mechanism Boyer, P.D, (1997) The ATP synthase-a splendidmolecularmachine Annu Reu Biochem 66,717-749. An accountof the historical developmentand current state of the binding-changemodel,written by its principal architect

An advanceddescriptionof respiratorycontrol Harris, D.A. & Das, A.M. (1991) Control of mrtochondrialATP synthesisin the heart Bzochem J. 28O,561-573 Advancedcliscussionof the regulationof ATP synthaseby Ca'* and other facbors. Klingenberg, M. & Huang, S,-G. (1999) Structure and function of the uncouplingprotein from brown adiposetisste B'i'ochi'm Bi.ophgs Acta 1416, 271-296. Nury, H,, Dahout-Gonzafez, C.rT)ezeguet, V., Lauquin, G.J.M., Brandolin, G., & Pebay-Pe5rroula,E. (2006) Relatronsbetween structure and function of the mitochondrialADP/ATPcarier Annu Reu Biochem 75, 713-741. Advancedreview Semenza, G,L, (2007) Oxygen-dependentregulationof mitochondrial respirationby hypoxia-induciblefactor L B'lochem J. 4O5,l-9. Intermediate-levelreview Simon, M,C. (2006) Comingup for arr: HIF-1 and mitochondrial oxygenconsumplion.Cell Metab 3, 150-151. Short, intermediatelevel review of the hypoxia-induciblefactor

Cabez6n, E., Montgomery, M.G., Leslie, A.G.W., & Walker, J,E. (2003) The structure of bovineF1-ATPasein complexwith its regulatoryproteinlFr Natr Stntct Bi,ol 10,744-750

Mitochondria

Hinkle, P,C.,Kumar, M.A., Resetar,A,, & Harris, D.L. (1991) Mechanisticstoichiometryof mitochondrialoxidativephosphorylation Bt ochemzstrUBO,3576-3582 A carefulanalysisof experimentalresults and theoreticalconsiderationson the questionof nonintegralP/O ratios.

Kroemer, G., Galluzzi, L,, & Brenner, C. (2007) Mitochondrial membranepermeabilizationin cell death Physi,ol Reu 87,99-163. Advanced,comprehensivereview of role of cytochromec in apoptosls

Khan, S. (1997) Rotary chemiosmoticmachines.Bzochi,m Bi,ophys Acta 1322,86-705 Detailedreview of the structuresthat underlie proton-dnvenrotary motion of ATP s;'nthaseand bacterialflagella Kresge, N., Simoni, R.D., & Hill, R.L. (2006)ATP synthesisand the binding changemechanism:the work of Paul D. Boyer J. Bi,oI Chem 281, e18,www.jbc.org A JBC ClassicArticle. Kresge, N., Simoni, R.D., & Hill, R.L. (2006) Unravelingthe enzymologyof oxidativephosphorylation:the work of Efraim Racker J B'i,oI Chem 281, e4,wwwjbc org A JBC ClassicArticle Nishizaka, T., Oiwa, K., Noji, H., Kimura, S., Muneyuki, E., Yoshida, M., & Kinosita, K., Jr. (2004) Chemomechanical coupling in F1-ATPaserevealedby simultaneousobservationof nucleotide kineticsandrotation Nat Sttttct Mol Bi,oLll.l42-148 Beautifttl demonstrationof the rotary motion of ATP synthase Sambongi, Y., Iko, Y., Tanabe, M., Omote, H., Iwamoto-Kihara, A., Ueda, I., Yanagida, T,, Wada, Y., & Futai, M. (1999) Mechanical rotation of the c suburutoligomerin ATP synthase(FnF1):direct observationSci,ence286, 1722-1724 The experimentalevidencefor rotation of the entire cylinder of c subunitsin FoFt Stock, D., Leslie, A.G.W.,& Walker, J.E. (1999)Molecular architectureof the rotary motor in ATP synthase.Science 286, 1700-1705 The first crystallographicview of the Fo subunit, in the yeast FoFr.SeealsoR H Fillingame'seditorial conunentin the sameissue of Sctence Weber, J. & Senior, A.E. (1997) Catal5,tic mechanism of F1ATPase Biochim Biophys Acta 1319, 1.9,58 An advanced review ofkinetic. structural. and biochemical evidence for the ATP slnthase mechanism. Regulation

of Oxidative

Phosphorylation

Brand, M.D. & Murphy, M.P. (1987) Control of electron flux through the respiratory chain in mitochondria and cells Bi.oL Reu Camb Philos Soc 62,141 193

in Thermogenesis, Steroid Synthesis,

and Apoptosis

McCord, J.M. (2002) Superoxidedismutasein agingand disease:an overiew. MethodsEnzgmol 349, 331-341. signaling Riedl, S.J. & Salvesen,G.S. (2007)The apoptosome: platform of cell death Nat Reu Mol CeLlBi,oI 8, 405-413 Intermediatelevel rer,.iew. Mitochondrial

Genes: Their Origin and Effects of

Mutations Abou-Sleirnan, P.M., Muqit, M.M.K., & Wood, N.W. (2006) Expandinginsightsof mitochondrialdysfunctionin Parkinson's diseaseNnl Reu Neurosci, 7,207-219 Boudina, S. & Abel, E.D. (2006) Mitochondrialuncoupling:a key contributer to reducedcardiacefficiencyin diabetes Phys'iology21, 250-258 Brandon, M., Baldi, P., & Wallace, D.C. (2006)Mitochondrial mutationsin cancer Oncogene25, 4647-4662 Chatterjee, A., Mambo, E., & Sidransky, D. (2006) Mitochondrial DNA mutationsin human cutcer Oncogene25, 4663 4674 Chen, Z.J. & Butoq R.A. (2005) The organizationand inheritance Nat Reu Genet 6,815-825. of themitochondrlalgenome intermediate-levelreview. de Duve, C. (2007) The origin of eukaryotes:a reappraisalNal Reu Genet 8,395-403. Intermediatelevel discussionof the evidencefor the endosr'rnbrotic originsof mitochondria andchJoroplasts Freeman, H., Shimomura, K., Horner, E., Cox, R.D., & Ashcroft, F.M. (2006) Nicotinamidenucleotidetranshydrogenase: a key role in insulin secretion CeIl Metab 3, 35 45. Houstek, J., Pickova, A., Vojtiskova, A., Mracek, T., Pecina, P., & Jesina, P. (2006) Mitochondrialdiseasesand geneticdefectsof ATP synthase. Bi,ochi,mBi,ophAsActa 1757,1400-1405 Neubauer, S. (2007) The failing heart-an engineout of fuel Neru EngI J Med 356,1140-1151 Intermediatelevel review of defeclsin oxidativephosphorylation and heart disease. Remedi, M.S., Nichols, C.G., & Koster, J.C. (2006)The mitochondriaand insulin release:Nnl just a passingrelationship Cell Metab 3,5-7

Further Readino fzo)__.t ' )_ Smeitink, J.A., Zeviani, M., Thrnbull, D.M., & Jacobs, H.T (2006) Mitochondriatmedicine:a metabolicperspectiveon the pathologyof oxidativephosphorylationdisorders.CeItMetab B, 9-13 Tbylor, R.W. & Thrnbull, D.M. (2005) MitochondrialDNA mutations in human disease.Nat Reu Genet 6, 389-402 Intermediatelevel review.

Ferreira, K.N., Iverson, T.M., Maghlaoul, K., Barber, J., & Iwata, S, (2004) Architecture ofthe photoslmtheticoxygen-evolving center Sci,ence303, 1831-1838 Fromrne, P., Jordan, P,, & Krauss, N. (2001) Structure ofphotosystemI. Bi,och'i,mBi,ophAsActa l5O7,5-31.

Wallace,D.C. (1999)Mitochondrialdiseasein man andmouse. Scr,ence283, 1482-1487

Hankamer, B., Barber, J., & Boekema, E.J. (1997) Structure and membraneorganizationof photosystemII in green plants.Annu Reu PlantPhysi,ol PlantMoL Bi,oL 48,541-571. Advancedreview

Wiederkehr, A. & Wollheim, C.B. (2006) Minireview:implication of mitochondriain insulin secretionand action.Endocrinologg 147, 2643-2649 Interrnediateleveldiscussionof mitochondrialfunction in 6 cells

Heathcote, P., Fyfe, P,K., & Jones, M.B. (2002) Reactioncentres: the structure and evolutionof biologicalsolarpowen Tlends Bi.ochemSci..27,79-87. Intermediatelevel review of photosystemsI and II.

PHOTOSYNTHESIS Light Absorption Cogdell, R.J., Isaacs, N.![r., Howard, T.D., Mcluskey, K., Fraser, N.J., & Prince, S.M. (1999) How photoslmtheticbacteria harvestsolarenergy..l Bacteriol 181,3869-3829. A short, intermediatelevel review of the structure and function of the light-harvestingcomplexof the purple bacteriaand exciton flow to the reaction center Green, B.R., Pichersky, E., & Kloppstech, K. (1991)Chlorophyll aZ-binding proteins:an extendedfamtly.Ttends Biochem Sci 16, 181-186. An intermedialeJeveldescriptionof the proteins that orient chlorophyllmoleculesin chloroplasts. Kargul, J., Nield, J., & Barber, J. (2003) Three-dimensional reconstructionof a light-harvestingComplexl-PhotosystemI (LHCI-PSI)supercomplexfrom the green algaChlamyd,omonas reinhardtr,i,J. Bi,oI Chem 278, 16,135-16,141 Zuber, H. (1986) Structure of light-harvestingantennacomplexes of photosyntheticbacteria,cyanobacteriaand red algae.Tiends Bzochem Sci, lL, 414-419 Light-Driven

Electron Flow

Amunts, A., Drory, O., & Nelson, N. (2007) The struclure of a plant photosystemI supercomplex at 3.4A resolutionNature 447, 58-63 Determinationof PSI structure by crystallography. Barber, J. (2002) PhotosystemII: a multlsubunit membrane protein that oxidizeswater Cum Opin Struct Bi,oL 12,523-b30 A short, intermediate-levelsummaryof the structure of PSII Barber, J. & Anderson, J,M. (eds). (2002)Photosystem II: Molecular Structure and Function Proceedingsof a Meeting,13-14 March 2002.Philos Tlans R Soc (Bi,oI Sci,) 357 (1426) A coilectionof 16 paperson photosystemIL B'iochim Biopttys ActaBioenerg (2007) 1767 (6). This iournal issuecontains10 reviewson the structure and function of photosystems Chitnis, P.R. (2001) PhotosystemI: function andphysiologyAnnu Reu PlantPhysioL PlantMoL BioI 52,593-626 An advancedand lengthyreview. Dau, H. & Haumann, M. (2007) Eight stepsprecedingO-O bond formation in oxygenicphotosynthesis-a basicreaction cycle of the photosystemII manganesecomplex.Biochim B,iophys Acta 1767, 472 483 One of severalpapersin this issuedealingwith modelsfor the water-splittingmechanism Deisenhofer, J. & Michel, H. (1991) Structuresof bacterrai photos],.nthetic reaction centersAnnu Reu CeLlB.iol 7,7-23 Descriptionof the structure of the reactioncenter of purple bacteriaand implicationsfor the function of bacterialand plant reacilon centers

Huber, R. (1990) A structural basisof light energyand electron transfer in biology.Eur J. Bi,ochem 187,283-305. Huber'sNobellecture, describingthe physicsand chemistryof phototransductions;an exceptionallyclear and weil-illustrateddiscussion,basedon crystallographicstudiesofreaction centers. Jensen, P,E., Bassi, R., Boekema, E.J., Dekker, J.P,, Jansson, S., Leister, D., Robinson, C., & Scheller, H.V. (2007) Structure, function and regulationof plant photosysteml B'ioch'im Bi,ophgs, Acta 1767,335-352. Jordan, P., Fromme, P,, Witt, H.T., Klukas, O., Saenger, T[., & Krauss, N. (2001) Three-dimensionalstructure of cyanobacterial photosystemI at 2 5 A Nclture 4ll, 909-917 Kamiya, N. & Shen, J.-R. (2003) Crystal structure of oxygenevolvingphotosystemII from Thermosgnechococcusuulcotntts at 3 7 A resolution Proc NatL Acad Sci,.LISA100, 98-103. Kargul, J., Nield, J., & Barber, J. (2003) Three-dimensional reconstructionof a light-harvestingcomplexI-photosystemI (LHCI-PSI)supercomplexfrom the green ilgaCh\amgdomonas reinhardti,'t: insightslnto light harvestingfor PSI J. Biol. Chem. 2 7 8 .1 6 . 1 3 5 - 1 6 . 1 4 1 Kok,8., Forbush, B., & McGloin, M. (1970) Cooperationof chargesin photosynthetic02 evolution: 1. A linear 4-step mechanismPhotochem Photob'tol.Ll, 45747 5. Classicexperimentshowingthe need for four photonsto split water Kramer, D.M., Avenson, T.J., & Edwards, G.E. (2007) governed Dynamicflexibility in the light reactionsof photosy'nthesis by both electron and proton transfer reactions.Tl"endsP\ant Sci,. 9,349-357 Intermediate-levelreview of regulationof state transitions. Vink, M., Ze4H., Alumot, N., Gaathon, A., Niyogi, K., Herrmann, R,G., Andersson, B,, & Ohad, I. (2004) Llghtmodulatedexposureof the light-haruestingcomplexII (LHCII) to protein kinase(s)and state transltion in Chlamydomonos reinhardti,i, xanthophyll mututts Biochem'i,strU 43, 7824-7833 Yano, J,, Kern, J., Sauer, K., Latimer, M.J., Pushkar, Y., Biesiadka, J., Loll, B., Saenger, T[., Messinger, J., Zouni, A., & Yachandra, V.K, (2006) Wherewater is oxidizedto dioxygen: stn-rctureof the photosyntheticMnaCacluster.Sci,ence314, 821-825 ATP Synthesis by Photophosphorylation Jagendorf, A.T. (1967) Acid-basetransitionsand phosphorylation by chloroplastsFed Proc 26, 1361-1369. Classicexperimentestablishingthe ability of a proton gradientto drive ATP synthesrsin the dark. The Evolution

of Oxygenic Photos).nthesis

Allen, J.F. & Martin, W (2007) Out of thin air. Nature 445, 610-612 Short, intermediatelevel discussionof how the modern Z schemeevolved

lDrrl 0 x i d a t i vPeh o s p h o r y l aatni odPn h o t o p h o s p h o r y l a t i o n Luecke, H. (2000) Atomic resolution structures ofbacterrorhodopsin photocycle intermediates: the role of discrete water molecules in the function of this ligfrt-driven ion pump B'iochi,m Bi.ophgs Acta 146O, 133-156 Advanced review of a proton pump that employs an internal chain of water molecules Luecke, H., Schobert, B., Richter, H.-T., Carteiller, J.-P., & Lanyi, J.K. (1999) Structural changes in bacteriorhodopsin during ion transport at 2 angstrom resolution. Sci,ence 286,255-264. This article, accompanled by an editoriai cornrnent in the same Sci,ence issue, describes the model for H+ translocation by proton hopping Poole, A.M. & Penny, D. (2007) Evaluating hypotheses for the origin of eukaryotes B'i,oEssays 29, 7 4-84 Intermediatelevel discussion of the endosl'rnbiont-origin theory Stiller, J.W. (2007) Plastid endosymbiosis, genome evolution and the origh ofgreen p\ants. Tlends Plont Sci 12, 391-396

Problems 1. Oxidation-Reduction Beactions The NADH dehydrogenasecomplex of the mitochondrial respiratory chain promotes the following seriesof oxidation-reductionreactions,in ,

.

,

-

q+

. -

9+

which Fe"- and Fe"- represent the iron in iron-sulfur centers, Q is ubiquinone, QH2 is ubiquinol, and E is the enz;.rne: (1)

NADH + H+ + E-FMN ------+NAD+ + E-FMNH2

(2)

E-FMNH2 + 2Fe3+ ------+E-FMN + 2Fe2+ + 2H+

(S)

2Fe2* + 2H* + Q ------+2Fe3" + qH,

Szm: NADH + H+ + Q -----+ NAD+ + QH2 For each of the three reactions catalyzed by the NADH dehydrogenase complex, identify (a) the electron donor, (b) the electron acceptor, (c) the conjugate redox pair, (d) the reducing agent, and (e) the oxidizing agent. 2. All Parts of Ubiquinone Have a Function In electron transfer, oniy the quinone portion of ubiquinone undergoes oxidation-reduction; the isoprenoid side chain remains unchanged. What is the function of this chain? 3. Use of FAD Rather Than NAD+ in Succinate Oxidation All the dehydrogenases of glycolysis and the citric acid cycle use NAD+ (E'' for NAD+/NADH is -0.32 V) as electron acceptor except succinate dehydrogenase, which uses covaiently bound FAD (E'' for FAD/MDH2 in this enz;rme is 0 050 V) Suggest why FAD is a more appropriate electron acceptor than NAD- in the dehydrogenation of succinate, based on the E'o values of fumarate/succinate (E'" = 0.031), NAD+ATADH, and the succinate dehydrogenase FAD/FADH2. 4. Degree of Reduction of Electron Carriers in the Respiratory Chain The degree of reduction of each carrier in the respiratory chain is determined by conditions in the mitochondrion

For example, when NADH and 02 are abundant, the steady-state degree of reduction of the carriers decreases as electrons pass from the substrate to 02. When electron transfer is blocked, the carriers before the block become more reduced and those beyond the block become more oxidized (see Fig. 19-6). For each of the conditions below, predict the

state of oxidation of ubiquinoneand cytochromesb, c1,c, and alas. (a) Abundant NADH and 02, but cyanideadded (b) Abundant NADH, but 02 exhausted (c) Abundant 02, but NADH exhausted (d) Abundant NADH and 02 5. Effect of Rotenone and Antimycin A on Electron Thansfer Rotenone, a toxic natural product from plants, stronglyinhibits NADH dehydrogenaseof insect and flsh mitochondria.Antimycin A, a toxic antibiotic, strongly inhibits the oxidation of ubiquinol. (a) Explain why rotenone ingestion is lethal to some insectand f,sh species. (b) Explain why antimycin A is a poison. (c) Giventhat rotenoneand antimycinA are equallyeffective in blocking their respectivesites in the electron-transfer chain,which wouid be a more potent poison?Explain. 6. Uncouplers of Oxidative Phosphorylation In normal mitochondriathe rate of electrontransfer is tightly coupledto the demandfor AIP. When the rate of use of ATP is relatively Iow, the rate of electron transfer is low; when demandfor AIP increases,electron-transferrate increases.Under these conditions of tight coupling,the number of AIP moleculesproduced per atom of oxygen consumedwhen NADH is the electron donor-the P/Oratio-is about2.5. (a) Predict the effect of a relatively low and a relatively high concentrationof uncouplingagent on the rate of electron transfer and the P/O ratio. (b) Ingestionof uncouplerscausesprofuse sweatingand an increasein body temperature.Explain this phenomenonin molecular terms. What happensto the P/O ratio in the presenceof uncouplers? (c) The uncoupler 2,4-dinitrophenolwas once prescribed as a weight-reducingdrug. How could this agent,in principle, serve as a weight-reducing aid? Uncoupling agents are no longer prescribed,becausesome deathsoccurredfollowing their use.Why might the ingestionof uncouplersleadto death? 7. Effects of Valinomycin on Oxidative Phosphorylation When the antibiotic valinomycinis addedto actively respiring mitochondria, several things happen: the yield of ATP decreases,the rate of 02 consumptionincreases,heat is reIeased,and the pH gradient across the inner mitochondrial membraneincreases.Doesvalinomycinact as an uncoupleror as an inhibitor of oxidative phosphorylation?Explain the experimental observationsin terms of the antibiotic'sability to transfer K* ions acrossthe inner mitochondrialmembrane. 8. Mode of Action of Dicyclohexylcarbodiimide (DCCD) When DCCD is added to a suspensionof tightly coupled,actively respiring mitochondria, the rate of electron transfer (measuredby 02 consumption)and the rate of AIP production dramaticallydecrease.If a solution of 2,4-dinitrophenolis now added to the preparation,02 consumptionreturns to normal but ATP production remahs inhibited. (a) What process in electron transfer or oxidative phosphorylationis affectedby DCCD?

?'il

Problems

(b) WhV does DCCD affect the 02 consumption of mitochondria?Explain the effect of 2,4-dinitrophenolon the inhibitedmitochondrialpreparation. (c) Which of the following inhibitors doesDCCDmost resemblein its action: antimycin A, rotenone,or oligomycin? 9. Compartmentalization of Citric Acid Cycle Components Isocitrate dehydrogenaseis found only ln the mitochondrion,but malate dehydrogenaseis found in both the cytosoland mitochondrion.What is the roie of cvtosolicmalate dehydrogenase? 10. The Malate-c-Ketoglutarate Thansport System The transport system that conveysmalate and a-ketoglutarate acrossthe inner mitochondrialmembrane(seeFig. 19-29) is inhibited by tz-butylmalonate.Supposen -butylmalonateis added to an aerobic suspensionof kidney cells using glucose exclusivelyas fuel Predict the effect of this inhibitor on (a) glycolysis,(b) oxygenconsumption,(c) lactate formation, and (d) ATP syrrthesis. 11. Cellular ADP Concentration Controls ATP Formation Although both ADP and P1are required for the sy'nthesis of ATP,the rate of synthesisdependsmainly on the concentration of ADP,not P1.Why'i 12. Tlme Scales of Regulatory Events in Mitochondria Comparethe likely time scalesfor the adjustmentsin respiratory rate causedby (a) increased[ADP]and (b) reducedpO2. What accountsfor the difference? 13. The Pasteur Effect When 02 is added to an anaerobic suspensionof cells consumingglucoseat a high rate, the rate of glucoseconsumptiondeclinesgreatlyas the 02 is usedup, and accumulationof lactate ceases.This effect, flrst observed by Louis Pasteurin the 1860s,is characteristicof most cells capableof both aerobicand anaerobicglucosecatabolism. (a) Why doesthe accumulationof lactate ceaseafter O, is added? (b) Why doesthe presenceof 02 decreasethe rate of glucoseconsumption? (c) How doesthe onsetof 02 consumptionslowdownthe rate of glucose consumption?Expiain in terms of specific enzymes. 14. Respiration-Deficient Yeast Mutants and Ethanol Production Respiration-deficient yeast mutants (p ; "petites") can be produced from wild-type parents by treatment with mutagenicagents The mutants lack cy'tochromeoxidase, a deficit that markedly affects their metabolic behavior.One striking effect is that fermentation is not suppressedby Ozthat is, the mutantslack the Pasteureffect (seeProblem13). Somecompaniesare very interestedin using thesemutants to fermen[ wood chips to ethanolfor energyuse.Explain the advantagesof using these mutants rather than wild-t5,peyeast for large-scaleethanolproduction.Why doesthe absenceof cy'tochromeoxidaseeliminatethe Pasteureffect? 15. Advantages ofSupercomplexes for Electron Tbansfer There is growing evidencethat mitochondrial Complexes I, II, III, and IV are part of a larger supercomplex.What might

be the advantageof having all four complexeswithin a supercomplex? 16. How Many Protons in a Mitochondrion? Electron transfer translocatesprotonsfrom the mitochondrialmatrix to the externalmedium, establishinga pH gradient acrossthe inner membrane(outside more acidic than inside). The tendency of protons to diffuse back into the matrix is the driving force for ATP synthesisby ATP synthase.During oxidative phosphorylationby a suspensionof mitochondriain a medium of pH 7.4,the pH of the matrix hasbeenmeasuredas 7.7. (a) Calculate[H-] in the extemal medium and in the matrix under these conditions. (b) What is the outside-to-inside ratio of [H+]?Cornrnent on the energyinherent in this concentrationdifference (Hint: S e eE q n 1 1 - 4 ,p . 3 9 6 . ) (c) Calcuiatethe number of protonsin a respiringliver mitochondrion, assuming its inner matrix compartment is a sphereof diameter1.5pr.m. (d) From these data,is the pH gradient alone sufficientto generateATP? (e) If not, suggesthow the necessaryenergyfor synthesis of ATP arises. 17. Rate of AI? Tbrnover in Rat Heart Muscle Rat heart muscleoperatingaerobicallyfills more than 90%o of its ATP needs gram phosphorylation. Each of tissue consumes02 by oxidative g.moVmin, with $ucoseasthe fuel source at the rate of 10.0 (a) Calculatethe rate at which the heart muscleconsumes glucoseand producesATP. (b) For a steady-stateconcentrationof ATP of 5.0 pmoVg of heart muscletissue,calculatethe time required (in seconds) to completelyturn over the cellularpool of AIP. What doesthis result indicate about the need for tight regulationof ATP production? (Note: Concentrationsare expressedas micromoles per grarn of muscletissuebecausethe tissueis mostly water.) 18. Rate of ATP Breakdown in Insect Flight Muscle ATP production in the flight muscleof the fly Luci,Li,aseri,cata results almost exclusively from oxidative phosphorylation. During flight, 187mL of O2/hr . g of body weight is neededto maintain an ATP concentrationof 7.0 pmoVg of flight muscle. of the weight of the Assumingthat flight musclemakestp 200/o fly, calculatethe rate at which the flight-muscleATP pool turns over.How long would the reservoir of ATP last in the absence of oxidative phosphorylation?Assume that reducing equivalents are transferredby the glycerol 3-phosphateshuttle and that 02 is at 25'C and 101.3kPa (1 atm). 19. Mitochondrial Disease end Cancer Mutations in the genes that encode certain mitochondrial proterns are associatedwith a high incidence of some t1-pesof cancer.How might defectivemitochondrialead to cancer? 20. Variable Severity of a Mitochondrial Disease Individualswith a diseasecausedby a speciic defectin the mitochondrial genomemay have slrnptoms ranging from mild to severe.Explain why. 21. Tbansmembrane Movement of Reducing Equivalents Under aerobic conditions, extramitochondrial NADH

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must be oxidizedby the mitochondrialelectron-transferchain. Considera preparationof rat hepatocytescontainingmitochondriaand all the cytosolicenzlmes. If [4-3H]NADHis introduced,radioactivitysoonappearsin the mitochondrialmatrix. However,if [7-]4C]NADHis introduced,no radioactivityappears in the matrix What do these observationsreveal about the oxidation of extramitochondrial NADH bv the electrontransfer chain?

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22, High Blood Alanine Level Associated with Defects in Oxidative Phosphorylation Most individuals with genetic defects in oxidative phosphorylation are found to have relatively high concentrations of alanine in their blood. Explain this in biochemical terms. 23. NAD Pools and Dehydrogenase Activities Although both p1'ruvate dehydrogenase and glyceraldehyde 3-phosphate dehydrogenase use NAD* as their electron acceptor, the two enzlrres do not compete for the same cellular NAD pool. Why? ,n.Diabetes as a Consequence of Mitochondrial E Il Defects Glucokinase is essential in the metabolism of glucose in pancreatic B cells Humans with two defective copies of the glucokinase gene exhibit a severe, neonatal diabetes, whereas those with only one defective copy of the gene have a much milder form of the disease (mature onset diabetes of the young, MODY2) Explain this difference in terms of the biology of the B cell

25. Effects of Mutations in Mitochondrial Complex II Singlenucleotidechangesin the genefor succinate dehydrogenase(Complex II) are associatedwith midgut carcinoidtumors. Suggesta mechanismto explain this observation 26. Photochemical Effieiency of Light at Different Wavelengths The rate of photosynthesis,measured by 02 production, is higher when a green plant is illuminated with Iight of wavelength680nm than with light of 700nm. However, illumination by a combinationof light of 680 nm and 700 nm givesa higher rate of photosynthesisthan light of either waveIength alone Explain. 27. Balance Sheet for Photosyrthesis In 1804Theodore de Saussureobservedthat the total weight of oxygenand dry organic matter produced by plants is greater than the weight of carbon dioxide consumedduring photosynthesisWhere doesthe extra weight comefrom? 28. Role of II2S in Some Photosynthetic Bacteria llluminated purple sulfur bacteria carry out photos;nthesis in the presenceof H2Oand laCOr,but only if HrS is addedand 02 is absent.Duringthe courseofphotosynthesis, measuredby for-

H2Sis convertedto elemental mation of IraC]carbohydrate, sulfur, but no 02 is evolved.What is the role of the conversion of H2Sto sulfur?Why is no 02 evolved? 29. Boosting the Reducing Power of Photosystem I by Light Absorption When photosystemI absorbsred light at 700 nm, the standardreduction potential of P700 changes from 0.40 V to about -I.2 V. What fraction of the absorbed light is trapped in the form of reducing power? 30. Electron Flow through Photosystems I and II Predict how an intiibitor of electronpassagethough pheophytinwotrld affect eiectronflow through (a) photosystemII and (b) photosystemI. Expiainyour reasoning. 31. Limited ATP Synthesis in the Dark In a Iaboratoryexperiment, spinachchloroplastsare illuminated in the absence of ADP and P1,then the light is turned off and ADP and P1are added.ATP is synthesizedfor a short time in the dark. Explain this finding. 32. Mode of Action of the Herbicide DCMU When chloroplasts are treated with 3-(3,4-dichiorophenyl)-1,1dimethylurea(DCMU,or diuron), a potent herbicide,02 evolution and photophosphorylationcease.Oxygenevolution,but not photophosphorylation,can be restored by addition of an externalelectronacceptor,or Hill reagent.How doesDCMU act as a weed killer? Suggesta locationfor the inhibitory action of this herbicide in the scheme shown in Figure 19-56. Explain. 33. Effect of Venturicidin on Oxygen Evolution Venturicidin is a powerful inhibitor of the chloroplastATP syrrthase, lnteracting with the CFo part of the enzyme and blocking proton passagethrough the CF.CFI complex. How would venturicidin affect oxygen evolution in a suspensionof wellllluminated chloroplasts?Would your answer change if the experiment were done in the presence of an uncoupling (DNP)?Explaln. reagentsuchas 2,4-dinitrophenol 34. Bioenergetics of Photophosphorylation The steadystate concentrationsof ATP,ADP, and P, in isolated spinach chloroplastsunder full illuminationat pH 7.0 are 120.0,6.0, and 700.0;.cu,respectively. (a) What is the free-energyrequirementfor the sy'r-rthesis of 1 moi of AIP under theseconditions? (b) The energyfor ATP synthesisis furnished by lightinduced electron transfer in the chloroplasts.What is the minimum voltagedrop necessary(during transfer of a pair of electrons) to synthesizeATP under these conditions? (Youmay needto refer to Eqn 13-7,p 515.) 35. Light Energy for a Redox Reaction Supposeyou have isolateda new photosyntheticmicroorganismthat oxidizes H2Sand passesthe electrons to NAD-. What wavelength of light would provide enough energy for H2Sto reduce NAD+ under standardconditions?Assume 100%efficiencyin the photochemicalevent, and use E'" of -243 mV for H2Sand -320 mV for NAD-. SeeFigure 19-46 for the energy equivalents of wavelengthsof light.

Problems lzzll 36. Eqnilifufium Constant for Water-Splitting Reactions The coenzS.,rne NADP* is the terminal elbctron acceptor in chloroplasts,accordingto the reaction 2H2O + 2NADP* -----+2NADpH + 2H+ + 02 Use the information in Table Ig-2 Io calcuiatethe equilibrium constantfor this reactionaL25"C. (The relationshipbetween Kin and AG'ois discussedonp 492.)How can the chtoroplast overcomethis unfavorableequilibrlum? 37. Energetics of Phototransduction During photos5,nthesis, eight photons must be absorbed(four by each photosystem)for every 02 moleculeproduced: 2H2O + 2NADP+ + 8 photons ----+ 2NADPH + 2H+ + 02 Assuming that these photons have a wavelength of 700 nm (red) and that the light absorptionand use of light energyare 100% efficient, calculate the free-energy change for the process. 38. Electron lbansfer to a Hill Reagent Isolatedspinach chloroplasts evolve 02 when illuminated in the presence of potassiumferricyanide (a Hill reagent), according to the equatron 2H2O + 4Fe3* -----+02 + 4H+ + 4Fe2+ whereFe3* representsferricyanideand Fe2*,ferrocyanide.Is NADPHproducedin this process?Explain. 39. How Ofiten Does a Chlorophyll Molecule Absorb a Photon? The amount of cilorophyil a (M, 892) in a spinachleaf is about20 p.g/cm2of leaf surface.In noondaysunligfit (average enerS/ reachingthe leaf is 54 J/cmz . min), the leaf absorbs about 50% ofthe radiation.How often doesa singleclrlorophyll moleculeabsorba photon?Giventhat the averagelifetime of an excitedchlorophyllmoleculein vivo is 1 ns,what fractionof the chlorophyllmoleculesare excitedat any onetime? 40. Effect of Monochromatic Light on Electron Flow The extent to which an electron carrier is oxidizedor reduced during photosyntheticelectron transfer can sometimesbe observed directly with a spectrophotometer.When chloroplasts are illuminated with 700 nm light, cytochrome;flpiastocyanh, and plastoquinoneare oxidized When chloroplastsare illuminated with 680 nm light, however,these electron carriers are reduced.Explain. 41. Function of Cyclic Photophosphorylation When the [NADPH]4NADP+lratio in chloroplastsis high, photophosphorylationis predominantlycyclic (see Fig. 19-56). Is 02 evolved during cyclic photophosphorylation?Is NADPH produced? Explain. What is the main function of cyclic photophosphorylation?

DataAnalysis Problem 42. Photophosphorylation: Discovery, Rejection, and Rediscovery In the 1930sand 1940s,researchers werebeginning to makeprogresstoward understandingthe mechanismof

photosyrrthesisAt the time, the role of "energy-richphosphate bonds" (today,"ATP") in glycolysisand cellularrespirationwas just becoming known. There were many theories about the especiallyaboutthe role of hght. mechanismof photosSmthesis, This problem focuseson what was then called the "primary photochemicalprocess"-that is, on what it is, exactly,that the energyfrom capturedlight producesin the photosyntheticcell. Interestingly,one important part of the modern model of photosy'nthesiswas proposedearly on, only to be rejected,ignored for severalyears,then finally revivedand accepted. \n 1944,Emerson, Stauffer, and Umbreit proposed that "the function of light energyin photosy'nthesis is the formation of 'energy-rich'phosphatebonds" (p. 107). In their model (hereafter,the "Emersonmodel"), the free energy necessary to drive both CO2fixation and redrction came from these "energy-richphosphatebonds" (i.e., ATP), produced as a result of light absorptionby a chlorophyll-containhgprotein. This model was explicitly rejected by Rabinowitch (1945). A.fter summarizingEmerson and coauthors'findings, Rabinowitchstated:"Until more positiveevidenceis provided, we are inclinedto consideras more convincinga generalargument againstthis hypothesis,which can be derived from energy is eminently a problem of energy considerations.Photos5,rrthesis good can be served,then, by converting accunruLatinn What iight quanta (even those of red light, which amount to about 43 'phosphatequanta' of on-ly10 kcal per kcai per Einstein) hto mote?This appearsto be a start in the wrong direction-toward dissi,pation rather than toward accumulationof energr" (Vol. I, p. 228). This argument,along with other evidence,led to the abandonmentof the Emersonmodelrurtilthe 1950s,whenit was found to be correct-albeit in a modifledform. For each piece of information from Emerson and coauthors'article presentedin (a) through (d) below, answer the following three questions: 1. How doesthis information support the Emersonmodel, in which light energyis used directly by chlorophyll lo make ATP, and the ATP then providesthe energyto drive CO2fixation and reduction? 2. How would Rabinowitchexplainthis information,basedon his model (andmost other modelsof the day), in which light eners/ is useddirectlyby clrlorophyllfrcmnke redtunttng contpounds?Rabinowitchwrote: "Theoretically,there is no reasonwhy all electronicenergr containedin moleculesexcited by the absorptionof light shouldnot be availablefor oxidation-reduction"ffol. I, p. 152).In this model,the reducing compoundsare then usedto fix and reduceCO2,and the ener€iyfor thesereactionscomesfrom the large amourts of releasedby the reductionreactions. ftee energ5r 3. How is this information explainedby our modern understandingof photosynthesis? (a) Chlorophyllcontains aM1'* ion, which is known to be an essentialcofactorfor many enzymesthat catalyzephosphorylation and dephosphoryiationreactions. (b) A crude "chlorophyll protein" isolatedfrom photos)rytthetic cells showedphosphorylatingactivity.

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(c) The phosphorylatingactivity of the "cNorophyll protein" was inhibited by light. (d) The levels of several different phosphorylatedcompounds in photosyrrthetic cells changed dramatically in responseto light exposure. (Emerson and coworkerswere not able to identily the speci-flccompoundsinvolved.) As it turned out, the Emerson and Rabinowitchmodels were both partly correct and partly incorrect. (e) Explain how the two models relate to our current model of photoslrrthesis, In his rejection of the Emerson model, Rabinowitch went on to say: "The difficulty of the phosphate storage theory appearsmost clearly when one considersthe fact that, in weak light, eight or ten quantaoflight are sufficient to reduce one moleculeof carbon dioxide. If each quantum should produce one molecule of high-energy phosphate,

the accumulated energy would be only 80-100 kcal per Einstein-while photosynthesisrequires at least I12 kcal per mole, and probably more, becauseof lossesin irreversiblepartial reactions" (Vol. 1, p.228). (f) How does Rabinowitch'svalue of 8 to 10 photons per moleculeof CO2reduced comparewith the value acceptedtoday?Youneed to consult Chapter20 for someof the information required here. (g) How would you rebut Rabinowitch'sargument,based on our current knowledgeabout photosS'nthesis? References Emerson,R.L., Stau-ffer,J.F., & Umbreit, W.W.(1944)Relationshipsbetweenphosphorylation andphotos5'nthesis in ChIoreLLa Am. J. Botang31,107-120. Rabinowitch,E.I. (1945)Photosgnthesi,s and RelatedProcesses, Interscience Publishers, NewYork

. . . the discoveryof the long-livedisotopeof carbon,carbon-14,Dy by SamueR l u b e na n d M a r t i nK a m e ni n 1 9 4 0 o r o v i d e dt h e i d e a lt o o l for for the tracingof the routealongwhich carbondioxidetravelson its way to carbohydrate. -Melvin Calvin, NobelAddress,

Carbohyd rateBiosynthesis inPlants andBacteria nutrients. They must have sufflcientmetabolicflexibility 20.1 PhotosyntheticCarbohydrateSynthesis 773 to allowthem to adaptto changingconditionsin the place 20,2 Photorespiration andthe(oandGMPathways 786 wherethey are rooted.Finally,plantshavethick cell walls made of carbohydratepol;,'rners,which must be assem20.3 Biosynthesis andSucrose of5tarch 791 bled outsidethe plasmamembraneand which constitute (ellulose a signi-ficantproportion of the cell'scarbohydrate. 20.4 Synthesis of(ellWall Polysaccharides: Plant The chapterbeginswith a descriptionof the process andBacterial Peptidoglycan 794

20.5 Integration ofCarbohydrate inthe Metabolism Plant Cell 797 e havenow reacheda turning point in our study of cellular metabolism.Thus far in Part II we havedescribedhow the maiormetabolicfuelscarbohydrates,fatty acids, and amino acids-are degradedthroughconvergingcatabolicpathwaysto enter the citric acid cycleand yield their electronsto the respiratory chain, and how this exergonicflow of electrons to oxygenis coupledto the endergonicsynthesisof ATP. We now turn to anaboli,cpathways,which use chemical energyin the form of ATP and NADH or NADPHto synthesizecellularcomponentsfrom simpleprecursormolecules. Anabolic pathways are generally reductive rather than oxidative.Cataboiismand anabolismproceed simultaneouslyin a dynamicsteadystate, so the energy-yieldingdegradation of cellular componentsis counterbalanced by biosyntheticprocesses,which create and maintain the intricate orderlinessof living cells. Plants must be especiallyversatilein their handling of carbohydrates,for severalreasons.First, plantsare autotrophs, able to convert inorgaruccarbon (as CO2)into organic compounds.Second,biosynthesisoccursprrmarily in plastids,membrane-bounded organellesuruqueto photosyntheticorganisms,and the movementof intermediatesbetweencellularcompartmentsis an important aspect of metabolism.Third, plants are not motile: they carmotmoveto find better suppiiesof water, sunlight,or

by which CO2is assimilatedinto trioses and hexoses, then considersphotorespiration,an important side reaction during CO2fixation, and the ways in which certain plantsavoidthis sidereaction.We then look at how the biospthesis of sucrose(for sugar transport) and starch (for energy storage)is accomplishedby mechanismsanalogousto those employedby animalcells to make glycogen.The next topic is the synthesisof the celluloseof plant cell walls and the peptidoglycanof bacterial cell walls, illustrating the problems of energydependentbiosynthesisoutsidethe plasmamembrane. Finally,we discusshow the various pathwaysthat share pools of common intermediates are segregatedwithin organellesyet integratedwith one another.

(arbohydrate Synthesis 20.1Photosynthetic The synthesis of carbohydratesin animal cells always employsprecursorshavingat leastthree carbons,all of which are less oxidized than the carbon in CO2.Plants and photosyntheticmicroorganisms,by contrast, can q,nthesizecarbohydratesfrom CO2and water, reducing CO2at the expenseof the energyand reducingpowerfurnishedby the ATP and NADPHthat are generatedby the Iight-dependentreactionsof photosynthesis(Fig. 2O-l). Plants (and other autotrophs) can use CO2as the sole sourceofthe carbonatomsrequiredfor the biosynthesis of celluloseand starch, lipids and proteins, and the many other organic componentsof plant cells. By contrast,heterotrophscannotbring aboutthe net reduction of CO2to achievea net s;mthesisof glucose.

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Green plants contain in their chloroplastsunique enzlrnatic machinery that catalyzes [he conversion of CO2 to simple (reduced) organic compounds, a process called CO2 assimilation. Thus process has also been called CO2 fixation or carbon fixation, but we reserve these terms for the specif,c reaction in which CO2 is incorporated (fixed) into a three-carbon organic compound, the triose phosphate 3-phosphoglycerate. This simple product of photosynthesis is the precursor of more complex biomolecules, including sugars, polysaccharides, and the metabolites derived from them, all of

Melvin Caivin,1911-1997

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which are synthesizedby metaboLicpathwayssimilar to those of animaltissues.Carbondioxide is assrmilatedvia a cyclic pathway,its key intermediates constantly regenerated. The pathway was elucidatedin the early 1950s by Melvin Calvin, Andrew Benson,and JamesA. Bassham,and is often calledthe Calvin cycle or, more descriptively, the photosynthetic carbon reduction cycle.

Carbohydrate metabolism is more complex in plant cells than in animal cells or in nonphotosynthetic microorganisms. In addition to the universal pathways of glycolysis and gluconeogenesis,plants have the unique reaction sequencesfor reduction of CO2 to triose phosphates and the associatedreductive pentose phosphate pathway-all of which must be coordinately regulated to ensure proper allocation of carbon to energy production and syrrthesis of starch and sucrose. Key enz;,rnes are regulated, as we shall see, bV (1) reduction of disulfide bonds by electrons flowing from photosystem I and (2) changes in pH and Mg2* concentration that result from illumination. When we look at other aspects of plant carbohydrate metabolism, we also flnd enzymes that are modulated bV (3) conventional allosteric regulation by one or more metabolic intermediates and (4) covalent modiflcation (phosphorylation).

Plaltids Are0rganelles Unique tellsandAlgae toFlant Most of the biosynthetic activities in plants (including CO2assimilation)occur in plastids, a family of selfreproducingorganellesboundedby a doublemembrane and containinga small genomethat encodessome of their proteins.Most proteins destinedfor plastidsare encodedin nuclear genes,which are transcribedand translatedlike other nuclear genes;then the proteins are importedinto plastids.Plastidsreproduceby binary flssion, replicating their genome (a single circular DNA molecule) and using their orn'nenzyrnesand ribosomes to synthesizethe proteins encodedby that genome. Chloroplasts (see Fig. 19-45) are the sites of CO2 assimilation. The enzymesfor this processare contained in the stroma,the solublephaseboundedby the inner chloroplastmembrane.Amyloplasts are colorlessplastids (that is, they lack chlorophyll and other pigments found in chloroplasts).They have no internal membranes analogousto the photosynthetic membranes (thylakoids) of chloroplasts,and in plant tissuesrich in starch these plastids are packed with starch granules (Fig. 2O-Z). Chloroplastscan be converted to

FIGURE 20-2 Amyloplastsfilled with starch (dark granules)are stainedwith iodine in this sectionof Ranunculusroot cells. Starch granules in varioustissues rangefrom 1 to 100 pm in diameter.

Synthesis 20.1Ph0t0synthetic(arbohydrate frril proplastids by the loss of their internal membranes and chlorophyll, and proplastidsare interconvertible with amyloplasts(Fig. 20-g). In turn, both amyloplasts and proplastidscan developinto chloroplasts.The relative proportions of the plastid t;,pes dependon the type of plant tissue and on the intensity of illumination. Cells of green leavesare rich in chloroplasts,whereasamyloplasts domrnatein nonphotos;,rrthetictissuesthat store starchin largequantities,suchaspotatotubers. The inner membranesof all types of plastidsare impermeable to polar and charged molecules. Ttaffic acrossthesemembranesis mediatedbv setsof snecif.c transporters.

[arbon Dioxide Assirnilation 0ccurE inThree Stages The flrst stagein the assimilationof CO2into biomolecules (Fig. 20-4) is the carbon-fixation reaction: condensation of CO2 with a five-carbon acceptor, ribulose 1,5-bisphosphate, to form two molecules of 3-phosphoglycerate. In the second stage, the 3-phosphoglycerate is reducedto triose phosphates. Overall,three moleculesof CO2are flxed to three molecules of ribulose1,5-bisphosphate to form six molecules of glyceraldehyde3-phosphate(18 carbons) in equilibrium with dihydroxyacetonephosphate.In the third stage,flve of the six moleculesof triosephosphate (15 carbons)are usedto regeneratethree moleculesof ribulose 1,5-bisphosphate (15 carbons),the starting material.The sixth molecule of triose phosphate,the net product of photosynthesis,can be used to make hexosesfor fuel and building materials, sucrosefor transport to nonphotosynthetictissues,or starch for storage.Thus the overall processis cyclical,with the continuous conversion of CO2 to triose and hexose phosphates.Fructose6-phosphateis a key intermediate in stage3 of CO2assimilation;it standsat a branch point, leadingeither to regenerationof ribulose l,5-bisphosphate or to synthesis of starch.The pathwayfrom hexosephosphate to pentosebisphosphateinvolvesmany of the same reactionsused in animal cells for the conversionof pentosephosphatesto hexose phosphatesduring the nonoxidativephaseof the pentose phosphate pathway (see Fig. 14-22).In the photosyntheticassimilationof CO2,essentiallythe sameset of reactionsoperatesin the other direction,convertinghexose phosphatesto pentosephosphates.This

FIGURE 20-3 Plastids:their originsand interconversions. All typesof plastidsare boundedby a doublemembrane, and some(notablythe The internal internalmembranes. haveextensive maturechloroplast) membranescan be lost (when a maturechloroplastbecomesa pro(as a proplastidgives rise to a pregranal plastid)and resynthesized plastidand then a maturechloroplast). Proplastids in nonphotosyn(such which contain root) give rise to amyloplasts, as thetictissues and theseorof starch.All plantcellshaveplastids, largequantities includingthe syntheganellesarethe siteof otherimportantprocesses, flavins, amino acids,thiamine,pyridoxalphosphate, sis of essential and vitaminsA, C, E,and K.

reductive pentose phosphate cycle uses the same enzyrnesasthe oxidativepathway,and severalmore enzymesthat make the reductive cycle irreversible.All 13 enzyrnesof the pathway are in the ciloroplast stroma.

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FIGURt 20-4 Thethreestagesof CO2assimilation in photosyntheticorganisms.Stoichiometries of threekey inter(numbersin parentheses) mediates revealthefateof carbon atomsenteringand leavingthe cycle.As shownhere,three CO2arefixedfor the netsynthesis of one moleculeof glyceraldehyde3-phosphate. This cycle is the photosynthetic carbonreductioncycle,or the Calvincycle.

Stage 1: Fixation

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(arbohydrate Biosynthesis inPlants andBacteria

Stage l: Fixation of CO2 into 3-Phosphoglycerate An important clue to the nature of the CO2-assimilation mechanismsin photosy'ntheticorganismscame in the late 1940s.Calvin and his associatesilluminated a suspensionof green algaein the presenceof radioactive carbon dioxide (tnC0r) for just a few seconds,then quickly killed the cells, extracted their contents,and with the help of chromatographicmethodssearchedfor the metabolitesin which the labeled carbon first appeared.The flrst compoundthat becamelabeledwas 3-phosphoglycerate, with the raC predominantly locatedin the carboxylcarbonatom.Theseexperiments strongly suggestedthat 3-phosphoglycerateis an early intermediate in photosynthesis.The many plants in which this three-carboncompoundis the first intermediate are called Cs plants, in contrast to the Caplants describedbelow. The enzyme that catalyzesincorporation of CO2 into an organic form is ribulose l,5-bisphosphate carboxylase/oxygenase, a narnemercifully shortened to rubisco. As a carboxylase,rubisco catalyzesthe covalent attachment of CO2 to the five-carbon sugar ribulose 1,5-bisphosphateand cleavageof the unstable six-carbon intermediate to form two molecules of 3-phosphoglycerate, one of which bears the carbon

Top view FIGURE 20-5 Structureof ribulose1,5-bisphosphate carboxylase (rubisco).(a) Top and sideview of a ribbon model of form I rubiscofrom spinach(PDBlD BRUC). Theenzymehaseightlarge subunits(blue)and eightsmallones(gray),tightlypackedinto a structureof M, 550,000 Rubiscois presentat a concentrationof about250 mglml in the chloroplast stroma,corresponding to an extraordinarily highconcentration of activesites(-4 mv). Amino acidresidues of theactivesiteareshownin yellow,Mg2* in green. (b) Ribbonmodelof form ll rubiscofrom the bacteriumRhodospirillum rubrum(PDBlD gRUB).Thesubunits arein grayandblue.A Lysresidueat the activesitethat is carboxylated to a carbamatein theactiveenzymeis shownin red.Thesubstrate, ribulose1,5-bisphosphate,is yellow; Mg2+rsgreen.

introduced as CO2in its carboxylgroup (Fig. 20-4). The enzyrne'soxygenaseactivity is discussedin Section20.2. There are two distinct forms of rubisco. Form I is found in vascularplants,algae,and cyanobacteria; form II is conined to certain photosyntheticbacteria.Plant rubisco, the crucial enzymein the production of biomass from CO2,has a complexform I structure (Fig. 20-5a), with eight identicallarge subunits(M,53,000;encoded in the chloroplastgenome,or plastome),eachcontaining a catalytic site, and eight identical small subunits (M, 14,000;encodedin the nuclear genome) of uncertain function. The form II rubisco of photosyntheticbacteria is simplerin structure, havingtwo subunitsthat in many respectsresemblethe largesubunitsof the plant enzyrne (Fig. 20-5b). This similarity is consistentwith the endoq.'rnbionthypothesisfor the origin of chloroplasts(p. 33). The plant erz;.'rnehasan exceptionallylow turnover number; only three moleculesof CO2are fixed per second per molecule of rubisco at 25 "C. To achievehrgh rates of CO2 fixation, plants therefore need large amountsof this enz;rme.In fact, rubiscomakesup almost 50o/oof soluble protein in chloroplastsand is probably one of the most abundantenz1rynes in the biosphere. Central to the proposedmechanismfor plant rubisco is a carbamoylatedLys side chain with a bound

Side view

20.1PhotosyntheticCarbohydrate5ynthesis ?rrl FIGURE 20-6 Centralrote of Mg2* in the catalyticmechanismof ruin a roughly from PDBlD lRXO)Mg2* is coordinated bisco.(Derived octahedralcomplexwith six oxygenatoms:one oxygenin the carbatwo at mateon Lys2o1; two in the carboxylgroupsof Clu2oaandAsp203; andone in the ribulose1,S-bisphosphate; C-2andC-3ofthe substrate, CO2.A watermoleculeoccupiesthe CO2-bindingsite othersubstrate, In thisfigurea CO2moleculeis modeledin its in thiscrystalstructure. place.(Residue numbersreferto thespinachenzyme.) Carbamoyl-Lys201

11i.294

Rubisco ^^nbP. .

Mg2* ion. The Mgz+ ion brings together and orients the reactantsat the active site (Fig. 20-6) and polarizes the CO2,openingit to nucleophilicattack by the five-carbon enediolatereaction intermediate formed on the enzyme(FiS. 20-7). The resulting six-carbon

Glu i, 'i

Ribulose 1,5bisphosphate forms an Lnediolate at the active site.

,.OJg) Cfr,

-oH Ribulo 12 1,5-bis

-fD Carbamoylated Lys side chain

o\ zo C I H COH

I

Glu

AsP... 3-Phosphoglycerate

,o-€ 'cfi"

i'

CHro-@ Carbanion protonation generates second molecule of 3-phosphoglycerate.

Glu

Asp..

OH ',H io. \H /?\ \?/

'

II 3-Phosphoglycerate

i Mg2t

I ra, * |

cH"o + TADP+ 7Pi QL-l) 7 malonyl-CoA

CHs-CH2-CH2-C-S Butyryl group

and reduction: then sevencyclesof condensation + 14NADPH+ 14H* --) Acetyl-CoA+ 7 malonyl-CoA palmitate+ 7CO"+ 8 CoA+ 14NADP*+ 6H2O (2I-2)

o.,

//o //c- cHz-c\

S-CoA

O

Note that only six net water moleculesare produced, becauseone is used to hydrolyzethe thioesterlinking the palmitate product to the enzyme. The overall process(the sumofEqns 21-1 and 2I-2) is

Malonyl-CoA

o

8 Acetyl-CoA+ 7 ATP + 14NADPH+ 14H- -+ + 6H2O (21-3) palmitate+ 8 CoA+TADP+ 7Pi+ 14NADP+

-o CH3-CH2

group, acting like the acetyl group in the first cycle, is Iinked to two carbons of the malonyl-ACPgroup with concurrentlossof CO2.The productof this condensation is a six-carbonacylgroup,covalentlyboundto the phos-SH group.Its B-ketogroupis reduced phopantetheine in the next three stepsof the sl'nthasecycle to yield the saturatedacyl group, exactly as in the first round of reactions-in this caseforming the six-carbonproduct. Sevencycles of condensationand reduction produce the 16-carbon saturated palmitoyl group, still bound to ACP.For reasonsnot well understood,chain elongationby the synthasecomplex generally stops at this point and free palmitateis releasedfrom the ACPby a hydrolytic activity (thioesterase;TE) in the multifunctional protein. We can considerthe overall reaction for the slmthesis of palmitatefrom acetyl-CoAin two parts.First, the formationof sevenmalonyl-CoAmolecules:

CHz-

o il

s -C--CH2-C

CHz CH2- CH3

o

0-Ketoacyl-ACP FIGURE 21-7 Beginningof the secondround of the fatty acid synthesiscycle.Thebutyrylgroupis on the Cys-SH group.The incom-SH ing malonylgroup is firstattachedto the phosphopantetheine step,the entirebutyrylgroupon the group Then,in the condensation for the carboxylgroupof the malonylresidue, Cys-SH is exchanged to step@ in Figrre which is lostas CO2(green). Thisstepis analogous groupi now containsfour 21-6 The product,a six-carbon B-ketoacyl and two derivedfrom the acetylcarbonsderivedfrom malonyl-CoA CoA that startedthe reaction.The B-ketoacylgroup then undergoes steps@ through@, as in Figure2.1-6.

The biosynthesisof fatty acidssuchaspalmitatethus requires acetyl-CoAand the input of chemical energy in two forms: the group transfer potential of ATP and the reducing power of NADPH. The ATP is required to attach CO2to acetyl-CoAto make malonyl-CoA;the NADPHis requiredto reducethe doublebonds. In nonphotosyntheticeukaryotesthere is an addibecauseacetyl-CoAis tional costto fatty acid s;,'nthesis, generatedin the mitochondriaand must be transported to the cy'tosol.As we will see,this extra step consumes two ATPs per moleculeof acetyl-CoAtransported,increasingthe energeticcost of fatty acid synthesisto three ATPsper two-carbonunit.

ofMany inthe(ytosol 0ccurs Aeid Synthesis Fatty ofPlants butinthe[hloroplasts 0rganisms In most higher eukaryotes,the fatty acid synthasecomplex is found exclusivelyin the c;,'tosol(I.ig. 21-8)' as are the biosynthetic enzymesfor nucleotides,amino acids,and glucose.This location segregatessynthetic processesfrom degradativereactions,many of which take placein the mitochondrialmatrix. Thereis a correspondingsegregationof the electron-carryingcofactors used in anabolism(generallya reductiveprocess)and thoseusedin catabolism(generallyoxidative).

Ft{

L i p iB di o s y n t h e s i s

Animal cells, yeast cells

&l:

Plant cells

Mitochondria o No fatty acid oxidation o Fatty acid oxidation o Acetyl-CoA production o Ketone body synthesis o Fatty acid elongation Endoplasmic reticulum . Phospholipidsynthesis o Sterol synthesis(late stages) o Fatty acid elongation o Fatty acid desaturation

Cytosol . NADPH production (pentosephosphate pathway; malic enzyme) . [NADpH]/[NADp*] high r Isoprenoidand sterol synthesis (early stages) o Fatty acid synthesis FIGURt yeastand 21-8 Subcellular localization of lipid metabolism. vertebrate cellsdifferfrom higherplantcellsin the compartmentation of lipid metabolism. Fattyacid synthesis takesplacein the compartmentin which NADPHis available for reductive (i.e, where synthesis

Usually, NADPH is the electron carrier for anabolic reactions, and NAD+ serves in catabolic reactions. In hepatocytes, the [NADpH]/[NADP+] ratio is very high (about 75) in the cytosol, furnishing a strongly reducing environment for the reductive synthesis of fatty acids and other biomolecules. The cytosolic [NADH]/[NAD"] ratio is much smaller (only about 8 X 10-4), so the NAD+-dependent oxidative catabolism of glucose can take place in the same compartment, and at the same time, as fatty acid synthesis. The [NADH]/[NAD+] ratio in the mitochondrion is much higher than in the cytosol, because of the flow of electrons to NAD+ from the oxidation of fatty acids, amino acids, pyruvate, and acetyl-CoA. This high mitochondrial [NADH]i[NAD+] ratio favors the reduction of oxygen via the respiratory chain. In hepatocy'tesand adipocytes, cy'tosolicNADpH is largely generated by the pentose phosphate pathway (see Fig. l4-2I) and by malic enzJrme (Fig. Zt-9a). The NADP-linked malic enzyme that operates in the carbon-assimilation pathway of C4 plants (see Fig. 20_28) is unrelated in function. The pyruvate produced in the reaction shown in Figure 21-9a reenters the mitochondrion. In hepatoc5,tes and in the mammary gland of lactating animals, the NADPH required for fatty acid biosynthesis is supplied primarily by the pentose phosphate pathway (Fig. 21-9b).

. NADPH, ATP production . [NADPH]/[NADP*] high o Fatty acid synthesis

o Fatty acid oxidation (--->H2o2) o Catalase, peroxidase: H2O2------->H2O

the tNADPHI/INADP-l ratiois high);thisisthecytosolin animalsand yeast,and the chloroplast in plants Processes in redtypearecovered in thischapter.

coo tl CHOH

coo C:O

ll

CH, l-

n r i r l i cc n z r r r r c

+ co2

CHa

cooMalate

Py'ruvate (a)

NADP*

Glucose 6-phosphate

NADP*

pentose phosphate pathway

Ribulose 5-phosphate

(b) FIGURE 21-9 Productionof NADPH.Two routesto NADPH, catalyzed6y (1) malic enzymeand (b) the pentosephosphatepathway.

In the photosyrrtheticcells of plants, fatty acid synthesis occursnot in the cytosolbut in the chloroplast stroma (Fig. 21-B). This makes sense, given that NADPH is producedin chloroplastsby the light reactions of photosynthesis: light

H2o + NADP*

\,

+ o 2 + N A D P H+ H *

Acids andEicosanoids ofFatty 21.1Biosynthesis | 813|

Acetate lsShuttled outofMitochondria as(itrate In nonphotosyr-rthetic eukaryotes,nearly all the acetylCoA used in fatty acid symthesisis formed in mitochondria from pyruvate oxidation and from the catabolismof the carbon skeletonsof amino acids Acetyl-CoAarising from the oxidation of fatty acids is not a significant source of acetyl-CoAfor fatty acid biosynthesisin animals, becausethe two pathwaysare reciprocally regulated,as describedbelow The mitochondrial inner membrane is impermeable to acetyl-CoA, so an indirect shuttle transfers acetyl group equivalentsacrossthe inner membrane (Fig. 2f-l0). Intramitochondrial acetyl-CoAfirst reacts with oxaloacetateto form citrate, in the citric acid cycle reaction catalyzedby citrate synthase (see Fig. 16-7). Citrate then passesthrough the inner membraneon the citrate transporter. In the cyInner membrane

tosol, citrate cleavageby citrate lyase regenerates acetyl-CoAand oxaloacetatein an ATP-dependentreaction. Oxaloacetatecannot return to the mitochondrial matrix directly, as there is no oxaloacetate transporter.Instead, cytosolic malate dehydrogenase reducesthe oxaloacetateto malate,which can return to the mitochondrialmatrix on the malate-a-ketoglutarate transporter in exchangefor citrate. In the matrix, malate is reoxidizedto oxaloacetateto complete the shuttle. However,most of the malate produced in the cytosol is used to generate cytosolic NADPH through the activity of malic enzyme(Fig. 21-9a). The pyruvateproducedis transportedto the mitochondria by the pyruvate transporter (Fig. 21-10), and converted back into oxaloacetateby pyruvatecarboxylase in the matrix. The resulting cycle results in the consumption of two ATPs (by citrate lyase and pyruvate

Outer rnembrane Cytosol

CoA-SH Fatty acid synthesis

r

ADP + P;

Acetyl-CoA

Acetyl-CoA (multiple sources) Oxaloacetate

Oxaloacetate +H+

+H*

Malate

,| t I I

ADP + P;

pyru\ iiLC carboxllasc

I t !

t. Malatea-ketoglutarate transporter

Pyruvate

+H*

Pyruvate

Pyruvate transpofier

FIGURE 21-10 Shuttlefor transferof acetyl groupsfrom mitochondria to the cytosol.The mitochondrialouter membraneis freelypermeableto all thesecompounds.Pyruvatederivedfrom amino acid in the mitochondrial matrix,or from glucoseby glycolysis catabolism in the cytosol,is convertedto acetyl-CoAin the matrix.Acetylgroups passout of the mitochondrionas citrate;in the cytosolthey are

deliveredas acetyl-CoAfor fatty acid synthesis.Oxaloacetateis rematrixand is ducedto malate,whichcan returnto the mitochondrial The major fate for cytosolicmalateis oxiconvertedto oxaloacetate. dation by malic enzymeto SeneratecytosolicNADPH; the pyruvate oroducedreturnsto the mitochondrialmatrix.

let!

LipiB d iosynrhesis

carboxylase) for every molecule of acetyl-CoA delivered to fatty acid synthesis. After citrate cleavage to generate acetyl-CoA, conversion of the four remaining carbons to pyruvate and CO2 via malic enzyme generates about half the NADPH required for fatty acid synthesis. The pentose phosphate pathway contributes the rest of the needed NADPH.

Fatty Acid Biosynthesis lsTightly Regulated When a cell or organism has more than enough metabolic fuel to meet its energy needs, the excess is generally converted to fatty acids and stored as lipids such as triacylglycerols The reaction catalyzed by acetyl-CoA carboxylase is the ratelimiting step in the biosynthesis of fatty acids, and this enzyme is an important site of regulation. In vertebrates, palmitoyl-CoA, the principal product of fatty acid synthesis, is a feedback inhibitor of the enzyme; citrate is an allosteric activator (Fig. 2l-lla), increasing 7.,,,-. Citrate plays a central role in diverting cellular metabolism from the consumption (oxidation) of metabolic fuel to the storage of fuel as fatty acids. When the concentrations of mitochondrial acetyl-CoA and ATP increase, citrate is transported out of mitochondria; it then becomes both the precursor of cytosolic acetyl-CoA and an allosteric signal for the activation of acetyl-CoA carboxylase At the same time, citrate inhibits the activity of phosphofructokinase-l (see Fig. 15-14), reducing the flow of carbon through glycolysis. Acetyl-CoA carboxylase is also regulated by covalent modification. Phosphorylation, triggered by the hormones glucagon and epinephrine, inactivates the

Citrate

,

Acetyl-CoA

enzymeand reducesits sensitivityto activationby citrate, thereby slowing fatty acid synthesis.In its active (dephosphorylated) form, acetyl-CoAcarboxylasepolymerizesinto long fi.laments(Fig. 21-1lb); phosphorylation is accompaniedby dissociationinto monomeric subunitsand loss of activity. The acetyl-CoAcarboxylaseof plants and bacteria is not regulated by citrate or by a phosphorylationdephosphorylationcycle. The plant enzymeis activated by an increasein stromalpH and [Mg'*], which occurs on illuminationof the plant (seeFig. 20-17). Bacteriado not use triacylglycerolsas energystores.In E coLi,,the primary role of fatty acid synthesisis to provide precursorsfor membranelipids;the regulationof this process is complex, involving guanine nucleotides (such as ppcpp) that coordinatecell growth with membraneformation (seeFigs 8-39,28-24). In additionto the moment-by-moment regulationof enzy'rnaticactivity, these pathwaysare regulated at the Ievelof geneexpression.For example,when animalsingest an excessof certain polyrrnsaturated fatty acids, the expressionof genes encodinga wide range of lipogenicenzyrnesin the liver is suppressed. The detailed mechanismby which these genesare regulatedis not yet clear. If fatty acid synthesisand B oxidation were to proceed simultaneously,the two processeswould constitute a futile cycle, wasting energy.We noted earlier (see Fig. 17-I2) that B oxidation is blocked by malonyl-CoA, which inhibits carnitine acyltransferase I. Thus during fatty acid synthesis,the production of the first intermediate,malonyl-CoA,shuts down B oxidation at the level of a transport systemin the mitochondrial inner membrane. This control mechanismillustrates another advantageof segregating synthetic and degradativepathwaysin different cellular compartments.

Long-(hain Saturated Fatty Acids Are Synthesized fromPalmitate

I I J J

'- Palmitoyl-CoA (a)

(b)

F I G U R E2 l - 1 1 R e g u l a t i o n o f f a t t y a c i d s y n t h e s i s .( a ) I n r h e c e l l s o f v e r t e b r a t e sb, o t h a l l o s t e r i cr e g u l a t i o n a n d h o r m o n e - d e p e n d e n tc o v a l e n t m o d i f i c a t i o n i n f l u e n c e t h e f l o w o f p r e c u r s o r si n t o m a l o n y l - C o A In plants, acetyl-CoA carboxylase is activated by the changes in [ M g 2 * ] a n d p H t h a t a c c o m p a n y i l l u m i n a t i o n ( n o t s h o w n h e r e ) .( b ) F i l aments of acetyl-CoA carboxylase (the active, dephosphorylated form) a s s e e nw i t h t h e e l e c t r o nm i c r o s c o o e .

Palmitate, the principal product of the fatty acid synthase system in animal cells, is the precursor of other long-chain fatty acids (Fig. 2f-f2). It may be lengthened to form stearate (18:0) or even longer saturated fatty acids by further additions of acetyl groups, through the action of fatty acid elongation systems present in the smooth endoplasmic reticulum and in mitochondria. The more active elongation system of the ER extends the 16-carbon chain of palmitoyl-CoA by two carbons, forming stearoyl-CoA. Although different enzyrne systems are involved, and coenz;.'rneA rather than ACP is the acyl carrier in the reaction, the mechanism of elongation in the ER is otherwise identical to that in palmitate synthesis: donation of two carbons by malonyl-CoA, followed by reduction, dehydration, and reduction to the saturated 1S-carbon product, stearoyl-CoA.

y c i dasn dE i c o s a n o i[dbst ; ] 2 1 . 1B i o s y n t h e0sf Fi sa t t A

a Requires Acids ofFatty Desaturation Oxidase Mixed-Function Palmitate 16:0

I

eloneation - l a t eI

v) Stearate 18:0

18:1(Ae)

I

desaturation V (in nlants I onlv I v

I

v Linoleate 1g:2(ae,1s) cle

/

(-

/ a-Linolenate 18r3(Ae,12,15)

II

J Other polyunsaturated fatty acids

ate 18.3(46,e,12) I eloneation 't Eicosatrienoate 20.3(A8,11,14) I Jdesaturation Arachidonate 20.4(A5,8,11,14)

is FIGURE 21-12 Routesof synthesis of other fatty acids.Palmitate fattyacids,aswell and longer-chain saturated the precursor of stearate andoleate.Mammalscanasthe monounsaturated acidspalmitoleate (shadedpink),which not convertoleateto linoleateor a-linolenate fatty of requiredin the dietasessential acids.Conversion aretherefore is outfattyacidsand eicosanoids linoleateto otherpolyunsaturated the numfattyacidsaresymbolized by indicating lined Unsaturated ber of carbonsand the numberand positionof the doublebonds,as i n T a b l e1 0 - 1.

o2+2}l*+ cH3- (c H2)" -cHr;911r-

(c H2)- -c\

Palmitateand stearateserveas precursorsof the two most commonmonounsaturatedfatty acids of animal 16:1(Ae),and oleate,18:1(Ae); tissues:palmitoleate, both of these fatty acidshave a singiecis doublebond betweenC-9 and C-10 (see Table 10-1). The double bond is introduced into the fatty acid chain by an oxidative reaction catalyzedby fatty acyl-CoA desaturase (Fig. 21-13), a mixed-function oxidase (Box 21-1). TWo different substrates,the fatty acid and NADH or NADPH, simultaneouslyundergo twoelectronoxidations.The path of electronflow includes a cytochrome(cytochromeb5) and a flavoprotein(cytochrome b5 reductase),both of which, like fatty are in the smoothER. Bacteria acyl-CoAdesaturase, have two cytochrome b5 reductases,one NADHwhich of dependentand the other NADPH-dependent; these is the main electron donor in vivo is unclear.In plants, oleate is producedby a stearoyl-AOPdesaturase in the chloroplaststroma that uses reduced ferredoxin as the electrondonor. Mammalianhepatocytescan readily introduce double bondsat the A'position of fatty acidsbut cannotintroduceadditionaldoublebondsbetweenC-10and the methyl-terminal end. Thus mammalscannot synthes?e or a-linolenate,18:3(Ae'12'15). linoleate,18:2(Ae'12), both; the desaturases synthesize can Plants,however, A12and A15positions at the bonds that introducedouble The ER enchloroplast. the ER and are Iocatedin the phospholipid, on a but fatty acids zymesact not on free that containsat least one oleate phosphatidylcholine, glycerol (Fig. 2f-f4). Both plants and linked to the bacteria must s1'nthesizepolyunsaturatedfatty acids to ensuremembranefluidity at reducedtemperatures. Becausethey are necessaryprecursorsfor the syrtthesisof other products,linoleateand a-linolenateare essential fatty acids for mammals;they must be obtained from dietary plant material. Once ingested,

,o S-CoA

saturated fatty acyl-CoA

2H2O + CH3-(CH2 )" -CItsC$-Monounsaturated fatty acyl-CoA

(CH2)- -C\

//O

(Fe3*)

(FADH,)

S-CoA

FIGURE 21-13 Electrontransferin the desaturationof fatty acids in vertebrates.Blue arrowsshow the path of electronsas two substrates-a fattyacyl-CoAand NADPH-undergo oxidationby molec-

take placeon the lumenalface of the ular oxygen.Thesereactions ocbut with differentelectroncarriers, smoothER.A similarpathway, c u r si n p l a n t s .

LttC

LipidBiosynthesis

In this chapterwe encounterseveralenz).rnesthat carry out oxidation-reductionreactionsin which molecular oxygenis a participant.The reactionthat introducesa doublebond into a fatty acyl chain (see Fig. 2t-13) is one suchreaction. The nomenclaturefor enz).rnesthat catalyzereactions of this generaltlpe is often confusingto students,as is the mechanismof the reactions.Oxidase is the general name for enz).rnesthat catalyzeoxidationstn which molecularoxygenis the electronacceptorbut oxygenatoms do not appearin the oxidizedproduct (however,there is an exceptionto this "rule,"aswe shallsee!).The enzyrne that createsa double bond rn fatty acyl-CoA during the oxidationof fatty acidsin peroxisomes (seeFig. lZ-13) is an oxidaseof this t;,pe; a secondexampleis the cytochrome oxidaseof the mitochondrialelectron-transfer chain (seeFrg. 19-14).In the first case,the transferof two electronsto H2Oproduceshydrogenperoxide,H2O2; in the second,two electronsreduce|O, to H2O.Many, but not all, oxidasesare flavoproteins. Oxygenases catalyzeoxidative reactions in which oxygen atoms are directly incorporated into the substrate molecule,forming a new hydroxyl or carboxyl group, for example.Dioxygenases catalyzereactions in which both oxygenatomsof 02 are incorporatedinto the organicsubstratemolecule.An exampleof a dioxygenaseis tryptophan2,3-dioxygenase, which catalyzes the openingof the five-memberedring of tryptophan in the catabolismof this amino acid. When this reaction takesplacein the presenceof 1802,the isotopicoxygen atomsare found in the two carbonylgroups of the product (shownin red). NH, I

Tryptophan

^l

(-.1.; :

I tI

trtr, rl,.ilL

2.:l (Ljo\\gcI)iLsf ]

J itu"

I -cHr-CH-COO

reductantsof the two oxygen atoms of 02. The main substrateacceptsone of the two oxygenatoms,and a cosubstratefurnishes hydrogen atoms to reduce the other oxygenatom to H2O.The generalreactionequation for monooxygenases is AH + BH2+ O-O--+A-OH + B + HrO where AII is the main substrateand BH2the cosubstrate. Becausemost monooxygenasescatalyzereactions in which the main substrate becomeshydroxylated, they are also called hydroxylases. They are also sometimes called mixed-function oxidases or mixed-function oxygenases, to indicate that they oxidize two different substratessimultaneously.(Note here the use of "oxidase"-a deviationfrom the generalmeaningof this term noted above.) There are dj-fferentclassesof monooxygenases, depending on the nature of the cosubstrate.Someuse reduced flaun nucleotides(FMNH2or MDH2), others use NADH or NADPH, and still others use a-ketoglutarate as the cosubstrate.The enz;'rnethat hydro:rylatesthe phenyl ring of phenylalarrineto fom tyrosineis a monooxygenasefor which tetrahydrobiopterin servesascosubstrate (seeFig. 18-23).Thisis the enzl'rnethat is defectivein the humangeneticdiseasephenylketonuria. The most numerousand most complexmonooxygenationreactionsare those employinga type of heme protein calledcytochrome P-450. This cytochromeis usuallypresentin the smoothER rather than the mitochondria.Like mitochondrialcytochromeoxidase,cytochrome P-450 can react with 02 and bind carbon monoxide,but it can be differentiatedfrom cy[ochrome oxidasebecausethe carbonmonoxidecomplexof its reduced form absorbslight strongly at 450nm-thus the nameP-450. CytochromeP-450 catalyzeshydroxylation reactionsin which an organicsubstrate,RH, is hydroxylated to R-OH, incorporatingone oxygen atom of 02; the other oxygenatomis reducedto H2Oby reducingequivalents that are furnished by NADH or NADPH but are usually passedto cy'tochromeP-450by an iron-sulfur protein. Figure 1 showsa simplifledoutline of the action of cy'tochromeP-450,which has intermediatestepsnot yet fully understood.

H NH-C\

RH

() N-Formylkynurenine

Monooxygenases,more abundantand more complex in their action, catalyzereactionsin which only one of the two oxygenatomsof 02 is incorporatedinto the organic substrate,the other being reduced to H2O. Monooxygenases require two substratestt_rserve as

o2 Hzo NADPT

FIGUR 1E

ROH

Acids andEicosanoids ofFatty 21.1Biosynthesis [ttN

CytochromeP-450is actuallya family of similar proteins; severalhundredmembersof this protein family are knov,n,eachwith a different substratespeciflcity.In the adrenal cortex, for example, a specific cytochrome P-450 participatesin the hydroxylationof steroidsto yield the adrenocorticalhormones(see Fig. 2l-46). Cytochrome P-450 is also important in the hydroxylation of many different drugs, such as barbiturates and other xenobiotics(substances foreignto the organism), particularly if they are hydrophobicand relatively insoluble. The environmental carcinogenbenzo[o]pyrene (found in cigarette smoke) undergoes cytochrome

o CH2-O-C

CH-

o rtl

cHr-o-ii-o-cH,

Phosphatidylcholinecontaining oleate,18:1(Ae)

A desatulase

-cH, -fr(cHr),

linoleatemay be convertedto certain other pollunsaturated acids, particularly 7-linolenate,eicosatrienoate, all of which can and arachidonate(eicosatetraenoate), be madeonly from linoleate (FLg.2l-12). Arachidonate, is an essentialprecursorof regulatory 20:4(45'8'11'1n1, lipids, the eicosanoids.The 20-carbonfatty acids are synthesizedfrom linoleate (and a-linolenate) by fatty acid elongationreactionsanalogousto those described on page814.

from20-Carbon AreFormed Eicosanoids Acids Fatty Polyunsaturated

j

cH2-o

o L? CH-O-Ci

CHr-O

I

CH - O

C O

P-450-dependenthydroxylationduring detoxification. Hydroxylation of xenobioticsmakesthem more soluble in water and allows their excretion in the urine. Unfortunately, hydroxylation of some compoundsconverts them to toxic substances,subvertingthe detoxiflcation system. Reactionsdescribedin this chapterthat are catalyzed oxidasesare those involved in fatty mixed-function by (Fig.21-13),Ieukotrienesynthedesaturation acyl-CoA plasmalogen synthesis(Fig. 21-30), (Fig. 21-16), sis (Fig. 2l-37), and to cholesterol of squalene conversion (FiC. 2l-46). synthesis steroidhormone

Phosphatidylcholine c,ontaining or-linolenate, 18'3(Ae'12'15)

in plants Desaturases 21-14 Action of plant desaturases. FIGURE fatty oleateto polyunsaturated oxidize phosphatidylcholine-bound fromthe phosphatidylcholine arereleased acids.Someof the products bv hvdrolvsis

Eicosanoidsare a family of very potent biologicalsignaling moleculesthat act as short-rangemessengers, affectingtissuesnear the cells that produce them. In responseto hormonal or other stimuli, phospholipase 42, present in most types of mammaliancells,attacks membranephospholipids,releasingarachidonatefrom the middle carbonof glycerol.Enzymesof the smooth ER then convert arachidonate to prostaglandins, beginning with the formation of prostaglandin H2 (PGH2), the immediate precursor of many other prostaglandinsand of thromboxanes(Fig. 2l-l5a). The two reactionsthat lead to PGH2are catalyzedby a bifunctional enzyme, eyclooxygenase (COX), also calledprostaglandin H2 synthase. In the first of two activity introducesmolecusteps,the cyclooxygenase Iar oxygento convert arachidonateto PGG2.The second step, ca|alyzedby the peroxidaseactivity of COX, convertsPGG2to PGH2. Mammalshave two isozymesof prostaglandinH2 s)'nthase,COX-I and COX-2.Thesehavedifferentfunctions but closelysimilaramino acid sequences(60% to 65% sequenceidentity) and similar reaction mechanisms at both of their catalytic centers.COX-1is responsiblefor the synthesisof the prostaglandinsthat regulatethe secretionof gastricmucin, and COX-2for the prostaglandinsthat mediate inflammation, pain, and fever.

rk'4 L i p i dB i o s y n t h e s i s o .ro-c...

Phospholipid containing arachidonate ' 'r' '1 r - t

CH,

II

Acetylated, inactivated COX

Lysophospholipid

l\

I

'\

-/coo-

+

+

Arachidonate, 20:4(45

8'r1'14)

o-c'

o //-

cooI 2"'-oE

ttl

CH3 ( \ iiLrr\!( illl:(, r ( L \ i t \ o l( O \

2 ---

aspirin,ibuprofen

Aspirin (acetylsalicylate)

Salicylate

CH. I

CH. CH"

.;( I

o

CHz

I

CH

CH

cf,\coo

cH3coo

Ibuprofen

Naproxen

(b)

,/ Other prostaglandins

Thromboxanes

(a) FIGURE 2l-15 The "cyclic', pathwayfrom arachidonateto prostaglandins and thromboxanes.(a) After arachidonateis releasedfrom phospholipids by the actionof phospholipase 42, the cyclooxygenase andperoxidase activities of COX(alsocalledprostaglandin H, synthase) catalyzethe productionof pCH2,the precursor of other

Rofecoxib (Vioxx)

prostaglandins (b)Aspirininhibitsthefirstreaction andthromboxanes. by acetyiating an essential Serresidueon the enzyme.lbuprofenand naproxeninhibitthesamestep,probablyby mimickingthestructure of the substrateor an intermediatein the reaction.(c) COX-2-specific cyclooxygenase inhibitorsusedas painrelievers (seetext). Valdecoxib (Bextra)

Pain can be relievedby inhibiting COX-2.The first drug widely marketedfor this purposewasaspirin (acetylsalicylate; Fig.21-15b).The nameaspirin(from o for acetyl andsp,ir for Spi,rsaure,the Germanword for the salicylates prepared from the plant Spi,raea u|marza) appearedin 1899 when the drug was introduced by the Bayer company.Aspirin irreversiblyinactivatesthe cyclooxygenase activity of both COXisozymes, by acetylatinga Ser residueand blocking each enz),Tne's active site. The slnthesis of prostaglandinsand thromboxanesis thereby inhibited. Ibuprofen, another widely used ?Lonsteroidal a,ntienflammatorydrug (NSAID; Fig. 21-15b),inhibitsthe samepair of enzymes.However,the inhibition of COX-1can result in undesiredside effects including stomachirritation and more serious conditions. In the 1990s,fotlowing discoveryof the crystal structuresof COX-1and COX-2,NSAIDcompoundsthat

Celecoxib (Celebrex)

(c)

had a greater specificity for COX-2 were developed as advanced therapies for severe pain. Three of these drugs were approved for use worldwide: rofecoxib (Vioxx), valdecoxib (Bextra), and celecoxib (Celebrex) (Fig. 2l-l5c). Launched in the late 1990s, the new compounds were initially a success for the pharmaceutical firms that produced them. However, enthusiasm turned to concern as fleld repofis and clinical studies connected the drugs with an increased risk of heart attack and stroke. The reasons for the problems are still not clear, but some researchers speculated that the COX-2 inhibitors were altering the fine balance maintained between the hormone prostacyclin, which dilates blood vessels,prevents blood clotting, and is reduced by COX2 inhibitors, and the thromboxanes, produced on the

y c i dasn dE i c o s a n o i[dt ts t ] 2 1 . 1B i o s y n t h eosf Fi sa t t A

Arachidonate

rivn"ue.n,,-""71 O"

or\oo*r*""u'"

cooooH

12-Hydroperoxyeicosatetraenoate (12-HPETE)

coo

5-Hydroperoxyeicosatetraenoate (5-HPETE)

multistep /

/ Otherleukotrienes

\uri.r"o Leukotriene Aa (LTAa)

\ I,TCA FIGURE 21-16 The"linear" pathwayfrom arachidonate to leukotrienes.

pathway invoMng COX-I, that help to form blood clots. Vioxx was withdrawn from the market in2004, and Bextra was withdrawn soonafter.As of early 2007,Celebrex is still on the market but usedwith increasedcaution. Thromboxane s5rnthase,presentin bloodplatelets (thrombocytes),convertsPGH2to thromboxane42, from which other thromboxanes are derived (Frg. 21-15a). Thromboxanesinduce constriction of blood vessels and platelet aggregation,early stepsin blood clotting. Low dosesof aspirin,taken regularly,reducethe probability of heart attacksand strokesby reducingthromboxaneproduction.I Thromboxanes,Iike prostaglandins,containa ring of flve or six atoms;the pathwayfrom arachidonateto these two classesof compounds is sometimescalled the "cyclic" pathway,to distinguishit from the "linear" pathway that leads from arachidonateto the leukotrienes, which are linear compounds(Fig. 21-16). Leukotriene symthesisbeginswith the action of severallipoxygenases that catalyzethe incorporationof molecularoxygeninto arachidonate.Theseenz),rnes, found in leukocytesand in heart, brain, lung, and spleen, are mixed-function oxidasesthat use cytochromeP-450(Box 21-1). The various leukotrienes differ in the position of the peroxide group introduced by the lipoxygenases.This linear pathway from arachidonate,unlike the cyclic pathway,is not inhibited by aspirin or other NSAIDs. Plantsalsoderiveimportant signalingmoleculesfrom fatty acids.As in animals,a key step in the initiation of signalinginvolvesactivationof a speciflcphospholipase. In plants, the fatty acid substratethat is releasedis aIinolenate.A lipoxygenasethen catalyzesthe first step in a pathway that convertslinolenateto jasmonate,a substanceknown to havesignalingrolesin insectdefense,resistance to fungal pathogens, and pollen maturation. Jasmonate(seeFig. 12-32) alsoaffectsseedgermination, root growth, and fruit and seeddevelopment.

\ LTD4

y cids i sa t t A 21 SUMMAR Y. 1 B i o s y n t h eosf F a n dE i c o s a n o i d s r

r

r

r

r

Long-chainsaturatedfatty acidsare synthesized from acetyl-CoAby a cytosolic systemof six enzymaticactivitiesplus acyl carrier protein (ACP). There are two types of fatty acid synthase. FASI, found in vertebratesand fungi, consistsof multifunctional polypeptides.FASII is a dissociated systemfound rn bacteria and plants. Both contain two t),pesof -SH groups (one furnished by the phosphopantetheineof ACP,the other by a Cys residue of B-ketoacyl-ACPslmthase) that function as carriers of the fatty acyl intermediates. Malonyl-ACP,formed from acetyl-CoA(shuttled out of mitochondria) and CO2,condenseswith an acetyl bound to the Cys-SH to yield acetoacetyl-ACP, with releaseof CO2.This is followedby reduction to the >B-hydroxy derivative,dehydrationto the tra, ts-L" -tnsaturatedacyl-ACP,and reduction to butl'nyl-ACP.NADPHis the electron donor for both reductions.Fatty acid sy'nthesisis regulatedat the level of malonyl-CoAformation. Six more moleculesof malonyl-ACPreact successivelyat the carboxyl end of the growing fatty acid chain to form palmitoyl-ACP-the end product of the fatty acid symthasereaction.Free palmitate is releasedby hydrolysis. Palmrtatemay be elongatedto the 18-carbon stearate.Palmitateand stearatecan be desaturated to yield palmitoleateand oleate,respectively,by the action of mtxed-functionoxidases. Mammalscannot make linoleate and must obtain it from plant sources;they convertexogenous linoleateto arachidonate,the parent compoundof

F"l

L i p i dB i o s y n t h e s i s

eicosanoids(prostaglandins, thromboxanes,and Ieukotrienes),a family of very potent signaling moiecules.The synthesisof prostaglandins and thromboxanesis inhibitedby NSAIDsthat act on the cyclooxygenaseactivity of prostaglandinH2 synthase.

21.2Biosynthesis ofTriacylglycerols

Glucose qlvcolvsrs

v

CH2OH

t-t-

C:O

CH'OH

O

CHOH

till cH2-o-P-o-l

cH2oH

Dihydroxyacetone Ophosphate

Most of the fatty acids synthesized or ingested by an organism have one of two fates: incorporation into triacylglycerols for the storage of metabolic energy or incorporation into the phospholipid components of membranes. The partitioning between these alternative fates depends on the organism's current needs. During rapid growth, synthesis of new membranes requires the production of membrane phospholipids; when an organism has a plentiful food supply but is not actively growing, it shunts most of its fatty acids into storage fats. Both pathways begin at the same point: the formation of fatty acyl esters of glycerol. In this section we examine the route to triacylglycerols and its regulation, and the production of glycerol 3-phosphate in the process of glyceroneogenesis.

--')

Glycerol I

a-Ketoglutarate l-glutamate

+ NADP* + H2O

We encountered this reaction in the catabolism of aminoacids(seeFig. 18-7). In eukaryoticcells,L-glutamate dehydrogenaseis located in the mitochondrial matrix. The reaction equilibrium favors the reactants, and the K^for NHf (-1 mvr)is so high that the reaction probably makes only a modest contribution to NHf, assimilation into amino acids and other metabolites. (Recall that the glutamate dehydrogenasereaction, in reverse(seeFig. 18-10),is one sourceofNHl destined for the urea cycle.) Concentrationsof NHf, high enough for the glutamate dehydrogenasereaction to make a significant contribution to glutamate levels generally occur only when NH3 is added to the soil or when organisms are grown in a laboratory in the presence of high NH3 concentrations.In general,soil bacteriaand plants rely on the two-enzymepathway outlined above (Eqns22-1,22-2).

lsa Primary Regulatory Synthetase 6lutamine Metabolism inNitrogen Point The activity of glutamine symthetaseis regulatedin virtually all organisms-not surprising, given its central metabolicrole as an entry point for reducednitrogen. In enteric bacteria such as,O.col'i, the regulationis unusually complex. The enzymehas 12 identical subunits of

i sm i nA o c i d sN,u c l e o t i daensdR , e l a t eMdo l e c u l e s L B s B _ B i o s y n t h eosf A M. 50,000 (Fig. 22-5) and is regulated both allosterically and by covalent modiflcation. Alanine, glycine, and at least six end products of glutamine metabolism are allosteric inhibitors of the enzyme (Fig. 22-6). Each inhibitor alone produces only partial inhibition, but the effects of multiple inhibitors are more than additive, and all eight together virtually shut down the enzyme. This control mechanism provides a constant adjustment of glutamine levels to match immediate metabolic requirements. Superimposed on the allosteric regulation is inhibition by adenylylation of (addition of AMP to) T\'r3e7,located near the enzyme's active site (Fig. 22-7). This covalent modification increases sensitivity to the allosteric inhibitors, and activity decreases as more

Glutamate

lt lv l tl

AMP -Glutamine Tryptophan

+

CTP Histidine

/\ Carbamoyl phosphate

Glucosamine 6-phosphate

FIGURE 22-6 Allostericregulationof glutaminesynthetase. The enzyme undergoescumulativeregulationby six end productsof glutaminemetabolism. Alanineand glycineprobablyserveas indicators of thegeneralstatusof aminoacidmetabolism in the cell.

FIGURE 22-5 Subunitstructureof glutaminesynthetase as determinedby x-raydiffraction.(PDBlD 2CLS)(a) Sideview.The 12 subunitsareidentical; theyaredifferently coloredto illustrate packingand placement.(b) Topview, showingactivesites(green).

subunits are adenylylated. Both adenylylation and deadenylylation are promoted by adenylyltransferase (AT in Fig. 22-T), part of a complex enzyrnatic cascadethat respondsto levelsof glutamine,a-ketoglutarate, ATP, and P1.The activity of adenylyltransferase is modulated by binding to a regulatory protein called Py1, and the activity of Pn, in turn, is regulatedby covalent modification (uridylylation), again at a Tyr residue.The adenylyltransferase complex with uridylylated Prr (Prr-UMP) stimulates deadenylylation, whereas the same complex with deuridylylated Pn stimulates adenylylationof glutamine synthetase.Both uridylylation and deuridylylation of P11zr€ brought about by a single enzyme, uridylyltransferase. Uridylylation is inhibited by binding of glutamine and P1to uridylyltransferaseand is stimulated by binding of a-ketoglutarateand ATP to P1. The regulationdoesnot stop there. The uridylylated P1 also mediates the activation of transcription of the gene encodingglutamine synthetase,thus increasing the cellular concentrationof the enzyme;the deuridylylated P1 brings about a decreasein transcription of the same gene. This mechanisminvolves an interaction of Prrwith additional proteins involved in gene regulation, of a type describedin Chapter28. The net result of this elaboratesystem of controls is a decreasein glutamine slmthetaseactivity when glutamine levels are high, and an increasein activity when glutaminelevelsare low and a-ketoglutarateand ATP (substratesfor the sy'nthetase reaction) are avai-lable. The multiple layers of regulation

2 2 . 10 v e r v i e w n etabolism 859 | o f N i t r o g eM

FIGURE 22-7 Secondlevel of regulation of glutamine synthetase: Tyr residue.(b) Cascade covalentmodifications.(a) An adenylylated (inactivation) leadingto adenylylation of glutaminesynthetase. AT represents adenylyltransferase; Uf uridylyltransferase Detailsof this cascadeare discussedin the text.

(b) a-Ketoglutarate

Glutamate

'--

S 'ffi-'s

\\

@

-1

el

--./

)

/ //

\ -(

. \\ /

AMP

Glutamine

permit a sensitiveresponsein which glutamine slnthesis is tailored to cellular needs.

Several Classes ofReaetions Play Special Roles inthe Biosynthesis ofAmino Acids andNutleotides The pathwaysdescribedin this chapterinclude a variety of interesting chemical rearrangements.Several of theserecur and deservespecialnote beforewe progress to the pathwaysthemselves.Theseare (1) transamination reactionsand other rearrangementspromotedby enzymescontaining pyridoxal phosphate; (2) transfer of one-carbon groups, with either tetrahydrofolate (usuallyat the -CHO and -CH2OH oxidationlevels) or S-adenosylmethionine(at the -CHe oxidation level) as cofactor; and (3) transfer of amino groups derived from the amide nitrogen of glutamine. Pyridoxal phosphate (PLP), tetrahydrofolate (Ha folate), and (adoMet)are describedin some S-adenosylmethionine detailin Chapter18 (seeFigs 18-6,l8-17, and 18-18). Here we focus on amino group transfer involving the amide nitrogen of glutamine.

More than a dozen known biosyntheticreactions use glutamine as the major physiologicalsource of amino groups,and most of these occur in the pathways catalyzoutlined in this chapter.As a class,the enzSrmes ing these reactionsare called glutamine amidotransferases. All have two structural domains:one binding glutamine, the other binding the second substrate, which selves as amino group acceptor (Fig. 22-8). A conseryedCys residuein the glutamine-bindingdomain is believedto act asa nucleophile,cleavingthe amidebond of glutamine and forming a covalent glutamyl-en4.rne intermediate. The NH3 produced in this reaction is not released,but instead is transferred through an "ammonia channel"to a secondactive site, where it reactswith the secondsubstrateto form the aminatedproduct. The covalentintermediate is hydrolyzedto the free enzyme and glutamate.If the second substratemust be activated,the usualmethodis the use of ATPto generatean acyl phosphateintermediate(R-OX in Fig. 22-8,wlth X as a phosphorylgroup). The enz;'rneglutaminaseacts in a similar fashion but uses H2O as the secondsubstrate, yielding NHf, and glutamate (see Fig. 18-8).

B60

B i o s y n t h eosfAi sm i n A o c i d sN,u c l e o t i daensdR , e l a t eMdo l e c u l e s

bacteria and in syrnbioticbacteria in the root nodulesof leguminousplants.

NHgacceptor domain

Glutaminebinding domain

r

The nitrogen cycle entailsformation of ammonia by bacterialfixation of N2,nitrification of arrunonia to nitrate by soil organisms,conversionof nitrate to arrunoniaby higher plants, synthesisof amino acids from ammoniaby all organisms,and conversionof nitmte to Nz by denitrifying soil bacteria.The ananunoxbacteria anaerobicallyoxidize anunonia to nitrogen,usingnitrite as an electronacceptor.

r

Fixation of N2 as NH3is carried out by the nitrogenasecomplex,in a reactionthat requires ATP.The nitrogenasecomplex is highly labile in the presenceof 02.

r

In living systems,reducednitrogen is incorporated first into amino acidsand then irto a variety of includingnucleotides.The key other biomolecules, entry point is the amino acid glutamate.Glutamate and glutamineare the nitrogen donorsin a wide range of biosynthetic reactions.Glutamine slrrthetase,which catalyzesthe formation of glutaminefrom glutamate,is a marnregulatory enzyrneof nitrogen metabolism.

r

The amino acid and nucleotidebiosynthetic pathwaysmake repeateduse of the biological cofactorspyridoxalphosphate,tetrahydrofolate, Pyridoxal phosphateis and S-adenosylmethionine. required for transaminationreactionsinvohrng glutamateand for other amino acid transformations. One-carbontransfersrequire S-adenosylmethionine and tetrahydrofolate.Glutamineamidotransferases catalyzereactionsthat incorporatenitrogen derivedfrom glutamine.

coo-

rl H3N-CH

I CHr

t-

^H* UYSGlutamine Glutamine amidotransferase The 7-amido nitrogen of glutemine (red) is released as NH3 in a reaction that proiably involves a covalent O gluta myl-enz;rme intermediate. The NHs travels through a channel to the secondactive site.

g1

7R-OH

\

or

,rC:O p2 Acceptor

Activated /substrate

*l

cooR-OX

H3N-cH CHO

Cvs-S-CO

tcH" t' o

*1"'

,.,-o*n' /

,rc:o

Rz

Glutamyl-enz5rme interrnediate NHs reacts with ary of several acceptors.

Acids 22.2Biosynthesis ofAmino -or RI

C:NH

+ H"O

g2 It4[(HANlsFrt FI6URE ?2-8 Proposedmechanismfor glutamineamidotransferases. Eachenzyme has two domalns The glutamine-binding domaincontainsstructural elements conserved amongmanyof these enzymes/includinga Cys residuerequiredfor activity The NH3acceptor(second-substrate) domainvaries. Twotypesof aminoacceptors are shown. X represents an activatinggroup,typically a phosphoryl group derivedfrom ATP,that facilitatesdisplacementof a hydroxyl groupfrom R-OH by NH3.

S U M M A R2Y2 . 1 0 v e r v i e o wf N i t r o g e n Metabolism r

The molecular nitrogen that makes up 80% of the earth's atmosphere is unavailable to most living organisms until it is reduced. This fixation of atmospheric N2 takes place in certain freeJiving

All amino acids are derived from intermediates in glycolysis, the citric acid cycle, or the pentose phosphate pathway (Fig. 22-9). Nitrogen enters these pathways by way of glutamate and glutamine. Some pathways are simple, others are not. Ten of the amino acids are just one or several steps removed from the common metaboIite from which they are derived. The biosynthetic pathways for others, such as the aromatic amino acids, are more complex. Organisms vary greatly in their abiJity to synthesize the 20 corrrmon amino acids. Whereas most bacteria and plants can synthesize all 20, mammals can synthesize only about half of them-generally those wrth simple pathways. These are the nonessential amino acids, not needed in the diet (see Table 18-1). The remainder, the essential amino acids, must be obtained from food. Unless otherwise indicated, the pathways for the 20 common amino acids presented below are those operative in bacteria. A useful way to organize these biosSmthetic pathways is to group them into six families correspondhg

F'!

2 2 . 2B i o s y n t h eosfA i sm i nA ocids

Glucose 6-

c-Ketoglutarate Glutamate Glutamine Proline Arginine

Pyruvate Alanine Valine* Leucine* Isoleucine*

3-Phosphoglyeerate Serine Glycine Cysteine

Phosphoenolpynrvate and erythrose 4-phosphate

Oxaloacetate Aspartate Asparagine Methionhe* Threonine* Lysine*

Phenylalanine* ryrosme

Thmf nnh an tt J yuvytLuL

*

Ribose 5-phosphate Histidine*

*Essential aminoacids lDerived fromphenylalanine in mammals.

Oxaloacetate

r"'""t\

KetoElutarate

x-.-/

PRPPis symthesized from ribose 5-phosphatederived from the pentosephosphatepathway (see Fig. 14-21), in a reaction catalyzed by ribose phosphate pyrophosphokinase: * ATP -> Ribose5-phosphate + AMP 5-phosphoribosyl-1-pyrophosphate This enzymeis allostericallyregulated by many of the biomoleculesfor which PRPPis a Drecursor.

Rise toGlutamate, {ry-Ketoglutarate 6ives Proline, Glutamine, andArginine

tIGURE 22-9 Overviewof aminoacid biosynthesis. Thecarbonskeleton precursors derivefrom threesources: glycolysis(pink),the citric acidcycle(blue),and the pentosephosphate pathway(purple).

to their metabolic precursors (Table 22-l), and we use this approachto structurethe detaileddescriptions that follow. In addition to these six precursors,there is a notable intermediate in severalpathways of amino acid and nucleotide synthesis: 5-phosphoribosytl-pyrophosphate (PRPP):

o

We have already described the biosynthesisof glutamate and glutamine. Proline is a cyclized derivative of glutamate(Fig. 22-f0). In the first step of proline synthesis,ATP reacts with the 7-carboxylgroup of glutamateto form an acyl phosphate,which is reducedby NADPH or NADH to glutamate 7-semialdehyde.This intermediateundergoesrapid spontaneouscyclization and is then reducedfurther to yield proline. Arginine is synthesizedfrom glutamate via ornithine and the urea cycle in animals(Chapter 18). In principle,ornithine could alsobe synthesizedfrom glutamate 7-semialdehydeby transamination, but the spontaneouscyclization of the semialdehydein the proline pathway precludesa sufficient supply of this

[ta{

Biosynthesis Molecules of Amino Acids, Nucleotides, andRelated

o I

CoA-SH

o.

HN-C-CH3

o" a

NHt

\\l

-o/c-cH2-cH2-cH-coo-

*tylgfita6"i"

"y"th*

cH-coo

/c-c}l2-cll2

o

N-Acetylglutamate

Glutamate

t "' t'*",lll]ll:: o

+

o.

NH'

\l

o

/c-c}l2-c}l2-cH-coo€,H

7-Glutamyl phosphate

I

7-glutamyl phosphate reductase

il HN-C-CH3 \\1

/ -C-CH,-CH9-CH-COO_

@-o

.]*r.

H*

\Nen(p)*

NAD(P)*

\pr

B

,+

o t1

T"'

o.\\ H

N-Acetyl-7-glutamyl phosphate

I .l nonenzymanc I J

HN-C-CH3

o.

/c-cll2-cHz-cH-cooGlutamate 7-semialdehyde

)-.",-"",-8"-.oo-

H

N-Acetylglutamate 7-semialdehyde

aminotransferase

a-Ketoglutarate

o HN-C-CH3

cH2-cH2-cH2-cH

coo

pp,

t-

OH I

.,,,,',. kH,C-C

2

r r c c t rl t r l n s l i , r ' l . c I

J'"coA-sH *l

S-CoA

coo-

l CHt

ll

Adenosine 5'-phosphosulfate (APS)

o-o

L-ATP J-ADP

I CH, I C:O

oo - o - sl t-tol - P - o -

OH OH

H3N-C-H

to

describedlater) from environmentalsulfates;the pathway is shown on the right side of Figure 22-13. Sulfate is activatedin two stepsto produce3'-phosphoadenosine 5'-phosphosulfate(PAPS),which undergoesan eightelectron reduction to sul-flde.The sulfldeis then used in the formation of cysteinefrom serinein a two-steppathway.Mammalssymthesize cysteinefrom two aminoacids: methionine furnishes the sulfur atom, and serine furnishesthe carbonskeleton.Methionineis first converted (see Fig. 18-18), which can to ,S-adenosylmethionine Ioseits methyl group to any of a number of acceptorsto (adoHcy). This demethyform S-adenosylhomocysteine lated product is hydrolyzedto free homocysteine,which urLdergoesa reaction with serine, catalyzedby cystathionine B-synthase, to yield cystathionine(Fig. 22-14). Finally, cystathionine y-lyase, a PlP-requiring enzyme, catalyzesremoval of ammonia and cleavageof cystathionineto yield free cysteine.

O-Acetylserine

o - c H, .H

ooH I - o- P:o I o-

3'-Phosphoadenosine 5'-phosphosulfate (PAPS)

nosine 5'-phosphate (PAP)

tlGURt22-13 Biosynthesis of cysteinefrom serinein bacteriaand plants.The originof reducedsulfur shownin the pathwavon the rieht,

22.2Biosynthesis ofAmino AcidsIoos ]

-ooc-cH-cH2-cH2-SH

Nlt, + HOCH2-Jn-COO-

ffi Homocysteine

Serine

o,stathionino /j srrrthase Birrg .t

-ooc-cH-cH2 -cH2 -

frtt.

r-a", -Jr-coo -

ffi Cystathionine

NH.

-ooc-c-cH2-cH3

t"

+ HS-CH2-CH-COO

o a-Ketobutyrate FIGURE22-14Biosynthesis ofcysteinefrom homocysteine and serine in mammals.The homocysteine is formedfrom methionine,as describedin the text

Three Nonessentialand Six[ssential Amino Acids AreSynthesized fromOxaloaeetate andPyruvate Oxaloacetate

I l-A'j"'tuIJl .''l\-\

lA.p"."st""l I M"tht".t.-l lLy.t"t I Th'"""l";l

lAr,"t"t \\/

(sreps @ to @1. lsa Keylnterrnediate intheSynthesis Ihnrismate Phenylalanine, ofTryptophan, andTyrosine

lv"u.-| |h".t""l #

;# "-'

Alanine and aspartate are synthesizedfrom pyruvate and oxaloacetate,respectively,by transaminationfrom glutamate.Asparagine is slmthesizedby amidation of aspartate,with glutaminedonatingthe NHf,. Theseare nonessentialaminoacids,and their simplebiosynthetic pathwaysoccurin all organisms. For reasonsincompletelyunderstood,the maligffi nant l;rrnphocytespresentin childhoodacute15.'rnIL phoblasticleukemia (ALL) require serurnasparaginefor growth. The chemotherapy for ALL is administered Eil

together wlth an t -asparaginasederived from bacteria, with the enz).rnefunctioningto reduceserurnasparagine. The combinedtreatmentresultsin a greaterthan 95%remissionrate in casesof childhoodALL (l-asparaginase treatment aloneproducesremissionin 40o/oto 60% of cases).However,the asparaginase treatmenthas some deleteriousside effects,and about I0o/oof patientswho achieveremissioneventuallysuffer relapse,with tumors resistant to drug therapy.Researchersare now developing inhibitors of human asparaginesy'nthetaseto augment thesetherapiesfor chjldhoodAl,L. r Methionine, threonine, lysine, isoleucine,valine, and leucineare essentialaminoacids.Their bios;,nthetic pathwaysare complexand intercomected (Fig. 22-15). In some cases,the pathways in bacteria, fungi, and plants differ significantly.Figure 22-15 showsthe bacterial pathways. Aspartate gives rise to methionine, threonine, and lysine. Branch points occur at aspartate Bsemialdehyde,an intermediate in all three pathways, and at homoserine,a precursor of threonine and methionine. Threonine,in turn, is one of the precursors of isoleucine.The valine and isoleucine_pathways share four enzymes(Fig. 22-15, steps @ ,o @l Pyruvate gives rise to valine and isoleucinein pathways that begin with condensationof two carbonsof pyruvate (in the form of hydroxyethyl thiamine pyrophosphate;seeFig. 14-14) with anothermoleculeof pyruvate(the valinepath) or with a-ketobutyrate(the isoleucinepath). The a-ketobutyrate is derived from threonine in a reaction that requires pyridoxal phosphate (Fig. 22-75, step Q!). An intermediate in the valinepathway,a-ketoisovalerate,is the starting point for a four-step branch pathway leading to leucine

Aromatic rings are not readily availablein the environment, even though the benzenering is very stable.The branchedpathwayto tryptophan, phenylalanine,and tyrosine, occurringin bacteria,fungi, and plants, is the main biologicalroute of aromatic ring formation. It proceedsthrough ring closureof an aliphatic precursor folIowed by stepwiseaddition of double bonds.The flrst

[tta]

Biosynthesis of Amino Acids, Nucleotides, andRelated Molecules

o

NH.

\\l

Aspartate

FIGURE 22-15 Biosynthesis of six essentialamino acidsfrom oxaloacetate and pyruvate in bacteria: methionine, threonine, lysine, isoleucine,valine,and leucine.Here,and in othermultisteppathways, the enzymesare listedin the key.Note that r,l-a,e-diaminopimelate, the productof step@ is symmetric.The carbonsderivedfrom pyruvate(andthe amino groupderivedfrom glutamate)are not tracedbeyond this point, becausesubsequentreactionsmay place them at eitherend of the lysinemolecule.

-o/C-CH2-CH-COO-

NHe f Threoninel

ott

t/\

//

q?

oH

T"'

Aspartyl-B-phosphate

NHi + nrO rLr

CH3-CH-CH-COO

|,--P,

/C-CH2-CH-COO-

@-o

@[""" N

l- "'o

@ NA.DP*

phosphohomoserine

@-o-crrr-a"r-f;1"oo-

R

1H'

CH2-CIL-CH-COO-

Homoserine

OH

^l (10)ls vl

Z'-Lu tt"

,^ f- Succinrl-Coa \9/ t coe J*

H"o

frn,

H + H-

NADP'

-oocAN*5oo-

,/

q,

Dihydropicolinate

CH2-CH2-CH-COO-

-oocScoo

O-Succinylhomoserine

O-Succinate

A 1-Piperidine-2,6dicarboxylate Succinyl-CoA - }Izo

^ ,( 1 2 )I

-

f- coe .-\

Cystathionine

ll

-ooc^o;f,cooI

Succinate

nl-J-

(14)l

*l

coo-

*l

coo-

coo-

H3N-C-H --

^---+

n-C-frn" qy

r,,r,-a,e-Diaminopimelate

Homocysteine

^ lr- aF-u"tfrrl I{o folate

ttt""tu*

tcoo-

T",

HS-CH2-CHr-CH-COO

F'ro

H3N-C-H I (qHr)3

+ NH3

N-Succinyl-2-amino6-keto-l-pimelate

N-Succinyl-r,,r,a,e-diaminopimelate

*

@r Ha folate f+

Ng,

(tHz)s

cH3-s-cH2-cH2-cH-coo

H3N-C-H

coomzso-a,e-Diaminopimelate

*NHa

lLy.t*l

lM"tltt""t""l

-

o c i d s| 8 6 7 | 2 2 . 2B i o s y n t h eosfAi sm i n A

O @

aspartokinase aspartate p-semialdehyde dehydrogenase homoserine dehydrogenase

cH3-e-coo

homoserine kinase synthase threonine @ @ homoserine acyltransferase

Pyruvate

A

cystathionine 7-synthase cystathionine B-lyase

^ITPP

(9)- co,

@ methionine s5'nthase @ dihydropicolinate synthase

f-_-1

cru-t*rPP I I

@ l1-pipericline-2,6-dicarboxylate dehydrogenase @ N-succinyl-2-amino-6-ketopimelate slmthase @ succinyl diaminopimelate aminotransferase

LOH] ------

cH3-cH2-c-coo I o

cHS-c-coo o

a-Ketobutyrate

@ succinyl diaminopimelate desuccinylase @ diaminopimelate epimerase

Pyruvate

( 1 8)

@ diaminopimelate decarboxylase (serine dehydratase) @ threonine dehydratase

@

6B) acetolactate s).nthase @ acetohydroxy acid isomeroreductase

CH.

a-Aceto-ahydroxybuty'r'ate

t"

CH$-f-?-COO

a-Acetolactate

ooH

I @l I

@ ainya"ory acid dehydratase @ va[ne aminotransferase synthase @ o-isopropylmalate @) isopropylmalate isomerase @ F-i.onronvl-alate dehydrogenase @ leucine aminotransferase

t ?",

| "e-?-f-.oo oHo L

CH

COO C-CHz-COO

CH3-CH

a-Isopropvlmalate

OH CH"

^l

I

?"' T Cl{a-d-i-g6g-tl

a,B-DihydroxyF-methVlvalerate

o,B-Dihydroxyisovalerate

OH OH

(23)I cH3 cooCH3-CH-CH

^l

CH-COO

B-Isopropvlmalate

OH

Q9f'H'o

?'. ?", CHs-q-q-COO ttl HO

d-KeLo-pmethYlvalerate

Il . -

@(,,, t\ |

---)

Y

CH CH,

CH,, CH

C

COO

o

ate

CH.

t" tcr{3*cI{-cH-coo CHO

lI."t"*t"a

CII,] ilIH3 CH3-CH

CIH CIOO

lv''l"El

@ gH.

*", cH3-cH clHr--cH coo

tl,"*-t.*l

n-KeioisocaProate

i sm i nA o c i d sN,u c l e o t i daensdR , e l a t eMdo l e c u l e s L B 6 B _ j B i o s y n t h eosf A

(o

3-Dehydroshikimate

Y

^ f- NelrH

o\ /coo-

-

C

Phosphoenolpyruvate (PEP)

ll

CHz

+H*

(4)t

F *oo"*

coo-

o.H \// C I QHOH I CHOH

@ Z-t "to-e-aeoxy-o-arabinoheptulosonate 7-phosphate s1'nthase d"hydroqoinate synthase @

Shikimate

Erythrose 4-phosphate

t^ cH,-o-{D

J.

@ S-d"hya.oquinate dehydratase @ shikimate dehydrogenase @ shikimate kinase

J-

@ s-enolpyrunylshikimate S-phosphate syntnase

a;\ r \9/ L

I

^lrHro

ul, J'"'

@ chorismate synthase

o. coo *c' I

Shikimate 3-phosphate

CHq

lHO-C-H

^l ,r,

(91, J-Pt

2-Keto-3-deoxy-oarabinoheptulosonate 7-PhosPhate

| H-C-OH n-d-on

cHr-o-@

@-o

^ lr** (?1, J" P'

5-Enolpynrvylshikimate 3-phosphate

)

)H .

S-Dehydroquinate

coo

/a\ L oJ-t'o Chorismate

3-Dehydroshikimate

four stepsproduceshikimate,a seven-carbon molecule derived from erythrose 4-phosphate and phosphoenolpytuvate (Fig. 22-16). Shikimate is converted to chorismate in three steps that include the addition of three more carbonsfrom anothermoleculeof phosphoenolpyruvate.Chorismateis the fust branch point of the pathway, with one branch leading to tryptophan, the other to phenylalanineand tyrosine. In the trSrytophan branch (Fig. 22-17), chorismate is convertedto anthranilatein a reaction in which glutamine donatesthe nitrogen that will becomepart of the indole ring. Anthranilate then condenseswith PRPP.

FIGURE 22-16 Biosynthesis of chorismate, an intermediatein the synthesis of aromaticaminoacidsin bacteriaand plants.AII car(lightpurple) bonsarederivedfromeithererythrose 4-phosphate (pink) Notethatthe NAD" requiredas or phosphoenolpyruvate a cofactorin stepQ) isreleased unchanged; it maybetransiently reducedto NADH duringthe reaction,with formationof an oxidizedreactionintermediate. Step@ is competitively inhibited by glyphosate(-COO-CH2-NH-CH2-POj), the active ingredient in thewidelyusedherbicideRoundup. Theherbicide is relativelynontoxicto mammals,which lackthisbiosynthetic pathway.The chemicalnamesquinate,shikimate,and chorismatearederivedfrom the namesof plantsin whichtheseintermediates havebeenfoundto accumulate

The indole ring of tryptophan is derived from the ring carbonsand amino group of anthranilate plus two carbons derived from PRPP.The final reaction in the sequence is catalyzedby tryptophan synthase. This enzyrnehas an a2p2subunit structure and can be dissociatedinto two a subunits anda B2unitthat catalyzedifferent parts of the overall reaction: Indole-3-glycerol phosphate ;oo; indole + glyceraldehyde 3-phosphate Indole * serine ----------------+ tryptophan + H2O p2 suDunlt

2 2 . 2B i o s y n t h eosfAi sm i nA o c i d s[ t a t ]

anthranilate synthase anthranilate phosphoribosyltransferase N-(5'-phosphoribosyl)-anthranilate isomerase indole-3-glycerol phosphate synthase tryptophan sSmthase

cooAnthranilate

N{5'-Phosphoribosyll anthranilate

@'| coo

HO OH

c-c-cH2-o

HO-

tl HH H

@ -J [.

Enol- 1-o-carboxyphenylamino-1deoxyribulose phosphate H2O + CO2

OH I

Indole-3-glycerol phosphate

The secondpart of the reactionrequirespyridoxal phosphate (Fig. 22-18).Indole formed in the first part is not releasedby the enz),rne,but insteadmovesthrough a channelfrom the d-subunit active site to one of the Bsubunit active sites, where it condenseswith a Schiff base intermediate derived from serine and PLP. Intermediate channelingof this type may be a feature of the entire pathway from chorismateto tryptophan. Erz;,.rne active sites catalyzingdifferent steps (sometimesnot sequentialsteps) of the pathway to tryptophan are found on single polypeptidesin some speciesof fungi and bacteria,but are separateproteinsin other species. In addition, the activity of some of these enzyrnesrequires a noncovalentassociationwith other enzyrnesof the pathway.These observationssuggestthat all the pathway enzyrnesare componentsof a large, multienzyme complex in both bacteria and eukaryotes. Such complexesare generallynot preservedintact when the enzymes are isolated using traditional biochemical methods,but evidencefor the existenceof multienz;rme complexesis accumulatingfor this and other metabolic pathways(p.619). In plants and bacteria, phenylalanine and tyrosine are synthesizedfrom chorismatein pathwaysmuch less complex than the tryptophan pathway. The common htermediateis prephenate(Fig. 22-f 9). The final step in both casesis transaminationwith glutamate. Animals can produce tyrosine directly from phenylalanine through hydroxylation at C-4 of the phenyl group by phenylalanine hydroxylase; this enzyme also participates in the degradation of phenylalanine (seeFigs 18-23, 18-24). Tj,rosineis considereda conditionally essentialamino acid, or as nonessentialinsofar as it can be synthesizedfrom the essentialamino acid phenylalanine.

Uses Precursors Biosynthesis Histidine Biosynthesis ofPurine Ribose S-phosphate

j lHt.ttdt.-l FIGURE 22-17 Biosynthesis of tryptophanfrom chorismatein bacteria and plants. ln E. coli, enzymescatalyzingsteps@ and @ are subunits of a singlecomplex.

The pathway to histidine in all plants and bacteria differs in severalrespectsfrom other amino acid biosynthetic pathways. Histidine is derived from three precursors(Fig.22-20): PRPPcontributesfive carbons,

870

B i o s y n t h eosfAi sm i nA o c i d sN,u c l e o t i daensdR , e l a t eMdo l e c u l e s

cooCHO

il-

o-c-cooH Chorismate

OH OH cH-cH-cHr-o-@

Hr-C-COO

o

Indole-3-glycerol phosphate

Prephenate An aldol cleavage produces indole and glyceraldehyde 3-phosphate; PLP is not required.

NAD-:

oH

o-

**";';; -y bo,,Z@

\\t

H

/c-cH-cHr-o-{9 Glyceraldehyde 3-phosphate

o o" / \ cHr-c-coo

\@\ co'- ou cHr-c-coo

Indole traverses tunnel between a and B subunits.

OH

ilrH. Dehvdration of

r r \ r ' r' l ' r " r '

Sgflne

tj sul,rrDrrs

IOfInS

I

PlP-aminoacrylate intermediate.

-cH,-cH-coo"l

t-

OH

/a\ \a/

Serine

cHr-cH-coo

HrO

cHr-cH-coo

r\

\>

iB'/

OH

l-T;r""t"a

lPh""y1"1""t"A @ chorismate mutase @ prephenate dehydrogenase @ prephenate dehydratase

tlGURt22-19 Biosynthesis of phenylalanineand tyrosinefrom cho. rismatein bacteriaand plants.Conversionof chorismateto prephenateis a rarebiologicalexampleof a Claisenrearrangement.

, PLP-aminoacrylate adduct I

Indole condenses with the aminoacrylate intermediate (2 steps).

@l J

HB,.

/-'u'

cH-coo

*NH

-tt

u6

@-o-cr, UHs

Quinonoid intermediate

Imine linkage joining tryptophan to PLP is hydrolyzed.

I{

Aldimine with tryptophan

MECHANISM FIGURE 22-18Tryptophan synthase reaction. Thisenzymecatalyzes a multistep reaction withseveral typesof chemical rearrangements. ThePLPJacilitated transformations occurattheg carbon

(C-3)of the amino acid, as opposedto the a-carbonreactionsdescribedin Fi e 18-6. The6 carbonof serineisattached to the indole ring system. TryptophanSynthaseMechanism

ocids 871 2 2 . 2B i o s y n t h eosfAi sm i nA

PPi

-C H

2_

@ [$ ' tx-'.'

d*

NI-5', -Phosphoribosyl-AMP

N 1 - 5 ' -PhosphoribosyI-ATP

^l H,O (UI

To purine biosyrrthesis

I

/a/\.. NN_

r1^N-l nrl-@ ox l{\ /

H2N-C

rv^rr-@@

\/ o'.la H2N-C

,. H,N-C _i/

NH'

N

H--N-c

HN CH

6-o-cu" v

I I c:o I

5-Aminoimidazole4-carboxamide ribonucleotide (AICAR)

H-C-H

Glutamine Glutamate

l\

H

Hc *\ lt .9H (1

|

H \c-Y

H-C-OH l H-C OH

1^f I

H

61 6o

t^ cHro(ry

Nr-5' -Phosphoribosylformrmrno5-aminoimidazole-4carboxamide ribonucleotide

N1-5'-Phosphoribulosylformimino-5-aminoimidazole-4-carboxamide ribonucleotide

glutamine amidotransferase imidazole glycerol 3-phosphate dehydratase

pyrophosphohydrolase

@ - t I' H,o

I H

l-..--\1 C.-H H:C

ATP phosphoribosyl transferase Imidazole glycerol 3-phosphate I

o

A

phosphoribosyl-AMP cyclohydrolase phosphoribosylformimino-5-aminoimidazole4-carboxamide ribonucleotide isomerase

l-histidinol phosphate aminotransferase histidinol phosphate phosphatase histidinol dehydrogenase

H

//

iN

C}lz

I

C:O

t^ cH,oQg) Imidazole acetol 3-phosphate

t -Histidinol phosphate

r,-Histidinol

(green).Notethatthe derivative of ATPremainingafterstep@ (AICAR) (seeFig.22-33, step (N), so in purinebiosynthesis is an intermediate

f IGURE 22-20 Biosynthesis of histidinein bacteriaand plants.Atoms derivedfrom PRPPandATPareshadedpink and blue,respectively. Two of the histidinenitrogens are derivedfrom glutamineand glutamate

ATPis rapidlyregenerated.

the purine ring of ATP contributesa nitrogen and a carbon, and glutamrnesupplies the second ring nitrogen. The key steps are condensationof ATP and PRPP,in which N-l of the purine ring is linked to the activatedC-l of lhe riboseof PRPP(stepe inFig.22-20);purinering operungthat ultimately leavesN-l and C-2 of.adenine

linked to the ribose (step @); and formation of the rmidazolering, a reaction in which glutamine donatesa nitrogen (step @;. The use of ATP as a metaboliterather cofactoris unusual-but not wasteful, than a Lugh-energy because it dovetails with the purine biosynthetic pathway.The remnant of ATP that is releasedafter the

[b'{

B i o s y n t h eosf A i sm i nA o c i d sN,u c l e o t i daensdR , e l a t eMdo l e c u l e s

transfer of N-l and C-2 is 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), an intermediate of purine biosynthesis(see Fig. 22-33) that is rapidly recycledto ATP.

Amino AcidBiosynthesis lsunder Allosteric Regulation As detailedin Chapter15,the control of flux through a metabolicpathway often reflects the activity of multiple enzyrnesin that pathway.In the caseof amino acid synthesis,regulationtakes placein part through feedback inhibition of the first reaction by the end product of the pathway.This first reaction is often catalyzedby an allosteric enzyme that plays an important role in the overall control of flux through that pathway.As an example, Figure 22-21 showsthe allostericregulationof isoleucine syrrthesisfrom threonine (detailed in Fig. 22-15). The end product,isoleucine,is an allostericinhibitor of the flrst reactionin the sequence.In bacteria, such allosteric modulation of amino acid synthesiscontributes to the minute-to-minuteadjustmentof pathway activity to cellular needs. Allosteric regulationof an individual enz;rmecan be considerablymore complex.An exampleis the remarkable set of allosteric controls exerted on glutamine synthetaseof E. coli,(Fig.22-6). Six productsderivedfrom glutamine serveas negativefeedbackmodulatorsof the enzyrne,and the overall effects of these and other modulators are more than additive. Suchregulationis called concerted inhibition. Additional mechanismscontribute to the regulation of the aminoacidbiosyntheticpathways.Becausethe 20 common amino acids must be made in the correct proportionsfor protein synthesis,cells have developed waysnot only of controlling the rate of sy'nthesisof indi-

vidual amino acids but also of coordinating their formation. Such coordination is especially well developed in fast-growing bacterial cells. Figure 22-22 shows how E. coli, cells coordinate the synthesis of lysine, methionine, threonine, and isoleucine, all made from aspartate. Several important types of inhibition patterns are evident. The step from aspartate to aspartyl-B-phosphate is catalyzed by three isozlrnes, each independently controlled by different modulators. This enzyme multiplicity prevents one biosynthetic end product from shutting down key steps in a pathway when other products of the same pathway are required. The steps from Aspartate

Aspartyl-p-phosphate

II I

+

Aspartate B-semialdehyde B2

B1

@* @*-- . Homose

'8 I

8* ',

I

3 stens I

I

Methionine +

NH, I

CHB-CH-CH-COOI OH +ft

Threonine

I t l r r , ' , r ' i r "l " l r r , r r . r r ' r . '

a-Ketobutyrate

Y

o CH3-CH2-C-COO

-

a-Ketobuty'rate

-

CHg FIGURE 22-21 Allosteric regulationof isoleucinebiosynthesis. The firstreactionin the pathwayfrom threonineto isoleucine is inhibited by the end product,isoleucine.This was one of the first examplesof allostericfeedbackinhibitionto be discoveredThe steosfrom aketobutyrate to isoleucinecorrespondto steps@ through@ in figure22-15 (fivesteps,because@ is a two-stepreaction).

----\-

-Isoleucine

FIGURE 22-22 Intertockingregulatorymechanisms in the biosynthesis of severalamino acids derived from aspartatein E. coli. Threeenzymes(A, B, C) haveeithertwo or three isozymeforms,indicatedby numericalsubscripts. In eachcase,one isozyme(Ar, B,, and Cz)has no allostericregulation;theseisozymesare regulatedby changesin (Chapter28). Synthesis the amountsynthesized of isozymes ,\2 and B1 is repressed whenmethionine levelsarehigh,andsynthesis of isozyme C2 is repressed when isoleucine levelsare high.EnzymeA is aspartokinase;B, homoserinedehydrogenase; C, threoninedehydratase.

F'{

ocids 2 2 . 3M o l e c u lDees r i v ef rdo mA m i nA

aspartateB-semialdehyde to homoserineand from threonineto c-ketobutyrate(detailedin Fig. 22-15) are also catalyzedby dual, independentlycontrolledisoz;rmes. Oneisozyrnefor the conversionof aspartateto aspartylB-phosphateis allostericallyinhibited by two different modulators,lysineand isoleucine,whoseactionis more than additive-another exampleof concertedinhibition. The sequencefrom aspartateto isoleucineundergoes multiple, overlappingnegativefeedbackinhibitions; for example,isoleucineinhibitsthe conversionof threonine to a-ketobutyrate(as describedabove),and threonine inhibits its own formation at three points: from homoserine,from aspartateB-semialdehyde, and from aspartate (steps@, @, and @in Fig.22-15).This overall regulatory mechanismis called sequential feedback inhibition Similarpatterns are evidentin the pathwaysleading to the aromatic amino acids The flrst step of the early pathwayto the commonintermediatechorismateis catalyzedby the enzyme2-keto-3-deoxy-l-arabinoheptulosonate 7-phosphate(DAHP) synthase (step @ in Fig. 22-16) . Most microorganismsand plants havethree DAHP synthaseisoz;.rnes.One is allostericallyinhibited (feedbackinhibition) by phenylalanine,anotherby tyrosine,and the third by tryptophan.This schemehelpsthe overallpathwayto respondto cellularrequirementsfor one or more of the aromaticaminoacids.Additional regulation takes place after the pathway branchesat chorismate. For example,the enzymescatalyzingthe first two steps of the tryptophan branch are subject to allostericinhibition by tryptophan.

SUMMAR 2Y 2 . 2 B i o s y n t h eosf A i sm i n o Acids r

Plants and bacteria symthesizeall 20 common amino acids. Mammals can synthesize about half; the others are required in the diet (essential amino acids).

r

Among the nonessential amino acids, glutamate is formed by reductive amination of a-ketoglutarate and serves as the precursor of glutamine, proline, and arginine. Alanine and aspartate (and thus asparagine) are formed from pyruvate and oxaloacetate,respectively,by transamination. The carbon chain of serine is derived frorn 3-phosphoglycerate.Serine is a precursor of glycine; the B-carbon atom of serine is transferred to tetrahydrofolate. In microorganisms, cysteine is produced from serine and from sulflde produced by the reduction of environmental sulfate. Mammals produce cysteine from methionine and serine by a series of reactions requiring S-adenosylmethionine and cystathionine.

r

Among the essential amino acids, the aromatic amino acids (phenylalanine, tyrosine, and tryptophan) form by a pathway in which chorismate occupies a

key branchpoint. Phosphoribosylpytophosphateis a precursorof tryptophanand histidine.The pathway to histidine is intercornected with the purine can alsobe formed synthetic pathway.TJzrosine by hydroxylation of phenylalanine(and thus is consideredconditionallyessential).The pathways for the other essentialamino acidsare complex. r

The aminoacid biosyntheticpathwaysare subjectto allostericend-productinhibition;the regulatoryenzymeis usuallythe first in the sequence.Regulation of the varioussyntheticpathwaysis coordinated.

Acids fromAmino 22.3Molecules Derived In addition to their role as the buiiding blocks of proteins, amino acids are precursorsof many specialized biomolecules,including hormones, coenzymes,nucleotides, alkaloids, cell wall polymers, porphyrins, antibiotics,pigments, and neurotransmitters.We describe here the pathways to a number of these amino acid derivatives.

lsa Precursor ofPorphyrins Glycine The biosynthesisof porphyrins, for which glycine is a majorprecursor,is our first examplebecauseof the central importance of the porphyrin nucleus in heme proteins such as hemoglobinand the cytochromes.The porphyrins are constructed from four moleculesof the monopyrrole derivative porphobilinogen, which itself is derived from two molecules of D-aminolevulinate. There are two major pathways to D-aminolemlinate. In higher eukaryotes(Fig. 22-23a), glycine reacts with succinyl-CoAin the first step to yield a-aminop-ketoadipate, which is then decarboxylated to 6-aminolevulinate.In plants, algae,and most bacteria, is formedfromglutamate(ng. 22-23b). 8-aminolevulinate The glutamate is first esterified to glutamyl-tRNAcr" (see Chapter27 on the topic of transferRNAs);reduction by NADPH converts the glutamate to glutamate l-semialdehyde,which is cleavedfrom the IRNA. An aminotransferaseconverts the glutamate 1-semialdehyde to 6-aminolevulinate. In all organisms,two moleculesof 6-aminolemlinate condenseto form porphobilinogenand, through a series reactions,four moleculesof porof complex enz).rynatic phobilinogen come together to form protoporphyrin (Fig. 22-24). The iron atom is incorporatedafter the protoporphytin has been assembled,in a step catalyzed by ferrochelatase.Porphyrin biosynthesisis regulatedin higher eukaryotesby the concentrationof the heme product, which servesas a feedback inhibitor of early steps in the synthetic pathway. Genetic defects in the biosynthesisof porphyrins can lead to the accumulation of pathway intermediates,causing a variety of human diseasesknown collectivelyas porphyrias (Box 22-2).

-ofAmino Acids, Nucleotides, andRelated Molecules 874_) Biosynthesis

(a)

cooI CH, CoA-SH tCH, + cH"-frH. rJ-aminolevulinate tt" synthase C-S-CoA coo tl o Succinyl-CoA lctl"t""l

cooI

coo-

CHo

CHo

t-

t-

CH,

CHO

CO, ^

t-

C:O

tc:o

d-aminolevulinate synthase

l*

cH-NH3 I

I

CH, t*NH,

coo

6-Aminolevulinate

a-Amino-pketoadipate

(b)

cooI

coo I CHo t-

CH.

I-

CH"

l-*

NADPH

CH"

gluranyl-tRNA synthetase

HC-NH3 I

coo CHO

t-

CHo

glutamyl-tRNA reductase

l-*

HC-NH3 I

coo

NADP+ TRNAGI''

l-*

HC-NH3 I C:O H

c-o t^,

tRNA"'" Glutamyl-tRNAGlu FIGURE (a) In mostanimals, 22-23 Biosynthesis of 5-aminolevulinate. includingmammals, 6-aminolevulinate issynthesized fromglycineand

Glutamate l-semialdehyde succinyl-CoATheatomsfurnishedby glycineareshownin red. (b) In bacteriaand plants,the precursorof 6-aminolevulinate is glutamate.

coo

NHe 6-Aminolevulinate

Porphobilinogen

Ac Pr Preuroporph5z'inogen

Pr Pr Uroporphyrinogen III

I @ l" nco" J

Vinyl group Pr CHs 2 COz

Fe'* CO,

Putrescine

cHr-s-]Iffiil Methylthioadenosine

Decarboxylated adoMet

++ HaN-(CH2)B-NH-(CH2)4-NHB

propyltrnrinotranslerase

FIGURE 22-30 Biosynthesis of spermidineand spermine.The plpdependentdecarboxylation stepsare shadedin pink. In thesereactions,S-adenosylmethionine (in its decarboxylated form) acts as a sourceof propylamino groups(shaded blue).

African sleeping sickness, or African trypanosomiasis, is caused by protists (single-celled eukaryotes) called trypanosomes (Fig. 1). This disease (and related trypanosome-caused diseases) is medically and economi_ cally significant in many developing nations. Until the late twentieth century, the disease was virtually incurable. Vaccines are ineffective because the parasite has a novel mechanism to evade the host rmmune system. The cell coat of trypanosomes is covered with a single protein, which is the antigen to which the immune system responds. Every so often, however, by a process of genetic recombination (see Table 28-1), a few cells in the population of infecting tr)?anosomes switch to a new protein coat, not recognized by the immune system. This process of "changing coats" can occur hundreds of times. The result is a chronic cyclic infection: the human host develops a fever, which subsides as the immune system beats back the first infection; trypanosomes with changed coats then become the seed for a second infection, and the fever recurs. This cycle can repeat for weeks, and the weakened person eventually dies. Some modern approaches to treating African sleeping sickness have been based on an understanding of enzymology and metabolism. In at teast one such approach, this involves pharmaceutical agents designed as mechanism-based enzyme inactivators (suicide

Spermidine

Il

nrti-(cttr), -rrrg-(cH2)4-NH-(GH/3 -fr1g, Spernine

FfGURE1 Trypanosomabrucei rhodesiense,one of several trypanosomes knownto causeAfricansleepingsickness.

inactivators;p.20Q. A vulnerablepoint in trypanosome metabolism is the pathway of polyamine biosynthesis. The polyamines spermine and spermidine, involved in DNA packaging,are required in large amounts in rapidly dividing cells.The first step in their synthesisis catalyzed by ornithine decarboxylase,a Plp-requiring enzyrne(seeFig. 22-30).In mammaliancells, ornithine decarboxylaseundergoesrapid turnover-that is, a constant round of enzyme degradation and synthesis. In sometrypanosomes,however,the enzyme(for reasonsnot well understood)is stable,not readilyreplaced by newly synthesizedenzyme.An inhibitor of ornithine

o c i d s| 8 8 1 | 2 2 . 3M o l e c u lDees r i v ef rdo mA m i nA

Ornithine

CO,

H2N-(CHz)a-

+H H3N-(CH2)3-CH I *NH

+

CH

CH

Putrescine

FIGURE 2 Mechanism reaction. of ornithinedecarboxylase

decarboxylasethat binds permanently to the enzyrne would thus have little effect on human cells, which could rapidly replace inactivated enzyme,but would adverselyaffect the parasite. The first few steps of the normal reaction catalyzed by ornithine decarboxylaseare showrlin Figure 2. Once CO2is released,the electronmovementis reversedand DFMO FF \./

@-o-cg, )-., .on VY7Y l=ft^cn, tl

OH

@-o-c

CHa

H Pyridoxal phosphate

Schiffbase

HON-(CHq)r ---l

C

NH

putrescine is produced (see Fig. 22-30). Basedon this mechanism,severalsuicide inactivators have been designed, one of which is difluoromethylornithine (DFMO). DFMO is relativelyinert in solution.When it binds to ornithine decarboxylase,however,the enzyme is quickly inactivated (FiS. 3). The inhibitor acts by providing an alternative electron sink in the form of two strategically placed fluorine atoms, which are excellentleavinggroups.Insteadof electronsmovinginto the ring structure of PLP, the reaction results in displacementof a fluorine atom.The S of a Cysresidueat the enzyme'sactive site then forms a covalent complex with the highly reactive PlP-inhibitor adduct in an essentially irreversible reaction. In this way, the inhibitor makesuse of the enzyme'sown reaction mechanisms to kiII it. DFMO has proved hgfily effective against African sleeping sicknessin clinical trials and is now used to treat African sleepingsicknesscausedby Tlypanosoma brucei gamb'i'ense.Approachessuch as this show great promise for treating a wide range of diseases.The designof drugsbasedon enzyrnemechanismand structure can complement the more traditional trial-anderror methodsof developingpharmaceuticals.

HrN-(CHr)3-? *NH

additional rearangements

CH OH FIGURE 3 tnhibition of ornithine bv DFMO. decarboxvlase

CHt

>

Stuckl

-ofAmino Acids, Nucleotides, andRelated Molecules tBB2I Biosynthesis

*l

coo *l

H3N-C-H CH"

NADPH, 02

I

jxaorH,o,

I

l-

CH,

I

I CHo

CH"

CH" | NH I

I

NH I C:N-OH

l* 9:NH,

H3N-C-H 1 CH"

CH,

t-

NH

coo-

*l

H3N-C-H I CH"

CH"

I NH, Arginine

coo

I

NH, Hydroxyarginine

c:o I

+NO' Nitric oxide

NH, Citrulline

tIGURE 22-31 Biosynthesis of nitric oxide.Bothstepsarecatalyzed by nitricoxidesynthaseThenitrogenof the NO is derivedfromthe guanidinium groupof arginine

Arginine lsthePrecursor forBiological Synthesis ofNitric Oxide A surprise finding in the mid-1980s was the role of nitric oxide (No)-previously known mainly as a component of smog-as an important biological messenger. This simple gaseoussubstancediffuses readily through membranes, although its high reactivity limits its range of diffusion to about a 1 mm radius from the site of slmthesis. In humans NO plays a role in a range of physiological processes, including neurotransmission, blood clotting, and the control of blood pressure. Its mode of action is describedin Chapter 12 @. aa|. Nitric oxide is synthesized from arginine in an NADPH-dependent reaction catalyzed by nitric oxide synthase (Fig. 22-31), a dimeric enz).rne structurally related to NADPH cytochrome p-450 reductase (see Box 21-1). The reaction is a five-electron oxidation. Each subunit of the enzyme contains one bound molecule of each of four different cofactors: FMN, FAD. telrahydrobiopferin,and Fe't heme NO is an unstable molecule and cannot be stored. Its synthesis is stimulated by interaction of nitric oxide synthase with Ca2+calmodulin (see Fig. Iz-It).

S U M M A R2Y2 . 3 M o l e c u l D e se r i v ef dr o m A m i n oA c i d s r

Many important biomolecules are derived from amino acids. Glycine is a precursor of porphyrins Degradation of iron-porphyrin (heme) generates bilirubin, which is converted to bile pigments, with several physiological functions.

r

Glycine and arginine give rise to creatine and phosphocreatine, an energy buffer. Glutathione, formed from three amino acicls, is an important cellular reducing agent.

r

Bacteria synthesize D-amino acids from L-amino acids in racemization reactions requiring pyridoxal

phosphate. l-Amino acids are commonly found in certain bacterial walls and certain antibiotics. r

The aromatic amino acids give rise to many plant substances.The PlP-dependent decarboxylation of some amino acids yields important biological amines, including neurotransmitters.

r

Arginine is the precursor of nitric oxide, a biological messenger.

22.4Biosynthesis andDegradation ofNucleotides As discussed in Chapter 8, nucleotides have a variety of important functions in all cells. They are the precursors of DNA and RNA. They are essential carriers of chemical energy-a role primarily of ATP and to some extent GTP. They are components of the cofactors NAD, FAD, S-adenosylmethionine, and coenz;.'rne A, as well as of activated biosynthetic intermediates such as UDpglucose and CDP-diacylglycerol. Some, such as cAMp and cGMP,are also cellular second messengers. Two types of pathways lead to nucleotides: the de novo pathways and the salvage pathways. De novo synthesis of nucleotides begins with their metabolic precursors: amino acids, ribose 5-phosphate, CO2,and NH3. Salvage pathways recycle the free bases and nucleosides released from nucleic acid breakdown. Both types of pathways are important in cellular metabolism and both are discussedin this section. The de novo pathways for purine and pyrimidine biosynthesis seem to be nearly identical in all Iiving organisms. Notably, the free bases guanine, adenine, thymine, cytidine, and uracil ate not intermediates in these pathways; that is, the bases are not synthesized and then attached to ribose, as might be expected. The purine ring structure is built up one or a few atoms at a time, attached to ribose throughout the process. The pyrrmidine ring is synthesized as orotate, attached to

Frrl

nucleotides 2 2 . 4B i o s y n t h easnidsD e g r a d a toi of N

ribose phosphate, and then converted to the common pyrimidine nucleotides required in nucleic acid synthesis. Although the free bases are not intermediates in the de novo pathways, they are intermediates in some of the salvage pathways. Several important precursors are shared by the de novo pathways for syrrthesis of p;,rimidines and purines Phosphoribosyl pyrophosphate (PRPP) is important in both, and in these pathways the structure of ribose is retajned in the product nucleotide, in contrast to its fate in the tq,ptophan and histidine biosynthetic pathways discussed earlier. An amino acid is an important precursor in each type of pathway: glycine for purires and aspaftate for pyrimidines. Glutamine again is the most important source of amino groups in five different steps in the de novo pathways. Aspartate is also used as the source of an amilo group in the purine pathways, in two steps. TWo other features deserve mention. First, there is evidence, especially in the de novo purine pathway, that the enzymes are present as large, multienzyme complexes in the cell, a recurring theme in our discussion of metabolism. Second, the cellular pools of nucleotides (other than ATP) are quite small, perhaps 1% or less of the amounts required to synthesize the cell's DNA. Therefore, cells must continue to synthesize nucleotides during nucleic acid synthesis, and in some cases nucleotide synthesis may limit the rates of DNA replication and transcription. Because of the importance ofthese processesin dividing cells, agents that inhibit nucleotide synthesis have become particularly importanLin medicine. We examine here the biosynthetic pathways of purine and pyrimidine nucleotides and their regulation, the formation of the deoxynucleotides, and the degradation of purines and pyrimidines to uric acid and urea We end with a discussion of chemotherapeutic agents that affect nucleotide synthesis.

Purine Nucleotide DeNovo Synthesis Begins withPRPP The two parent purine nucleotidesof nucleic acids are adenosine 5'-monophosphate (AMP; adenylate) and guanosineS'-monophosphate (GMP;guanylate),contain-

john Buchanan

ing the purine basesadenine and guanine. Figure 22-32 shows the origrn of the carbon and nitrogen atoms of Lhe purine nng system, as detemined by John Buchanan usi4g isotopic tracer experiments in birds The detailed pathway of purine biosynthesis was worked out primarily by Buchanan and G. Robert Greenbergin the 1950s. In the first committed step of the pathway, an amino group

Coz Aspartate

v/

*zc-'r-\ 1l t-**-t=# Formate

,

.C -

GlYcine

Formate

il

22-32 Origin of the ring atomsof purines.Thisinformation FIGURE prewith 14C-or t5N-labeled wasobtainedfrom isotopicexperiments is suppliedin theformof Nro-formyltetrahydrofolate cursorsFormate

donated by glutamine is attached at C-l of PRPP (Fig.

is 22-:13). The resulting 5-phosphoribosylamine highly unstable, with a halflife of 30 seconds at pH 7.5. The purine ring is subsequently built up on this structure The pathway described here is identical in all organisms, with the exception of one step that differs in higher eukaryotes as noted below. The second step is the addition of three atoms from glycine (trig.22-33, step @). An ATP is consumed to activate the glycine carboxyl group (in the form ofan acyl phosphate) for this condensation reaction. The added gtycine amino group is then formylated byNlo-formyltetrahydrofolate (step @), and a nitrogen is contributed by glutamine (step (!), before dehydration and ring closure yield the five-membered imidazole ring of the purine nucleus, as 5-aminoimidazole ribonucleotide (AIR; step Q)). At this point, three of the six atoms needed for the second ring in the purine structure are in place To complete the process, a carboxyl group is flrst added (step @). This carboxylation is unusual in that it does not require biotin, but instead uses the bicarbonate generally present in aqueous solutions. A rearrangement transfers the carboxylate from the exocyclic amino group to position 4 of the imidazole ring (step @). Steps @ and @ are found only in bacteria and fungi. In higher eukaryotes, including humans, the 5-aminoimidazole ribonucleotide product of step (!) is carboxylated directly to carboxyaminoimidazole ribonucleotide in one step instead of two (step @1. ffre enzyrne caI'alyzing this reaction is AIR carboxylase. Aspartate now donates its amino group in two steps (@ and @): formation of an amide bond, followed by elimination of the carbon skeleton of aspartate (as fumarate). (Recall that aspartate plays an analogous role in two steps of the urea cycle; see Fig. 18-10.) The flnal carbon is contributed by N10-formyltetrahydrofolate (step @), and a second ring closure takes place to yield the second fused ring of the purine nucleus (step purine ring @1. fne flrst intermediate with a complete is inosinate (IMP).

f

l

I BB4

B i o s y n t h eosfAi sm i nA o c i d sN,u c l e o t i daensdR , e l a t eMdo l e c u l e s

o.-_ H

@-o-9n

(-l\

--\1

.d

fl

HCO.r

./\^

i'\J'o-gt-g l-l OH ^ l

5-Phosphoribosyl l-pyrophosphate(PRPP)

OH

ATP

l/'

ADP + Pi

t

H

arNt> v

@o

N5-Carboxyaminoimidazole ribonucleotide (N5-CAIR)

's-eno"prro-B>ribosylamine OH

OH

l.e

^f

(2) lz-

I

Carboxyaminoimidazole ribonucleotide (CAIR)

Pi

J't H -e C - ' _ 1 O:C

Glycinamide ribonucleotide(GAR)

R

coo I

Nto-FormYl Ho folate ^ f'\Y/[ J> Ha folate H H,C

C-H,' li O

O:C

Formylgiycinamide ribonucleotide(FGAR) P

^t

R

(9)l\

Fumarate

U

R

H c-H^' Y d

H"C -l HN:C

^ l0 or

lbrmylglycinamidine ribonucleotide(FGAM)

Nro-Formyl Hn folate

vI

J> Ha folate

o

R N-Formylaminoimidazole4-carboxamide ribonucleotide GAICAR)

n /R\[

,yl\

R

t->

^t

(1Dl\ H"O

o R FIGURE 22-33 De novo synthesis of purine nucleotides:construction of the purinering of inosinate(lMp).Eachadditionto the purinering is shadedto match Figure22-32. After step @, R symbolizesthe 5-phospho-o-ribosyl groupon whichthepurineringis built.Formation of S-phosphoribosylamine tstep @) is the first committedstep in purinesynthesis. Note that the productof step@, AICAR,is the remnantofATPreleased duringhistidine (seeFig.22_20,slep biosynthesis (!). Abbreviations are givenfor most intermediates to simplifythe namingof the enzymes.Step@ is the alternative path from AIR to CAIRoccurringin highereukaryotes.

o-T-o-1H2.o o

KH

H

@ Slutamine-PRPP amidotransferase @ Con synthetase GAR t""tt.formylase Q (9 FGAR amidotransferase @ FGAM cyctase (AIR synthetase) .^trs-cam synthetase @ AIR carboxvlase Q (7) No-CAIRmutase @ S,UCan s1'nthetase

Q

Inosinate (IMP)

selcentru."

q9 AICAR transformylase @ frur synthase

n u c l e o t i d e[ tst L 2 2 . 4B i o s y n t h easnidsD e g r a d a toi of N

H -ooc-cH2-c-cooI

NH

Fumarate

o Adenylate (AMP)

Adenylosuccinate

AMP + PPi

u

Inosinate (IMP)

(

/

o

XN{P-glutnmine amidotransIerase

FIGURE 22-34 Biosynthesis of AMPandCMPfromlMP.

Guanylate (GMP)

Xanthylate (XMP)

As in the tryptophan and histidine biosynthetic pathways,the enz5,'mes of IMP synthesisseemto be organizedas large, multienzymecomplexesin the cell. Onceagain,evidencecomesfrom the existenceof single polypeptideswith severalfunctions, some catalyzing nonsequentialsteps in the pathway.In eukaryotic cells rangingfrom yeastto fruit flies to chickens,steps@, @, and @ in Figure 22-33 are catalyzed,by a multifunctional protein. An additional multifunctional protein catalyzes steps @ and @ t.r humans, a multifunctional enz).rnecombinesthe activities of AIR carboxylaseand SAICARs;'nthetase(steps@ and@). In bacteria,these activities are found on separateproteins, but the proteins may form a large noncovalentcomplex.The channeling of reactionintermediatesfrom one enz;,.rne to the next permitted by these complexesis probably especially important for unstable intermediates such as 5phosphoribosylamine. Conversionof inosinateto adenylaterequfuesthe insertion of an amino group derived from aspartate (FiS. 22-34); this takesplacein two reactionssimilar to those used to introduceN-l of the purine ring (Fig. 22-33,steps@and @). e crucialdifferenceis that GTP rather than ATP is the source of the high-energyphosphate in synthesizingadenylosuccinate.Guanylateis formed by the NAD+-requiringoxidation of inosinateat C-2, followed by addition of an amino group derived from glutamine. ATP is cleavedto AMP and PP1in the final step (Fie.22-34).

Purine Nucleotide Biosynthesis lsRegulated byFeedback Inhibition Three major feedback mechanisms cooperate in regulating the overall rate of de novo purine nucleotide synthesis and the relative rates of formation of the two end products, adenylate and guanylate (Fig. 22-35).

Ribose5-phosphate I

i:?:;:llil:i?;l$ ,,TPRPP |6 synthetaset

ADP ATP

-

dc

cDp

(}a

dTTP €--(*dUDP

A

#

a

CI

_ _ \\

tu'ol"op dcrp

dUDP+)+dlTP

aR

/

\/

\ l

dGTP

dGDP

GDP

dGTP

_,/ dATP

dADP Products

I

Y

OA

ADP Substrates

FIGURE 22-42 Regulation of ribonucleotide reductase by deoxynucleoside triphosphates. Theoverallactivityof theenzymeis affected bybinding attheprimary regulatory site(left)Thesubstrate specificity of theenzymeisaffected bythenatureof theeffector molecule bound

Three classes of ribonucleotide reductase have been reported. Their mechanisms (where known) generally conform to the scheme in Figure 224l,but they differ in the identity of the group suppllng the active-site radical and in the cofactors used to generate it. The E co\i, enzlme (class I) requires oxygen to regenerate the tyTosyl radical if it is quenched, so this erzl'rne functions only in an aerobic envirorunent. Class II enz),.rnes,found in other microorganisms, have 5'-deoxyadenosylcobalamin (see Box l7-2) rather than a binuclear iron center. Class III el\z],rnes have evolved to function in an anaerobic envirorunent. E. co|z contains a separate class III ribonucleotide reductase when grown anaerobically; this enz)'rne contains an iron-sulfur cluster (structurally distinct from the binuclear iron center of the class I enz;nne) and requires NADPH and S-adenosylmethionine for activity. It uses nucleoside triphosphates rather than nucleoside diphosphates as substrates. The evolution of different classes of ribonucleotide reductase for production of DNA precursors in different environments reflects the importance of this reaction in nucleotide metabolism. Regulation of E. coli, ribonucleotide reductase is unusual in that not only its acti,ui,t!/ but its substrate spec,ifi,ci,ty is regulated by the binding of effector molecules. Each Rl subunit has two types of regulatory site (Fig. 22-40). One type affects overall enz).rne activity and binds either ATP, which activates the enz;.'rne,or dAfP, which inactivates it. The second type alters substrate specificity in response to the effector molecule-ATp, dAIP, dTTP, or dGTP-that is bound there (Fig. ZZ-42). When ATP or dATP is bound, reduction of UDP and CDp is favored. When dTTP or dGTP is bound, reduction of GDP or ADP, respectively, is stimulated. The scheme is designed to provide a balanced pool of precursors for

dADP

dATP

Products

at the secondtype of regulatorysite,the substrate-specificity site (right). Thediagramindicates inhibitionor stimulation of enzymeactivity with the four differentsubstrates. The pathwayfrom dUDP to dTTPis described later(seeFigs22-43,22-44)

DNA synthesis.ATP is also a general activator for biosynthesisand ribonucleotidereduction. The presence of dATP in small amountsincreasesthe reduction of pyrimidine nucleotides.An oversupplyof the pyrimidine dNTPs is signaledby high Ievels of dTTP, which shifts the specificityto favor reduction of GDP.High levels of dGTP,in turn, shift the specificity to ADP reduction, and high levels of dATP shut the enzy'rnedown. These effectors are thought to induce several distinct enzymeconformationswith altered speciflcities.

Thymidylate lsDerived fromd(DP anddUMP DNA containsthymine rather than uracil, and the de novo pathway to thymine involves only deoxyribonucleotides.The immediate precursor of thymidylate (dTMP) is dUMP.In bacteria, the pathway to dUMP beginswith formation of dUTP,either by deamination of dCTP or by phosphorylation of dUDP (FiS. 22-43). The dUTP is convertedto dUMP by a dUTPase. The latter reaction must be efficient to keep dUTP pools low and prevent incorporationof uridylate into DNA. Conversion of dUMP to dTMP is catalyzed by thymidylate synthase. A one-carbonunit at the hydroxymethyl (-CH2OH) oxidation level (see Fig. 18-17) is transferred from AF,Mo-methylenetetrahydrofolateto dUMP,then reducedto a methylgroup (Fig. 2244).The reduction occursat the expenseof oxrdationof tetrahydrofolate to dihydrofolate,which is unusualin tetrahydrofolate-requiring reactions. (The mechanism of this reaction is shown in Frg. 22-50.) The dihydrofolate is reducedto tetrahydrofolateby dihydrofolate reductasea regenerationthat is essentialfor the manyprocessesthat

F"l

2 2 . 4B i o s y n t h easnidsD e g r a d a toi of N nu c l e o t i d e s

dCDP ribonuclcotide

dcitminase

d iphosphate

rcductase

UDP

dCTP nucleoside kinase

dUDP

v dUTP clflTPasc

v dUMP vmidvlaLe nrnasc

J

dTMP FIGURE 22-43 Biosynthesis of thymidylate(dTMP).The parnwaysare shownbeginning with the reactioncatalyzed by ribonucleotide reductase.Figure22-44 givesdetailsof the thymidylatesynthasereaction

dUMP

requiretetrahydrofolate.In plantsand at leastoneprotist, th).'rmdylates),Tlthaseand dihydrofolate reductasereside on a singlebifunctional protein. About l0o/oof the human population (and up to 50%o of peoplein impoverishedcommunities)suffers from folic acid deflciency.Whenthe deflciencyis severe, the s;.rnptomscan include heart disease,cancer, and some types of brain dysfunction. At least some of these symptomsarise from a reduction of thymidylate synthesis,leadingto an abnormalincorporationof uracil into DNA. Uracil is recognizedby DNA repair pathways (describedin Chapter25) and is cleavedfrom the DNA. The presence of high levels of uracil in DNA leads to strand breaks that can greatly affect the function and regulation of nuclear DNA, ultimately causing the observed effects on the heart and brain, as well as inthat leadsto cancer.r creasedmutagenesis

HN

dTMP

€Fo-qu,

@o-qH, H

oA*

H

thvmiclvlate synthasc

H

.::::t::::a'l:il',,:

:j,fi{,;1i;, N5,N10-Methylene-

7,8-Dihydrofolate

Tetrahydrofolate FIGURE 22-44 Conversionof dUMP to dTMP by thymidylatesynthaseand dihydrofolatereductase.Serinehydroxymethyltransferase is requiredfor regeneration of the Ns,N1o-methylene form of tetrahydro-

folate. In the synthesisof dTMB all three hydrogensof the added methyl group are derived from Ns,Nto-methylenetetrahydrofolate (pinkand gray).

i sm i nA o c i d sN,u c l e o t i daensdR , e l a t eMdo l e c u l e s i _ 8 9 2] B i o s y n t h eosfA

Degradatlon ofPurines andPyrimidines PrCIdu(es tlricAcid andUrea, Respectively Purine nucleotidesare degradedby a pathwayin which they lose their phosphatethrough the action of 5'-nucleotidase (Fig. 22-45). Adenylate yields adenosine, which is deaminatedto inosine by adenosine deaminase, and inosine is hydrolyzedto hypoxanthine(its purine base)and >ribose. Hypoxanthineis oxidizedsuccessivelyto xanthineand then uric acid by xanthine oxidase, a flavoenz;.'rne with an atom of molybdenumand four iron-sulfur centersin its prostheticgroup.Molecular oxygenis the electronacceptorin this complexreaction. GMPcatabolismalsoyieldsuric acidas endproduct. GMP is first hydrolyzed to guanosine,which is then cleavedto free guanine.Guanineundergoeshydrolytic removal of its amino group to yield xanthine, which is

convertedto uric acid by xanthine oxidase(Frg.22-45). Uric acid is the excretedend product of purine catabolismin primates,birds, and someother animals.A healthy adult human excretesuric acid at a rate of about 0.6 g/24 h; the excretedproduct arisesin part from ingested purines and in part from turnover of the purine nucleotides of nucleic acids. In most mammals and many other vertebrates,uric acid is further degradedto allantoin by the action of urate oxidase. In other organismsthe pathway is further extended, as shown in Figure 22-45. The pathwaysfor degradationof py'rimidinesgenerally lead to NHf production and thus to urea synthesis. Thgnine, for example,is degradedto methylmalonylsemialdehyde(Fig.22-46), an intermediateof valine catabolism.It is further degradedthrough propionyl-CoAand methylmalonyl-CoAto succinyl-CoA (see Fig. 18-27). Excreted by:

AMP

l- H"o Iufl(,()tl(lrst

I

rT_:^ urlc ^-:r aclo

J"R

GMP

Adenosine

Primates, birds, reptiles, insects

l- H,o -

l- H"O

rl,r,,-r, ( l ( , i l D l l r l i r s[N t

, - n t r e l',, 1i 1 . , - t I

J" P'

I, NH3

Guanosine

Inosine

l-r'o

l-H,o V'

r r , l ,' - , l r - , I

llll( l('():l(llLs(r I

N

J'Ribot"

J-

oo

Rlbot"

rry\*s

"T\*s

Guanine

Allantoate

Bony frshes

(keto rorm)

I , , , , , , r,, ,l ,, - H 2 O+ 0 2

HzO

-.(

Most mammals

Hypoxanthine

\*Ail'

n,N^lrAry'

Allantoin

NHa \---Z

'' '''

o

J'",o,

grlLnirrr, \

rkir'ri.,rsr' -

Xanthine

'/ (ketoform)

"T/}*S

noAxA xrrrthinr,f0xl0llsc

N

l"

H2O + 02 Hzoz Amphibians, cartilaginous frshes

Uric acid

FIGURI 22-45 Catabolism of purinenucleotides. Notethatprimates excretemuchmorenitrogenasureavia the ureacycle(Chapter.l8) thanas

Marine invertebrates

uric acid from purinedegradation. Similarly, fishexcretemuch more nitrogenas NHj than as urea producedby the pathwayshown here.

F"-l

2 2 . 4B i o s y n t h easnidsD e g r a d a toi of N nu c l e o t i d e s

o -cHN' -?-.n,

Thymine

lated in a sterile"bubble"environment.ADA deflciency was one of the flrst targets of human gene therapy trials (seeBox 9-2). t

Purine Bases Arefiecycled andFyrimidine by5alvaqe Pathways

OzC'-*,'CH H

Dihydroth}.mine

Free purine and pyrimidine bases are constantly released in cells during the metabolic degradation of nucleotides. f'ree purines are in large part salvaged and reused to make nucleotides, in a pathway much simpler than the de novo slmthesis of purine nucleotides described earlier. One of the primary salvage pathways consists of a single reaction catalyzed by adenosine phosphoribosyltransferase, in which free adenine reacts with PRPP to yield the corresponding adenine nucleotide: Adenine + PRPP --+ AMP + PPi

H2N-C-NH

__CI."-?H-C(

p-Ureidoisobutyrate

ocH:o HrO NH; +HCO;

oo .r/o ic-cH-c\

H

cH,

o-

Methylmalonylsemialdehyde FIGURE 22-46 Catabolismof a pyrimidine.Shownhereis the pathway for thymine.The methylmalonylsemialdehyde is furtherdegradedto succinyl-CoA

Geneticaberrationsin humanpurinemetabolism have been found, some with serious consequences.For example,adenosine deaminase (ADA) deficiency leads to severeimmunodeflciencydisease in which T lymphocytesand B lymphocytesdo not developproperly.Lack of ADA leadsto a 100-foldincrease in the cellular concentrationof dATP,a strong inhibitor of ribonucleotidereductase(Fig. 22-42). High levelsof dATPproducea generaldeficiencyof other dNTPsin T lymphocytes.The basis for B-lymphocytetoxicity is less clear. Individuals with ADA deficiency lack an effective immune system and do not survive unless iso-

Free guanine and hypoxanthine (the deamination product of adenine;Fi9.2245) are salvaged in the same way phosphoribosyltransby hypoxanthine-guanine ferase. A similar salvage pathway exists for pyrimidine bases in microorganisms, and possibly in mammals. A genetic lack of hypoxanthine-guanine phosphoE ribosyltransferase activity, seen almost exclusively E in male children, results in a bizarre set of s;'rnptoms called Lesch-Nyhan syndrome. Children with this genetic disorder, which becomes manifest by the age of 2 years, are sometimes poorly coordinated and mentally retarded. In addition, they are extremely hostile and show compulsive self-destructive tendencies: they mutilate themselves by biting off their fingers, toes, and lips. The devastating effects of Lesch-Nyhan slmdrome illustrate the importance of the salvage pathways. Hypoxanthine and guanine arise constantly from the breakdorm of nucleic acids. In the absence of hypoxanthine-guanine phosphoribosyltransferase, PRPP levels rise and purines are overproduced by the de novo pathway, resulting in high levels of uric acid production and goutlike damage to tissue (see below). The brain is especially dependent on the salvage pathways, and this may account for the central nervous system damage in children with Lesch-Nyhan sy'rLdrome.This s;mdrome was another target of early trials ur gene therapy (see Box 9-2).t

UricAcid Causes Gout Excess Long thought, erroneously,to be due to "high living," gout is a diseaseof the joints causedby an elevatedconcentrationof uric acid in the blood and tissues.Thejoints becomeinflamed,painful,and arthritic, owing to the abnormaldepositionof sodium urate crystals.The kidneysare alsoaffected,as excessuric acid is depositedin the kidney tubules.Gout occurspredominantlyin males.Its precisecauseis not known,but it often involves an underexcretion of urate. A genetic of purine metabodeflciencyof one or another enz),Tne cases. Iismmay alsobe a factor in some

894

B i o s y n t h eosfAi sm i nA o c i d sN,u c l e o t i daensdR , e l a t eMdo l e c u l e s

OH

Allopurinol

OH

Hypoxanthine (enol form)

OH (-H N'"-c-crr.

N lll ,.6-N' Ho-"_-N_ H Oxypurinol tl6URt 22-47 Allopurinol,an inhibitorof xanthineoxidase.Hypoxanthineis the normalsubstrate of xanthineoxidaseOnly a slightal(shadedpink) yieldsthe terationin the structureof hypoxanthine medicallyeffective enzymeinhibitorallopurinolAt theactivesite,allopurinolis convertedto oxypurinol,a strongcompetitiveinhibitor thatremainstightlyboundto the reducedformof the enzyme Gout is effectively treated by a combination of nutritional and drug therapies Foods especially rich in nucleotides and nucleic acids, such as liver or glandular products, are r dthheld from the diet Major alleviation of

the symptoms is provided by the drug allopurinol (Fig. 22-47), which inhibits xanthine oxidase, the enzyme that catalyzes the conversion of purines to uric acid. Allopurinol is a substrate of xanthine oxidase, which converts allopurinol to oxypurinol (alloxanthine). Oxypurinol inactivates the reduced form of the enz;.'rne by remaining tightly bound in its active site When xanthine oxidase is inhibited, the excreted products of purine metabolism are xanthine and hypoxanthine, which are more water-soluble than uric acid and less likely to form crystalline deposits. Allopurinol was developed by Gertrude Elion and George Hitchings, who also developed acyclovir, used in treating people \ rith genital and oral herpes infections, and other purine analogs used in cancer chemotherapy I

Many{herinotherapeutic Agents Target Enzymes Pathways intheFlucleotide Biosynthetic The growth of cancer celis is not controlled in the same way as cell growth in most normal tissues. Cancer ceils have greater requirements for nucleotides as precursors of DNA and RNA, and consequently are generally more sensitive than normal cells to inhibitors of nucleoti.de bioslnthesis. A growing array of important chemotherapeutic agents-for cancer and other diseases-act by inhibiting one or more enzymes in these pathways. We describe here several well-studied examples that illustrate productive approaches to treatment and help us understand how these enzymes work. The first set of agents includes compounds that inhibit glutamine amidotransferases. Recall that glutamine is a nitrogen donor in at least half a dozen separate reactions in nucleotide biosynthesis. The binding sites for glutamine and the mechanism by which NHf is extracted are quite similar in many of these enzymes.Most are strongly inhibited by glutamine analogs such as azaserine and acivicin (FiS. 22-48). Azaserine, characterized by John Buchanan in the 1950s, was one of the first examples of a mechanism-basedenzyme inactivator (suicide inactivator; p. 204 and Box 22-3). Acivicin shows promise as a cancer chemotherapeutic agent. Other useful targets for pharmaceutical agents are thpnidylate synthase and dihydrofolate reductase, enzymes that provide the only cellular pathway for thyrnine synthesis (Fig. 22-49). One inhibitor that acts on th;.midylate synthase, fluorouracil, is an important chemotherapeutic agent. Fluorouracil itself is not the enzyme inhibitor. In the cell, salvage pathways convert it to the deoxynucleoside monophosphate FdUMP, which binds to and inactivates the enzyme. Inhibition by FdUMP (Fig. 22-50) is a classic example of mechanism-based enzyme inactivation. Another prominent chemotherapeutic agent, methotrexate, is an inhibitor of dihydrofolate reductase. This folate analog acts as a competitive inhibitor; the enzyme binds methotrexate wrth about 100 times higher affurity than dihydrofolate. Aminopterin is a related compound that acts similarly.

Tn'

N--r,*

Tn'

1r-\

CH"-C-COO NHsl--CHl-l llHllH /,Q-QH2

CH'-C-COO

C-O

ci'

d

Glutamine

Azaserine

N-

_o'CH-C

ill c-cH2

NU, COO

H

Cl Acivicin

C e r t r u dE e l i o n( 1 9 1 8 - 91 9 9 )a n o C e o r g eH i t c h i n g(s1 9 0 5 - 91 9 8 )

FIGURE 22-48 Azaserine andacivicin,inhibitorsof glutamineamidoTheseanalogsof glutamineinterferein severalamino transferases. acidand nucleotide pathways biosynthetic

2 2 . 4B i o s y n t h easnidsD e g r a d a toi of N n u c l e o t i d e[ tst {

FdUMP

dUMP

FdI,JMP

dTMP

s

-AP,N1o-Methylene Ha folate

; i

rhvnldylatr st nthlrsc

H'

H'

7,8-Dihydrofolate

N5,Nlo-Methylene H4 folate

Enzyme thiolate adds at C-6 of dUMR a Michaeltype addition; N10 is protonated and

+Ht

Glycine diltrclr ofirlltc r c r d u rt r r s e

r,6

.

lon

ls

formed from methylene-H4 folate

H4 folate NADP+

Serine (a)

:B

oAT'ti' oI

It

.

lv"-lmlnlum

-\F HN' Y

1l

O r-".,]i,-r-

oA,o-/1-tt

:B

o,'l-^-lr-t

:B

I

R\

HsCO

I

C-5 carbanion adds to M-iminium ion.

HsCO

o4N/

H Fluorouracil

Trimethoprim

H

i.\

Methylidene is forrned at C-5 of pyrimidine; N5 is eliminated to form Ha folate

Methotrexate

ft) FIGURE 22-49 Thymidylate synthesis andfolatemetabolism (a)Duringthymidylate astargetsof chemotherap!. synthesis,

Ns,Nlo-methylenetetrahydrofolate is convertedto 2,8-dihydrofolate; the Ns,NlO-methylenetetrahydrofolate is regenerated in two steps (see Fig. 22-44).This cycle is a major targetof severalchemotherapeutic agents.(b) Fluorouraciland methotrexateare important chemotherapeutic agents. In cells,fluorouracil is converted to FdUMP, which inhibitsthymidylate synthaseMethotrexate, a structural analog of tetrahydrofolate, inhibitsdihydrofolate reductase; the shadedamino and methylgroupsreplacea carbonyloxygenand a proton,respectively,in folate (seeFig. 22-44) Anotherimportantfolateanalog, aminopterin,is identicalto methotrexate exceptthat it lacksthe shadedmethylgroup.Trimethoprim, a tight-binding inhibitorof bacterialdihydrofolate reductase, wasdeveloped asan antibiotic.

Dead-end covalent conplex

I + @ 1l''. Enz,"rne ' l - > 'dihvdrofolate '.' "' I o

,r*\tn. \ ) o 'N-

1,8 hydride shift generates dTMP and dihydrofolate'

R

dTMP MECHANISM FIGURE 22-50 Conversion of dUMPto dTMPand its inhibitionby FdUMP.Theleftsideis the normalreactionmechanism of thymidylate synthase. The nucleophi Iic sulfhydrylgroupcontributed by the enzymein step@ and the ringatomsof dUMPtakingpart in the reactionare shownin red; :B denotesan aminoacid sidechain that actsas a baseto abstracta protonafterstep @ fne hydrogens derivedfrom the methylenegroup of N5,N1o-methylenetetrahydrofo-

lateareshadedin gray.The1,3hydrideshift(step@), movesa hydride ion (shadedpink)from C-6 of H+ folateto the methylgroupof thymiThis to dihydrofolate. dine,resultingin the oxidationof tetrahydrofolate hydrideshiftis blockedwhen FdUMPis the substrate lright).Steps@ of but resultin a stablecomplex-consisting and @ proceednormally, FdUMPlinkedcovalentlyto the enzymeand to tetrahydrofolate-that Mechanism the enzyme I ThymidylateSynthase inactivates

laooj

B i o s y n t h eosf A i sm i nA o c i d sN,u c l e o t i daensdR , e l a t eMdo l e c u l e s

The medicalpotential of inhibitors of nucleotide biosynthesisis not limited to cancer treatment. All fast-growingcells (including bacteria and protists) are potential targets. T[imethoprim, an antibiotic developedby Hitchingsand Elion, binds to bacterial dihydrofolatereductasenearly 100,000times better than to the mammalianenzyme.It is used to treat certain urinary and middle-earbacterial infections. Parasitic protists, such as the trypanosomesthat causeAfrican sleepingsickness(African trypanosomiasis),lack pathwaysfor de novo nucleotidebiosynthesis and are particularly sensitiveto agents that interfere with their scavengingof nucleotides from the surrounding environment using salvagepathways. Allopurinol (Fig. 22-47) and several similar purine analogshave shown promisefor the treatment of African trypanosomiasisand related afflictions. See Box 22-3 for another approach to combating African trypanosomiasis, made possibleby advances in our understanding of metabolism and enzyme mechanisms.r

KeyTerms Terms in bold are defined i,n the glossary

nitrogen cycle 852 nitrogen fixation 852 anammox 852 s5'rnbionts 852 nitrogenasecomplex 854 leghemoglobin 856 glutamine synthetase 857 glutamatesynthase 857 glutamine amidotransferases 859 5-phosphoribosyl-1pyrophosphate (PRPP) 861 tryptophansynthase 868 porphyrin 873 porphyria 873 bilirubin 875 phosphocreatine 876 creatine 876 glutathione (GSH) 876 SUMMAR 2Y 2 . 4 B i o s y n t h e sDi seagnrda d a t i o nauxin 878 dopamine 878 ofNucleotides norepinephrine 878 The purine ring system is built up step-by-step epinephrine 878 beginning with 5-phosphoribosylamrne. The amino 7-aminobuty'rate acids glutamine, glycine, and aspartate furnish all (GABA) 878 the nitrogen atoms of purines. TWo ring-closure serotonin 878 steps form the purine nucleus. histamine 878 clmetldine 879 Pyrimidines are synthesized from carbamoyl spermine 879 phosphate and aspartate, and ribose S-phosphate is then attached to yield the pyrimidine ribonucleotides. Nucleoside monophosphates are converted to their triphosphates by enz}..rnaticphosphorylation reactions. Ribonucleotides are converted to deoxyribonucleotides by ribonucleotide reductase, an enzyrne with novel mechanistic and regulatory characteristics. The thyrnine nucleotides are derived from dCDP and dUMP. Uric acid and urea are the end products ofpurine and pyrimidine degradation. Free purines can be salvaged and rebuilt into nucleotides. Genetic deflcienciesin certain salvage enz].rnes cause serious disorders such as Lesch-Nyhan syndrome and ADA deficiency. Accumulation of uric acid crystals in the joints, possibly caused by another genetic deficiency, results in gout Enzlnnes of the nucleotide biosynthetic pathways are targets for an array of chemotherapeutic agents used to treat cancer and other diseases.

qnermidina

R7q

ornithine decarboxylase 879 de novo pathway 882 salvage pathway 882 inosinate(lMP) 883 carbamoylphosphate synthetaseII 886 aspartate transcarbamoylase887 nucleoside monophosphate kinase 888 nucleoside diphosphate kinase 888 ribonucleotide reductase 888 thioredoxin 888 thyrnidylatesy'nthase 890 dihydrofolate reductase 890 adenosinedeaminase deflciency 893 Lesch-Nyhan syndrome 893 allopurinol 894 azaserine 894 acivicin 894 fluorouracil 894 methotrexate 894 aminopterin 894

Further Reading Nitrogen Fixation Arp, D.J. & Stein, L.Y. (2003) Metabolismof inorganicN compoundsby ammonia-oxidizingbacteria Crit Reu Biochem MoI Bi.oI 38,491 495 Burris, R.H. (1995) Breakingthe N-N bond.,4nnu Reu Plant Ph11si.ol PlantMol BioI 46,1-19. Fuerst, J.A. (2005) Intracellularcompartmentationin planctomycetes Annu Reu Mi,crobi,ol 59,299-328 Igarishi, R.Y. & Seefeldt, L.C. (2003) Nitrogenflxation: the mechanism of the Mo-dependentnitrogenaseCrit Reu Bi,ochem MoL BioL 88,351-384 Patriarca, E.J., Thte, R., & Iaccarino, M. (2002) Key role of bacterial NHj metabolismin rhizobium-plants;rnbiosis.Mzcrobi,ol MoL Bi.oI Reu 66,203-222. A good overwiewof ammoniaassrmilationin bacterialsystems and its regulation Prell, J. & Poole, P. (2006) Metabolicchangesof rhizobiain legume nodules TtrendsMr,crobi,ol 14, 161 168 A good summaryof the intricate s;anbloticrelationshipbetween rhizobialbactenaand their hosts Sinha, S.C. & Smith, J.L. (2001) The PRT protein family.Cum Or:in Stntct Bi,ol. ll. 733-739

Probtems [ssz--l L/ Descriptionof a proternfamily that includesmany amtdotransferases,with channelsfor the movementof NHr from one active site to another Amino Acid Biosynthesis Frey, P.A. & Hegeman, A.D. (2007)Enzgmati,c Rectcti,onMechanzsrzs,Oxford UniversityPress,NewYork An updated sufiunaryof reactionmechanisms,including onecarbonmetabolismand pyridoxal phosphateenzlmes. Neidhardt, F.C. (ed.). (1996)EscherichiacoliandSalmonella: Cellulctr and MolecularBto\ogg,2ndedn,ASMPress,Washington, DC Volume 1 of this two-volumeset has 13 chaptersdevotedto detailed descriptlonsof amino acid and nucleotidebiosynthesisin bacteria. The web-basedversionat www.ecosalorg is updatedregularly. A valuableresource Pan P.,Woehl, E., & Dunn, M.F. (1997)Protetnarchitecture,dynamicsand allosteryin tryptophan synthasechanneling.Thends Biochem Sci, 22,22-27 Richards, N.G.J. & Kilberg, M.S. (2006) Asparaginesyrrthetase chemotherapy.,nnu Reu Bi,ochem 75,629 654 Compounds Derived from Amino Acids ,{ioka R.S., Phillips, J.D., & Kushner, J.P. (2006)Biosynthesis of hemein mammalsBzochim Btophys Acta MoI CeIIRes 1763, 723-736 Bredt, D.S. & Snyder, S.H. (1994) Nitric oxide: a physiologicmessengermoleculeAn;nu Reu Btochem 63,175-195. Meister, A. & Anderson, M.E. (1983)GlutathioneAnnu Reu Btochem 52,71I-760 Morse, D. & Choi, A.M.K. (2002)Hemeoxygenase-l-the "emergingmolecule"hasarrivedArn J. Resp CeIIMoLBioI 27,8 16. Rondon, M.R., Thzebiatowski, J.R., & Escalante-Semerena, J.C. (1997)Biochemistryand moleculargeneticsof cobalamin biosynthesisProg Nu,cLeitAcidResMol BioL 56,347-384. Stadtman, T.C. (1996)Selenocysteine Annu Reu Biochem 65, 83-100

Molecular Bases of Inhemted Di,sease,Sth edn, McGraw-Hill Professional, New York This four-volume set has good chapters on disorders of amino acid, porphyrin, and heme metabolism See also the chapters on inborn errors of purine and pyrimidine metabolism.

Problems 1. ATP Consumption by Root Nodules in Legumes Bacteria residing in the root nodules of the pea plant consume more than 20o/o of the ATP producedby the plant. Suggestwhy consume so much ATP. thesebacteria 2. Glutamate Dehydrogenase and Protein Synthesis The bacterium Methy\ophi\us rnethAlotrophus can s)'nthesize protein from methanol and ammonia.RecombinantDNA techniqueshave improved the yeld of protein by introducing into M. methAlotroph?rsthe glutamate dehydrogenasegene from O colx Why doesthis geneticmanipulationincreasethe protein fleld? 3. PLP Reaction Mechanisms Pyridoxal phosphatecan help catalyze transformations one or two carbons removed from the a carbon of an amino acid. The enz''rne threonine slnthase (seeFig. 22-15) promotesthe PlP-dependentconto threonine Suggesta mechaversionof phosphohomoserine nism for this reaction. 4. Transformation ofAspartate to Asparagine There are two routes for transformingaspartateto asparagineat the expense of ATP. Many bacteria have an asparagineslrlthetase that usesamnonium ion as the nitrogen donor.Mammalshave an asparaginesynthetasethat usesglutamine as the nitrogen donor Giventhat the latter requiresan extra ATP (for the synthesisof glutamine),why do mammalsuse this route?

Nucleotide Biosynthesis Carreras, C.W. & Santi, D.V. (1995)The catall'ticmechanismand structureof th5.-rnidylate s)'nthaseAnnu Reu Btochem 64,72I-762 Holmgren, A. (1989) Thioredoxinand glutaredoxinsystemsJ Bzol Chetn 264, 13,963-13,966 Kappock, T.J,, Ealick, S.8., & Stubbe, J. (2000)Modularevolupathway.Curr Opin Chem BioL 4, tion of the purine bios5,.nthetic 567-572 Kornberg, A. & Baker, T.A. (1991)DNA Repli,cati,olx,2nd edn, W H Freemanand Company,New York This text includesa good summaryof nucleotidebiosynthesis Licht, S., Gerfen, G.J., & Stubbe, J. (1996)Ttlyl radicalsin ribonucleotrde reductasesSctence271, 477-481 Nordlund, P. & Reichard, P. (2006) Ribonucleotidereductases Annu Reu Bzochem 75,681-706 Schachman, H.K. (2000) Stiil looking for the ivory tower Annu Reu Biochem 69,1-29 A lively descriptionof researchon aspartatetranscarbamoylase, accompaniedby delightful tales of scienceand politics Stubbe, J. & Riggs-Gelasco, P. (1998) Harnessingfree radicals: formation and function of the tyrosyl radicalin ribonucleotidereductase Trends Biochem Sci 28,438-443 Genetic Diseases Scriver, C.R., Beaudet, A.L., Valle, D., Sly, W.S.,Childs,8., Kinzle4 L.W., & Vogelstein, B. (eds). (2001) The Metabo\ic and

5. Equation for the Synthesis of Aspartate from Glucose Write the net equation for the synthesisof aspartate(a nonessentialamino acid) from glucose, carbon dioxide, and anunorua. 6. Asparagine Synthetase Inhibitors in Leukemia Therapy Mammalian asparagine synthetase is a glutamine-dependentamidotransferase.Efforts to identify an effective inhibitor of human asparaginesynthetasefor use in chemotherapyfor patients with leukemiahas focusednot on the amino-terminalglutaminasedomain but on the carboxylterminal synthetaseactive site Explain why the glutaminase domainis not a promisingtarget for a useful drug. 7. Phenylalanine Hydroxylase Deficiency and Diet T!'rosine is normallya nonessentialaminoacid,but individualswith a geneticdefect in phenylalaninehydroxylaserequire tlrosine in their diet for normal growth. Explain 8. Cofactors for One-Carbon Tbansfer Beactions Most one-carbontransfersare promoted by one of three cofactors: (Chapter biotin, tetrahydrofolate,or,S-adenosylmethionine is generallyusedas a methyl group 18). S-Adenosylmethionine donor;the transferpotential of the methyl group in AF-methyltetrahydrofolateis insufflcientfor most biosyntheticreactions.

lhr*]

B i o s y n t h eosfAi sm i nA o c i d sN,u c l e o t i daensdR , e l a t eMdo l e c u l e s

However, one example of the use of ,Al-methyltetrahydrofolate in methyl group transfer is in methionine formation by the methionine s;mthase reaction (step @ of Fig. 22 15); methionine is the immediate precursor of ,S-adenosylmethionine (see Fig. 18-18) Explain how the methyl group of S-adenosylmethionine can be derived from M-mefhyltetrahydrofolate, even though the transfer potential of the methyl group in M-methyltetrahydrofolate is one one-thousandth of that in S-adenosylmethionine 9. Concerted Regulation in Amino Acid Biosynthesis The glutamlne sy-nthetaseof E coli, is independently modulated by various products of glutamine metabolism (see Fig. 22-6) .In tltis concerted inhlbition, the extent of enzy'rne inhibition is greater than the sum of the separateinhibitions causedby each product. For E coli grou.n in a medium rich in histidine, what would be the advantage of concerted inhlbitlon?

10. Relationship between Folic Acid Deficiency and Anemia Folic acid deflciencv,believedto be the most common vitamin deflciency, causes a type of anemla in which hemoglobin synthesis is impalred and erythrocytes do not mature properly. What is the metabolic relationship between henioglobin synthesis and folic acid deflciency? 11. Nucleotide

Biosynthesis

in Amino Acid Auxotrophic Bacteria Mld-type E coli cells can svnthesize all 20 common amino acids, but some mutants, called amino acid auxotrophs, are unable to synthesize a speclflc amino acid and require its addition to lhe cuiture medium for optimal growth Besides their rolc in protein synthesis, some amino acids are also precursors for other nitrogenous cell products. Consider the three amino acld auxotrophs that are unable to synthesize glycine, glutamine, and aspartate, respectively For each mutant, what nitrogenous products other than proteins would the cell fail to synthesize? 12. Inhibitors ofNucleotide Biosynthesis Suggest mechanisms for the inhibition of (a) alanine racemaseby l-fluoroalanine and (b) glutamine amidotransferases bv azaserine. 13. Mode of Action of Sulfa Drugs Some bacteria require p-aminobenzoate in the culture medium for normal growth, and their growth is severely inhibited by the additlon of sulfanilamide, one of the earliest sulfa drugs. Moreover, in the presence of this drug, 5-aminoimldazole-4-carboxamide ribonucleotide (AICAR; see Flg. 22-33) accumulates in the culture medium These effects are reversed bv addition of excess p-aminobenzoate

14. Pathway of Carbon in Pyrimidine Bios5mthesis Pre14Cin dict the locations of orotate isolated from cells growrr on a small amount of uniformly labeled [laClsuccinate Justify your predlction 15. Nucleotides as Poor Sources ofEnergy Under starvation conditions, organisms can use proteins and amino acids as sources of energy. Deamination of amino acids produces carbon skeletons that can enter the glycolytic pathway and the citric acid cycle to produce energy h the form ofAIP Nucleotides, on the other hand, are not similarly degraded for use as energylrelding fuels. What observations about cellular physiology support this statement? What aspect of the structure of nucleotides makes them a relatively poor source of energy?

16. Theatment of Gout Allopurinol (seeFig 22-47), an inhibitor of xanthine oxidase, is used to treat chronic gout Explain the biochemical basis for this treatment Patients treated with allopurinol sometimes develop xanthine stones in the kidneys, although the incidence of kidney damage is much lower than in untreated gout. Explain this observation in the light of the following solubilities in urine: uric acid, 0.15 g/L; xanthine, 0 05 g/L; and hypoxanthine, 1.4 g/L 17. Inhibition of Nucleotide Synthesis by Azaserine The diazo compound O-(2-drazoacetyl)-l-serine, knovm also as azaserine (see Fig. 2248), is a powerful inhibitor of glutamine amidotransferases If growing cells are treated with azaserine, what intermediates of nucleotide biosvnthesis will accumulate? Explain

DataAnalysis Problem 18. Use of Modern Molecular Techniques to Determine the Synthetic Pathway of a Novel Amino Acid Most of the biosynthetic pathways described in this chapter were determined before the development of recombinant DNA technology and genomics, so the techniques were quite different from those that researchers would use today. Here we explore an example of the use of modern molecular techniques to investigate the pathway of syrrthesis of a novel amino acid, (2S)-4-amino-2hydrorybutytate (AHBA). The techniques mentioned here are described in various places in the book; this problem is designed to show how they can be integrated in a comprehensive study. AHBA is a 7-amino acid that is a component of some aminoglycoside antibiotics, inciuding the antibiotic butirosin. Antibiotics modified by the addition of an AHBA residue are often more resistant to inactivation by bacterial antibiotic-resistance enzymes. As a result, understanding how AHBA is synthesized and added to antibiotics is useful in the design of pharmaceuticals. In an article published in 2005, Li and coworkers

p-Aminobenzoate

Sulfanilamide

(a) What is the role of p-aminobenzoate in these bacteria? (Hint: See Fig. 18-16.) (b) WhV does AICAR accumulate in the presence of sulfanilamide? (c) S4ry are the inhibition and accumulation reversed by additlon of excessp-aminobenzoate?

describe how they determined the synthetic pathway of AHBA from glutamate.

fru,

r"\c"'\-"\czrln ill oo Glutamate

OH ,i.)

NHi^^C--" I o AHBA

Problems fassl (a) Briefly describethe chemicaltransformationsneeded to convert glutamate to AHBA. At this point, don't be concernedaboutthe order ofLhereactions. Li and colleaguesbeganby cloningthe butirosin biosyrithetic gene cluster from the bacterium Baci,llus circulans, wlidn makeslargequantitiesof butirosin. They identiied flve genesthat are essentialfor the pathway:btrl, btrJ, btrK, btrO, and btrv. They clonedthese genesnto E. coli, plasmidsthat allow overexpressionof the genes,producingproteinswith "histidinetags" (seep. 314) fusedto their aminotermini to facilitatepuriflcation The predicted amino acid sequenceof the BtrI protein showed strong homologyto known acyl carrier proteins (see Fig.21-5). Usingmassspectrometry(seeBox 3-2), Li and colleaguesfound a molecularmassof 11,812for the purifiedBtrI protein (including the His tag) When the purifled Btrl was incubatedwith coenzl'rneA and an enz)'rnekno'"mto attach CoA to other acyl carrier proteins, the majority molecular species had anM. of 12,153. (b) How would you use these data to argue that Btrl can function as an acyl carrierprotein with a CoAprostheticgroup? Using standardterminology,Li and coauthorscalled the form of the protein lackingCoAapo-Btrl and the form with CoA (iinked as in Fig 21-b) holo-Btrl. When holo-Btrl was incubated with glutamine,ATP,and purifled BtrJ protein, the holoBtrl speciesof M, 12,153was replaced with a speciesof M, 12,281,corresponding to the thioesterof glutamateand holoBtrI Basedon these data, the authorsproposedthe followhg stmcturefor the M. 12,281species(7-glutamy1-S-Btrl):

(c) What other structure(s) is (are) consistentwith the data above? (d) Li and coauthors argued that the structure shornn here (y-glutamyl-S-Btrl) is likely to be correct becausethe acarboxyl group must be removed at some point in the synthetic process.Explain the chemical basis of this argument. (Hint: SeeFig. 18-6c) The BtrK protein showedsignificanthomologyto PLP-dependentaminoacid decarboxylases, and BtrK isolatedfrom-O col'lwas found to containtightly bound PLP When 7-glutamylS-BtrI was incubated with purifled BtrK, a molecular species of M, 12,240was produced. (e) What is the most likely structure of this species? (f) Interestingly,when the investigatorsincubated glutamate and ATP with purifled BtrI, BtrJ, and BtrK, they found a molecularspeciesof M, 12,370.What is the most likely structure of this species?Hint: Rememberthat BtrJ can useATP to 7-glutamylatenucleophilicgroups. Li and colleaguesfound that BtrO is homologousto monooxygenaseenzymes(see Box 21-1) that hydroxylate alkanes,uslng FMN as a cofactor,and BtrV is homologousto an NAD(P)H oxidoreductase.TWoother genesin the cluster, btrG and btrH, probably encode enzymes that remove the 7-giutamyl group and attach AHBA to the target antibiotic molecule. (g) Basedon these data, proposea plausiblepathway for the sy'nthesisof AHBA and its addition to the target antibiotic. Include the enz5.rnesthat calalyze each step and any other substratesor cofactorsneeded(ATP,NAD, etc.) Reference Li, Y., Llewellyn, N.M., Giri, R., Huang,F., & Spencer'J.B'

BtrI 7-Glutarnyl-S-Btrl

(2005) Biosynthesis of the unique amino acid side chain of butirosin: possible protective-group chemistry in an acyl carrier protein-mediated pathway Chem Bi'oL 12, 665-675

We recognizethat eachtissueand, moregenerally,eachcell of the organismsecretes. . . specialproductsor fermentsinto the blood which therebyinfluenceall the othercellsthus integrated with eachotherby a mechanismotherthan the nervoussystem. -Charles EdouardBrown-56quard and I d'Arsonval,article in ComptesRendusde la Soci6t6de Biologie,/B9/

Hormonal Regulation and Integration ofMammalian Metabolism 23.1 Hormones: Diverse Structures forDiverse Functions 901 23.2 Tissue-Specific Metabolism: TheDivision ofLabor 912 23.3 Hormonal Regulation ofFuel Metabolism 922 23,4 Obesity andtheRegulation ofBody Mass 930 23.5 0besity, theMetabolic Syndrome, and Type 2 Diabetes 938 n I Chapters13 through 22wehave discussedmetaboI lism at the level of the individual cell, emphasizing I central pathwayscorrrmonto almost all cells, bacterial, archaeal,and eukaryotic. We have seen how metabolic processeswithin cellsare regulatedat the level of individual enzyrnereactions,by substrateavailability,by allostericmechanisms, and by phosphorylationor other covalentmodiflcationsof enz;rmes. To appreciatefully the significanceof individual metabolic pathwaysand their regulation,we must view these pathwaysin the context of the whole organism.An essential characteristic of multicellular organismsis cell djfferentiationand divisionof labor.The specialized firnctionsof the tissuesand organsof complexorganismssuch as humansimposecharacteristicfuel requirementsand pattems of metabolism.Hormonalsignalsintegrateand coordinate the metabolic activities of djfferent tissues and optimize the allocationof fuelsand precursorsto eachorgan. In this chapterwe focus on mammals,looking at the specializedmetabolismof severalmajor organsand tissuesand the integration of metabolismin the whole organism. We begin by examining the broad range of

hormonesand hormonal mechanisms,then turn to the tissue-specificfunctions regulated by these mechanisms. We discussthe distribution of nutrients to various organs-emphasizingthe central role played by the liver-and the metabolic cooperation among these organs.To illustrate the integrative role of hormones,we describethe interplay of insulin, glucagon,and epinephrine in coordinatingfuel metabolismin muscle,Iiver,and adiposetissue.The metabolicdisturbancesin diabetes further illustrate the importanceof hormonalregulation of metabolism.We discussthe long-termhormonalregulation of body mass and, finally, the role of obesity in developmentof the metabolic s;mdromeand diabetes.

for Diverse Structures 23.'lHormones: Functions Diverse Virtually every processin a complex organismis regulated by one or more hormones:maintenanceof blood pressure,blood volume, and electrolytebalance;embryogenesis;sexual differentiation,development,and reproduction;hunger, eating behavior,digestion,and fuel allocation-to namebut a few.We examinehere the methods for detecting and measuringhormonesand their interactionwith receptors,and considera representativeselectionof hormonetypes. The coordination of metabolism in mammals is achievedby the neuroendocrine system. Individual cellsin one tissuesensea changein the organism'scircumstancesand respondby secretinga chemicalmessenger that passesto another cell in the same or differenttissue,where the messengerbinds to a receptor moleculeand triggers a changein this second "9t t 19011

l

Regulation andIntegration ofMammalian Metabolism L902] Hormonal neuronalsignaling(Fig. 23-la), the chemrcalmessenger (neurotransmitter;acetylcholine,for example)may travel only a fractionof a micrometer,acrossthe s;,rrapticcleft to the next neuron in a network. In hormonalsignaling,the messengers-hormones-are carried in the bloodstream to neighboringcells or to distant organsand tissues;they may travel a meter or more before encountprinothpir (a) Neuronal signaling

I .i'

;.

.

target cell (FiC.23-1b). Except for this anatomicdrfference, these two chemicalsignalingmechanismsare remarkably similar. Epinephrine and norepinephrine,for example,serveas neurotransmittersat certain slmapses of the brain and neuromuscularjunctionsof smoothmuscle,and ashormonesthat regulatefuel metabolismin liver and muscle,The followingdiscussionof cellularsignaling emphasizeshormone action, drawing on discussionsof fuel metabolismin earlierchapters,but most of the fundamental mechanismsdescribedhere also occur in neurotransmitteraction.

,/

TheDetection andPurifiration ofHormones Requires a Bioassay

Target cells

r

/

Contraction

Metabolic change

rl

Secretion

F

tt

T

(b) Endocrine sigrraling FIGURE23-lSignaling by the neuroendocrine system.(a)In neuronal signaling, electrical signals(nerveimpulses) originatein the cell body ofa neuronandtravelveryrapidlyoverlongdistances to theaxontip, whereneurotransmitters arereleased and diffuseto thetargetcell.The targetcell (anotherneuron,a myocyte,or a secretory cell) is only a fractionof a micrometeror a few micrometersaway from the site of neurotransmitter release(b) In the endocrinesystem,hormonesare secreted intothebloodstream, whichcarriesthemthroughout thebodyto targettissues that maybe a meteror moreawayfrom the secretingcell. Bothneurotransmitters and hormonesinteractwith specificreceptors on or in theirtargetcells,triggering responses.

How is a hormone detected and isolated? First, researchers find that a physiological process in one tissue depends on a signal that originates in another tissue. Insulin, for example, was first recognized as a substance that is produced in the pancreas and affects the volume and composition of urine (Box 23-I). Once a physiological effect of the putative hormone is discovered, a quantitative bioassay for the hormone can be developed.In the case of insulin, the assayconsisted of injecting extracts of pancreas (a crude source of insulin) into experimental animals deficient in insulin, then quantifying the resulting changes in glucose concentration in blood and urine. To isolate a hormone, the biochemist fractionates extracts containing the putative hormone, with the same techniques used to purify other biomolecules (solvent fractionation, chromatography, and electrophoresis), and then assays each fraction for hormone activity. Once the chemical has been purified, its composition and structure can be determined. This protocol for hormone characterization is deceptively simple. Hormones are extremely potent and are produced in very small amounts. Obtaining sufficient hormone to allow its chemical characterization often involves biochemical isolations on a heroic scale. When Andrew Schally and Roger Guillemin independently purified and characterized thyrotropin-releasing hormone (TRH) from the hypothalamus, Schally's group processed about 20 tons of hypothalamus from nearly two million sheep, and Guillemin's group extracted the hypothalamus from about a million pigs! TRH proved to be a simple derivative of the tripeptide Giu-His-Pro (FiS. 23-2). Once the structure of the hormone was known, it could be chemically synthesized in large quantities for use in physiological and biochemical studies. For their work on hypothalamic hormones, Schally and Guillemin shared the Nobel Prize in Physiology or Medicine in 1977, along with Rosalyn Yalow, who (with Solomon A. Berson) developed the extraordinarily sensitive radioimmunoassay (RIA) for peptide hormones and used it to study hormone action. RIA revolutionized

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o srD i v e r sFeu n c t i o n s 2 3 . 1H o r m o n D e si v: e r sSet r u c t u rf e

Millions of people with type 1 diabetesmellitus inject themselvesdaily with pure insulin to compensatefor the Iack of production of this critical hormoneby their own pancreaticB cells.Insulininjectionis not a cure for diabetes,but it allowspeoplewho otherwisewould have died young to lead long and productivelives.The discovery of insulin, which beganwith an accidentalobservation, illustratesthe combinationof serendipityand careful experimentationthat led to the discoveryof manyof the hormones. In 1889, Oskar Minkowski,a young assistantat the MedicalCollegeof Strasbourg,and Josefvon Mering, at the Hoppe-Seyler Institutein Strasbourg, had a friendly disagreementabout whether the pancreas, known to contain lipases,was important in fat digestion in dogs.To resolvethe issue,they beganan experiment on the digestion of fats. They surgically removedthe pancreasfrom a dog,but beforetheir experiment got any farther, Minkowskinoticed that the dog was now producingfar more urine than normal (a common symptom of untreated diabetes).Also, the dog's urine had glucoselevels far above normal (another symptom of diabetes). These findings suggestedthat lack of some pancreaticproduct caused diabetes. Minkowskitried unsuccessfullyto prepare an extract ofdog pancreasthat wouldreversethe effectofremoving the pancreas-that is, would lower the urinary or blood glucoselevels.We now know that insulin is a protein,and that the pancreasis very rich in proteases (trypsin and chirmotrypsin),normally releaseddirectly into the small intestineto aid in digestion.Theseproteasesdoubtlessdegradedthe insulinin the pancreatic extracts in Minkowski'sexperiments. Despiteconsiderableeffort, no signiflcantprogress was made in the isolation or characterizationof the "antidiabeticfactor" until the summer of 1921,when Frederick G. Banting, a young scientist working in the

Frederick C Banting, 1891-1 941

J J. R Macleod,1876-1935

Iaboratory of J, J. R. Macleod at the University of Toronto,and a studentassistant,CharlesBest,took up the problem. By that time, severallines of evidence pointed to a group of specializedcells in the pancreas (the isletsof Langerhans;seeFig.23-27) as the source of the antidiabetic factor, which came to be called insulin (from Latin i,nsula, "island"). Taking precautionsto prevent proteolysis,Banting and Best (later aided by biochemistJ. B. Collip) succeededin December I92I in preparing a puri-fledpanof experimental creatic extract that cured the s5rmptoms diabetesin dogs.On January25,1922 fiust one month later!), their insulin preparation was injected into LeonardThompson,a 14-year-oldboy severelyill with diabetesmellitus. Within days, the levels of ketone bodiesand glucosein Thompson'surine dropped dramatically;the extract savedhis life. In 1923,Banting and Macleod won the NobelPrize for their isolationof insulin. Bantingimmediatelyannouncedthat he would share his prize with Best; Macleod shared his with Collip. By 1923,pharmaceuticalcompanieswere supplying thousandsof patients throughout the world with insulin extracted from porcine pancreas.With the development of genetic engineeringtechniques in the 1980s(Chapter9), it becamepossibleto produceunIimited quantities of human insulin by inserting the cloned human gene for insulin into a microorganism, which was then cultured on an industrial scale.Some patients with diabetesare now fitted with implanted insulin pumps, which release adjustableamounts of insulin on demand to meet changingneeds at meal times and during exercise. There is a reasonable prospectthat, in the future, transplantationof pancreatic tissue will provide diabetic patients with a sourceof insulin that respondsas well as normal pancreas, releasinginsulin into the bloodstreamonly when bloodglucoserises.

CharlesBest,1899-1978

J. B. Collip,1892-1965

904

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(a)

Radiolabeled hormone

VVV VYY VVY

U

C-NH

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HC-N,

,cH

Pyroglutarnate Histidine pyroGlu-His-Pro-NH,

R o g eC r uillemin

Rosalyn S Yalow

gvv oe,

o

U

Antibody Prolylamide

FIGURE 23-2 Thestructureof thyrotropin-releasing hormone(TRH). Purified(byheroicefforts) fromextracts of hypothalamus, TRHproved to be a derivative of the tripeptideClu-His-ProThe side-chain carboxyl groupof the amino-terminal Clu formsan amide (red bond) with the residue's a-aminogroup,creatingpyroglutamate, and the carboxylgroupof the carboxyl+erminal Prois converted to an amide (red-NHr). Suchmodrfications arecommonamongthe smallpeptide hormones. In a typicalproteinof M, ^-50,000, the charges on the amino-and carboxyl{erminal groupscontributerelativelylittleto the overallcharge on the molecule,but in a tripeptidethesetwo charges dominatethe propertiesof the molecule.Formationof the amide derivatives removes thesecharces

AndrewV Schally

v g v

hormone research by making possible the rapid, quantitative, and specific measrirement of hormones in minute amounts. H o r m o n e - s n e cl i c a n t i b o d ies are the key to the radio-immunoassay. Purified hormone, injected into rabbits, elicits antibodies that bind to the hormone with very high affinity and specificity. When a constant amount of isolated antibody is incubated with a fixed amount of the radioactively labeled hormone, a certain fraction of the radioactive hormone binds to the antibody (FiS. 2:]-:t) If, in addition to the radiolabeled hormone, unlabeled hormone is also present, the unlabeled hormone competes with and displaces some of the labeled hormone from its binding site on the antibody. This binding competition can be quantifled by reference to a standard curve obtained with known amounts of unlabeled hormone. The degree to which labeled hormone is displaced from antibody is a measure of the amount of (unlabeled) hormone in a sample ofblood or tissue extract. By using very highly radioactive hormone, researchers can make

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10

100

1000

Unlabeled ACTH added (pg)

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(RlA).(a) A low concentratron FIGURE 23-l Radioimmunoassay ot radiolabeled hormone(red)is incubated with @ a fixedamountof antibodyspecificfor thathormoneor @ a fixedamountof antibodyand variousconcentrations of unlabeled hormone(blue).In the Iattercase, unlabeled hormonecompetes with labeledhormonefor bindingto the antibody;theamountof labeledhormoneboundvariesinversely with the concentration of unlabeledhormonepresent (b) A radioimmunoassayfor adrenocorticotropic hormone (ACTH; also called corticotropin) A standard curveof the ratiolbound]/[unbound] radiolabeledACTHvs. [unlabeled ACTHadded](ona logarithmic scale)is constructed andusedto determine theamountof (unlabeled) ACTHin an unknownsample.If an aliquotcontaining an unknownquantityof unlabeled hormonegives,say,a valueof 0.4 for the ratiotboundl/[unboundl(seearrow),the aliquotmustcontainabout20 pg of ACTH.

the assay sensitive to picograms of hormone in a sample. A newer variation of this technique, enz),Tne-linked im-

munosorbentassay(ELISA),is illustratedin Figure5-26b.

Hormones ActthrouEh Speeifie Fligh-Affinity Cellular Receptors As we saw in Chapter 12, all hormones act through highly specific receptors in hormone-sensitive target cells, to which the hormones bind with high affinity (see

Functions forDiverse Diverse Structures 23.1Hormones: Ft{ Fig. 12-1a). Each cell type has its own combination of hormone receptors, which define the range of its hormone responsiveness Moreover, two cell fipes with the same type of receptor may have different intracelltrlar targets of hormone action and thus may respond drfferently to the same hormone. The specificity of hormone action results from structural complementarity between the hormone and its receptor; this interaction is extremely selective, so structurally similar hormones can have different effects. The hrgh affinity of the hteraction allows cells to respond to very low concentrations of hormone. In the design of drugs rntended to intervene in hormonal regulation, we need to know the relative specificity and affinity of the drug and the natural hormone Recall that hormone-receptor interactions can be quantrf,ed by Scatchard analysis (see Box i2-1), which, under favorabie conditions, yields a quantitative measure of affinity (the drssociation constant for the complex) and the mrmber of hormone-binding sites in a preparation of receptor. The locus of the encounter between hormone and receptor may be extracellular, cytosolic, or nuclear, depending on the hormone type. The intracellular consequencesof hormone-receptor hteraction are of at Ieast six general types: (1) a second messenger (such as cAMP or inositol trisphosphate) generated inside the celi acts as an allosteric regulator of one or more enzyrnes; (2) a receptor t;,'rosine kinase is activated by the extracellular hormone; (3) a receptor guanylyl cyclaseis activated and produces the second messenger cGMP; (4) a change in membrane potential results from the opening or closing of a hormone-gated ion channel; (5) an adhesion receptor on the celi surface interacts with molecules in the extraceliular matrix and conveys information to the cytoskeleton; or (6) a steroid or steroidlike molecule causesa change rn the level of expression (transcription of DNA into mRNA) of one or more genes,mediated by a nuclear hormone receptor protein (see Fig. I2-2). Water-solublepeptide and amine hormones (insulin and epinephrine, for example) act extracellularly by binding to cell surface receptors that span the piasma membrane (Fig. 23-4). When the hormone binds to its extracellular domain, the receptor undergoes a conformational change analogous to that produced in an allosteric enzyrne by binding of an effector molecule. The conformational change triggers the downstream effects of the hormone A single hormone molecuie, in forming a hormonereceptor complex, activates a catalyst that produces many molecules of second messenger, so the receptor serves not only as a signal transducer but also as a signal amplifier. The signal may be further amplified by a signaling cascade, a series of steps in which a catalyst activates a catalyst, resulting in very large ampliflcations of the original signal. A cascade of ttus tlpe occurs in the regulation of glycogen synthesis and breakdown by epinephrine (see Fig. l2-T). trpinephrine activates (through its receptor) adenylyl cyclase, which produces many molecules of cAMP for each molecule of receptorbound hormone Cvclic AMP in turn activates cAMP-

Peptide or amine hormone binds to receptor on the outsideofthe cell; acts through receptor without entering the cell.

Steroid or thyroid hormone enters the cell; hormonereceptor complex acts in the nucleus.

Altered transcription

Altered activity of preexisting enzynne

Altered amount of newly s5'nthesized proteins

of hormoneaction.ThepepFIGURE 23-4 Twogeneralmechanisms hormones arefasteractingthansteroidandthyroid "" ihli:. dependent protein kinase (protein kinase A), which activates glycogen phosphorylase b kinase, which activates glycogen phosphorylase b. The result is signal ampliflcation: one epinephrine molecule causes the production of many thousands of molecules of glucose l-phosphate from glycogen. Water-insoluble hormones (steroid, retinoid, and thyroid hormones) readily pass through the plasma membrane of their target cells to reach their receptor proteins in the nucleus (Fig. 23-4). With this class of hormones, the hormone-receptor complex itself carries the message;it interacts with DNA to alter the expression of specifi.cgenes, changing the enz;,nnecomplement of the cell and thereby changing cellular metabolism (see Fig. 12-29). Hormones that act through plasma membrane receptors generally trigger very rapid physiological or biochemical responses. Just seconds after the adrenal medulla secretes epinephrine into the bloodstream, skeletal muscle responds by accelerating the breakdov,n of glycogen. By contrast, the thyroid hormones and the sex (steroid) hormones promote maximal responses in their target tissues only after hours or even days. These differences in response time correspond to different modes of action. In general, the fast-acting hormones lead to a change in the activity of one or more preexisting enzymes in the cell, by allosteric mechanisms or

F'.1

H o r m o nRael g u l a t iaonndI n t e g r a t ioofnM a m m a l iM a ne t a b o l i s m

covalentmodif,cation.The slower-acting hormonesgenerallyalter geneexpression,resultingin the synthesisof more (upregulation) or less (downregulation)of the regulatedprotein(s).

Hormones AreChemically Diverse Mammals have several classesof hormones, distinguishable by their chemical structures and their modes of action (Table 23-I). Peptide, amine, and eicosanoid hormones act from outside the target cell via surface receptors. Steroid, vitamin D, retinoid, and thyroid hormones enter the cell and act through nuclear receptors. Nitric oxide also enters the cell, but activates a cy'tosolic enz),Tne,guanylyl cyclase (see Fig. 12-20). Hormones can also be classifledby the way they get from their point of release to their target tissue. Endocrine (from the Greekendon, "within," andkri,nei,n, "to release") hormones are released into the blood and carried to target cells throughout the body (insulin and glucagon are examples). Paracrine hormones are released into the extracellular space and diffuse to neighboring target ceils (the eicosanoid hormones are of this type). Autoerine hormones affect the same cell that releasesthem, binding to receptors on the cell surface. Mammals are hardly unique in possessinghormonal signaling systems. Insects and nematode worms have highly developed systems for hormonal regulation, with fundamental mechanisms similar to those in mammals. Plants, too, use hormonal signals to coordinate the activities of their tissues (Chapter 12). The study of hormone action is not as advanced in plants as in animals, but we do know that some mechanisms are shared. To illustrate the structural diversity and range of action of mammalian hormones, we consider representative examples of each major class listed in Table 23-1. Peptide Hormones Peptide hormones may have from 3 to 200 or more amino acid residues. They include the pancreatic hormones insulin, glucagon, and

somatostatin;the parathyroidhormonecalcitonin;and all the hormonesof the hypothalamusand pituitary (describedbelow). Thesehormonesare synthesizedon ribosomes in the form of longer precursor proteins (prohormones),then packagedinto secretoryvesicles and proteolyticallycleavedto form the active peptides. Insulin is a smallproten (M,5,800) with two pollpeptide chains,A and B, joined by two drsulfldebonds.It is snthesizedin the pancreasas an inactive sin$e-chainprecursor,preproinsulin(Fig. 23-5), with an amino-terminal "signal sequence"that drects its passageinto secretory vesicles.(Signalsequencesare discussedin Chapter27; see Fig. 27-38.) Proteolytic removal of the signal sequenceand formation of ttLreedrsulfldebonds produces proinsulin,wtuch is stored in secretorygranulesur pancreaticB cells Whenbloodglucoseis elevatedsufflciently to trigger insulin secretion,proinsulinis convertedto active insulin by specificproteases,which cleavetwo peptide bondsto form the matureinsulinmolecule In some cases,prohormoneproteins, rather than flelding a singlepeptidehormone,produceseveralactive (POMC)is a spectacuhormones.Pro-opiomelanocortin Iar example of multiple hormonesencodedby a single gene.The POMCgeneencodesa largepolypeptidethat is progressivelycarvedup rnto at least nine biologicallyactive peptides(FiS. 23-6). In manypeptidehormonesthe termrnalresiduesare modified,asin TRH (Fig. 23-2). The concentrationof peptidehormonesin secretory granulesis so high that the vesiclecontentsare virtually crystalline;when the contentsare releasedby exocytosis,a large amount of hormoneis releasedsuddenly.The capillaries that serve peptide-producing endocrine glands are fenestrated (and thus permeableto peptides), so the hormone molecules readily enter the bloodstreamfor transportto target cells elsewhere.As noted earlier,all peptide hormonesact by binding to receptorsin the plasmamembrane.They causethe generation of a second messengerin the cytosol, which changesthe activity of an intracellular enz;.'rne,thereby alteringthe cell'smetabolism.

$pe

Example

Syntheticpath

Peptide

Insulin, glucagon

Proteoifiic processingof prohormone

Catecholamhe

Epinephrhe

From tyrosine

Eicosanoid

PGEr

From arachidonate (20:4 fatty acid)

Steroid

Testosterone

From cholesterol

Vitamin D

1,25-Dihydroxycholecalciferol

From cholesterol

Retinoid

Retinoic acid

From vitamin A

Thyroid

Ttiiodothl'ronine (T3)

From T\rr in thlr'oglobulin

Nitric oxide

Nitric oxide

From arginine * 02

Modeof action

Plasmamembranereceptors;second messengers

Nuclearreceptors;transcriptional regulation

Cytosoiic receptor (guany$ cyclase) and second messenger (cGMP)

Functions[rtt] for Diverse Diverse Structures 23.1Hormones: Preproinsulin

Proinsulin

Mature insulin

23-5 Insulin.Matureinsulinis formedfrom FIGURE its largerprecursorpreproinsulinby proteolyticprocessing.Removalof a 23 amino acid segment(the signalsequence)at the amino terminusof preproinsulinandformationof threedisulfidebondsproduces proinsulin.Furtherproteolyticcutsremovethe C peptide from proinsulinto producematureinsulin,composedof A and B chains.Theamino acid sequenceof bovineinsulinis shownin Figure3-24.

Pro-opiomelanocortin (POMC) gene

I ,1,

DNA

mRNA

Signal peptide

B-Lipotropin

FIGURE 23-6 Proteolyticprocessing of the pro-opiomelanocortin (POMC)precursor.The initial geneproductof the POMCgeneis a long polypeptidethat undergoescleavageby a seriesof specific proteases to produceACTH, B- and 7-lipotropin,a-, B-, and 7CLIP MSH (melanocyte-stimulating hormone,or melanocortin), (corticotropin-like intermediarypeptide),B-endorphin,and Metenkephalin.The pointsof cleavageare pairedbasic residues, Arg-Lys,Lys-Arg,or Lys-Lys.

Cateeholamine Hormones The water-solublecompounds epinephrine (adrenaline) and norepinephrine (noradrenaline) are eatecholamines, namedfor the structurally related compound catechol. They are synthesizedfrom tyrosine. Tyrosine -----+ uDopa -----+ Dopamine --> Norepinephrine -+ Epinephrine

producedin the brain and in other neural Catecholamines tissues function as neurotransmitters,but epinephrine

B-Endorphin I

I Y Met-enkephalin

and norepineph-rineare also hormones,synthesizedand secreted by the adrenal glands. Like the peptide horare highly concentratedin secremones,catecholamines tory vesiclesand releasedby exocytosis,and they act through su-rfacereceptorsto generateintracellular second messengerc.They mediatea wide variety of physiologicalresponsesto acutestress(seeTable23-6). Eicosanoid Hormones The eicosanoidhormones (prostaglandins,thromboxanes,and leukotrienes) are

Regulation andlntegration ofMammalian Metabolism L90B_]Hormonal derived from the 20-carbonpoll'unsaturatedfatty acid arachidonate Phospholipids

I

J Arachidonate Q0:4)

Prostaglandins

Thromboxanes

Leukotrienes

Unlikethe hormonesdescribedabove,they are not synthesized in advanceand stored; they are produced, when needed,from arachidonateenzlmaticallyreleased from membrane phospholipidsby phospholipase42. The enzymesof the pathway leading to prostaglandins and thromboxanes(seeFig. 21-15) are very widely distributed in mammaliantissues;most cells can produce these hormone signals,and cells of many tissuescan respond to them through specific plasma membrane receptors.The eicosanoidhormonesare paracrile hormones,secretedinto the interstitial fluid (not primarily into the blood) and actingon nearbycells. Prostaglandins promote the contraction of @ E smooth muscle, including that of the intestine and uterus (and can thereforebe used medicallyto induce labor). They alsomediatepain and inflammationin all tissues.Many antiinflammatorydrugs act by inhibiting steps in the prostaglandinsynthetic pathway (see Fig. 21-15). Thromboxanesregulate platelet function and therefore blood clotting. LeukotrienesLTCa and LTDaact throughplasmamembranereceptorsto stimulate contractionof smoothmusclein the intestine,pulmonary airways, and trachea. They are mediators of anaphylaxis,a severe,detrimentalimmuneresponse.I Steroid Hormones The steroid hormones(adrenocortical hormonesand sex hormones)are synthesized from cholesterolin severalendocrinetissues. Cholesterol

J

Progesterone

./i\ Cortisol (glucocorticoid)

I

J

Testosterone

Aldosterone (mineralocorticoid)

I

Estradiol (sexhormones) They travel to their target cells through the bloodstream, bound to carrier proteins. More than 50 corticosteroid hormones are produced in the adrenal cortex by reactions that remove the side chain from the D ring of cholesterol and introduce oxygen to form keto and

hydroxyl groups.Many of these reactionsinvolve cy(see Box 21-1). The steroid tochromeP-450enz}.'rnes hormones are of two general types. Glucocorticoids (such as cortisol) prtmarily affect the metabolismof carbohydrates;mineralocorticoids(such as aldosterone) regulatethe concentrationsof electrol;,'tesin the blood. Androgens(testosterone)and estrogens(suchas estradiol; see Fig. 10-19) are synthesizedin the testesand ovaries.Their synthesisalsoinvolvescytochromeP-450 enzyrnesthat cleavethe side chain of cholesteroland introduce oxygen atoms. Thesehormonesaffect sexual development,sexualbehavior,and a variety of other reproductive and nonreproductivefunctions. All steroid hormonesact through nuclear receptors to changethe level of expressionof specificgenes (p. a56). They can alsohavemore rapid effects,probably mediatedby receptorsin the plasmamembrane. Vitamin D Hormone Calcitriol (1,25-dihydroxycholecalciferol)is produced from vitamin D by enzymecatalyzedhydroxylation in the liver and kidneys (see Fig. 10-20a). Vitamin D is obtainedin the diet or by photolysisof 7-dehydrocholesterol in skin exposedto sunlight. 7-Dehydrocholesterol I uu righ,

J

Vitamin D3 (cholecalciferol)

I

J 25-Hydroxycholecalciferol

I

J

1,25-Dihydroxycholecalciferol

Calcitriol works in conceft with parathyroid hormone in Caz* homeostasis, regulating[Ca2+]in the blood and the balancebetweenCa2+depositionand Caz+mobilizationfrom bone.Acting through nuclearreceptors,calcitriol activatesthe synthesisof an intestinal Ca2*-bindingprotein essentialfor uptakeof dietary Ca2+. InadequatedietaryvitaminD or defectsin the biosynthesis of calcitriol result in seriousdiseasessuch as rickets, in which bonesareweakandmalformed(seeFig. 10-20b).r Retinoid Hormones Retinoidsare potent hormones that regulatethe growth, survival,and differentiation of cells via nuclear retinoid receptors.The prohormone retinol is synthesizedfrom B-carotene,prrmarrlyin liver fseeFig. 10-21), and many tissuesconvert retinol to the hormoneretinoicacid (RA). B-Carotene

J

VitaminAl (retinol)

J Retinoic acid

Functions forDiverse Structures Diverse 23.1Hormones: [rtt-] All tissuesare retinoid targets,as all cell types haveat least one form of nuclearretinoid receDtor. In adults, the most significant targets include cornea,skin, epitheliaof the lungsand trachea,and the immunesystem.RA regulatesthe synthesisof proteins essentialfor growth or differentiation. Excessivevitamin A can causebirth defects,and pregnantwomen are advisednot to use the retinoid creamsthat have been developedfor treatmentof severeacne.I Thyroid Hormones The thyroid hormones Ta (thyroxine) and T3 (triiodothyronine) are s1'nthesizedfrom the precursorproteinthyroglobulin(M. 660,000).Up to 20 Tjr residuesin thyroglobulin are enzymaticallyiodinated in the thyroid gland, then two iodotyrosine residuescondenseto form the precursorto thyroxine. Whenneeded,thyroxineis releasedby proteolysis.Condensation of monoiodotyrosinewith diiodothyronine producesT3, which is also an activehormonereleased hrz nrntenlrrqiq

Thyroglobulin-Tyr

', Thyroglobulin - Tyr - I (iodinated Tyr residues) jn.ot"otv.i. Thyroxine (Ta), triiodothyronine (T3)

The thyroid hormonesact throughnuclearreceptorsto stimulate energy-yieldingmetabolism,especiallyin Iiver and muscle,by increasingthe expressionof genesencodingkey catabolicenzyrnes. Nitric Oxide (NO) Nitric oxide is a relatively stable free radical synthesizedfrom molecularoxygenand the guanrdiniumnitrogen of arginine (see Fig. 22-31) in a reactioncatalyzedby NO synthase. Arginine+ I+NADPH+ 2O2-----+ NO + citrulline -r 2H2o+ U NADP+ This enzlme is found in manytissuesand cell types:neurons, macrophages,hepatoc5,'tes, myoc),tesof smooth muscle,endothelialcells of the blood vessels,and epithelial cells of the kidney. NO acts near its point of release,enteringthe target cell and activatingthe cytosolic enzyrneguanylyl cyclase,which catalyzesthe formation cGMP(seeFig. 12-20). of the secondmessenger

of Neuronal Hormone Release lsRegulated bya hllerarchy andl{CIrmCInal Signals The changinglevelsof speciflchormonesregulatespeciflc cellularprocesses,but what regulatesthe level of each hormone?The brief answer is that the central nervous system receivesinput from many internal and external sensors-signals about danger,hunger,dietary intake,blood compositionand pressure,for exampleand orchestratesthe productionof appropriatehormonal

signalsby the endocrinetissues.For a more complete answer, we must look at the hormone-producing systemsof the humanbody and someof their functional interrelationships. Figure 23-7 showsthe anatomiclocation of the major endocrineglandsin humans,and Figure 23-8 representsthe "chainof command"in the hormonalsignaling hierarchy.The hypothalamus, a small region of the brain (FiS. 23-9), is the coordinationcenterof the endocrinesystem;it receivesand integratesmessages from the central nervoussystem.In responseto these the hypothalamusproducesregulatoryhormessages, mones (releasing factors) that pass directly to the nearby pituitary gland, through specialblood vessels and neuronsthat connectthe two glands(FiS.23-9b). The pituitary gland has two functionally distinct parts. The posterior pituitary containsthe axonalendingsof many neurons that originate in the hypothalamus. These neurons produce the short peptide hormones oxytocin and vasopressin(Fig. 23-10), which move down the axon to the nerve endings in the pituitary, wherethey are storedin secretorygranulesto awaitthe signalfor their release, The anterior pituitary respondsto hypothalamic hormones carried in the blood, producing tropic hormones, or tropins (from the Gteektropos,"turn"). These relatively long polypeptidesactivate the next rank of endocrineglands(Fig.23-B),which includesthe adrenalcortex,thyroid gland,ovaries,and testes.These glandsin turn secretetheir speciflchormones,which are carriedin the bloodstreamto the target tissues.For

Hypothalamus

Pituitary

Thyroid Parathyroids (behind the thyroid)

Adipose tissue Adrenals Pancreas Kidneys Ovaries

Theglandsareshadedpink. glands. tlGURE23-7Themajorendocrine

isrO'

H o r m o nRael g u l a tai onndI n t e g r a t oi ofM n a m m a lM i aent a b o l i s m

+

Sensoryinput from environment

FIGURE 23-8 The major endocrinesystemsand their target tissues.Signalsoriginatingin the centralnervoussystem(top) passvia a series (bottom). of relaysto the ultimatetargettissues In additionto the systems shown,the thymus,pinealglano,ano groupsof cellsin thegastrointestinal tractalsosecretehormones. Dashedlinesrepresent neuronalconnections

Neuroendocrine origins of sigrrals

Corticotropin (ACTH)

Second

targets

Follicle- Luteinizing Somatotropin Prolactin s t l n l u l a r l n g h u r r n o n e (growth hormone) M,22,0O0 normone .u.20.;00 M.2t,500 M,21.000

Thyrotropin M.28,000

d

tarEets

.\4"nY tissues

Vasopressin (antidiuretic

Blood glucose

Ovaries/testes

Cortisol. Thyroxine corticosterone. (T4), triiodo_ aldosterone thyronine (TB)

ultimate

Oxytocin M,1,007

Mgscles. liver

lil

Insulin,

ProgesLerone, e-qtradrol Testosterone

Reproductive

Epinephrine ."i,,\:fiff;;,"

Mammary glands'

organs

Smooth muscle,

lil kidney

muscles

mammary glands

example,corticotropin-releasing hormonefrom the hypothalamusstimulatesthe anterior pituitary to release ACTH,which travelsto the zonafasciculataof the adrenal cortex and triggersthe releaseof cortisol.Cortisol, lhe ultimate hormonein this cascade,acts through its

Liver, muscles, heart

receptor in many types of target cells to alter their metabolism.In hepatocl'tes,one effect of cortisolis to increasethe rate of gluconeogenesis. Hormonalcascadessuch as thoseresponsiblefor the releaseof cortisoland epinephrineresult in large

A{ferent nerve signals to

Hypothalamus -q\

Anterior pituitary Posterior pituitary

(a)

FIGURE 23-9 Neuroendocrine originsof hormonesignals.(a) Locationof the hypothalamus andpituitary gland.(b) Detailsof thehypothalamus-pituitary system. signalsfromconnecting neurons stimulate the hypothalamus to secrete releasing factorsinto a bloodvesselthatcarriesthe hormones directlyto a capillarynetwork in the anteriorpituitary. In response to eachhypothalamic releasing factor, the anteriorpituitaryreleases the appropriate hormoneintothe generalcirculation.Posterior pituitaryhormones aresynthesized in neurons arisingin the hypothaiamus, transported alongaxonsto nerveendingsin the posterior pituitary, and storedthereuntilreleased intothe bloodin response to a neuronal signal

Anterior pituitary - Capillary ' network

Posterior pituitary

Releaseof anterior pituitary hormones (tropins)

Release ofposterior pituitary hormones (vasopressin,oxytocin) Veins carry hormones to systemic blood

(b)

Functions forDiverse Structures Diverse 23.1Hormones: fsr I

fru,

NH.

t"

Cvs------r

t"l TVr Il Ile

rl GIn 4.' tl

I

Infection

rvr

l"l

Phe

S

S

GIn

S

I

4.. tl

I

Pro

tl

Pain.4

Leu

I

Glv

t" c:o I

t"

--t

Hypothalamus

II I

Corticotropin-releasing hormone (CRH) (tg)

NHz Human vasopressin (antidiuretic hormone)

I I

+ 0

Thesetwo horboxylterminusis commonin shortpeptidehormones). (shaded), mones,identicalin all but two residues haveverydifferent biologicaleffectsOxytocinactson the smoothmuscleof the uterus and mammarygland,causinguterinecontractions duringlaborand (alsocalledanpromotingmilk releaseduringlactation. Vasopressin tidiuretichormone)increases water reabsorption in the kidneyand promotesthe constriction blood of blood vessels, therebyincreasing pressure.

i,8

Anterior pituitary

II

+ Adrenocorticotropic hormone (ACTH) \llg)

I I

+

of the initial signal and allow exquisite

fine-tuning of the output of the ultimate hormone (FiS. 23-11). At each level in the cascade,a small signalelicits a larger response.For example,the initial electricalsignalto the hypothalamusresults in the releaseof a few nainogramsof corticotropin-releasing hormone,which elicits the releaseof afew m'icrogrclrns of corticotropin.Corticotropinacts on the adrenalcortex to causethe releaseof mi,Ili,gramsof cortisol,for an overall ampliflcationof at least a millionfold. At eachlevel of a hormonalcascade,feedbackinhibition of earlierstepsin the cascadeis possible;an unnecessarilyelevatedlevel of the ultimatehormoneor of an intermediatehormoneinhibits the releaseof earlier hormonesin the cascade.Thesefeedbackmechanisms accomplishthe sameend as thosethat limit the output of a biosynthetic pathway (compare Fig, 23-11 with Fig. 6-33): a product is synthesized(or released)only until the necessaryconcentrationis reached.

I

+

FIGURE 23-10 Two hormonesof the posteriorpituitary gland.The c a r b o x y l - t e r m i n arle s i d u e o f b o t h p e p t i d e s i s g l y c i n a m i d e , -NH-CH2-CONH2 (asnotedin Fig.23-2, amidationof the car-

amplifications

Hypoglycemia

o

Pro

I

Human oxytocin

,/Hemorrhage

+

CvsJ

C:O

NHz

I

Central nervous€ system ! I

I ArE t" Glv t-

I

I *

I

S

9v.---I

Fear 0""'

9t'___-] TI

0 Adrenel,gland

II * - Cortisol (*g.t

/lr

Mritisle,r tt&{t

. ft$ii#'

23-1 I Cascadeof hormonereleasefollowingcentralnervous FIGURE tissuealongthe In eachendocrine systeminputto the hypothalamus. amplified,and a stimulusfrom the levelaboveis received, pathway, of the nexthormonein the cascadeThe into the release transduced cascadeis sensitiveto regulationat severallevelsthroughfeedback Theproduct inhibitionby the ultimatehormone(in thiscase,cortisol). inhibition of feedback as in production, its own thereforeregulates withina singlecell. pathways biosynthetic

23.1 H o r m o n D e si v: e r s e SUMMARY S t r u c t u rfeosrD i v e r s e Functions r

secretedby Hormonesare chemicalmessengers certaintissuesinto the bloodor interstitialfluid, servingto regulatethe actMty of other cellsor tissues.

r

r

and ELISA are two very Radioimmunoassay sensitivetechniquesfor detectingand quantifying hormones, Peptide,amine,and eicosanoidhormonesact outsidethe target cell on specificreceptorsin the plasmamembrane,alteringthe level of an intracellularsecondmessenger.

fI

912

r

r

r

Hormonal Regulation andIntegration ofMammalian Metabolism

Steroid,vitaminD, retiroid, and thyroid hormones enter target cellsand alter geneexpressionby interactingwith spectficnuclearreceptors. Hormonalcascades,in which catalystsactivate catalysts,amplify the initial stimulusby several orders of magnitude,often in a very short time (seconds). Nerveimpulsesstimulatethe hypothalamusto send speciic hormonesto the pituitary gland,thus stimulating (or inhibiting) the releaseof tropic hormones.The anterior pituitary hormonesin turn stimulateother endocrineglands(thyroid, adrenals, pancreas)to secretetheir characteristichormones, which in turn stimulatespeci_fic target tissues.

fats,which serveasfuel throughoutthe body;in the brain, cells pump ions acrosstheir plasmamembranesto produceelectricalsignals.The liver playsa centralprocessing and distributing role in metabolismand furnishesall other organsandtissueswith an appropriatemix of nutrientsvia the bloodstream.The fur-rctionalcentrality of the liver is indicated by the conunonreferenceto all other tissues and organsas"extrahepatic"or "peripheral."Wetherefore begin our discussionof the divisionof metaboliclabor by consideringthe transformationsof carbohydrates,amino acids,and fats ur the mammalianliver.This is followedby brief descriptionsof the primary metabolicfunctions of adiposetissue,muscle,brain, and the mediumthat interconnectsall others:the blood.

TheLiver Proresses andDistributes Nutrients

23.2Tissue-5pecific Metabolism: TheDivision ofLabor Each tissue of the human body has a specialized function, reflected in its anatomy and metabolic activity (FiS. 23-12). Skeletal muscle allows directed motion; adipose tissue stores and releases energy in the form of

During digestionin mammals,the three main classesof nutrients (carbohydrates,proteins,and fats) undergo enzymatic hydrolysis into their simple constituents. This breakdownis necessarybecausethe epithelialcells Iining the intestinal lumen absorb only relatively small molecules.Many of the fatty acids and monoacylglycerols releasedby digestionof fats in the intestine are Brain

Secretesinsulin and glucagon in responseto changes in blood glucose concentration.

Pancreas

Processesfats, carbohydrates, proteins from diet; synthesizes and distributes Iipids, ketone bodies,and glucose for other tissues; converts excessnitrogen to urea.

Transports ions to maintain membrane potential; integrates inputs from body and surroundings; sends signals to other organs.

Carries lipids from intestine to liver.

Adipose

Portal vein Carries nutrients from intestine to liver. Small intestine Absorbs nutrients from the diet, moves them into blood or Iyrnphatic system.

tIGURE23-12 Specializedmetabolicfunctionsof mammaliantissues.

Synthesizes, stores, and mobilizes triacylglycerols. (Brown adipose tissue: carries out thermogenesis.)

23.2 I i s s u e - S p e c i f i c M e t a b o l i s m :LTahbeoD Ir Si vrilsIi o n o f reassembledwithin these epithelial cells into triacylglycerols(TAGs). After being absorbed,most sugarsand amino acids and some reconstituted TAGs pass from intestinal epithelial cells into blood capillaries,and travel in the bloodstreamto the liver; the remainingTAGs enter adipose tissue via the lymphatic system.The portal vein is a direct route from the digestiveorgansto the liver, and liver thereforehasfirst accessto ingestednutrients.The liver has two main cell types. Kupffer cells are phagocytes, important in immune function. Hepatocytes, of primary interest here, transform dietary nutrients into the fuels and precursorsrequiredby other tissues,and exportthemvia the blood.The kindsand amountsof nutrients suppliedto the liver vary with severalfactors, including the diet and the time between meals. The demandof extrahepatictissuesfor fuelsand precursors varies among organs and with the level of activity and overall nutritional state of the individual.

To meet these changingcircumstances,the liver has remarkablemetabolicflexibiJity.For example,when the diet is rich in protein, hepatocytes supply themselves with high tevelsof enz;,rnesfor aminoacid catabolismand gluconeogenesis.Within hours after a shift to a highcarbohydratediet, the levelsof theseenz)'rnesbeginto drop and the hepatocytesincreasetheir synthesisof enzyrnesessentialto carbohydratemetabolismand fat s5mthesis. Liver enzy'rnesturn over (are synthesizedand degraded)at 5 to 10timesthe rate of enz;rmetumover in other tissues,such as muscle.Extrahepatictissuesalso can adjust their metabolismto prevailingconditions,but none is as adaptableasthe liver, and none is so centralto the organism'soverall metabolism. What follows is a survey of the possiblefates of sugars,amino acids,and tipids that enter the liver from the bloodstream.To help you recall the metabolictransformationsdiscussedhere, Table2S-2showsthe major pathwaysand processesand indicates by figure number where each pathway is

Pathway

Figurereference

Ci,tri,cdcid cycle: acetyl-CoA--+ 2CO2 Onidatiue phosphorglati,on: NIP slnthesis

r6-7 t9-20

Carbohydrate catabolism GlgcogenolEsis:glycogen ------+glucose1-phosphate-----+ biood glucose Herose entry i,nto glgcolysi,s:fructose,mannose,galactose-----+ glucose6-phosphate GIgcolg si,s:glucose ---+ pyruvate acetyl-CoA Pgntu ate dehydrogenase react'ion: p)ryuvate-+ Lacti,c acidJernlentat'ion: glucose -----+ lactate + ZATP Pentosephosphate pathway:glucose 6-phosphate-----+ pentosephosphates+ NADPH

15-25;15-26 r4-10 r4-2 t6-2 r4-3 I4-2I

Carbohydrate anabolism Gluconeogeneszs:citric acid cycle intermediates ------+glucose Glucose-alani,necgcle: $ucose ------+pytuvate ------+alanine -----+ glucose Glycogensgnthesi,s:glucose6-phosphate-----+ glucosel-phosphate --+ glycogen Amino acid and nucleotide metabolism Ami,no acid degrad,at'ion:aminoacids ----+ acetyl-CoA,citric acid cycle intermediates Ami,no acid synthesi,s Urea cgcle: NH3 -----+ urea Glucose-alotti,ne cgcle: alanine ------+glucose Nucleotide synthesis: amino acids ----+ purines,pgimidines Hormpne atndneurotrantsnxitter sgnth,esi,s

r+t6 18-9 15€0 18-15 22-9 18-10 18-9 22-33;22-36 22-29

Fat catabolism --r acetyl-CoA B On'idati,onoJJatty acids: falty acid ------+acetyl-CoA -----+ COzvia citric acid cycle Onid,ati,onoJketone bod.i,es: B-hydroxybutlrate

17-8 T7-19

Fat anabolism Fattg acid sgnth,esis:acetyl-CoA -----+ fatty acids Tir[acyLgLg cerol sgnthesi,s: acetyl_coA -----+ fatty acids -----+ triacylglycerol Ketone bodyJormati,on: acetyl-CoA------+acetoacetate,B-hydroxybutl'rate Cholesteroland,cholesterylestersgntheses:acetyi-CoA------)cholesterol-----+ cholesterylesters phospholipids Phospholipid sgnthes'ts:fatty acids ->

2r-6 2l-18;21-19 17-18 2l-33 to 21-37 2I-17;2I-23to2I-28

-

l

[914 ]

H o r m o nRael g u l a t iaonndI n t e g r a t ioofnM a m m a l iM a ne t a b o l i s m

presented in detail. Here, we provide summaries of the pathways, referring to the numbered pathways ancl reactions in Figures 23-13 to 23-tb. Sugars The glucose transporter of hepatocytes (GLUT2) is so effective that the concentration of glucose in a hepatocyte is essentiallythe same as that in the blood. Glucose entering hepatocy'tesis phosphorylated by hexokinase IV (glucokinase) to yield glucose 6-phosphate. Glucokinase has a much higher K,. for glucose (10 mu) than do the hexokinase isozymes in other cells (p. 58a) and, unlike these other iso4'rnes, it is not inhibited by its product, glucose 6-phosphate. TlLe presence of glucokinase allows hepatocytes to continue phosphorylating glucose when the glucose concentration rises well above levels that would overwhelm other hexokinases. The highK., ofglucokinase also ensures that the phosphorylation of glucose in hepatocytes is minimal when the glucose concentration is low, preventing the liver from consuming glucose as fuel via glycolysis. This spares glucose for other tissues. Fructose, galactose, and mannose, all absorbed from the small intestine, are also converted to glucose 6-phosphate by enzymatic pathways examined in Chapter 14. Glucose 6-phosphate is at the crossroads of carbohydrate metabolism in the liver. It may take any of several major metabolic routes (Fig. 23-13), depending on the current metabolic

Liver

cogen

Hepatoc5rte

(E (!) Glucose6- -+

needsof the organism.By the actionof variousallosterically regulatedenzl'rnes,and through hormonal regulation of enzyrnesynthesisand activity, the liver directs the flow of glucoseinto one or more of thesepathways. @ Glucose 6-phosphateis dephosphorylatedby glucose 6-phosphataseto yield free glucose (see Fig. 15-28), which is exportedto replenishblood glucose.Export is the predominantpathwaywhen glucose 6-phosphateis in limited supply, becausethe blood glucose concentrationmust be kept sufficiently high (4 mu) to provide adequateenergy for the brain and other tissues.@ Glucose6-phosphatenot immediately neededto form blood glucoseis convertedto Jiverglycogen, or has one of severalother fates.Followingglycolysis and the pyruvate dehydrogenasereaction,@ the acetyl-CoAso formed canbe oxidizedfor energyproduction by the citric acid cycle,with ensuingelectrontransfer and oxidative phosphorylation yielding ATp. (Normally,however,fatty acidsare the preferredfuel for energy production in hepatocytes.)@ Acetyl-CoA can also serve as the precursor of fatty acids,which are incorporatedinto TAGsand phospholipids, and of cholesterol. Much of the lipid synthesizedin the liver is transportedto other tissuesby bloodlipoproteins.@ elternatively,glucose6-phosphatecan enter the pentose phosphate pathway, yielding both reducing power (NADPH), neededfor the biosynthesisof fatty acidsand cholesterol,and n-ribose5-phosphate,a precursorfor nucleotidebiosymthesis. NADPHis alsoan essentialcofactor in the detoxification and elimination of many drugs and other xenobioticsmetabolizedin the liver. Amino Acids

Amino acids that enter the liver follow

phosphate

COZ

*idati"u phosphorylation

FIGURE23-13 Metabolic pathways for glucose 6-phosphate in the I i v e r . H e r e a n d i n F i g u r e s2 3 1 4 a n d 2 3 - 1 5 , a n a b o l i c p a t h w a y sa r e g e n e r a l l ys h o w n l e a d i n g u p w a r d , c a t a b o l i c p a t h w a y s l e a d i n g d o w n _ ward, and distribution to other organs horizontally The numbered p r o c e s s e si n e a c h f i g u r e a r e d e s c r i b e di n t h e t e x t .

discussedin Chapter27. The Iiver constantlyrenewsits own proteins,which have a relatively high turnover rate (averagehalf-life of hours to days), and is alsothe site of biosynthesisof most plasmaproteins. @ Alternatively, aminoacidspassin the bloodstreamto other organs,to be used in the synthesisof tissue proteins.@ Other amino acids are precursors in the biosynthesisof nucleotides,hormones,and other nitrogenouscompounds in the liverand othertissues. @ Amino acidsnot neededas biosyntheticprecursors are transaminatedor deaminatedand degradedto yield pyruvate and citric acid cycle intermediates,with various fates;@ the ammoniareleasedis converted to the excretory product urea. @ Pyruvate can be conv_erted to glucoseand glycogenvia gluconeogenesis, or it can be convertedto acetyl-CoA,which has several @ possiblefates:@ oxidationvia the citric acid cycleand @ oxidativephosphorylationto produceATp,or @ conversionto lipids for storage.@ Citrlc acid cycle intermediatescan be siphonedoff into glucosesynthesisby gluconeogenesis. The liver alsometabolizesamino acidsthat arrive intermittentlyfrom other tissues.The bloodis adeouatelv

Division ofLanorl-ors] Metabolism:The 23.2Tissue-Specific

por

Plasma proteins

-l

Amino acids in blood

Amino acids

Tissue proteins

-,

gluconeogenesis @ Lipids

+ \-

(

Fattv u.16s

+ \-

AcetyI-CoA

FIGURE 23-14 Metabolism of aminoacidsin the liver.

supplied wrth glucose just after the digestion and absorption of dietary carbohydrate or, between meals, by the conversion of liver glycogen to blood glucose. During the interwalbetween meals, especiallyif prolonged, some muscle protein is degraded to amino acids. These amino acids donate their amino groups (by transamination) to pyruvate, the product of glycolysis, to yield alanine, which Q! is transported to the liver and deaminated. Hepatocytes convert the resulting pyruvate to blood glucose (via glucoreogenesis@), and the ammonia to urea for excretion (p One beneflt ofthis glucose-alaninecycle (see FiC. 18-9) is the smoothing out of fluctuations in blood glucose between meals. The amino acid deficit incurred in muscles is made up after the next meal by incoming dietary amino acids. Lipids The fatty acid components of lipids entenng hepatocytes also have several different fates (Fig. 23-15). @ Some are converted to liver lipids. @ Under most circumstances, fatty acids are the primary oxidative fuei tn the liver. Free fatty acids may be activated and oxidized to yield acetyl-CoA and NADH. @ ffLe acetyl-CoA is further oxidized via the citric acid cycle, and @ oxidations in the cycle drive the s5,erthesisof ATP by oxtdative phosphorylation.

Liver lipids

lfepatocyte PIasma Iipoproteins Free fatty acids in blood

Steroid hormones

02

HzO

oxidative phosphorylation

FIGURE 23-15 Metabolismof fattv acidsin the liver.

9 1 6 , H o r m o nRael g u l a tai onndI n t e g r a t oi ofM n a m m a lM i aent a b o l i s m

@ Excess acetyl-CoA, not required by the liver, is converted to acetoacetateand B-hydroxybutyrate; these ketone bodies circulate in the blood to other tissues, to be used as fuel for the citric acid cycle. Ketone bodies may be regarded as a transport form of acetyi groups They can supply a signi-ficantfraction of the energy in some extrahepatic tissues-up to one-third in the heart, and as much as 60% to 70o/oin the brain during prolonged fastmg @ Some of the acetyl-CoA derived from fatty acids (and from glucose) is used for the biosyrrthesisof cholesteroi, which is required for membrane synthesis.Cholesterol is also the precursor of all steroid hormones and of the bile salts, which are essential for the digestion and absorption of lipids. The other two metabolic fates of lipids involve specialized mechanisms for the transport of insoluble lipids in blood. @ natty acids are converted to the phospholipids and TAGs of plasma lipoproteins, which carry lipids to adipose tissue for storage as TAGs @ Some free fatty acids are bound to serum albumin and carried to the heart and skeletal muscles, which take up and oxidize free fatty acids as a major fuel Serum albumtn is the most abundant plasma protein; one molecule can carry up to l0 molecules of free fatty acid. The liver thus serves as the body's distribution center, exporting nutrients in the correct proportions to other organs, smoothing out fluctuations in metabolism caused by intermittent food intake, and processingexcessamino groups into urea and other products to be disposedof by the kidneys. Certain nutrients are stored in the liver, including Fe ions and vitamin A The liver also detoxiies foreign organic compounds, such as drugs, food additives, preservatives, and other possibly harmful agents with no food value. Detoxiflcation often involves the cl,tochrome P-450-dependent hydroxylation of relativelv insoluble orgaruccompounds,making them sufficiently soluble for further breakdown and excretion (see Box 21-1) (a)

Ae{ip*se Ti..,sues St*re andSuppty Ffitty A{ids There are two distrnct types of adipose tissue, white and brown, with quite distinct roies, and we focus first on the more abundant of the two. White adipose tissue QryAT) (Fig. 23-16a) is amorphous and widely distributed in the body: under the skin, around the deep blood vessels, and in the abdominal cavity. The adipocytes of WAT are large (diameter 30 to 70 g.m), spherical cells, completely fllled with a single large Iipid (TAG) droplet that constitutes about 650/oof the cell mass and squeezesthe mitochondria and nucleus into a thin layer against the plasma membrane (Fig. 23-16b). In humans, WAT typically makes up about l5o/o of the mass of a healthyyoung adult. The adipocytes are metabolicallyvery active, responding quickly to hormonal stimuli in a metabolic interplay with the Jiver, skeletal muscles, and heafi. Like other cell types, adipocytes have an active glycoly'tic metaboJism, oxidize p;,ruvate and fatty acids via the citric acid cycle, and carry out oxidative phosphorylation. Durrng periods of high carbohydrate intake, adipose tissue can convefi glucose (via pyruvate and acetyi-CoA) to fatty acids, convert the fatty acids to TAGs, and store the TAGs as large fat globules-although, in humans, much of the fatty acid s;,nthesis occurs tn hepatocy'tes. Adipocytes store TAGs arriving from the liver (carried in the blood as WDLs; see Fig. 2I-40a) and from the intestinal tract (carried in chylomicrons), particularly after meals rich in fat. When the demand for fuel rises, lipases in adipocytes hydrolyze stored TAGs to release free fatty acids, wliich can travel in the bloodstream to skeletal muscle and the heart. The release of fatty acids from adipocy'tesis greatly accelerated by epinephrine, which stimulates the cAMP-dependent phosphorylation of perilipin and thus gives the hormone-sensitive lipase access to TAGs in the lipid droplet (see Fig I7-3). Hormonesensitive lipase is also stimulated by phosphorylation, (b) White adipocyte

(c)

Brown adipocyte

Nucleus Mitochondria

Nucleus

Lipid droplet

16lF I G U R E2 3 - 1 6 A d i p o c y t e s o f w h i t e a n d b r o w n a d i p o s e t i s s u e . ( a ) C o l o r i z e d s c a n n i n g e l e c t r o n m i c r o g r a p h o f h u m a n a d i p o c y t e si n w h i t e a d i p o s e t i s s u e ( W A T ) . I n f a t t i s s u e s ,c a p i l l a r i e s a n d c o l l a g e n f i b e r sf o r m a s u p p o r t i n gn e t w o r k a r o u n d s p h e r i c a la d i p o c y t e s A l m o s t t h e e n t i r e v o l u m e o f e a c h o f t h e s em e t a b o l i c a l l ya c t i v ec e l l s i s t a k e n u p by a fat droplet. (b) A typical adipocyte from WAT and (c) an adipocyte

from brownadiposetissue(BAT).In BATcells,mitochondria aremuch moreprominent, the nucleusis nearthe centerof the cell,and multiple fat dropletsarepresentWhiteadipocytes are largerand containa singlehugeIipid droplet,which squeezes the mitochondria and nucleusagainst the plasmamembrane

Division ofLabor,2"tr1 Metabolism:The 23.2Tissue-5pecific but this is not the main cause of increasedlipolysis. Insulin counterbalancesthis effect of epinephrine, decreasingthe activity of the lipase The breakdownand synthesisof TAGs in adipose tissue constitute a substratecycle; up Io 70o/oof the fatty acids releasedby hormone-sensitivelipase are reesterifledin adipocytes,re-formingTAGs.Recallfrom Chapter15 that suchsubstratecyclesallowfine regulation of the rate and direction of flow of intermediates througha bidirectionalpathway.In adiposetissue,glycerol li.beratedby hormone-sensitivelipase cannot be reused in the synthesisof TAGs,becauseadipocytes lack glycerol kinase. Instead, the glycerol phosphate required for TAG synthesisis made from pyruvate by glyceroneogenesis, involving the cytosolic PEP carboxykinase(seeFig. 2l-22). In addition to its central function as a fuel depot, adiposetissueplays an important role as an endocrine organ,producingand releasinghormonesthat signalthe state of energyreservesand coordinatemetabolismof fats and carbohydratesthroughout the body.We return to this function Iater in the chapteras we discussthe hormonalregulationof body mass

Brown Adipose Tissue lsThermogenic In small vertebratesand hibernating animals,a signi-ficant proportion of the adiposetissueis brown adipose tissue (BAT), distinguishedfrom WAT by its smaller (diameter 20 to 40 pm), differently shaped (polygonal, not round) adipocy'tes(FiS.23-l6c). Like white adipocytes,brown adipoc;'tesstoretriacylglycerols, but in severalsmallerIipid droplets per ceil rather than as a singlecentral droplet. BAT cellshavemore mitochondria and a richer supply of capillariesthan WATcells,and it is the cytochromesof mitochondriaand the hemoglobinin capillariesthat give BAT its characteristicbroum color. A unique feature of brov,n adipocytesis their strong expressionof the gene Ul/C1, which encodesthermogenin, the mitochondrialuncouplingprotein (see Fig. 19-34). Thermogeninactivity is responsiblefor the principal function of BAT:thermogenesis. In brown adipocytes,fatty acids stored in lipid droplets are released,enter mitochondria,and undergo completeconversionto CO2via B oxidation and the citric acid cycle. The reduced FADH2and NADH so generatedpasstheir electronsthrough the respiratory chain to molecular oxygen. In WAT, protons pumped out of the mitochondria during electron transferreenter the matrix throughATP synthase,with the energyof electrontransfer conservedin ATP synthesis. In BAT, thermogenin provides an alternative route for protons to reenter the matrix that bypasses ATP synthase;the energy of the proton gradient is thus dissipatedas heat, which can maintain the body (especiallythe nervoussystemand viscera) at its optimal temperature when the ambient temperature is relativelylow.

FIGURE 23-17 Distributionof brown adiposetissuein a newborn infant. At birth, human infantshave brown fat distributedas shown here,to protectthe ma.jorblood vesselsand the internalorgans.This of brown fat recedesover time, so that an adult hasno maior reserves brownadipose.

In the human fetus, differentiation of fibroblast "preadipocy'tes"into BAT begins at the twentieth week of gestation,and at the time of birth BAT represents1% of total body weight.The brorm fat depositsare located can ensure where the heat generatedby thermogenesis that vital tissues-blood vesselsto the head,major abdominal blood vessels,and the viscera,including the pancreas,adrenal glands,and kidneys-are not chilled as the newbornentersa world of lower ambienttemperature (Fig. 23-17). At birth, WATdevelopmentbeginsand BAT begins to disappear.By adulthoodhumans have no discrete deposits of BAT, although brown adipocytes remain scatteredthroughoutthe WAT,makingup about 1% of all adipocytes.Adults alsohavepreadipocytesthat can be induced to differentiate into BAT during adaptation to chronic cold exposure.Humanswith pheochromocytoma(tumors of the adrenalgland) overproduce epinephrineand norepinephrine,and one effect is differentiation of preadipocytesinto discrete regions of BAT,Iocalizedroughly as in newborns.In the induced adaptationto chronic cold, and in the normal differentiation of WATand BAT,the nuclear transcription factor PPART(describedlater in the chapter) plays a central role.

D"l

H o r m o nRael g u l a t iaonndI n t e g r a t ioofnM a m m a l iM a ne t a b o l i s m

Muscles Use ATP forMechanicalWork Metabolismin the cells of skeletalmuscle-myocytesis specializedto generateATP asthe immediatesourceof energy for contraction. Moreover,skeletal muscle is adaptedto do its mechanicalwork in an intermittent fashion, on demand.Sometimesskeletalmusclesmust work at their maximumcapacityfor a short time, as in a 100m sprint; at other timesmoreprolongedwork is required,as in rurminga marathonor in extendedphysicallabor. There are two general classesof muscle tissue, which differ in physiological role and fuel utilization. Slow-twitch musele, also calledred muscle,provides relatively low tensionbut is highly resistantto fatigue.It producesATP by the relativelyslowbut steadyprocess of oxidative phosphorylation.Red muscleis very rich in mitochondria and is served by very dense networks of blood vessels,which bring the oxygenessentialto ATP production.Fast-twitch muscle, or white muscle,has fewer mitochondriathan red muscleand is lesswell supplied with bloodvessels,but it can developgreatertension,and do so faster.White muscleis quickerto fatigue becausewhen active,it usesATP faster than it can replaceit. Thereis a geneticcomponentto the proportion of red and white musclein any individual; with training, the enduranceof fast-twitch muscle can be improved. Skeletalmusclecanusefreefatty acids,ketonebodies,or glucoseas fuel, dependingon the degreeof muscular activity (Fig. 23-18). In resting muscle, the primary fuels are free fatty acids from adipose tissue andketonebodiesfrom the liver.Theseare oxidizedand degradedto fleld acetyl-CoA,which enters the citric acid cycle for oxidation to CO2.The ensuingtransfer of electronsto 02 pror,rdesthe energyfor ATP synthesisby oxidative phosphorylation.Moderatelyactive muscle usesbloodglucosein additionto fatty acidsand ketone

bodies.The glucoseis phosphorylated,then degraded by glycolysisto pyruvate, which is convertedto acetylCoA and oxidizedvia the citric acid cycle and oxidative phosphorylation. In maximally active fast-twitch muscles,the demand for ATP is so great that the blood flow cannotprovide 02 and fuels fast enoughto supply sufflcientATP by aerobic respiration alone. Under these conditions, storedmuscleglycogenis brokendownto lactateby fermentation (p. 530). Each glucoseunit degradedyields three ATP, becausephosphorolysisof glycogenproducesglucose6-phosphate(via glucose1-phosphate), sparing the ATP normally consumedin the hexokinase reaction.Lactic acid fermentationthus respondsmore quickly than oxidative phosphorylationto an increased need for ATP, supplementingbasal ATP production by aerobic oxidation of other fuels via the citric acid cycle and respiratory chain. The use of blood glucoseand muscleglycogenas fuels for muscularactivity is greatly enhancedby the secretionof epinephrine,which stimuIatesboth the releaseofglucosefrom liver glycogenand the breakdor,r'n of glycogenin muscletissue. The relatively small amount of glycogen(about 1% of the total weight of skeletalmuscle) limits the amount of glycolytic energy availableduring all-out exertion. Moreover,the accumulationof lactate and consequent decreasein pH in maximally active musclesreduces their efficiency. Skeletal muscle, however, contains anothersourceof ATP,phosphocreatine (10 to 30 mrr,r), which can rapidly regenerateATP from ADP by the creatinekinasereaction:

o-

- O - P I: O

I l*

N-H C:NHo

t-

cHg-ry

Bursts of

I

CH,

tcooPhosphocreatine

Muscle contraction FIGURE 23-18 Energysourcesfor musclecontraction.Differentfuels are usedfor ATPsynthesis duringburstsof heavyactivityand during lightactivityor rest.Phosphocreatine can rapidlysupplyATp

+ ADP --

during activitv

during recovery

NHo

ATP +

l1

C:NHz CH3-N -l

I

CHO

tcooCreatine

During periodsof activecontractionand glycolysis,this reaction proceedspredominantly in the direction of ATP synthesis;duringrecoveryfrom exertion,the same enzymeresynthesizesphosphocreatinefrom creatine and ATP. Given the relatively high levels of ATP and phosphocreatinein muscle,these compoundscan be detectedin intact muscle,in real time, by NMR spectroscopy(Fig. 23-f9). After a period of intensemuscularactivity,the individual continues breathing heavily for some time, using much of the extra 02 for oxidativephosphorylation in the liver. The ATP produced is used for gluconeogenesis(in the liver) from lactate that has been carried in the blood from the muscles.The glucose thus formed returns to the musclesto replenishtheir

2 3 . 2T i s s u e - 5 p eM c ief ti ca b o l i s m :DTihvei s i oonf L a b o r[ r t f - ]

b!e

Muscle: ATP produced by glycolysis for rapid contraction.

Exercise -5 -10 -15 -20 0 Chemical shift (parts per million) (identity of the compound) tIGURE23-19 Phosphocreatine buffersATP concentrationduring (ofrlP) showing A "stackplot"of magnetic exercise. resonance spectra (P;),phosphocreatine (PCr),and ATP(eachof its inorganicphosphate threephosphates givinga signal). Theseries of plotsrepresents the passageof time, from a periodof restto one of exercise,and then of recovery.Notethatthe ATPsignalhardlychanges duringexercise, kept highby continuedrespiration andby the reservoir of phosphocreatine, whichdiminishes duringexercise. Duringrecovery, whenATPproduction by catabolism is greaterthanATPutilizationby the (nowresting) muscle,the phosphocreatine reservoir is refilled

Blood glucose

Blood lactate

Lactate --7;#Glucose

t glycogen,completingthe Cori cycle (Fig. 23-20; see alsoBox 15-4). Actively contractingskeletalmuscle generatesheat as a b),productof imperfect couphngof the chemicalenergyof AIP with the mechanicalwork of contraction.Ttus heat production can be put to good use when ambient temperatureis low: skeletalmusclecarriesout shivering thermogenesis, rapidly repeatedmuscle contraction that producesheat but little motion, helpingto maintain the body at its preferredtemperatureof 37 oC. Heart musclediffersfrom skeletalmusclein that it is continuously active in a regular rhythm of contractionand reiaxation,and it has a completely aerobic metabolism at all times. Mitochondria are much more abundantin heart musclethan in skeletal muscle,making up aimosthalf the volume of the cells (Fig. 2:i-21). The heartusesmainlyfreefatty acids,but alsosomegiucoseand ketonebodiestakenup from the blood,as sourcesof energy;thesefuels are oxidizedvia the citric acid cycle and oxidativephosphorylationto generateATP Like skeletalmuscle,heart muscledoes not store lipids or glycogenin large amounts.It does have small amountsof reserveenergy in the form of phosphocreatine, enoughfor a few secondsof contraction, Becausethe heart is normallyaerobicand obtains its energyfrom oxidativephosphorylation,the failure of

FIGURE 23-21 Electronmicrographof heart muscle.In the profuse mitochondria of hearttissue,pyruvate(fromglucose), fattyacids,and ketonebodiesareoxidizedto driveATPsynthesis. Thissteadyaerobic metabolism allowsthe humanheartto pumpbloodat a rateof nearly 6 L/min,or about350 Lihr-or 200 x 106L over70 years.

ATP Liver: ATP used itr'sjmthesrs of glucose(gluconeogenesis) during recovery.

-.ri'

F lit,}. FIGURE 23-20 Metabolic cooperationbetweenskeletalmuscleand the liver: the Cori cycle. Extremelyactive musclesuse glycogenas some Duringrecovery, via glycolysis. lactate energysourceigenerating to the liver and convertedto glucosevia of this lactateis transported to to the bloodand returned Thisglucoseis released gluconeogenesis. The overallpathway the musclesto replenishtheir glycogenstores. (glucose---rlactate--; glucose)constitutes the Cori cycle

Lp^

T-l

Regulation andIntegrati0n ofMammalian Metabolism 1920] Hormonal 02 to reacha portion of the heartmusclewhenthe blood vesselsareblockedby lipid deposits(atherosclerosis) or bloodclots (coronarythrombosis)can causethat region of the heart muscle to die. This is what happensin myocardialinfarction, more conunonlyknown as a heart attack. r

TheBrain Uses Energy forTransmission of Electrical lmpulses The metabolismof the brain is remarkablein several respects.The neurons of the adult mammalianbrain normallyuse only glucoseas fuel (Fig.23-22). (Astrocytes,the other major cell type in the brain, can oxidize fatty acids.) The brain has a very active respiratory metabolism (Fig. 23-23); it uses 02 at a fairly constant rate, accountingfor almost 200/oof the total 02 consumedby the body at rest. Becausethe brarn contains very little glycogen, it is constantly dependent on incomingglucosein the blood. Shouldblood glucosefall significantlybelow a critical level for even a short time, severe and sometimesirreversible changesin brain function may result. Although the neurons of the brain cannot directly usefree fatty acidsor lipids from the blood as fuels,they can, when necessaryuse B-hydroxybutlrate (a ketone body), formed from fatty acidsin the liver. The capacity ofthe brain to oxidizeB-hydroxybutyratevia acetyl-CoA becomesimportant during prolonged fasting or starvation, after liver glycogenhas been depleted,becauseit allowsthe brain to use body fat as an energy source. This sparesmuscle proteins-until they become the brain'sultimate sourceof glucose(via gluconeogenesis in the liver) during severestarvation. Neuronsoxidize glucoseby glycolysisand the citric acid cycle,and the flow ofelectronsfrom theseoxidations through the respiratorychainprovidesalmostall the ATP Starvation

(b)

12.00

2.00 mg/100g /min

FIGURE 23-23 Glucosemetabolismin the brain.The technioueof (PET)scanningshowsmetabolicactivpositronemissiontomography ity in specificregionsof the brain.PETscansallow visualization of isotopically labeledglucosein precisely localizedregionsof the brain of a Iivingperson,in real time.A positron-emitting glucoseanalog (2-lt8F]-fluoro-2-deoxy-o-glucose) is inlectedinto the bloodstream; a few secondslater,a PETscan showshow much of the glucosehas beentakenup by eachregionof the brain-a measureof metabolicactivity.Shownhereare PETscansof front-to-backcrosssectionsof the brainat threeIevels,from the top (at the left)downward(to the right). The scanscompareglucosemetabolismwhen the experimental subject(a) is restedand (b) hasbeendeprivedof sleepfor 48 hours.

used by these cells. Energy is required to create and maintain an electrical potential across the neuronal plasmamembrane.The membranecontainsan electrogenic ATP-drivenantiporter,the Na*K* MPase, which simultaneouslypumps 2 K+ ions into and 3 Na+ ions out of the neuron (seeFtg. 11-37). The resuitingtransmembranepotential changestransientlyas an electricalsignal (actionpotential) sweepsfrom one end of a neuronto the other (see Fig. 12-25). Action potentials are the chief mechanismof informationtransferin the nervoussystem, so depletionof ATP in neuronswould havedisastrouseffects on all activities coordinatedby neuronal signaling.

Blood Carries Oxygen, Metaboliter, andHormones

Electrogenictransport by Na+K+ ATPase FIGURE 23-22 The fuels that supply ATp in rhe brain. The energy sourceusedby the brainvarieswith nutritionalstate.The ketonebody usedduringstarvationis B-hydroxybutyrate. Electrogenic transportby the Na+K*ATPase potentialessential maintains the transmembrane to information transfer amongneurons.

Blood mediatesthe metabolicinteractionsamongall tissues.It transports nutrients from the small intestine to the liver, and from the liver and adiposetissue to other organs;it alsotransportswasteproductsfrom the extrahepatic tissuesto the liver for processingand to the kidneys for excretion. Oxygen moves in the bloodstream from the lungsto the tissues,and CO2generatedby tissue respirationreturns via the bloodstreamto the lungs for exhalation.Blood also carrieshormonal signalsfrom one tissueto another.In its role as signalcarrier, the circulatory system resemblesthe nervous system;both regulate and integrate the activities of different organs.

TheDivision ofLab0r] szr I Metabolism: 23.2Iissue-5pecific

Inorganic components (107o) NaCl,bicarbonate, phosphate, CaCl2,MgC12, KCl,Na2SOa Organic metabolites and waste products (207o) glucose, amino acids, lactate, pyruvate, ketone bodies, citrate, urea, uric acid

Plasma proteins (707o) Majorplasmaproteins:serumalbumin,very-low-density lipoproteins(VLDL),low-densitylipoproteins(LDL), (hundreds high-densitylipoproteins(HDL),immunoglobulins of kinds),fibrinogen,prothrombin,manyspecialized transport proteinssuchastransferrin FIGURE 23-24 Thecomposition of blood.Whole bloodcan oe separatedinto blood plasmaand cells by centrifugation. About 10o/oof blood plasmais solutes,of which about 10% consistsof inorganic salls,2oo/osmallorganicmolecules,and TOohplasmaproteins. The major dissolvedcomponents are listed.Bloodcontainsmany other substances, oftenin traceamounts. Theseincludeothermetabolites, enzymesihormones,vitamins,trace elements,and bile pigments. Measurements of the concentrations of components in blood plasma areimportantin the diagnosis andtreatment of manydiseases.

The average adult human has 5 to 6 L of blood. Almost iulf of this volume is occupied by three types of blood cells (Fig.23-24): erythrocytes (red cells), filled with hemoglobin and specialized for carrying 02 and CO2; much smaller nurnbers of leukocytes (white cells) of several types (including lymphoc)'tes, also found in lymphatic tissue), which are central to the immune system that defends against infections; and platelets, which help to mediate blood clotting. The liquid portion is the blood plasma, whuch is 900/owater and 10% solutes. Dissolved or suspended in the plasma is a large variety of proteins, Iipoproteins, nutrients, metabolites, waste products, inorganic ions, and hormones. More than 70o/oof the plasma solids are plasma proteins, primarily immunoglobu-lins (circulating antibodies), serurn albumin, apolipoproteins involved in the transport of lipids, transferrin (for iron transport), and blood-clottu€ proteins such as flbrinogen and prothrombin. The ions and low molecular weight solutes in blood plasma are not fixed components but are in constant flLlx between blood and various tissues. Dietarv uotake of the

inorganicions that are the predominant electroly'tesof blood and c),tosol(Na*, K*, and Caz+) is, in general, by their excretionin the urine. For many colmterbalanced blood components,something near a dl'namic steady state is achieved:the concentration of the component changeslittle, althougha continuousflux occrrs between the drgestivetract, blood,and urine. The plasmalevelsof '+ r'+ , ^ 2.+ Na-, K-, and Ca"- remaincloseto I40,5,andz.1lnM,respectively,with little changein responseto dietarytntake. Any significantdeparturefrom thesevaluescan result in seriousillnessor death.The kidneysplay an especiallyimportant role in maintainir€ ion balanceby selectivelyflltering waste products and excessions out of the blood while preventingthe loss of essentialnutrients and ions. The human erythrocyte losesits nucleus and mitochondria during differentiation.It therefore relies on glycolysisalone for its supply of ATP. The Iactate producedby glycolysisreturnsto the liver,wheregluconeogenesisconvertsit to glucose,to be storedas glycogen or recirculatedto the peripheraitissues.The erythroc],'tehas constantaccessto glucosein the bloodstream. The concentrationof gJucosein plasmais subject SF s$r to tight regulation. We have noted the constant ff requirementof the brain for glucoseand the role of the liver in maurtainingbloodglucosein the normal range,60 to 90 mg/100mL of wholeblood(-4.5 mu). (Becauseerythrocy'tesmakeup a signiflcantfraction of bloodvolulne, their removalby centrifugationleavesa supernatantfluid, the plasma,containingthe "blood glucose"in a smaller volume.To convertblood glucoseto plasmaglucoseconcentration,multiply the blood glucoselevel by 1.14.) When blood glucosein a human drops to 40 mg/100mL (the hypoglycemiccondition),the person experiences discomfoft and mental confusion(Fig. 23*25); further FIGURE23-25 Physiologicaleffects of low bloodglucosein humans. Bloodglucoselevels of 40 mg/100 mL and below constitutesevere hypoglycemia.

Normal range Subtle neurological signs; hunger Releaseof glucagon, epinephrine, cortisol Sweating, trembling

Lethargy Convulsions,coma

Permanent brain damage (if prolonged) Death

f_l

p22)

Hormonal Regulation andIntegration ofMammalian Metabolism

reductions lead to coma, convulsions,and, in extreme hypo$ycemia,death.Maintainingthe normal concentration of glucosein blood is thereforea very high priority of the organism,and a variety of regulatory mechanisms haveevolvedto achievethat end.Amongthe most important regulatorsof bloodglucoseare the hormonesinsulin, glucagon,and epnephrine,as discussedin Section23.3.t

usesmost of its ATP for the active transport of Na* and K- to maintain the electricalpotential across the neuronalmembrane. r

The bloodtransfersnutrients,wasteproducts,and hormonal signalsamongtissuesand organs.

23.3Hormonal Regulation ofFuel Metabolism S U M M A2 R3Y. 2 T i s s u e - S p e Mcei ft iacb o l i s m :The mhute-by-minute adjustmentsthat keep the blood glucoselevel near 4.5 mn involve the combinedactions T h eD i v i s i o n fL a b o r r

In mammalsthere is a division of metaboliclabor amongspecializedtissuesand organs.The liver is the central distributing and processingorganfor nutrients. Sugarsand amino acidsproduced in digestioncrossthe intestinal epithelium and enter the blood, which carriesthem to the liver. Some triacylglycerolsderived from ingestedlipids also make their way to the liver, where the constituent fatty acidsare usedin a variety of processes.

r

Glucose6-phosphateis the key intermediatein carbohydratemetabolism.It may be polymerized into glycogen,dephosphorylated to bloodglucose, or convertedto fatty acidsvia acetyl-CoA.It may undergooxidation by glycolysis,the citric acid cycle, and respiratory chain to yield ATP,or enter the pentosephosphatepathwayto yield pentoses and NADPH.

r

Amino acidsare used to synthesizeliver and plasma proteins,or their carbonskeletonsare converted to $ucoseand glycogenby gluconeogenesis; the ammoniaformedby deaminationis convertedto urea. The liver convertsfatty acidsto triacylglycerols, phospholipids,or cholesteroland its esters,for transportas plasmalipoproteinsto adiposetissue for storage.Fatty acidscan alsobe oxidizedto yreld ATP or to form ketone bodies,which are circulated to other tissues.

r

r

White adiposetissuestoreslargereservesof triacylglycerols,and releasesthem into the blood in responseto epineptLrineor $ucagon.Brown adipose tissueis specializedfor thermogenesis,the result of fatty acid oxidationin uncoupledmitochondria.

r

Skeletalmuscleis specializedto produceand use ATP for mechanicalwork. During strenuousmuscular activity, glycogenis the ultimate fuel, supplying ATP through lactic acid fermentation.During recovery,the lactate is reconverted(through gluconeogenesis) to glycogenand glucosein the liver. Phosphocreatineis an immediatesourceof ATP during active contraction.

r

Heart muscle obtainsnearly all its ATP from oxidativephosphorylation.

r

The neurons of the brain use only glucoseand B-hydroxybutyrateas fuels, the latter being irnportant during fasting or starvation.The brain

of insulin, glucagon,epinephrine,and cortisol on metabolic processesin many body tissues,but especiallyin Iiver, muscle,and adiposetissue.Insulin signalsthese tissuesthat bloodglucoseis higherthan necessary;as a result, cellstake up excessglucosefrom the blood and convert it to glycogen and triacylglycerolsfor storage. Glucagonsignalsthat blood glucoseis too low, and tissues respond by producing glucosethrough glycogen breakdown and (in the liver) gluconeogenesisand by oxidizingfats to reducethe use of glucose.Epinephrine is releasedinto the bloodto preparethe muscles,Iungs, and heart for a burst of activity. Cortisol mediatesthe body's responseto longer-term stresses.We discuss these hormonal regulationsin the context of three normal metabolic states-well-fed, fasted, and starvingand look at the metabolic consequencesof diabetes mellitus, a disorder that results from derangementsin the signalingpathwaysthat control glucosemetabolism.

(ounters Insulin HighBlood Glucose Acting through plasma membrane receptors (see Figs 12-15, 12-16), insulin stimulatesglucoseuptake by muscle and adipose tissue (Table 23-3), where the glucose is converted to glucose 6-phosphate.In the liver, insulin alsoactivatesglycogenslmthaseand inactivatesglycogenphosphorylase, so that much of the glucose6-phosphateis channeledinto glycogen. Insulin also stimulatesthe storageof excessfuel as fat in adiposetissue (FiS.23-26). In the liver, insulin activatesboth the oxidation of glucose6-phosphateto pyr"uvatevia glycolysisand the oxidation of pyruvate to acetyl-CoA.If not oxidized further for energy production, this acetyl-CoAis usedfor fatty acid synthesis,and the fatty acidsare exportedfrom the liver asthe TAGsof plasmalipoproteins(VLDLs) to adiposetissue.Insulin stimulatesthe synthesisof TAGs in adipocytes,from fatty acids releasedfrom the VLDL triacylglycerols. Thesefatty acidsare ultimately derived from the excess glucose taken up from blood by the liver. In sununary, the effect of insulin is to favor the conversionof excess blood glucoseto two storageforms: glycogen(in the liver and muscle) and triacylglycerols (in adipose tissue) (Table 23-3). Besides acting directly on muscle and liver to change their metabolism of carbohydratesand fats, insulin can also act in the brain to signal these tissues indirectly, as describedlater.

f-l

l etabolism Rle g u l a t i oonf F u eM 2 3 . 3H o r m o n a [923__]

.- Insulin - to brain, adipose,muscle

// \ \ -

Pancreas

'

I

:Glucose

J Amino acids f+NH3 - {J1s2 a-Keto acids J Protein synthesis J

1v

TAG

TAG

Lymphatic system

'

, Adipose , tissue

ATP

FIGURE 23-26 Thewell-fedstate:the lipogenicliver.lmmediately after a calorie-rich meal,glucose, fattyacids,andaminoacidsenterthe liver. Insulinreleased in response to the high bloodglucoseconcentration stimulatesglucoseuptakeby the tissues.Someglucoseis exportedto the brainfor its energyneeds,and someto adiposeand muscletissue. In the liver,excessglucoseis oxidizedto acetyl-CoA,which is usedto

in VLDLsto adipose fattyacidsfor exportastriacylglycerols synthesize isobtained for lipidsynthesis TheNADPHnecessary andmuscletissue. Excess phosphate pathway. glucose in the pentose of by oxidation amino acidsare convertedto pyruvateand acetyl-CoA,which arealso Dietaryfatsmovevia the lymphaticsystem,as usedfor lipid synthesis. to muscleandadiposetissues. fromthe intestine chylomicrons,

Metaboliceffect

Ta.rgetenzyme

t Glucoseuptake (muscle,adipose) 1 Glucoseuptake (liver)

1 Glucosetransporter (GLUT4) t Glucokinase(increasedexpression)

t Grycogensynthesis(liver, muscle)

1 Glycogens,.nthase J Grycogenphosphorylase

J GlycogenbreakdownQiver,muscle) 1 Glycolysis,acetyl-CoAproduction (liver, muscle) 1 Patty acid synthesis(liver) (adiposetissue) I TfiacylglycerolsSrnthesis

PancreaticB Cells Secrete Insulin inResponse to(hanges inBlood Glucose Whenglucoseentersthe bloodstreamfrom the intestine after a carbohydrate-richmeal,the resulting hcrease in bloodglucosecausesincreasedsecretionofinsulin (and

t pnx-t 1uyt PFK-2) I Pyruvatedehydrogenasecomplex I Acetyl-CoAcarboKylase I Lipoprotein lipase

decreasedsecretionof glucagon)by the pancreas.Insulin releaseis largely regulatedby the level of glucose in the blood supplying the pancreas.The peptide hormonesinsulin, glucagon,and somatostatinare produced by clusters of specializedpancreatic cells, the islets of Langerhans(Fig. 23-27 ). Each cell t)?e of the islets

ft

a ne t a b o l i s m 1 9 2 4 I H o r m o nRael g u l a t iaonndl n t e g r a t ioofnM a m m a l iM

glycolysis.With the higher rate of glucosecatabolism, @ IATpt increases,causingthe closilrgof ATP-gated K+ channels in the plasma membrane.@ Reduced efflux of K+ depolarizesthe membrane.(Recall from Section12.6that exit of K+ through an open K+ channel hyperpolarizesthe membrane;closingthe K+ channel therefore effectively depolarizesthe membrane^.) Membrane depolarizationopens voltage-gatedCa'* channels,and @ the resulting increasein cytosolic lCa"*l triggers@ ttre releaseof insulin by exocytosis. Parasympatheticand sympathetic nervous system signals also affect (stimulate and inhibit, respectively) insulin release.A simple feedbackloop limits hormone release:insulinlowersbloodglucoseby stimulatingglucoseuptakeby the tissues;the reducedbloodglucoseis detectedby the B cell as a diminishedflux through the hexokinasereaction;this slows or stopsthe releaseof insulin. This feedbackregulationholds blood glucose concentrationnearly constant despite large fluctuationsin dielaryintake. The activity of ATP-gatedK+ channelsis centralto ffi E the regulationof insulin secretionby B cells.The charLnelsare octamersof four identical Kir6.2 subunits and four identical SUR1 subunits, and are constructed alongthe samelines as the K+ chamels of bactenaand those of other eukaryoticcells (see Figs 11-48, 1149, and 11-50). The four Kir6.2 subunitsform a conearound the K+ channeland function as the selectivityfllter and ATP-gatingmechanism(Fig. 23-29). When [AIP] rises (indicatingincreasedblood glucose),the K+ channels close,depolarizingthe plasmamembraneand triggering insulin releaseas shownin Fiqure 23-28.

(glucason) a cell (glucagon)

/r '/

B cell (insulin)

Blood vessels

cell (somatostatin)

FIGURE 23-27 The endocrinesystemof the pancreas.The pancreas containsboth exocrinecells(seeFig.1S-3b),which secrete digestive enzymesin the formof zymogens, and clusters of endocrine cells,the isletsof Langerhans Theisletscontaina, B, and6 cells(alsoknownas A, B, and D cells,respectively), eachcell typesecretinga specificpeptide hormone

producesa singlehormone:d ceUsproduceglucagon;B cells,insulin;and 6 cells,somatostatin. As shown in Figure 23-28, when blood glucose rises,@ GLUT2 transporterscarry glucoseinto the p cells, where it is immediately converted to glucose 6-phosphateby hexokinaseIV (glucokinase)and enters

'ter Extracellular

space

Glucose 6-phosphate

I grycorvsts J 1Crtnc . . acrd cycle I

.t FIGURE 23-28 Glucoseregulation of insulin secretionby pancreaticp cells.When the bloodglucoselevelis high,activemetabolism of glucosein the B cell raisesintracellular in the plasmamem[ATP],closingK* channels braneand thusdepolarizing the membraneIn response to the changein membrane potential, voltage-gated Ca2* channelsopen, allowing Ca2*to flow intothecell (Ca2+isalsoreleased fromthe endoplasmic reticulum,in response to the initial elevationof [Ca2+]in the cytosol) Cytosolic[Ca2*]is now highenoughto trigger insulin releaseby exocytosis. The numbered processes are discussedin the text.

I oxidative ohosnhovlation I

@ reretr '

tu ! i

+

i;:l.l.i ."u{r,

depolarization

Insulin

rE"l

F u eM l etabolism 2 3 . 3H o r m o nRael q u l a t ioofn

The sulfonylureasare sometimeused in combination with injected insulin, but often sufflcealonefor controlling type 2 diabetes.

(a)

Glyburide

oO

ilA

S-N,' II H

(b)

o

KirG 2

-N H

Glipizide of B cellsare, Mutationsin the ATP-gatedK+ charLnels in conthat result in Kir6.2 fortunately,rare. Mutations (red in Fig. 23-29b) residues stantly open K+ channels lead to neonatal diabetes mellitus, with severehyperglycemiathat requires insulin therapy. Other mutations in Kir6.2 or SUR1 (blue residues in Fig. 23-29b) produce permanently closed,K+ channelsand continuous release of insulin. If untreated, individuals with these mutations developcongenitalhlperinsulinemia (h1perinsulinismof infancy); excessiveinsulin causessevere hypoglycemia (low blood glucose) leading to irreversiblebrain damage.One effectivetreatment is surgical removal of part of the pancreasto reduce insulin production.I FIGURE 23-29 ATP-gated K+ channelsin B cells.(a) The octameric structure of the channel,viewedperpendicular to the membrane. The channelis formedby four identicalKi162 subunits, and outsidethem arethefour SURl (sulfonylurea receptor) subunits(b)Thestructure of the Kir6.2portionof the channel,viewedin the planeof the membrane.Forclarity,onlytwo transmembrane domainsandtwo cytosolic domainsareshown.ThreeK* ions(green) areshownin the regionof (shownin the selectivity filter.Mutationin certainaminoacidresidues red) leadsto neonataldiabetes;mutationin others(shownin blue) leadsto hyperinsulinism of infancyThisstructure(coordinates courtesyof Frances Ashfordand hercolleagues at the University of Oxford) was not obtaineddirectlyby crystallography, but by mappingthe known Ki16.2sequence onto the crystalstructures of a bacterialKir l ;P D Bl D l P Z B )a n d t h e a m i n oa n d c a r b o x ydl o c h a n n e(l K i r B a c. . 1 m a i n so f a n o t h e K , i r 3 . l ( P D Bl D 1 U 4 E ) C o m p a r et h i s r i r p r o t e i nK .l.l-50 with thegatedK* channelin Figure structure

The sulfonylurea drugs, oral medicationsused in the treatment of type 2 diabetesmellitus, bind to the SUR1 (sulfonylzreareceptor) subunitsof the K+ channels,closing the channelsand stimulating insulin release.The first generationof thesedrugs (tolbutamide, for example)was developedin the 1950s.The secondgenerationdrugs,including glyburide (Micronase),glipizide (Glucotrol),and glimepiride (Amaryl), are more potent and have fewer side effects. (The sulfonylurea moiety is screenedpink in the following structures.)

Glucose LowBlood [ounters Glucagon Severalhours after the intake of dietary carbohydrate, blood glucoselevels fall slightly becauseof the ongoing oxidationof glucoseby the brain and othertissues.Lowered blood glucosetriggerssecretionof glucagon and insulinrelease(fig. 23-30). decreases Glucagoncausesan increasein blood glucoseconcentrationin severalways (Table23-4). Like epinephrine, it stimulatesthe net breakdownof liver glycogen by activatingglycogenphosphorylaseand inactivating glycogensynthase;both effectsare the result of phosphorylation of the regulated enzymes,triggered by cAMP Glucagoninhibits glucosebreakdownby glycolysis in the liver, and stimulatesglucose synthesisby gluconeogenesis. Both effectsresult from loweringthe an alconcentrationof fructose 2,6-bisphosphate, losteric inhibitor of the gluconeogenicenzyme fruc(FBPase-1)and an activator tose 1,6-bisphosphatase phosphofructokinase-1. Reglycolytic enzyme of the conis ultimately 2,6-bisphosphate] call that [fructose trolled by a cAMP-dependentprotein phosphorylation reaction (see Fig. 15-17). Glucagonalso inhibits the glycolytic enzymepyruvate kinase (by promoting its cAMP-dependentphosphorylation),thus blocking the conversionof phosphoenolpyruvateto pyruvate and preventing oxidation of pyruvate via the citric acid

[rr.]

H o r m o nRael g u l a t iaonndI n t e g r a t ioofnM a m m a l iM a ne t a b o l i s m

FIGURE 23-30 The fastingstate: the glucogenic liver.After some hourswithout a meal, the liver becomesthe principalsourceof glucosefor the brain Liverglycogenis brokendown,andtheglucose 1-phosphateproducedis convertedto glucose 6-phosphate, then to free glucose,which is releasedinto the bloodstream. Amino acidsfrom the degradation of proteinsin liver and muscle, and glycerolfrom the breakdownof TACsin adipose tissue,are used for gluconeogenesis. The liverusesfattyacidsas its principalfuel,and excessacetyl-CoAis convertedto ketonebodiesfor exportto othertissues; the brainis especially dependenton thisfuel whenglucoseis in shortsupply (seeFig.23-22\.

Pancreas

cycle. The resulting accumulationof phosphoenolpyruvate favors gluconeogenesis.This effect is augmented by glucagon'sstimulation of the synthesisof the gluconeogenicenzymePEP carboxykinase.By stimulating glycogenbreakdown,preventingglycolysis,and promoting gluconeogenesis glucagonenables in hepatocy'tes, the liver to exportglucose,restoringbloodglucoseto its normal level. Although its primary target is the liver, glucagon (like epinephrine)also affects adiposetissue,activating TAG breakdown by causing cAMP-dependent phosphorylationof perilipin and hormone-sensitivelipase. The activated lipase liberates free fatty acids, which are exported to the liver and other tissues as fuel, sparing glucosefor the brain. The net effect of

glucagonis therefore to stimulate glucose synthesis and release by the liver and to mobilize fatty acids from adiposetissue,to be used insteadof glucoseby tissuesother than the brain (Table23-4). All these effects of glucagonare mediated by cAMP-dependent protein phosphorylation.

During Fasting andStarvation, Metabolism 5hifts to Provide Fuel fortheBrain The fuel reservesof a healthy adult human are of three types: glycogenstored in the liver and, in smaller quantities, in muscles;large quantities of triacylglycerolsin adiposetissues;and tissue proteins,which can be degradedwhen necessaryto provide fuel (Table 23-5).

Metaboliceffect

Effect on gucosemetabolism

Targetenzyme

t Glycogenbreakdown (liver)

Glycogen------+glucose

1 Glycogenphosphorylase

J GlYcogens}'nthesis(liver) J Gtycolysis(liver)

Lessglucosestored as glycogen Lessglucoseused as fuel in liver

J prx-r

(tiver) 1 Gluconeogenesis

Amino acids I Glycerol | Oxaloacetate )

1 FBPase-2 J Pyruvatekhase t PEP carboxykinase

1 Fatty acid mobilization(adiposetissue)

----+ glucose

J Glycogensy'nthase

Lessglucoseused as fuel by liver, muscle

1 Hormone-sensitivelipase

Providesalternativeto glucoseas energy sourcefor brain

J Acetyl-CoAcarboxylase

l Pre (periupi"-@) 4--

I I{etogenesrs

Metabolism 23.3Hormonal Regulation ofFuel ,lr1

ftpe of tuel

Caloric equivalent (thousandsof kcal (kJ))

weight(kg)

Normal-weight, 70 kg man Tfiacyl$ycerols (adiposetissue)

141(58e) 24 (r00) 0.e0(3.8) 0.10(0.42)

lo

Proteins (mainly muscle)

o

Glycogen(muscle,liver)

0.225 0.023

Circulatingfuels (giucose,fatty acids, triacyiglycerols,etc.)

Estimatedsurvival (months)x

166(694)

Total Obese, 140 kg man Ttiacylglycerols(adiposetissue) Proteins (mainJymuscle)

752(3,t40) 32 (r34) o.e2(3.8) 0.11(0.46)

80 8 023 0.025

Giycogen(muscle,liver) Circulatingfuels

785(3,280)

Total

I4

*Survival timeis calculated on theassumption of a basalenergy expenditure of 1,800kcal/day.

In the first two hours after a meal, the blood glucose level is diminished slightly, and tissues receive glucosereleasedfrom liver glycogen.There is Iittle or no synthesisof lipids. By four hours after a meal,blood glucosehasfallenfurther, insulin secretionhasslowed, and glucagonsecretionhas increased.Thesehormonal signals mobilize triacylglycerols,which now become the primary fuel for muscle and liver. Figure 23-31

16''"'_l 16"#-1 I is exported | | I to the kidneyl I I and excreted| |

is exported I ro the brain I via the I

L_l_11"". I Dle$l""1rn l

showsthe responsesto prolongedfasting. @ To provide glucosefor the brain, the liver degradescertain proteins-those most expendablein an organismnot ingesting food. Their nonessentialamino acids are transaminatedor deaminated(Chapter 18), and@ the extra amino groups are convertedto urea, which is exported via the bloodstreamto the kidneys and excreted.

Fattyaclds (imported from adiposetissue) are oxidized as fuel, producing acetyl-CoA. @

Ketonebodiesare exported via the bloodstreamto the brain, which uses them as fuel.

Lack ofoxaloacetate prevents acetyl-CoAentry into the citric acid cycle; acetyl-CoAaccumulates.

FIGURE 23-31 Fuel metabolismin the liver duringprolongedfastingor in uncontrolleddiabetesmellitus.After depletionof storedcarbohydrates,proteins become an important sourceof glucose,producedfrom glucogenic (@ to @). amino acidsby gluconeogenesis Fattyacidsimportedfrom adiposetissueare convertedto ketonebodiesfor export to the reacbrain(@ to @). Brokenarrowsrepresent tionswith reducedflux undertheseconditions. Thesteosarefurtherdescribedin the text.

Regulation andIntegration ofMammalian Metabolism lrtU-t Hormonal Also in the liver, the carbon skeletonsof glucogenic amino acids are convertedto pyruvate or intermediatesof the citric acid cycle.@These intermediates(as well as the glycerol derived from TAGsin adiposetissue) providethe startingmaterialsfor gluconeogenesis in the liver, @ yreldingglucosefor export to the brain @ patty acids are oxidizedto acetyl-CoA,but as oxaloaceteis depletedby the use of citric acid cycleintermediatesfor gluconeogenesis, @ entry of acetyl-CoA into the cycleis inhibitedand acetyl-CoAaccumulates. @ ffris favors the formation of acetoacetyl-CoAand ketonebodies.After a few daysof fasting,the levelsof ketonebodiesin the bloodrise (Fig. 23-32) asthey are exported from the liver to the heart, skeletalmuscle, and brain, which use these fuels instead of glucose (Fig.23-31,@). Acetyl-CoAis a cntical regulatorof the fate of pymvate;it allostericallyinlLibitspyruvatedehydrogenase and stimulatespy'uvate carboxylase(seeFig. 15-20).ln these waysacetyl-CoApreventsit own further productionfrom pyruvate whjle stimr-rlaturgthe conversionof p5,.mvateto oxaloacetate,the first stepin gluconeogenesis, Ttiacylglycerolsstored in the adiposetissue of a normal-weightadult could provide enoughfuel to maintain a basalrate of metabolismfor aboutthreemonths;a very obeseadult hasenoughstoredfuel to endurea fast of more than a year (Table 23-5). When fat reservesare gone,the degradationof essentialproteinsbegins;this leadsto lossof heartandliver functionand,in prolonged starvation,to death. Stored fat can provide adequate energy (calories) during a fast or rigid diet, but vitamins and minerals must be provided, and sufflcient dietary glucogenicamino acids are needed to replace those being used for gluconeogenesis. Rationsfor those on a weight-reduction diet are commonlyfortified with vitamins,minerals,and aminoacidsor proteins.

Epinephrine Signals lmpending Activity When an animal is confronted with a stressful situation that requires increased activity-flghting or fleeing, in

Inmediate effect Physiological t Heart rate I lJlooopressure t Dilation of respiratorypassages Metabolic 1 Glycogenbreakdown(muscle,liver) J Glycogenslmthesis(muscle,liver) (liver) t Gluconeogenesis t Glycolysis(muscle) T Fatty acid mobi-lization(adiposetissue) t Glucagonsecretion J Insutin secretion

FO

CR

!

t4

a'/ cd-

02468 Days ofstarvation FIGURE 23-32 Plasmaconcentrations of fatty acids,glucose,and ketone bodiesduringthe first weekof starvation.Despitethe hormonal mechanisms for maintaining the levelof glucosein blood,glucose beginsto diminishafter2 daysof fasting.The levelof ketonebodies, almostunmeasurable beforethe fast,risesdramatically after2 to 4 days of fasting.Thesewater-solubleketones,acetoacetateand Bhydroxybutyrate, supplement glucoseas an energysourceduringa long fast.Fattyacidscannotserveas a fuel for the brain;they do not crossthe blood-brain barrier

the extreme case-neuronal signalsfrom the brain trigger the releaseof epinephrineand norepinephrinefrom the adrenalmedulla. Both hormonesdilate the respiratory passages to facilitatethe uptakeof 02, increasethe rate and strengthof the heartbeat,and raisethe blood pressure,therebypromotingthe flow of 02 and fuelsto the tissues(Table23-6). Epinephrineacts primarily on muscle,adipose,and liver tissues.It activatesglycogenphosphorylaseand inactivatesglycogens;mthaseby cAMP-dependentphosphorylationof the enz;.'rnes, thus strmulatingthe conversion of liver glycogento blood glucose,the fuel for anaerobicmuscularwork. Epinephrinealso promotes

0verall effect

,.,.."ur" delivery of 02 to fissues(muscle) ]

Increaseproduction of glucosefor fuel IncreasesATP production in muscle Increasesavailabilityof fatty acidsas fuel Rein-forcemetaboliceffects of epinephrine

F u eM l etabolism 2 3 . 3H o r m o nRael g u l a t ioofn lbrr]

the anaerobicbreakdown of muscle glycogenby lactic acid fermentation,stimulatingglycolytic ATP formation. The stimulation of glycolysisis accomplishedby raising the concentrationoffructose2,6-bisphosphate, a potent allosteric activator of the key glycolytic enz),-rne phosphofructokinase-l(seeFigs 15-16,15-17).Epinephrine alsostimulatesfat mobilizationin adiposetissue,activating (by cAMP-dependentphosphorylation)hormonesensitivelipase and moving asidethe perilipin covering the lipid droplet surface (see Fig. I7-3). Finally, epinephrine stimulatesglucagonsecretionand inhibits insulin secretion,reinforcing its effect of mobilizingfuels and inhibiting fuel storage.

(ortisol Signals Stress,lncluding LowEload Glucose A variety of stressors (anxiety, fear, pain, hemorrhage, infection, low blood glucose, starvation) stimulate release of the corticosteroid hormone cortisol from the adrenal cortex. Cortisol acts on muscle, liver, and adipose tissue to supply the organism with fuel to withstand the stress Cortisol is a relatively slow-acting hormone that alters metabolism by changing the kinds and amounts of certain enzyrres s;mthesized in its target cells, rather than by regulating the activity of existing enz;rne molecules In adipose tissue, cortisol leads to an increase in the release of fatty acids from stored TAGs. The exported fatty acids serve as fuel for other tissues, and the glycerol is used for gluconeogenesis in the liver. Cortisol stimulates the breakdown of muscle proteins and the export of amino acids to the liver, where they serve as precursors for gluconeogenesis. In the liver, cortisol promotes gluconeogenesis by stimulating synthesis of the key enzyme PEP carboxykinase (see Fig. 7a-I7b); glucagon has the same effect, whereas insulin has the opposite effect. Glucose produced in this way is stored in the Iiver as glycogen or exported immediately to tissues that need glucose for fuel. The net effect of these metabolic changes is to restore blood glucose to its normal level and to increase glycogen stores, ready to support the fight-or-flight response commonly associated with stress. The effects of cortisol therefore counterbalance those of insulin. During extended periods of stress, the continued release of cortisol loses its positive adaptive value and begins to cause damage to muscle and bone, and to impair endocrine and immune function.

insulin-dependentdiabetesmellitus (IDDM), and type 2 diabetes, or non-insulin-dependentdiabetesmellitus (NIDDM),alsocalledinsulin-resistantdiabetes. TJlpe1 diabetesbeginsearly in life, and symptoms quickly become severe.This diseaserespondsto insulin injection, becausethe metabolicdefect stems from an autoimmunedestructionof pancreaticB cells and a consequentinability to produce sufficient insulin. T}rpe 1 diabetes requires both insulin therapy and careful,lifelongcontrol of the balancebetweendietary intake and insulin dose.Characteristicsymptoms oftype 1 (and type 2) diabetesare excessivethirst and frequent urination (polyuria), Ieadingto the intake of Iarge volumes of water (polydipsia) ("diabetesmellitus" means "excessiveexcretion of sweet urine"). These symptoms are due to the excretion of large amountsof glucosein the urine, a conditionknown as glucosuria. TVpe 2 diabetes is slow to develop (typically in older, obeseindividuals), and the s;'rnptomsare milder and often go unrecognizedatf,rst. This is really a group of diseasesin which the regulatory activity of irsulin is disordered:insulinis produced,but somefeatureof the insulin-responsesystemis defective.Individualswith this disorder are insulin-resistant.The connectionbetween type 2 diabetesand obesity(discussedbelow) is an activeand excitingareaof research. Individuals with either type of diabetesare unable to take up glucose efficiently from the blood; recall that insulin triggers the movementof GLUT4 glucose transportersto the plasma membranein muscle and adiposetissue (seeFig. 12-16). Another characteristic metabolicchangein diabetesis excessivebut incomplete oxidation of fatty acids in the liver. The acetylCoA produced by B oxidation cannot be completely oxidized by the citric acid cycle, because the high INADHI/INAD+lratio producedby B oxidationinhibits the cycle (recall that three steps of the cycle convert NAD+ to NADH). Accumulationof acetyl-CoAleadsto overproductionof the ketonebodies,acetoacetateand B-hydroxybutyrate,which cannot be used by extrahepatic tissuesas fast as they are madein the liver In addition to p-hydroxybutyrate and acetoacetate,the blood of individuals with diabetesalso contains acetone, which resultsfrom the spontaneousdecarboxylation of acetoacetate:

oo cH3-c-cH2-coo

Diabetes Mellitus Arises fromDefeets inInsulin Production orAction Diabetes mellitus is a relatively common disease:nearly 60/oof the U.S. population shows some degree of abnormality in glucose metabolism that is indicative of diabetes or a tendency toward the condition. There are two major clinical classes of diabetes mellitus: type I diabetes, sometimes referred to as

Acetoacetate

+ H2o --------> cH3-c-cH3 + HCO; Acetone

Acetone is volatile and is exhaled,and in uncontrolled diabetesthe breath has a characteristicodor sometimesmistaken for ethanol. A diabetic individual who is experiencingmentalconfusiondue to high blood glucoseis occasionallymisdiagnosedas intoxicated,an error that can be fatal. The overproductionof ketone bodies, called ketosis, results in greatly increased

f

930

l

H o r m o nRael g u l a t iaonndI n t e g r a t ioofnM a m m a l iM a ne t a b o l i s m

concentrations of ketone bodies in the blood (ketonemia) and urine (ketonuria). The ketone bodies are carboxylic acids, which ionize, releasing protons. In uncontrolled diabetes this acid production can overwhelm the capacity of the blood's bicarbonate buffering system and produce a lowering of blood pH called acidosis or, in combination with ketosis, ketoacidosis, a potentially life-threatening condition. Biochemical measurements on blood and urine samples are essential in the diagnosis and treatment of diabetes. A sensitive diagnostic criterion is provided by the glucose-tolerance test. The individual fasts overnight, then drinks a test dose of 100 g ofglucose dissolved in a glass ofwater. The blood glucose concentration is measured before the test dose and at 30 min interuals for several hours thereafter. A healthy individual assimilates the glucose readily, the blood glucose rising to no more than about 9 or 10 mv; little or no glucose appears in the urine. In diabetes, individuals assimilate the test dose ofglucose poorly; their blood glucose level rises dramatically and returns to the fasting Ievel very slowly Becausethe blood glucose levels exceed the kidney threshold (about 10 mlr), glucose also appears in the urine. I

S U M M A R2Y3 . 3 H o r m o n R a le g u l a t i o n f F u eM l etabolism r

The concentration of glucose in blood is hormonally regulated. Fluctuations in blood glucose (normally 60 to 90 mg/100 mL, or about 4.5 ml,r) due to dietary intake or vigorous exercise are counterbalanced by a variety of hormonally triggered changes in metabolism in several organs.

r

High blood glucose elicits the release of insulin, which speeds the uptake of glucose by tissues and favors the storage offuels as glycogen and triacylglycerols, while inhibiting fatty acid mobilization in adipose tissue.

r

Low blood glucose triggers release ofglucagon, whrch stimulates glucose release from liver glycogen and shifts fuel metabolism in liver and muscle to fatty acid oxidation, sparing glucose for use by the brain. In prolonged fasting, triacylglycerols become the principal fuel; the liver converts the fatty acids to ketone bodies for export to other tissues, hcluding the brain.

r

Epineptrine prepares the body for increased activity by mobilizing glucose from glycogen and other precursors, releasing it into the blood.

r

Cortisol, released irt response to a variety of stressors (including low blood glucose), strmulates gluconeogenesis from amino acids and glycerol in the liver, thus raising blood glucose and counterbalancing the effects of insulin.

r

In diabetes,insulinis either not producedor not recognizedby the tissues,and the uptakeof blood glucoseis compromised.Whenbloodglucoselevels are high,glucoseis excreted.Tissuesthen depend on fatty acidsfor fuel (producing ketone bodies) and degradecellular proteins to provide glucogenic amino acidsfor glucoses;nthesis. Uncontrolled diabetesis characterizedbyhigh glucoselevels in the blood and urine and the production and excretionof ketonebodies.

23.40besity andtheRegulation of BodyMass In the U.S.population,30%of aduttsare obeseand another35o/o arc overweight,asdefinedin terms of body mass index (BMI), calculated as (weight in kg)/(height in m)'. A BMI below 25 is considerednormal; an individualwith a BMI of 25 to 30 is overweight;a BMI greater than 30 indicatesobesity. Obesityis life-threatening.It sigfficantly increasesthe chancesof developing tWe 2 diabetesas well as heart attack, stroke, and cancers of the colon, breast,prostate,and endometrium. Consequently,there is great interest in understanding how body massand the storageof fats in adiposetissue are regulated.I To a first approximation,obesityis the result of taking in more caloriesin the diet than are expendedby the body's energy-consumingactivities. The body can deal with an excessof dietarycaloriesin threeways:(1) convert excessfuel to fat and store it in adiposetissue, (2) burn excessfuel by extra exercise,and (3) "waste" fuel by diverting it to heat production (thermogenesis) by uncoupledmitochondria.In mammals,a complex set of hormonal and neuronal signalsacts to keep fuel intake and energyexpenditurein balance,so as to hold the amount of adiposetissue at a suitablelevel. Dealing effectively with obesity requires understanding these various checks and balancesunder normal conditions, and how thesehomeostaticmechanismscan fail.

Adipose Tissue Haslmportant Endorrine Functions One early hypothesis to explain body-mass homeostasis, the "adiposity negative-feedback" model, postulated a mechanism that inhibits eating behavior and increases energy consumption whenever body weight exceeds a certain value (the set point); the inhibition is relieved when body weight drops below the set point (Fig. 23-33 ). This model predicts that a feedback signal originating in adipose tissue influences the brain centers that control eating behavior and activity (metabolic and motor activity). The first such factor, leptin, was discovered in 1994, and subsequent research revealed that adipose tissue is an important endocrine organ that produces peptide hormones, known as adipokines. Adipokines may act locally (autocrine and paracrine

y a s s[ n r t 2 3 . 40 b e s i tayn dt h eR e g u l a t ioofnB o d M

Food fat synthesis

Adipose tissue

+Leptin

fat P oxidatio

Energy, heat tIGURE 23-'33 Set-pointmodelfor maintaining constantmass.When (dashed themassof adipose tissueincreases leptininoutline),released hibitsfeedingand fat synthesis and stimulates oxidationof fattyacids (solidoutline),a loweredlepWhenthe massof adiposetissuedecreases favorsa greater food intakeand lessfattyacidoxidation tin production action) or systemically (endocrine action), carrying i.nformation about the adequacy of the energy reserves

(TAGs) stored in adipose tissue to other tissues and to the brain. Normally, adipokines produce changes in fuel metabolism and feeding behavior that reestablish adequate fuel reserves and maintain body mass. When adipokines are over- or underproduced, the resulting dysregulation may result in life-threatening disease. Leptin (Greek Leptos, "thin") is an adipokine (167 amino acids) that, on reaching the brain, acts on receptors in the hypothalamus to curtail appetite. Leptin was first identified as the product of a gene designated OB (obese) in iaboratory mice. Mice with two defective copies of this gene (ob/ob genotype; lowercase letters signify a mutant form of the gene) show the behavior and physioiogy of animals in a constant state of starvation: their plasma cortisol Ievels are elevated; they are unable to stay warm and they grow abnormally, do not reproduce, and exhibit unrestrained appetite. As a consequence of the last effect, they become severely obese, weighing as much as three times more than normal mice (Fig. 23-34) They also have metabolic disturbances very similar to those seen in diabetes, and they are insulin-resistant When leptin is injected into oblob mice, they lose weight and increase their locomotor activity and thermogenesis. A second mouse gene, designated DB (diabetic), has also been found to have a role in appetite regulation. Mice with two defective copies (db/db) are obese and diabetic The DB gene encodes the leptin receptor. When the receptor is defective, the signaling function of leptin is lost. The leptin receptor is expressedprimarily in regions of the brain known to regulate feeding behavior-

tl6URE?3-34 Obesity caused by defective leptin production. Both of t h e s em i c e , w h i c h a r e t h e s a m e a g e , h a v e d e f e c t si n t h e O B g e n e .T h e m o u s e o n t h e r i g h t w a s i n i e c t e dd a i l y w i t h p u r i f i e d l e p t i n a n d w e i g h s 3 5 g T h e m o u s e o n t h e l e f t g o t n o l e p t i n , a n d c o n s e q u e n t l ya t e m o r e f o o d a n d w a s l e s sa c t i v e ; i t w e i g h s 5 7 g .

neurons of the arcuate nucleus of the hypothalamus (l'ig. Z:t-:l5a). Leptin carries the messagethat fat reselves are sufficient, and it promotes a reduction in fuel intake and increased expenditure of energy. Leptin-receptor interaction in the hypothalamus alters the release of Paraventricular nucleus

(a)

Arcuate nucleus

, ,I

Neuronal signal via sympathetic neuron

Leptin (via blood) Anterior pituitary

_

Adipose trssue

(b) B3-Adrenergic

Adenylyl cyclase

Nucleus

23*15 Hypothalamicregulationof food intake and energy FIGURE (a)Anatomyof thehypothalamus with and itsinteraction expenditure. betweenthe hypothalaadiposetissue.(b) Detarlsof the interaction musand an adioocvte, described laterin thetext

.

\

TAG

Lipid droplet

Perilipin

I

Regulation andlntegration ofMammalian Metabolism L932_lHormonal neuronalsignalsto the regionofthe brain that affectsappetite. Leptin also stimulates the s}rmpatheticnervous system,increasingblood pressure,heart rate, and thermogenesis by uncoupling the mitochondria of white adipocytes (Frg. 23-35b). Recall that thermogenin, or UCP,forms a charmelin the inner mitochondrial membrane that allows protons to reenter the mitochondrial matrix without passingthrough the ATP slmthasecomplex. This permits continualoxidationof fuel (fatty acids in an adipocyte) without ATP synthesis,dissipatingenergy as heat and consumingdietary calories or stored fats in potentially very large amounts. Arcuate nucleus

Leptin Stimulates Production ofAnorexigenic Peptide Hormones TWotypes of neuronsin the arcuatenucleuscontrol fuel intake and metabolism(Fig. 23-36). The orexigenic (appetite-stimulating)neurons stimulate eating by producing and releasing neuropeptide Y (NPY), which causesthe next neuron in the circuit to send the signal to the brain: Eat! The blood level of NPY rises during starvation,and is elevatedin both ob/ob anddb/db mice. The high NPY concentration presumablyunderlies the obesiWof these mice, who eat voraciously.

Muscle, adipose tissue, liver

Pancreas

FIGURE 23-36 Hormonesthat control eating.In the arcuatenucleus, two setsof neurosecretory cellsreceivehormonalinputand relayneuronal signalsto the cells of muscle,adiposetissue,and liver.Leptin and insulin are releasedfrom adiposetissueand pancreas,respectively,in proportionto the massof body fat.Thetwo hormonesact on anorexigenic neurosecretorycells to trigger release of a-MSH (melanocortin); this producesneuronalsignalsto eat lessand metabolize more fuel. Leptinand insulinalso act on orexigenicneurosecre-

tory cellsto inhibitthe releaseof NPI reducingthe ,,eat,,signalsentto the tissues.As describedlater in the text,the gastrichormoneghrelin stimulatesappetiteby activatingthe NPY-expressing cells;PYY3_ru, releasedfrom the colon, lnhlbltstheseneuronsand therebydecreases appetite.Eachof the two typesof neurosecretory cells inhibits hormone productionby the other,so any stimulusthat activatesorexigenic cells inactivatesanorexigeniccells, and vice versa.This strengthens the effectof stimulatoryinputs.

MassFrl ofBody 23.4 0besity andtheRegulation The anorexigenic (appetite-suppressing) neurons in the arcuate nucleus produce a-melanocyte(a-MSH; also knoum as stimulating hormone melanocortin), formed from its polypeptide precursor pro-opiomelanocortin (POMC; Fig. 23-6). Release of a-MSH causesthe next neuron in the circuit to send the signal to the brain: Stop eating! The amount of leptin released by adipose tissue depends on both the number and the size of adipocytes. When weight loss decreasesthe mass of lipid tissue, Ieptin levels in the blood decrease, the production of NPY falls, and the processesin adipose tissue shown in Figure 23-35 are reversed. Uncoupling is dimrnished, slowing thermogenesis and saving fuel, and fat mobilization slows in response to reduced signaling by cAMP. Consumption of more food combined with more efflcient utiIization of fuel results in replenishment of the fat reserve in adipose, bringing the system back into balance. Leptin may also be essential to the normal development of hypothalamic neuronal circuits. In mice, the outgrowth of nerve flbers from the arcuate nucleus during early brain development is slower in the absence of Ieptin, affecting both the orexigenic and (to a lesser extent) anorexigenic outputs of the hypothalamus. It is possible that the leptin levels durzng deuelopment of these circuits determine the details of the hardwiring of this regulatory system.

(aseade Leptin Triqgers ThatHegulates a5ignaling Expression 6ene The leptin signal is transducedby a mechanismalso usedby receptorsfor interferonand growth factors,the JAK-STATsystem(Fig. 23-37; see Fig. 12-18). The leptin receptor,which has a singletransmembranesegment, dimerizeswhen leptin binds to the extracellular domainsof two monomers.Both monomersare phosphorylated on a Tlr residueof their intracellular domain by a Janus kinase (JAK) The @-IVr residuesbecome docking sites for three proteins that are signal fransducersand activatorsof franscription(STATs3, 5, and 6; sometimescalledfat-STATS).The dockedSTATs are then phosphorylatedon Tlr residuesby the same JAK. After phosphorylation,the STATsdimerizethen move to the nucleus,where they bind to specificDNA and stimulatethe expressionoftarget genes, sequences including the gene for POMC, from which a-MSH is produced. The increasedcatabolismand thermogenesistriggeredby Ieptin are due in part to increasedsynthesisof the mitochondrial uncoupling protein thermogenin (product of the UCPi gene) in adipocytes.Leptin stimulatesthe synthesisof thermogeninby altering synaptic transmissionsfrom neuronsin the arcuatenucleusto adiposeand other tissues.In thesetissues,leptin causes which actsthrough increasedreleaseof norepinephrine, transcription of the receptors to stimulate B3-adrenergic

Plasma membrane

DNA mRNA

mechanismof leptinsignaltransduction 23-37 TheIAK-STAT FIGURE of the lepLeptinbindinginducesdimerization in the hypothalamus. catof specificTyr residues, tin receptor,followedby phosphorylation alyzedby Januskinase(JAK).STATsbound to the phosphorylated on throughtheirSH2domainsarenow phosphorylated leptinreceptor binding dimerize, The STATs of activity JAK by a separate Tyrresidues and enterthe nucleusHere,they bind eachother's@-Tyr residues, of cerin the DNA andalterthe expression regions regulatory specific theorganof thesegenesultimatelyinfluence Theproducts tarngenes. ism'sfeedingbehaviorandenergyexpenditure

LICPl gene. The resuiting uncoupling of electron transfer from oxidative phosphorylation consumes fat and is thermogenic (FiS. 23-35). Might human obesity be the result of insufficient leptin production, and therefore treatable by the injection of leptin? Blood levels of leptin are in fact usually much hi,gher in obese animals (including humans) than in animals of normal body mass (except, of course, in oblob mutants, which cannot make leptin). Some downstream element in the leptin response system must be defective in obese individuals, and the elevation in leptin is the result of an (unsuccessful) attempt to overcome the leptin resistance. In those very rare humans with extreme obesity who have a defective leptin gene (OB), Ieptin injection does result in dramatic weight loss. In the vast majority of obese individuals, however, the OB gene is intact. In clinical trials, the injection of leptin did not have the weightreducing effect observed in obese ob/ob mice' Clearly, most cases of human obesity involve one or more factors in addition to lePtin.

Fr-]

H o r m o nRael g u l a tai onndI n t e g r a t oi ofM n a m m a lM i aent a b o l i s m

TheLeptin System MayHave Evolved toRegulate the Starvation Response The leptin systemprobablyevolvedto adjustan animal's activity and metabolismduring periods of fasting and starvatron,not as a meansto restrict weight gain. The reducti,on in leptin level triggered by nutritional deflciencyreversesthe thermogenicprocesses iilustratedin Figure23-35,allowingfuel conservation. Leptin (acting in the hypothalamus)also triggers decreasedproduction of thyroid hormone (slowingbasalmetaboJism),decreased production of sex hormones (preventing reproduction),and increasedproductionof glucocorticoids (mobilizingthe body'sfuel-generatingresources). By minimizing energy expenditure and maximizing the use of endogenousreservesof energy,these leptinmediated responsesmay allow an animal to survrve periods of severe nutritional deprivation. In liver and muscle,leptin stimulatesAMP-activated protein kinase (AMPK), and through its action inhibits fatty acid synthesisand activatesfatty acid oxrdation,favoring processes. energy-producing

InsulinActsintheArcuate Nucleus to Regulate Eatingand Energy Conservation Insulin secretionreflectsboth the size of fat reserves (adiposity)and the current energybalance(blood glucoselevel). Insulin acts on insulin receptorsin the hypothalamus to inhibit eating (Fig. 23-36). Insutin receptorsin the orexigenicneuronsof the arcuatenucleusinhi,bi,fthe releaseof NPI and insulin receptorsin the anorexigenicneuronssti,mu\atea-MSHproduction, therebydecreasingfuel intakeandincreasingthermogenesis.By mechanismsdiscussedin Section23.3,insulin alsosignalsmuscle,liver,and adiposetissuesto rncrease the conversionof glucoseto acetyl-CoA,providingthe starting material for fat syrrthesis. Leptin makes the cells of Iiver and muscle more sensitiveto insulin. One hypothesisto explain this effect suggestscross-talkbetween the protein tyrosine kinasesactivatedby leptin and those activatedby insulin (Fig. 23-38); commonsecondmessengers in the two signalingpathwaysallowleptin to trigger someof the same dor,l'nstreamevents that are triggered by insulin, through insulin receptorsubstrate-2(IRS-2)and phosphoinositide3-kinase(PI-3K) (Fig. 12-16).

Adiponectin Acts through AMPK toIncrease Insulin Sensitivity Adiponectin is a peptide hormone (224 amino acids) produced almost exclusively in adipose tissue, an adipokine that sensitizes other organs to the effects of insulin, protects against atherosclerosis, and inhibits mflarnmatory responses (monoclte adhesion, macrophage transformation, and the proliferation and migration of the

Insulin receptor

j Inhibition offood intake

FIGURE 23-38 A possiblemechanismfor cross-talkbetweenreceptors for insulinandleptin,Theinsulinreceptor hasintrinsic Tyrkinaseactivity (seeFig 12-1 5),andthe leptinreceptor, whenoccupiedby itsligand, is phosphorylated by a solubleTyrkinaseUAK).One possible explanation for the observedinteractionbetweenleptinand insulinis that both mayphosphorylate the samesubstrate-inthe caseshownhere,insulin (lRS-2)When phosphorylated, receptorsubstrate-2 IRS-2activatesPl3K,whichhasdownstream consequences thatincludeinhibition of food intake.IRS-2serveshereasan integratorofthe inputfromtwo receptors.

cells of vascular smooth muscle). Adiponectin circulates in the blood and powerfully affects the metabolism of fatty acids and carbohydrates in liver and muscle. It increases the uptake of fatty acids from the blood by

myoc),'tesand the rate at which fatty acids undergo B oxidationin muscle.It alsoblocksfatty acid sytthesisand gluconeogenesis in hepatocy'tes,and strmulatesglucose uptakeand catabolismin muscleand liver. These effects of adiponectinare indirect and not fully understood,but AMPK clearly mediatesmany of them. Acting through its plasmamembranereceptor, adiponectin triggers phosphorylationand activation of AMPK. Recall (see Fig. 15-6) that AMPK is activated by factors that signal the need to shift metabolism away from biosynthesisand toward energy production (Fig. 23-39). When activated,AMPK phosphoryiates target proteins critical to the metabolismof lipids and carbohydrates, with profoundeffectson the metabolism of the wholeanimal(Fig. 23-40). Adiponectinreceptors are also present in the brain; the hormone activates AMPK in the hypothalamus,stimulatingfood intake and reducingenergyexpenditure. One enz;.'rneregulatedby AMPK in the liver and in white adiposetissue is acetyl-CoAcarboxylase,which producesmalonyl-CoA,the flrst intermediatecommitted to fatty acid synthesis.Malonyl-CoAis a powerful inhibitor of the enzyme carnitine acyltransferaseI,

Mass[ntu-l ofBody andtheRegulation 23.4 0besity 23-39 The role of AMP-activatedprotein kinase(AMPK)in FIGURE regulatingATP metabolism'ADP producedin syntheticreactionsis convertedto AMP by adenylatekinase.AMP activatesAMPK,which keyenby phosphorylating anabolicandcatabolicpathways regulates (see Fig.23-40). zymes

Anabolic processes

Cholesterol synthesis is also inhibited

Fig. 17-12).

by

and inactivatesHMG-CoA AMPK,which phosphorylates reductase,an enzymein the path to cholesterol(see Fig 21-34). Similarly,AMPK inhibits fatty acid s),'nthase and acyl transferase,effectively blocking the synthesis of triacylgiycerols. Mice with defectiveadiponectingenesare less !E sensitive to insulin than those with normal S adiponectin,and they showpoor glucosetolerance:ingestion of dietary carbohydratecausesa long-lasting rise in bloodglucose.Thesemetabolicdefectsresemble those of humanswith type 2 diabetes,who are similarly insulin-insensitive and clear glucose from the blood

Catabolicprocesses

E

which startsthe processof B oxidationby transporting fatty acidsinto the mitochondrion(see Fig. 17-6). By phosphorylatingand inactivatingacetyl-CoAcarboxyIase,AMPK inhibits fatty acid synthesiswhile relieving the inhibition (by malonyl-CoA) of B oxidation (see

Cardiac glycolysis

Glucose transport

t

I

I

PFK-2

a tt ------T-----

I GLUTl GLUT4

' Lons fast: ------+ starvatron

Heart fatty acid oxidation, glucoseuptake, glycolysis

Fatty acid synthesis

Fatty acid oxidation

Li polys

a

-

+v Feeding behavior

t,

a

GPAT

HMGR

..rt\l//2.

,r,

Brain

I

I

I

AMPK

--

Glycogen synthesis

tI

tI

HSL

Adipose tissue snnnKs

a

a

tI

FAS I ACC

Skeletal muscle fatty acid oxidation, glucoseuptake

Cholesterol isoprenoid Triacylglycerol synthesis synthesis

3' direction, with the free 3, OH as the point at which the DNA is elongated (the b, and 3,

(a)

:)

(b)

Unidirectional

Bidirectional

$r

Replication forks

.l

I Origin

,t^'

.L -'a

I

t t

FIGURE 25-3 Visualization of bidirectional (a) RepticaDNA replication. tion of a circularchromosome producesa structureresembling the Creeklettertheta,d, asboth strandsarereplicatedsimultaneousry tnew strands shownin pink).(b) Replication couldbe eitherunidirectional or bidirectional, andthesecanbe distinguished by autoradiography: when tritium(3H)is addedfor a shortperiodjust beforereplicationis stopped, label (pink)would be found at one or both replicationforks,respectively.This techniquehas revealedbidirectionalreplicationin E. coli, Bacillussubtilis,and other bacteriaThe autoradiogram here showsa replicationbubblefrom B.subtilis.fheheaviest graindensity(arrows)is at the two ends,wherereplicationis occurring.Theunreplicated partof thechromosome, outsidethe bubble,is unlabeled andthusnotvisible

ends of a DNA strand are defined in Fig. B-Z). Because the two DNA strands are antiparallel, the strand serving as the template is read from its 3' end toward its b, end. If synthesis always proceeds in the 5'-+S' direction, how can both strands be synthesized simultaneously? If both strands were s;mthesized continuouslg while the replication fork moved, one strand would have to undergo 3'->5' synthesis. This problem was resolved by

1\r4

Replication 2 5 . 1D N A

o

5

Direction of movement ofreplication fork Okazaki fragments \

B, S,

speci-ficnucleotidesequences(suchasthe restriction endonucleasesthat are so important in biotecturology;see Chapter 9, Fig. 9-2). You will encountermany flpes of nucleasesin this and subsequentchapters.

Leading strand

5,

3'

o

Polymerases hyDNA lsSynthesized DNA

3'

Laggrng strand

tIGURE25-4 Defining DNA strandsat the replicationfork. A new in the 5'--+3'directionThe DNA strand(red)is alwayssynthesized 3'-+5' Theleadingstrandis templateis readin theoppositedirection, fork. in thedirectiontakenby the replication continuously synthesized in discontinuously Theotherstrand,the laggingstrand,is synthesized in a directionoppositeto that in shortpieces(Okazakifragments) are spliced which the replication fork moves.TheOkazakifragments togetherby DNA ligase.In bacteria,Okazakifragmentsare -1,000 to long.In eukaryotic cells,they are 150 to 200 nu2,000nucleotides cleotideslong.

Reiji Okazaki and colleagues in the 1960s. Okazaki found that one of the new DNA strands is symthesized in short pieces, now called Okazaki This work fragments. ultimately led to the conclusion that one strand is synthesized continuously and the other discontinuously

(Fig. 25-4). The continuous strand, or leading strand, is the one in which 5'->3' synthesis proceeds in the same dftection as replication fork movement. The discontinuous strand, or lagging strand, is the one in which 5'->3' synthesis proceeds in the direction oppos'ite to the direction of fork movement. Okazaki fragments range in length from a few hundred to a few thousand nucleotides, depending on the cell type. As we shall see later, Ieading and lagging strand syntheses are tightly coordinated.

lsDegraded byNucleases DNA To explain the erzymology of DNA repJication, we first introduce the erz;rmes that degrade DNA rather than slmthesize it. These enzyrnes are known as nucleases, or DNases if they are specific for DNA rather than RNA. Every cell contains several different nucleases, belonging to two broad classes: exonucleases and endonucleases. Exonucleases degrade nucleic acids from one end ofthe molecule. Many operate in only the 5'-+3' or the 3'-+5' direction, removing nucleotides only from the 5' or the 3' end, respectively, of one strand of a double-stranded nucleic acid or of a single-stranded DNA. Endonucleases can begrn to degrade at specific internal sites in a nucleic acid strand or molecule, reducirg it to smaller and smaller fragments. A few exonucleases and endonucleases degrade only single-stranded DNA. There are a few important classes of endonucleases that cleave only at

ArthurKornberg,1918-2007

The searchfor an enz;rmethat could sl.nthesizeDNA beganin 1955. Work by Arthur Kornberg and colleaguesled to the purification and characterization of a DNA polyrnerasefrom E. coli, cells, a single-polyPePtide enz;'rnenow called DNA pol5rmerase | (M, 103,000; encoded by the poLA gene). Much later, investigatorsfound that E. coli, contains at least four other distinct DNA polymerases,describedbelow

Detailed studies of DNA poll'rnerase I revealed features of the DNA synthetic process that are now known to be common to all DNA polyrnerases. The fundamental reaction is a phosphoryl group transfer. The nucleophile is the 3'-hydroxyl group of the nucleotide at the 3' end of the growing strand. Nucleophilic attack occurs at the o phosphorus of the incoming deoxynucleoside 5'triphosphate (FiS. 25-5). Inorganic pyrophosphate is released in the reaction. The general reaction is (dNMP)" + dNTP -) DNA

(dNMP),*t + PPl Lengthened DNA

Q\-I)

where dNMP and dNTP are deoxynucleoside 5'monophosphate and 5'-triphosphate, respectively' The reaction seems to proceed with only a minimal change in free energy, given that one phosphodiester bond is formed at the expense of a somewhat less stable phosphate anhydride. However, noncovalent base-stacking and base-pairing interactions provide additional stabiIization to the lengthened DNA product relative to the free nucleotide. Also, the formation of products is facilitated in the cell by the 19 kJ/mol generated in the subsequent hydrolysis of the pyrophosphate product by the enz).rne plryophosphatase (p. 508). Early work on DNA poll'rnerase I led to the definition of two central requirements for DNA polymerization. First, all DNA pol),'rnerasesrequire a template. The polymerization reaction is guided by a template DNA strand according to the base-pairing rules predicted by Watson and Crick: where a guanine is present in the template, a cytosine deoxynucleotide is added to the new strand, and so on. This was a particularly important discovery, not only because it provided a chemical basis for accurate semiconservative DNA replication but also because it represented the first example of the use of a template to guide a bioslrrthetic reaction'

f

Metabolism [eB0 ] DNA (a)

ft)

Template DNA strand

Incoming dNTP is attacked at the o phosphate by the 3' hydroxyl of the growing DNA chain.

Growing DNA strand (primer)

Deoxy'ribose

o

,.1 Incoming deoxynucleoside 5'-triphosphate

"') 9r

-e

t

o s b

'.!(

OH

o-

o-

oH

I -o-P:o I

t9. ?

.( -\-rldl."1 H)

J

&

MICHANISM tlGURE 25-5 Elongation of a DNA chain.(a) DNA polymeraseIactivityrequiresa singleunpairedstrandto act as template and a primerstrandto providea free hydroxylgroupat the 3, end, to whicha new nucleotide unit is added.Eachincomingnucleotide is selectedin partby basepairingto the appropriate nucleotide in thetemplatestrand. Thereactionproducthasa newfree3, hydroxyl, allowing the additionof anothernucleotide. (b)Thecatalyticmechanism likely involvestwo Mg2+ ions,coordinatedto the phosphategroupsof the

incomingnucleotidetriphosphate and to threeAsp residues, two of whicharehighlyconserved in all DNA polymerases. TheMg2* ion depicted on the right facilitatesattackof the 3'-hydroxylgroup of the primeron the a phosphate of the nucleotide triphosphate; the other Mg2* ion facilitatesdisplacement of the pyrophosphateBoth ionsstabilize the structureof the pentacovalent transitionstate.RNA polymerasesuse a similarmechanism(seeFig 26-1b\. $ Nucleotide Polymerizationby DNA Polymerase

Second,the pol5.'rnerases rcqufueaprimer. Apnmeris a strand segment(complementaryto the template) with a free 3'-hydro>ryIgroup to which a nucleotidecanbe added; the free 3' end of the primer is calledthe primer terminus. In other words,par[ of the new strandmust alreadybe in place:all DNApolymerases canonlyaddnucleotidesto a preexistingstrand. Many primers are oligonucleotidesof RNA rather than DNA, and specializedenzyrness),Trthesize primers when and where they are requted. After adding a nucleotideto a growing DNA strand, a DNA pol].meraseeither dissociates or movesalongthe templateand addsanothernucleotide.Dissociationand reassociationof the polymerasecan limit the overall polymerization rate-the process is generally faster when a polymeraseadds more nucleotides without dissociatingfrom the template. The averagenumber of nucleotidesaddedbefore a polyrnerasedissociatesdefines its processivity. DNA polymerasesvary greatly in processivity;someaddjust a few nucleotidesbeforedissociating,othersadd many thousands.I Nucleotide poly-

10eto 1010nucleotidesadded.For the E coti,chromosomeof -4.6 x 106bp, this meansthat an error occurs only onceper 1,000to 10,000replications.During polymerization, discrimination between correct and incorrect nucleotidesrelies not just on the hydrogenbonds that specifythe correct pairing betweencomplementary basesbut alsoon the commongeometryof the standard A:T and G:C basepairs (Fig. 25-6). The activesite of DNA polymeraseI accommodatesonly base pairs with this geometry.An incorrect nucleotidemay be able to hydrogen-bondwith a base in the template,but it genemlly will not flt into the active site. Incorrect bases can be rejected before the phosphodiesterbond is formed. The accuracy of the pol}.'rnerizationreaction itself, however,is insufficientto accountfor the high degreeof fidelity in replication. Careful measurementsin vitro have shown that DNA pol;.'rnerases insert one incorrect nucleotidefor every 104to 105correctones.Thesemistakessometimesoccur becausea baseis briefly in an unusual tautomeric form (see Fig. B-9), allowing it to hydrogen-bondwith an incorrect partner.In vivo, the error rate is reducedby additionalenzymaticmechanisms. One mechanismintrinsic to virtually all DNA polymerasesis a separate3'-+5' exonucleaseactivity that double-checkseach nucleotideafter it is added. This

merizationby DNA Polymerase

Replication lsVery Arcurate Replication proceeds with an extraordinary degree of fldelity. In E. coli,, a mistake is macle only once for everv

Replication 25.1DNA [rut] DNApolymerase I DNA polymerase active site 3'-+5' (proofreading) exonuclease active site

(a)

(b) FIGURE 25-6 Contributionof base-pairgeometryto the fidelity of DNA replication.(a)ThestandardA:T and Q-:( lsss psi15haveverysimilar geometries, and an activesitesizedto fit one (blueshading)will generally accommodatethe other.(b) Thegeometryof incorrectlypairedbases can excludethem from the activesite,as occurson DNA polymerase.

nucleaseactivity permits the enzSrme to removea newly added nucleotide and is highly specificfor mismatched base pairs (Fig. 25-7). If the polyrnerasehas added the wrong nucleotide, translocation of the enzyme to the position where the next nucleotide is to be added is inhibited. This kinetic pause provides the opportunity for a correction. The 3'->5' exonucleaseactivity

25-7 An exampleof error correction by the 3'--+5' exonuFIGURE cleaseactivity of DNA polymerasel. Structuralanalysishas located the exonucleaseactivityaheadof the polymeraseactivityas the enzyme is orientedin its movementalongthe DNA. A mismatchedbase of DNA polymeraseI to (here,a C-A mismatch)impedestranslocation the mistakewith corrects the enzyme backward, site. Sliding next the activityin activity,then resumesits polymerase its 3'--+5'exonuclease the 5'->3' direction.

removesthe mispaired nucleotide, and the polymerase begins again. This activity, known as prd)fteading, is not simply the reverse of the polymerization reaction (Eqn 25-1), becausepyrophosphateis not involved. The polymerizing and proofreadingactivities of a DNA polyrnerasecan be measured sepaxately.Proofreading improves the inherent accuracy of the polymerization

[rui

DNA Metabolism

reaction 102-to 103-fold.In the monomericDNA polymerase I, the polymerizing and proofreading activities haveseparateactivesiteswithin the samepolypeptide. When base selection and proofreadingare combhed, DNA pol5.rnerase leavesbehindone net error for every 106to 108basesadded.Yet the measuredaccuracy of replication in E. coli, is higher still. The additionalaccuracyis pronded by a separateenz;rmesystem that repairs the mismatchedbasepairs remaining after replication. We describe this mismatch repair, along with other DNA repairprocesses, in Section25.2.

f. roliHas atLeast Five DNA Polymerases More than 90o/oof the DNA polymeraseactivity observedinE co|i,extractscan be accountedfor by DNA pol}rmeraseI. Soonafter the isolation of this enzl'rnein 1955,however,evidencebeganto accumulatethat it is not suited for replication of the large E. cole chromosome.First, the rate at which it addsnucleotides(600 nucleotides/min)is too slow (by a factorof 100or more) to accountfor the rates at which the replicationfork movesin the bacterialcell. Second,DNA poly.rnerase I has a relatively low processivity.Third, genetic studies have demonstratedthat many genes, and therefore many proteins, are involved in replication: DNA polymeraseI clearly does not act alone.Fourth, and most important,in 1969JohnCairnsisolateda bacterialstrain with an altered gene for DNA polymeraseI that produced an inactive enz),rne.Although this strain was abnormally sensitiveto agentsthat damagedDNA, it was nevertheless viable! A searchfor other DNA polSnnerases led to the discovery of E. coli, DNA polymerase II and DNA polymerase III in the early 1970s.DNA polymeraseII is an enz).rneinvolved in one type of DNA repair (section 25.3). DNA pol;.'rnerase III is the principal replicationenzymernE. coli,.Thepropertiesof thesethree DNA potymerasesare comparedin Table 25-1. DNA pol;'rnerases

FIGURE 25-8 l-arge(Klenow)fragmentof DNA polymerasel. This polymerase is widely distributedin bacteria.The Klenowfragment, producedby proteolytictreatmentof the polymerase, retainsthe polymerizationand proofreading activitiesof the enzyme.The Klenow fragmentshown here is from the thermophilicbacteriumBacillus (PDB lD 3BDP) The activesite for additionof stearothermophlius nucleotides is deep in the creviceat the far end of the bound DNA (blue;the darkbluestrandis the template).

IV and V, identifled

in 1999, are involved

DNApolymerase

m

II Structural gene* Subunits(number of different types) M, 3' --+5' Exonuclease(proofreading) 5'-+3' Exonuclease Polymerizationrate (nucleotides/s) Processivity(nucleotidesadded before pol;nnerasedissociates)

in an unusual

form of DNA repair (Section25.2). DNA polymeraseI, then, is not the primary enzyme of replication; instead it performs a host of clean-upfunctions during replication,recombination, and repair. The polymerase'sspecialfunctions are enhanced by its 5'-+3' exonucleaseactivity. This activity, distinct from the 3'->5' proofreadingexonuclease (Fig. 25-7), is located in a structural domain that can be separatedfrom the enzymeby mild proteasetreatment. When the 5'-+3' exonucleasedomain is removed,the remainingfragment (M. 68,000),the large fragment or Klenow fragment (Fig. 25-8), retains the polymerization and proofreading activities. The 5'-->3' exonucleaseactivity of intact DNA polymerase I can replacea segmentof DNA (or RNA) paired to the

poIA l

poLB 7

poIC (dnaE) >10

103,000

88,0001

Yes

Yes

Yes

No

r0-20

40

250-1,000

3-200

1,500

>500,000

791,500 Yes No

+Forenzymes withmorethanonesubunit, thegenelistedhereencodes thesubunitwithoolymerization activitvNotethat dnaEis an earlierdesignation for the genenowreferredto as polc. tPolymerization subunitonly.DNApolymerase ll shares several subunits withDNApolymerase lll, including thep, 7,5,6,, (seeTable 25-2). 1, andry'subunits

Replication 25.1DNA Ltu Nick

25-9 Nick translation.In this process,an RNAor DNA strand FIGURE degradedby the 5'--+3' pairedto a DNA templateis simultaneously replacedby the polypolymerase I and of DNA activity exonuclease meraseactivityof the sameenzyme.Theseactivitieshave a role in DNA repairand in the removalof RNA primersduring replication (bothdescribedlater).Thestrandof nucleicacid to be removed(either

,/ OH

I

5

.....,".",--.,-.B,

3',

1s',

Template DNA strand

strandin red. DNA DNA or RNA)is shownin green,the replacement bond, leavinga synthesisbeginsat a nick (a broken phosphodiester I extendsthe free 3'hydroxyl and a free 5'phosphate).Polymerase nontemplateDNA strandand movesthe nick along the DNA-a A nick remainswhere DNA polyprocesscalled nick translation. and is latersealedby anotherenzyme. meraseI dissociates,

DNA polymeraseI OH

5',

'\

3',

3',

D D

, Nick

I oH/i) \

D 3,

_"

. _ r . - . _ : . . - . . . t " - _ . . . . ". i . . . - . .

/

al

. .: =:.., i-._:-..,.r,.._,i:.-..i._,:..-.,.,-.- !,_,.._,.r_.

b,

template strand, in a processknown as nick translation (Fig. 25-g). Most other DNA polymeraseslack a 5' -->3'exonucleaseactivity. DNA polymeraseIII is much more complex than DNA polSrmerase I, having 10 types of subunits (Table 25-Z). Its pol;'rnerizationand proofreadingactivities

Numberof subunitsper Subunit holoenzyme l/.ofsubunit q

a

a

0

2 a

n

1

a

l

D'

I

X

I

a

1

B

4

Gene

reside in its a and e (epsilon) subunits,respectively. The 0 subunit associateswith a and e to form a core pol;rmerase,which canpolyrnerizeDNA but with Iimited processivity.TWocore polymerasescan be linked by another set of subunits,a clamp-loadingcomplex, or y complex, consisting of five subunits of four different are linked through types,12766'.The core polyrnerases (tau) subunits. T\vo additional subunits, X (chi) the r (psi), are bound to the clampJoadingcomplex. and 'y' The entire assemblyof 13 protein subunits (nine differIII* (Fig. 25-f 0a). ent types)is calledDNA pol;nnerase pol}rmerize DNA, but with DNA pol}'meraseIII* can a much lower processivitythan onewould expect for the organizedreplication of an entire chromosome.The necessaryincreasein processivityis providedby the addition of the B subunits,four of which completethe DNA polymeraseIII holoenzyme.The B subunitsassociatein pairs to form donut-shapedstructures that encircle the DNA and act like clamps(Fig. 25-10b). Each dimer associateswith a core subassemblyof polymeraseIII* (one dimeric clamp per core subassembly)and slides

Functionof subunit

129,900 27,500

poIC (dnaE)

Poly.merizationactivity

dnaQ (mutD)

3'-+5' Proofreading exonuclease

8,600 71,100

holE

Stabilizationof e subunit

dnaX

Stabletemplatebinding; core enzyrnedimerization

dnoX*

Clamploader

hol,A

Clampopener

47,500 38,700 36,900 16,600 15,200 40,600

hnLB

Ciamploader

hoLC

Interaction with SSB Interaction with y and X

hoLD dnaN

DNA clamp required for optimal processivity

*The subunitis encoded The7 asthe 7 subunit. 66%of ther subunithasthesameaminoacidsequence bya portionof thegeneforther subunit, suchthattheamino-terminal 7 termination. (seep.-) thatleadsto premature translational subunitis generated bya translational frameshifting mechanism

lEt-l

DNA Metabotism

Core (oeO) Core (oe0J

B clamp

tIGURE 25-10 DNA polymerase ttt. (a)Architecture of bacrerial DNA pofymerase lll Two coredomains,composedof subunits a, e, and0, are linkedby a five-subunit clamp-loading complex(alsoknown as the 7 complex)with the composition 12766'. They and z subunits are encodedby thesamegeneThe7 subunitis a shortened versionof the r subunit;r thuscontainsa domainidenticalto 7 alongwith an additional segmentthat interactswith the core polymerase. The othertwo subunits of DNA polymerase lll*,X andry'(notshown),alsobindto the clamp-loading complexTwop clampsinteract with thetwo-coresubassembly, eachclampa dimerof theB subunitThecomplexinteracts with the DnaBhelicase throughther subunits(b)TwoF subunits of E coll polymerase lll forma circularclampthatsurrounds the DNA.The clampslidesalongthe DNA molecule,increasing the processivity of the polymerase lll holoenzyme to greaterthan500,000nucleotides by preventing itsdissociation fromthe DNA.Theendview showsthetwo subunits as gray and light-blue ribbon structures B surroundinga space-filling modelof DNA. In the sideview,surfacecontourmodels (gray)surrounda stickrepresentation of theB subunits of a DNA double helix(lightand darkblue)(derivedfrompDB tD 2pOL)

along the DNA as replication proceeds. The B sliding clamp prevents the dissociation of DNA polymerase III from DNA, dramatically increasing processivity-to greater than 500,000 (Table 25-I).

DNA Replication Requires Many Enzymes andProtein tactors Replicationin E. coli, requfuesnot just a single DNA pollmerase but 20 or more different enz).rnesand proteins, eachperforrrunga specifictask. The entire complex has been termed the DNA replicase system or replisome. The enz}.'rnaticcomplexity of replication reflects the constraintsimposedby the structureof DNA and by the requirementsfor accuracy.The main classes of replication enz).rnesare consideredhere in terms of the problemsthey overcome. Accessto the DNA strands that are to act as templatesrequiresseparationofthe two parent strands.This is generally accomplishedby helicases, enzyrnesthat move along the DNA and separatethe strands, using

End view

Jide view

ft) chemical energy from ATP. Strand separation creates topologicalstressin the helical DNA structure (see Fig. 24-12), which is relieved by the action of topoisomerases. The separatedstrandsare stabilizedby DNAbinding proteins. As noted earlier, before DNA pol;rmerasescan begin slrrthesizingDNA, primers must be presenton the template-generally short segmentsof RNA synthesizedby enzyrnesknown as primases. Ultimately, the RNA primers are removedand replacedby DNA; in E col'i, this is one of the many functionsof DNA pol;.'rnerase I. After an RNA primer is removedand the gapis fllled in with DNA, a nick remainsin the DNA backbonein the form of a brokenphosphodiesterbond.These nicks are sealedby DNA ligases. All theseprocessesrequire coordinationand regulation,an interplaybest characterizedin the.B.cor?system.

Replication 25.1DNA Fttl

Replication ofthef. ro#Chromosome Froceeds in5tages The slnthesis of a DNA molecule can be divided into three stages: initiation, elongation, and termination, distinguished both by the reactions taking place and by the enzymes required. As you will find here and in the next two chapters, slnthesis of the major informationcontaining biological polymers-DNAs, RNAs, and proteins-can be understood in terms of these same three stages, with the stages of each pathway having unique characteristics. The events described below reflect information derived primarily from in vitro experiments using purified E col'i proteins, although the principles are highly conserved in all replication systems. Initiation The E colz replication origin, oriC, consists of 245 bp and contains DNA sequence elements that are highly conserved among bacterial replication origins. The general arrangement of the conserved sequences is illustrated in Figure 25-11. TWo types of sequences are of special interest: flve repeats of a 9 bp sequence (R sites) that serve as binding sites for the key initiator protein DnaA, and a region rich in A:T base pairs called the DNA unwinding element (DUE). There are three additional DnaA-binding sites (I sites),

Binding sites for DnaA protein, frve 9-bp sequences, consensussequence TT(A/DTNCACC

Tandem array of three 13-bp sequences, consensussequence GATCTNTTNTTTT

'----]-], DUE

and binding sites for the proteins IHF (integration host factor) and FIS (factor for inversion stimulation). These two proteinswere discoveredas required components of certain recombinationreactions describedlater in this chapter,and their namesreflect those roles. Another DNA-bindingprotein, HU (a histonelike bacterial protein originally dubbedfactor U), alsoparticipatesbut doesnot have a speciflcbinding site. At least l0 different enzymesor proteins (summarized in Table 25-3) participate in the initiation phase of replication.They open the DNA helix at the origin and establisha prepriming complex for subsequentreactions.The crucialcomponentin the initiationprocess is the DnaA protein, a member of the AAA* ATPase protein family (-ATPasesassociatedwith diverse celluMany AAA+ ATPases,including DnaA, lar cr,ctivities). form oligomers and hydrolyze ATP relatively slowly. This ATP hydrolysis acts as a switch mediating interconversionof the protein between two states.In the case of DnaA, the ATP-bound form is active and the ADP-boundform is inactive. Eight DnaA protein molecules,all in the ATP-bound state,assembleto form a helicalcomplexencompassing the R and I sites in ori,C (FiS. 25-12). DnaA has a higher affinity for the R sites than I sites, and binds R sites equallywell in its ATP- or ADP-boundform. The

v R1

vvVV

IHF

R5

F I G U R E 2 5 - 1A1r r a n g e m e n t osfe q u e n c e s i n t h e f . c o l i r e p l i c a t i o n origin, oriC. Consensus sequences(seep. 102) for key repeatedelementsare shown N represents any of the four nucleotidesThe horizontalarrowsindicatethe orientations of the nucleotidesequences (left{o-rightarrowdenotessequencein top strand;right-to-left, bottom

11

12

R2

FIS

R3

I3

R4

strand).FlsandlHFarebindingsitesforproteinsdescribedinthetext. R sitesare bound by DnaA. I sitesare additionalDnaA-bindingsites (with differentsequences), bound by DnaA only when the protein is with ATP complexed

DnaA-ATP

oriC

DUE

the DIIE region

FfGURt25-12 Model for initiationof replicationat the E coli origin, oriC. EightDnaA proteinmolecules, eachwith a boundATP,bind at .l the R and I sitesin the origin(seeFig.25-1 ). The DNA is wrapped a r o u n d t h i s c o m p l e x , w h i c h f o r m s a r i g h t - h a n d e ds h t reulci ct ua rl e . T h e A:T-rich DUEregionis denatured asa resultof thestrainimpartedby the adjacentDnaAbinding Formation of the helicalDnaAcomplexis

DnaC+ADP+P1

facilitatedby the proteinsHU, lHF,and FlS,which are not shownhere becausetheir detailedstructuralroles have not yet been defined. Hexamers of the DnaB proteinbind to eachstrand,with the aid of D n a C p r o t e iT nheDnaBhelicaseactivityfurtherunwindstheDNAin for primingand DNA synthesis. preparation

tbt.l

DNA Metabotism

I sites, which bind only the ATP-bound DnaA, allow discrimination between the active and inactive forms of DnaA. The tight right-handed wrapping of the DNA around this complex introduces an effective positive supercoil (see Chapter 24). The associated strain in the nearby DNA leads to denaturation in the A:T-rich DUE region. The complex formed at the replication origin also includes several DNA-binding proteins-Hu, IHR and FIS-that facilitate DNA bending. The DnaC protein, another AAA+ ATPase, then Ioads the DnaB protein onto the separated DNA strands in the denatured region. A hexamer of DnaC, each subunit bound to ATP, forms a tight complex with the hexameric, ring-shaped DnaB helicase. This DnaC-DnaB interaction opens the DnaB ring, the process being aided by a further interaction between DnaB and DnaA. Thro of the ring-shaped DnaB hexamers are loaded in the DUE, one onto each DNA strand. The ATP bound to DnaC is hydrolyzed, releasing the DnaC and leaving the DnaB bound to the DNA. Loading of the DnaB helicase is the key step in replication initiation. As a replicative helicase, DnaB migrates along the single-stranded DNA in the 5'-+3' direction, unwinding the DNA as it travels. The DnaB helicases loaded onto the two DNA strands thus travel in opposite directions, creating two potential replication forks. All other proteins at the replication fork are linked directly or indirectly to DnaB. The DNA pol1.'rneraseIII holoenzyme is Iinked through the r subunits; additional DnaB interactions are described below. As replication begins and the DNA strands are separated at the fork, many molecules of single-stranded DNA-binding protein (SSB) bind to and stabilize the separated strands, and DNA gyrase (DNA topoisomerase II) relieves the topological stress induced ahead of the fork by the unwinding reaction.

Protein DnaA protein

M,

Numberof subunits

52,000

Initiation is the only phaseof DNA replicationthat is known to be regulated,and it is regulated such that replication occurs only once in each cell cycle. The mechanismof regulationis not yet entirely understood, but genetic and biochemicalstudies have provided insightsinto severalseparateregulatory mechanisms. Once DNA polymeraseIII has been loaded onto the DNA, alongwith the B subunits (signalingcompletion of the initiation phase),the protein Hda binds to the B subunits and interacts with DnaA to stimulate hydrolysisof its bound ATP.Hda is yet another AAA+ ATPaseclosely related to DnaA (its name is derived from homologousto DnaA).This ATP hydrolysisleads to disassemblyof the DnaA complex at the origin. Slowreleaseof ADP by DnaA and rebindingof ATP cycles the protein between its inactive (with bound ADP) and active (with bound ATP) forms on a time scaleof 20 to 40 minutes. The timing of replication initiation is affected by DNA methylation and interactionswith the bacterial plasmamembrane.The ori,C DNA is methylated by the Dam methylase(Table25-3), which methylatesthe Nt position of adeninewithin the palindromic sequence (5')GATC. (Dam is not a biochemical expletive; it standsfor DNA adenine nzethylation.)The ori,C region of E coli,is highly enrichedin GATCsequences-it has 1l of them inilts245bp, whereasthe averagefrequency of GATCin the E. coLi,chromosomeas a whole is 1 in 256bp. Immediatelyafter replication,the DNA is hemimethylated: the parent strands have methylated ori,C sequencesbut the newly slnthesizedstrandsdo not. The hemimethylatedorzC sequencesare now sequestered by interaction with the plasmamembrane (the mechanism is unknown) and by the binding of the protein

Funetion Recognizesori sequence;opensduplex at specific cifaq

DnaB protein (helicase) DnaCprotein HU FIS IHF Primase(DnaGprotein) Single-strandedDNA-binding protein (SSB) DNA gy'rase(DNA topoisomeraseII) Dam methylase *Subunits in thesecasesareidenticat.

300,000 174,000 19,000 22,500 22,000 60,000 75,600 400,000 32,000

in

nrioin

6*

Unwinds DNA

6*

Requiredfor DnaB binding at origin

Z

Histonelikeprotein; DNA-bindingprotein; stimulates initiation

2+

DNA-bindingprotein; stimulatesinitiation

2

DNA-bindingprotein; stimulatesinitiation

1

S5mthesizes RNA primers

4*

Binds single-strandedDNA

4

Relievestorsional strain generatedby DNA unwinding Methylates(5')GATC sequencesat ori,C

I

Replication 2s.1DNA lbtl SeqA.After a time, ori,C is releasedfrom the plasma membrane,SeqAdissociates,and the DNA must be fully methylated by Dam methylasebefore it can again bind DnaA and initiate a new round of replication. Elongation The elongationphase of replication includes two distinct but related operations: Ieading strand synthesis and lagging strand s;erthesis.Several enzyrnesat the replicationfork are important to the slrrthesis of both strands.Parent DNA is first unwound by DNA helicases,and the resultingtopologicalstressis relievedby topoisomerases. Eachseparatedstrandis then stabrlizedby SSB.From this point, synthesisof leading and laggingstrandsis sharply different. Leading strand synthesis,the more straightforward of the two, beginswith the synthesisby primase (DnaG protein) of a short (10 to 60 nucleotide) RNA primer at the replicationorigrn.DnaGinteractswith DnaBhelicase to carry out this reaction,and the primer is slnthesizedin the direction oppositeto that in which the DnaBhelicase is moving.In effect, the DnaB helicasemovesalong the strandthat becomesthe laggingstrandin DNA synthesis; however,the first primer laid down in the first DnaGDnaB interaction serves to prime leading strand DNA synthesisin the oppositedirection.DeoxS,ribonucleotides are addedto this primer by a DNA polymeraseIII complex linked to the DnaBhelicasetetheredto the opposite

DNA topoisomeraseII (DNA gyrase)

DNA strand.Leadingstrandslrrthesisthen proceedscontinuously,keepingpacewith the unwindingof DNA at the replicationfork. Lagging strand s;mthesis,as we have noted, is accomplishedin short Okazakifragments (Fig. 25-l3a). First, an RNA primer is symthesizedby primase and, as III binds to in leadingstrand qmthesis,DNA poly.merase primer and adds deoxyribonucleotides(Fig. the RNA 25-13b). On this level, the synthesisof each Okazaki fragment seemsstraightforward,but the reality is quite complex. The complexity lies in the coord'inat'ion of Ieading and lagging strand synthesis.Both strands are produced by a single as;..mmetricDNA polf.rneraseIII dimer; this is accomplishedby looping the DNA of the Iaggingstrand as shown in Figure 25-14, bringing together the two points of polSrmerization. The synthesisof Okazakifragments on the lagging strand entails some elegant enzyrnatic choreography. DnaB helcase and DnaGprimaseconstitutea functional unit within the replication complex, the primosome. DNA polymeraseIII usesone set of its core subunits(the the leading strand concore pol5rmerase)to sSmthesize tinuously, while the other set of core subunits cycles from one Okazakifragmentto the next on the loopedlagging strand. DnaB helicase,bor-ndin front of DNA polymeraseIII, unwindsthe DNA at the replicationfork (Fig. 25-I4a) as it travels along the lagging strand template

Leading strand synthesis (DNA polymerase III) J

5', t

fork movement

J

5', SSB RNA prrmer

RNA primer from previous Okazaki fragment

Lagging strand synthesis (DNA polymerase III)

FIGURE 25-13 Synthesis of Okazakifragments. (a) At intervals,primasesynthesizes an RNA primerfor a new Okazakifragment.Notethat if we considerthe two templatestrandsas lying formally sideby side,laggingstrandsynthesis proceedsin the oppositedirectionfrom fork movement.(b) Eachprimeris extendedby DNA continues polymeraselll. (c) DNA synthesis untilthe fragmentextendsas far as the primer of the previouslyadded Okazaki fragment A nearthe replication new primeris synthesized fork to beginthe processagain.

Metaborism Lbtd DNA

ClampJoading complex with open B sliding clamp Lagging strand RNA primer of previous Okazaki fragment

in the 5'-+3' direction. DnaG primase occasionallyassociates wrth DnaB helicase and slmthesizes a short RNA primer (Fig. 25-14b). A newB sliding clamp is then positioned at the primer by the clampJoading complex of DNA pol}rmerase III (Fig. 25-14c). When syr-rthesisof an Okazaki fragment has been completed, replication halts, and the core subunits of DNA pol;rmerase III dissociate from their B sliding clamp (and from the completed Okazaki fragment) and associate with the new clamp (Fig. 25-14d, e). This initiates synthesis of a new Okazaki fragment. As noted earlier, the entire complex responsible for coordinated DNA synthesis at a replica-

FIGURE 25-14 DNA synthesis on the leadingand laggingstrands. Eventsat the replicationfork are coordinatedby a single DNA polymerase lll dimer,in an integrated complexwith DnaB helicase.This figureshowsthe replicationprocessalreadyunderway (parts(a; through(e) are discussedin the text).The Iaggingstrand is looped so that DNA synthesisproceedssteadilyon both the Ieadingand laggingstrandtemplatesat the sametime. Redarrows indicatethe 3'end of the two new strandsand the directionof DNA synthesis. The heavyblack arrowsshow the directionof movementof the parentDNA throughthe complex.An Okazaki fragmentis beingsynthesized on the laggingstrand.The subunit colorsand the functionsof the clamp-loading complexare explainedin Figure25-15 ONn Synthesis $

tion fork is known as the replisome.The proteins acting at the replicationfork are surunarizedinTable2S-4. The clampJoadingcomplex of DNA poly.rnerase III, consistingof parts of the two r subunits along with the 7, 6, and 6' subunits,is alsoan AAA* MPase This complex binds to ATP and to the new B sliding clamp. The binding imparts strain on the dimeric clamp, openingup the ring at one subunit interface (Fig. 25-f5). The newly primed lagging strand is slipped into the ring through the resulting break. The clamp loader then hydrolyzesATP,releasingthe B sliding clamp and allowing it to closearoundthe DNA.

25.1DNA Replication lbtr]

hotein

Numberof subunits

M"

SSB DnaB protein (helicase) Primase(DnaGprotein) DNA polymeraseIII DNA polymeraseI DNA ligase DNA gy'ase (DNA topoisomeraseII)

75,600 300,000 60,000 791,500 103,000 74,000 400,000

Funetion

o

Binding to single-strandedDNA DNA unwinding;primosomeconstituent

1

RNA primer slnthesis; primosomeconstituent

A

1

New strand elongation Filling of gaps;excisionof primers

1

Ligation

A

Supercoiling

t7

Source:ModifiedfromKornberg, A. (79821Supplement to DNAReplication, NewYork. TableS11-2,W.H.Freeman andCompany,

Clamp loader

B clamp

ATP

-+

ArS: +3P1

The replisome promotes rapid DNA synthesis, adding-1,000 nucleotides/s to eachstrand(leadingand lagging). Once an Okazaki fragment has been completed, its RNA primer is removed and replaced with DNA by DNA polymeraseI, and the remaining nick is sealedby DNA ligase(Fig. 25-16). DNA ligase cata\yzesthe formation of a phosphodiester bond between a 3' hydroxyl at the end of one Lagging strand

5'

J

5',*,'-***-*,."*$" 4 I

I

Nick

NMPs dNTPs

DNA polvnrcrasc I

Ntr c.Nj\$NsN!ilr.

ATP (or NAD*) DNA lJgasc

AMP + PP, (or NMN)

FIGURE 25-16 Finalstepsin the synthesis of laggingstrandsegments. RNA primersin the laggingstrandare removedby the 5'+3'exonucleaseactivityof DNA polymerase I and arereplacedwith DNA by the sameenzyme.Theremaining nickis sealedby DNA ligase. Theroleof ATPor NAD- is shownin Fisure25-17.

FIGURE 25-15 The DNA polymeraselll clamp loader.The five subunitsof the clamp-loading complexare the 7, 6, and 6' subunitsand the amino-terminal domain of each r subunit(see Fig.25-10).Thecomplexbindsto threemolecules of ATPand to a dimericB clamp.This binding forcesthe B clamp open at one of its two subunit interfaces. Hydrolysisof the boundATPallowsthe clamp to closeagainaroundthe DNA. B

DNA strand and a 5' phosphateat the end of another strand. The phosphatemust be activated by adenylylation. DNA ligasesisolated from viruses and eukaryotes use ATP for this purpose.DNA ligasesfrom bacteria are unusual in that many use NAD*-a cofactor that normally functions in hydride transfer reactions (see Fig. l3-24)-as the source of the AMP activating group (FiS. 25-17) DNA ligase is another enzyrneof DNA metabolismthat has becomean important reagentin recombinantDNA experiments(seeFig. 9-1). Termination Eventually,the two replication forks of the circular.E. col'i chromosomemeet at a terminus region containingmultiple copies of a 20 bp sequence called Ter (Fig. 25-18). The Ter sequencesare arrangedon the chromosometo create a trap that a replicationfork can enter but cannotleave.The Ter sequences function as binding sites for the protein Tirs (terminus utilization substance).The Tl-rs-Tercomplex can arrest a replication fork from only one direction. Only one Ttrs-Tercomplex functions per replication cycle-the complexfirst encounteredby either replication fork. Given that opposingreplication forks generally halt when they collide, Ter sequenceswould not seemto be essential,but they may preventoverreplication by one fork in the event that the other is delayedor halted by an encounter with DNA damage or some other obstacle. So, when either replicationfork encountersa functional Tus-Ter complex. it halts; the other fork halts

[ttt]

DNA Metaborism

? n-O-i,-O-l @ Adenylylationof DNA ligase

niU*"]]

ea""i*l

O^;NiAD-'C-:'^tiMIti AMP from ATP (R - DP.r i'

ppi (from ATp) or NMN (from NAD+)

o /-----\

+

QnzymefNH2

I

--

FIGURE 25-17 Mechanismof the DNA ligasereaction.In eachof the threesteps,one phosphodiester bond is formed at the expenseof another.Steps@ and@ leadto activation of the 5' phosphatein the nick.An AMP group is transferred firstto a Lysresidueon the enzymeand then to the 5' phosphatein the nick. In step@, the 3'-hydroxylgroup atracks this phosphate AMP,producinga phosphodiand displaces esterbondto sealthe nick. In the E.coll DNA ligasereaction, AMP is derivedfrom NAD*. The DNA ligasesisolatedfrom some viral and eukaryoticsourcesuse ATP ratherthan NAD*, and they releasepyrophosphate ratherthan nicoti(NMN)in step@ namidemononucleotide

--l

@ Displacement of AMP seals nick

P O lRibosef Adenine

o

-o

Enzyme-AMP

o P-o I

o-

@ Activation of 5' phosphatein nick

"*--*=I.]=.-='=-.::--"----*.:--..1-T.

'',oHO.O

o DNA ligase

\./ P ,/\

oPo I

o

o-o

SealedDNA

Nick in DNA

P -o"o@@

Clockwise fork

FfGURE 25-18 Terminationof chromosomereplication in E. coli. (TerAthroughTerDarepositionedon the chromoTheTersequences somein two clusterswith oppositeorientations.

FIGURE 25-19 Role of topoisomerases in replication termination. Replicationof the DNA separatingopposingreplicationforks leaves joined as catenanes, the completedchromosomes or topologicallyinterlinkedcirclesThe circlesare not covalentlylinked,but because theyare interwoundand eachis covalentlyclosed,theycannotbe separated-except by the action of topoisomerases. In E. col/, a type ll topoisomerase knownas DNA topoisomerase lV playsthe primaryrole in the separation of catenatedchromosomes, transientlybreakingboth DNA strands of onechromosome andallowingthe otherchromosome to passthroughthe break.

Counterclockwise fork

Clockwise fork

Catenated chromosomes I)NA topoisomerase IV

Separated chromosomes

lbrl

2 5 . 1D N A Reolication

when it meets the first (arrested) fork. The final few hundred basepairs of DNA betweenthese large protein complexesare then replicated Cbyan as yet unknown mechanism), completing two topologically interlinked (Fig. 25-19). DNA (catenated)circular chromosomes circleslinked in this way are known as catenanes. Separationof the catenatedcirclesinE. coLi,requirestopoisomeraseIV (a type II topoisomerase). The separated chromosomesthen segregateinto daughter cells at cell division.The termtnalphaseof replicationof other circular chromosomes,including many of the DNA viruses that infect eukaryoticcells,is similar.

Replication inEukaryotic tellslsBoth (omplex Similar andMore The DNA moleculesin eukaryoticcellsare considerably Iargerthan thosein bacteriaand are organizedinto complex nucleoproteinstructures(chromatin;p. 962). The essentialfeaturesof DNA replicationare the samein eukaryotes and bacteria, and many of the protein complexesare functionally and structurally conserved. However,eukaryoticreplicationis regulatedand coordinated with the cell cycle, introducingsome additional complexities. Origins of replication have a well-characterized structurein someIower eukaryotes,but they are much lessdefinedin highereukaryotes.In vertebrates,a variety of A:l-rich sequencesmay be usedfor replication initiation, and the sites may vary from one cell division to the next. Yeast(SaccharoTrlACes cereu,is,iae)has defined replication origins calledautonomouslyreplicating sequences(ARS), or replicators. Yeast replicators span -150 bp and containseveralessential,conserved sequences. About 400 replicatorsare distributedamong the 16 chromosomes of the haploidyeastgenome. Regulationensuresthat all cellularDNA is replicated onceper cell cycle.Much of this regulationinvolvesproteins called cyclins and the cyclin-dependentkinases (CDKs)with which they form complexes(p. 469).The cyclins are rapidly destroyedby ubiquitin-dependentproteolysisat the end of the M phase(mitosis),andthe absence of cyclins allows the establishmentof pre-replicative complexes (pre-RCs) on replicationinitiation sites.In rapidly growing cells,the pre-RCforms at the end of M phase.In slow-growingcells,it doesnot form until the end of Gl Formationof the pre-RCrendersthe cell competent for replication,an eventsometimescalledlicensing. As in bacteria, the key event in the initiation of replicationin all eukaryotesis the loadingof the replicative helicase,a heterohexamericcomplexof minichromosome maintenance (MCM) proteins (MCM2 to MCMT).The ring-shapedMCM2-7helicase,functioning much like the bacterial DnaB helicase,is loaded onto the DNA by another six-proteincomplex called ORC (origin recognition complex) (Fig. 25-2O). ORC has flve AAA+ MPase domainsamongits subunits and is functionally analogousto the bacterial DnaA. TWo other proteins, CDC6 (cell division cycle) and CDT1

FIGURE 25-20 Assemblyof a pre-replicative complexat a eukaryotic replicationorigin.The initiationsite (origin)is bound by ORC,CDC6, promoteloadandCDT1. Theseproteins,manyof themAAA+ ATPases, helicase,MCM2-7,in a reactionthat is analogous ing of the replicative to the loadingof the bacterialDnaBhelicaseby DnaCprotein.Loading of the MCM helicasecomplexonto the DNA formsthe pre-replicative complex,or pre-RC,and is the key stepin the initiationof replication.

(CDCl0-dependenttranscript 1), are also required to Ioad the MCM2-7 complex, and the yeast CDC6 is another AAA+ ATPase. Commitment to replication requires the synthesis and activity of S-phasecyclin-CDK complexes(such as the cyclin E-CDK2 complex;seeFig. 1245) and CDCTDBF4.Both typesof complexeshelp to activatereplication by binding to and phosphorylatingseveralproteins in the pre-RC. Other cyclins and CDKs function to inhibit the formation of more pre-RO complexesonce replicationhas been initiated. For example,CDK2binds to cyclin A as cyclin E levels decline during S phase,inhibiting CDK2and preventingthe Iicensingof additional pre-RCcomplexes.

lb"l

DNA Metabolism

The rate of movementof the replication fork in eukaryotes(-50 nucleotides/s)is only one-twentieththat observedin E. co\i,.At this rate, replication of an averagehuman chromosomeproceedingfrom a singleorigin would take more than 500 hours.Replicationof human chromosomesin fact proceeds bidirectionally from many origins,spaced30 to 300 kbp apart. Eukaryotic chromosomesare almost alwaysmuch larger than bacterial chromosomes,so multiple origins are probably a universalfeature of eukaryotic cells. Like bacteria,eukaryoteshaveseveraltypesof DNA polymerases. Somehavebeenlinked to particularfunctions,suchasthe replicationof mitochondrialDNA. The replication of nuclear chromosomesinvolvesDNA polymerasea, in associationwith DNA polymerase5. DNA pol5rmerase c is typically a multisubunit enz;rrnewith similar structure and properties in all eukaryotic cells. One subunit has a primaseactivity, and the largest subunit (M. -180,000) containsthe pol}.'rnerization activity. However,this polymerasehas no proofreading3'-+5' exonucleaseactivity, making it unsuitable for highfidelity DNA replication. DNA pol;.'rnerase a is believed to functiononly in the synthesisof short primers(either RNA or DNA) for Okazakifragments on the lagging strand.Theseprimers are then extendedby the multisubunit DNA polymerase 5. This enzymeis associated with and stimulatedby proliferating cell nuclear antigen (PCNA;M,29,000), a protein found in largeamountsin the nuclei of proliferatingcells.The three-dimensional structure of PCNA is remarkablysimilar to that of the p subunitof E co|i,DNA poly.rnerase III (Fig. 25-10b),although primary sequencehomology is not evident. PCNAhas a function analogousto that of the B subunit, forming a circular clamp that greatly enhancesthe processivityof the pol;rmerase.DNA polymerase6 has a 3'-+5' proofreadingexonucleaseactivity and seemsto carry out both leading and laggingstrand synthesisin a complex comparableto the dimeric bacterial DNA polymeraseIII. Yet another polymerase,DNA polymerase e, replacesDNA polynnerase 6 in somesituations,suchas in DNA repair. DNA pol}..rnerase s may alsofunction at the replicationfork, perhapsplayinga role analogousto that of the bacterialDNA polymeraseI, removingthe primers of Okazakifragmentson the laggingstrand. TWoother protein complexesalso function in eukaryotic DNA replication.RPA (replication protein A) is a eukaryotic single-strandedDNA-binding protein, equivalentin function to the E. coli, SSB protein. RFC (replication factor C) is a clamploaderfor PCNAand facilitatesthe assemblyof active replicationcomplexes. The subunits of the RFC complex have significant sequencesimilarity to the subunitsof the bacterial clamploading (7) complex. The termination of replication on linear eukaryotic chromosomesinvolvesthe synthesisof special structures called telomeres at the ends of each chromosome,as discussedin the next chapter.

ViralDNAPolymerases Provide Targets forAntiviralTherapy Many DNA viruses encode their own DNA polymerases,and someof thesehavebecometargets for pharmaceuticals.For example,the DNA pol}rmerase of the herpes simplex virus is inhibited by acyclovir, a compound developedby Gertrude Elion (p. 894). Acyclovir consistsof guanineattachedto an incomplete ribosering. o

Acyclovir

It is phosphorylatedby a virally encodedth;.rnidine kinase;acyclovirbinds to this viral enzymewith an affinity 200-fold greater than its binding to the cellular thyrnidine kinase. This ensuresthat phosphorylationoccurs mainly in virus-infected cells. Cellular kinases convert the resulting acyclo-GMPto acyclo-GTP,which is both an inhibitor and a substrate of DNA polymerases;acyclo-GTP competitivelyinhibits the herpes DNA polymerase more strongly than cellular DNA polymerases. Becauseit lacksa 3' hydroxyl,acyclo-GTPalsoactsas a chain terminator when incorporatedinto DNA. Thus viral replication is inhibited at severalsteps.r

SUMMAR 2Y 5 . 1 D N AR e p l i c a t i o n r

Replicationof DNA occurswith very high fldelity and at a designatedtime in the cell cycle. Replicationis semiconservative,eachstrand acting as template for a new daughter strand. It is carried out in three identiflablephases:initiation, elongation,and termination. The processstarts at a singleorigin in bacteria and usually proceedsbidirectionally.

r

DNA is sS.nthesized in the 5'-+3' directionby DNA poll'rnerases.At the replication fork, the leading strand is symthesizedcontinuouslyin the same direction as replication fork movement;the laggng strand is symthesizeddiscontinuouslyas Okazaki fragments,which are subsequentlyligated.

r

The fidelity of DNA replicationis maintainedby (1) baseselectionby the polymerase,(2) a 3'-->5' proofreadingexonucleaseactivity that is part of most DNA polymerases,and (3) specificrepair systemsfor mismatchesleft behind after replication.

r

Most cellshaveseveralDNA polymerases.lnE.coli,, DNA polymeraseIII is the primary replicationenzfne. DNA pol}.'rnerase I is responsiblefor specialfunctions dudng replication,recombination,and repair.

r

The separateinitiation, elongation,and termination phasesof DNA replication involve an array of enz),rnesand protein factors, many belongingto the AAA* MPase family.

25.2 DNARepair

r

F"l

The replication proteins in bacteriaare organized into replication factories,in which template DNA is spooledthroughtwo replisomestetheredto the bacterialplasmamembrane.

25.2DNA Repair Most cells have only one or two sets of genomicDNA. Damagedproteinsand RNA moleculescan be quickly replacedby usinginformationencodedin the DNA,but DNA moleculesthemselvesare irreplaceable.Maintaning the integrity of the hformation in DNA is a cellular imperative, supported by an elaborateset of DNA repair systems. DNA canbecomedamagedby a varietyof processes, some spontaneous,others catalyzedby environmental agents (Chapter8). Replicationitself canvery occasionallydamage the information content in DNA when errors introducemismatchedbasepairs (suchas G pairedwith T). The chemrstryof DNA damageis diverseand complex. The cellular responseto this damageincludes a wide range of enz;,rnatic systems that catalyzesome of the most interesting chemical transformationsin DNA metabolism.We first examinethe effectsof alterationsin DNA sequenceand then considerspeci-fi.c repair systems.

Mutations AreLinked toCancer The best way to illustrate the importance of DNA repairis to considerthe effectsof tntrepa'ired DNA damage(a lesion).The most seriousoutcomeis a change in the basesequenceof the DNA, which, if replicatedand transmittedto future generationsof cells,becomesperrnanent. A permanentchangein the nucleotidesequenceof DNA is called a mutation. Mutations can involve the replacementof onebasepair with another(substitutionmutation) or the additionor deletionof oneor morebasepairs (insertion or deletionmutations).If the mutation alfects nonessentialDNA or if it hasa neffible effect on the function of a gene,it is known as a silent mutation. Rarely,a mutation conferssomebiologicaladvantage.Most nonsilent mutations,however,are neutralor deleterious. In mammalsthere is a strong correlation between the accumulationof mutationsand cancer.A simpletest developedby Bruce Ames measuresthe potential of a given chemical compound to promote certain easily detectedmutationsin a specializedbacterialstrain (Fig. 25-21). Few of the chemicalsthat we encounterin daily Iife scoreas mutagensin this test. However,of the compoundskno'omto be carcinogenicfrom extensiveanimal trials, more than 90% are also found to be mutagenicin the Ames test. Becauseof this strong correlationbetween mutagenesisand carcinogenesis, the Ames test for bacterialmutagensis widely usedas a rapid and inexpensivescreenfor potentialhumancarcinogens. The genome of a t;,pical mammaliancell accumuIatesmany thousandsof lesionsduring a24-hot;rrperiod. However,asa resultof DNA repair,fewerthan I in 1,000 becomea mutation. DNA is a relatively stablemolecule,

(d) 25-21 Amestestfor carcinogens, basedon their mutagenicity. FIGURE typhimuriumhavinga mutationthat inactivates A strainol Salmonella an enzymeof the histidinebiosyntheticpathwayis plated on a medium.Few cells grow. (a) The few small coloniesof histidine-free S. typhimuriumthat do grow on a histidineJreemediumcarryspontapathwayto that permitthe histidinebiosynthetic neousback-mutations operate.Three identical nutrientplates (b), (c), and (d) have been inoculatedwith an equal numberof cells.Eachplatethen receives lowerconcentrations of a a diskof filterpapercontainingprogressively and the rateof back-mutation mutagen.Themutagengreatlyincreases Theclearareasaroundthe filterpaper hencethe numberof colonies. of mutagenis so high that it is lethal indicatewherethe concentration to the cells As the mutagendiffusesaway from the filter paper,it is that promote back-mutation. diluted to sublethalconcentrations of their effecton mutation compared on the basis Mutagenscan be rate.Becausemany compoundsundergoa varietyof chemicaltransformationsafter enteringcells, compoundsare sometimestested for mutagenicityafterfirst incubatingthem with a liver extract.Some havebeenfoundto be mutageniconly afterthis treatment. substances

but in the absenceof repair systems,the cumulative effect of many infrequent but damagingreactionswould make life impossible.r

Systems DNA Repair Multiple All(ellsHave The numberand diversityof repair systernsreflect both the impor[ance of DNA repair to cell sl-rviva]and the diverse sourcesof DNAdamage(Thble25-5). Somecommont),pes of lesions,suchaspy'imidinedimers(seeFig.8-31), canbe repaired by severaldistinct systems.Many DNA repair processesalso seemto be extraordinarily inefficient energetically-an exceptionto the pattern obselvedin the vast majority of metabolicpathways,where everyAIP is generally accountedfor and usedoptimally.Whenthe integriff of the geneticinformationis at stake,the amount of chemical enersi investedin a repair processseemsalmostirrelevant. DNA repair is possiblelargelybecausethe DNA moleculeconsistsof two complementarystrands.DNA damage in one strand can be removed and accurately replacedby usingthe undamagedcomplementarystrand

lEr*l

DNA Metabotism /^1Lr l

Enzymes/proteins

Tlpe ofdamage

ATC D

o

o

Mismatch repair

5'

TAG I

cHc

Dam methylase MutH, MutL, MutS proteins DNA helicaseII SSB DNA Pol5..rnerase III Exonuclease I ExonucleaseVII RecJnuclease ExonucleaseX DNA ligase

I replication

cHs ATC

5',

.''' crAG

"

Base-excision repair DNA glycosylases AP endonucleases DNA polymeraseI DNA ligase

Abnormalbases(uracil, hypoxanthine,xanthine); alkylatedbases;in some other organisms, pyrimidine dimers

GATC

o

r1

I I )

O

TAG

.,dm*.

Nucleotide-excision repair ABC excinuclease DNA pol5'rnerase I DNA ligase

+:

a,

For a short period following replication, the template strand is methylated and the new strand is not.

DNA lesionsthat cause large structural changes (e.g.,py'imidine dimers)

Direct repair

cHs

DNA photolyases

Pynmrdhe dimers

O6-Methylguanine-DNA methyltransferase

06-Methylguanine

AlkB protein

1-Methylguanine, 3-methylcytosine

o

as a template.We considerhere the principal types of repair systems,beguuLingwith those that repair the rare nucleotidemismatchesthat are left behindby replication. Mismateh Repair Correction of the rare mismatches left after replicationtn.E coli,trnprovesthe overallfldelity of repJicationby an additionalfactor of 102to 103.The mismatchesare nearly alwayscorrectedto reflect the informationin the old (template) strand,so the repair system must somehowdiscriminatebetween the template and the newly syrrthesizedstrand.The cell accomplishes this by taggrngthe templateDNA with methyl groupsto distinguishit from newly synthesizedstrands.The mismatch repair systemof E. coli,rncludesat least 12protein components(Table 25-5) that function either in strand discnmrnationor in the repair processitself. The strand discriminationmechanismhas not been worked out for most bacteriaor eukaryotes,but is well nnderstoodfor E co\i,and somecloselyrelatedbacterial species.In these bacteria,strand discriminationis basedon the action of Dam methylase,which, asyou will recall, methylatesDNA at the Af position of all adenines within (5')GATCsequences. Immediatelyafter passage ofthe replicationfork, there is a shortperiod (a few seconds or minutes) during which the template strand is methylated but the newly synthesizedstrand is not (Fig. 25-22). The transient unmethylated state of

t;

5', Ir

ATC

jL"

3', 5'

CT Hemimethylated DNA

5',

GATC

3',

3,]..:.c-T"iG.'"'.'-.]]'.-,.:...|..]'..1'.'.".l I

{[I,g]

,0Hq E', "

GATC

3', F l

C TAG CI{^ ---;J

cHs s', 3'

GATC

crAG I cHa

3', 5'

FIGURE 25-22 Methylationand mismatchrepair.Methylationof DNA strandscan serveto distinguishparent(template)strandsfrom newly synthesized strandsin E coli DNA, a functionthat is critical to mismatchrepair(seeFig 25-23).The methylationoccursat the N6 of adeninesin (5')CATCsequences. Thissequence is a palindrome(see Fig.B-18), presentin oppositeorientations on thetwo strands.

I-I

RepairLeesl 25.2DNA GATC sequences in the newly slmthesized strand permits the new strand to be distinguished from the template strand. Replication mismatches in the vicinity of a hemimethyiated GATC sequence are then repaired according to the information in the methylated parent (template) strand. Tests in vitro show that if both strands are methylated at a GATC sequence, few mismatches are repaired; if neither strand is methylated, repair occurs but does not favor either strand. The cell's methyl-directed mismatch repair system efflciently repairs mismatches up to 1,000 bp from a hemimethylated GATC sequence. How is the mismatch correction process directed by relatively distant GATC sequences?A mechanism is illustrated in Figure 25-23. MutL protein forms a complex with MutS protein, and the complex binds to all mismatched base pairs (except C-C). MutH protein binds to MutL and to GATC sequences encountered by the MutL-MutS complex DNA on both sides of the mismatch is threaded through the MutL-MutS complex, creating a DNA loop; simultaneous movement of both legs of the Ioop through the complex is equivalent to the complex moving in both directions at once along the DNA. MutH has a site-specific endonuclease activity that is inactive until the complex encounters a hemimethylated GATC sequence. At this site, MutH catalyzes cleavage of the unmethylated strand on the 5' side of the G in GATC, which marks the strand for repair. Further steps in the pathway depend on where the mismatch is located relative to this cleavage site (Fig. 25-24). When the mismatch is on the 5' side of the cleavage site (Fig. 25-24, right side), the unmethylated strand is unwound and degraded in the 3'-+5' direction from the cleavage site through the mismatch, and this segment is replaced with new DNA. This process requires the combined action of DNA helicase II, SSB, exonuclease I or exonuclease X (both of which degrade strands of DNA in the 3'-+5' direction), DNA polymerase III, and DNA ligase. The pathway for repair of mismatches on the 3' side of the cleavage site is simiiar (Fig 25-24,left), except that the exonuclease is either exonuclease VII (which degrades singlestranded DNA in the 5'-+3' or 3'-+5' direction) or RecJ nuclease (which degrades single-stranded DNA in the 5'-+3' direction). Mismatch repair is a particularly expensive process for E colz in terms of energy expended. The mismatch may be 1,000 bp or more from the GATC sequence.The degradation and replacement of a strand segment of this length require an enormous investment in activated deoxynucleotide precursors to repair a szngle mismatched base. This again underscores the importance to the cell of genomic integrity. All eukaryotic cells have several proteins structurally and functionally analogous to the bacterial MutS and MutL (but not MutH) proteins. Alterations in human genes encoding proteins of this type produce some of the most common inherited cancer-susceptibility

,rstril,,$.' I GATC

cHs

CTAG

Mismatched base pair ''

K

D

3

\ raa

tffi, t

mis25-23 A modelfor the early stepsof methyl'directed FIGURE match repair.The proteinsinvolvedin this processin E. coli have been purified (see Table 25-5). Recognitionof the sequence functionsof the MutH (5')CATCand of the mismatcharespecialized The MutL proteinformsa complex and MutSproteins,respectively. with MutSat the mismatch.DNA is threadedthroughthis complex along in bothdirections suchthatthe complexmovessimultaneously at a hemimethyprotein bound MutH a it encounters the DNA until strandon the tatedCATCsequenceMutH cleavesthe unmethylated 5 ' s i d e o f t h e C i n t h i ss e q u e n c eA. c o m p l e xc o n s i s t i nogf D N A h e then degradesthe unlicasell and one of severalexonucleases methylatedDNA strandfrom that point towardthe mismatch(see Fie.25-24).

[n,

DNA Meraborism

cHr

cHs 5' 3',

ADP+Pte'

cHa

cHr

cHg I

ATP ADP+P,

(tr

CHT

CH3

au

un3

cHa

cHs

s,

FIGURE 25-24 Completingmethyl-directed mismatchrepair.The combinedactionof DNA helicasell, SSB,and one of four different exonucleases removesa segmentof the new strandbetweenthe MutH cleavage siteand a pointjust beyondthe mismatch. Theexonuctease

that is useddependson the locationof the cleavagesite relativeto the mismatch,as shown by the alternativepathwayshere.The resulting gap is filled in (dashedline)by DNA polymerase lll, and the nick is sealedby DNA ligase(notshown).

slmdromes(seeBox 25-I , p. 1003), further demonstrating the value to the organismof DNA repair systems. The main MutS homologsin most eukaryotes,from yeast to humans,are MSH2 (MutS foomolog2), MSH3, and MSH6.Heterodimersof MSH2and MSH6generally bind to singlebase-pairmismatches,and bind lesswell to slightly longer mispairedloops. In many organisms the longer mismatches(2 to 6 bp) may be bound instead by a heterodimerof MSH2 and MSH3, or are bound by both types of heterodimersin tandem. Homologsof MutL, predominantlya heterodimerof MLHI (MutL izomolog1) and PMS1 (post-meiotic segregation), bind to and stabilizethe MSH complexes.Many details of the subsequentevents in eukaryotic mismatch repair remainto be worked out. In particular,we do not know the mechanismby which newly synthesizedDNA strandsare identified,althoughresearchhas revealedthat this strandidentiflcationdoesnot involve GATCsequences.

site in the DNA, commonlyreferred to as an AP site or abasic site. Each DNA glycosylaseis generallyspeciflc for one type of lesion, Uracil DNA glycosylases,for example, found in most cells, specifically remove from DNA the uracil that resultsfrom spontaneousdeaminationof cytosine. Mutant cells that lack this enzyme have a high rate of G:C to A:T mutations.This glycosylasedoesnot remove uracil residues from RNA or thymine residues from DNA. The capacityto distinguishthymine from uracil, the product of cytosine deamination-necessary for the selectiverepair of the latter-may be one reasonwhy DNA evolvedto containthymine insteadof uracil (p. 289). Mostbacteriahavejust onetype of uracil DNA glycosylase,whereashumanshaveat leastfour types,with different specificities-an indicator of the importance of uracil removal from DNA. The most abundant human uracil glycosylase,UNG, is associatedwith the human replisome,where it eliminatesthe occasionalU residue insertedin place of a T during replication.The deamination of C residuesis 100-foldfaster in single-stranded DNA than in double-strandedDNA, and humanshavethe enzlrne hSMUGl,which removesany U residuesthat occur in single-strandedDNA during replication or transcription.TWoother human DNA glycosylases, TDG and

Base-Excision Repair Every cell has a classof enzyrnescalledDNA glycosylases that recognizeparticularly common DNA lesions (such as the products of cytosineand adeninedeamination;seeFig. 8-30a) and remove the affected base by cleavingthe N-glycosyl bond. This cleavagecreatesan apurinic or apyrimidinic

25.2 DNARepair

MBD4,removeeither U or T residuespairedwith G, generated by deaminationof cytosine or 5-methylcy'tosine, respectively. OtherDNA glycosylases recognizeand removea variety of damagedbases,including formamidopy'rimidine and 8-hydroxyguanine(both arising from purine oxidation), hypoxanthine (arising from adenine deamination), and alkylatedbasessuchas 3-methyladenine and 7-methylguanine. Glycosylases that recognizeother lesions,including pyrimidine dimers,havealsobeen identified in someclassesof organisms.Rememberthat AP sites also arise from the slow,spontaneoushydrolysisof the N-glycosylbondsin DNA (seeFig. 8-30b). Oncean AP site has been formed by a DNA glycosylase,another type of enzymemust repair it. The repair is not made by simply inserting a new base and re-formingthe N-glycosylbond. Instead,the deoxyribose5'-phosphateleft behind is removedand replaced with a new nucleotide.This processbeginswith one of the AP endonucleases, enzymesthat cut the DNA strand containingthe AP site. The positionof the incisionrelativeto the AP site (5'or 3'to the site) varies with the type of AP endonuclease. A segmentof DNA including the AP site is then removed,DNA pol;.rnerase I replacesthe DNA, and DNA ligasesealsthe remaining nick (Fig. 25-25). In eukaryotes,nucleotidereplacement is carried out by specializedpolymerases,as describedbelow. Nucleotide-Exeision Repair DNA lesionsthat cause largedistortionsin the helicalstructure of DNA generally are repairedby the nucleotide-excision system,a repair pathway critical to the survival of all free-living organisms. In nucleotide-excision repair (Fig. 25-26), a (excinuclease) hydrolyzes two multisubunit enz;.'rne phosphodiester bonds,one on either side of the distortion causedby the lesion.ln E. coli,and other bacteria, the enzymesystemhydrolyzesthe f,fth phosphodiester bond bond on the 3' sideand the eighthphosphodiester on the 5' side to generatea fragmentof 12 to 13 nucleotides(dependingon whether the lesioninvolvesone or two bases).In humansand other eukaryotes,the enz;.'rnesystemhydrolyzesthe sixth phosphodiesterbond on the 3' side and the twenty-secondphosphodiester bond on the 5' side,producinga fragmentof 27 to 29 nucleotides.Following the dual incision, the excised oligonucleotidesare releasedfrom the duplex and the resulting gap is filled-by DNA po\rmeraseI tn E. coli, and DNA pol;.rnerasee in humans. DNA ligase seals the nick. ln E. coli,, the key enz;rmaticcomplex is the ABC excinuclease,which has three subunits, UvrA (M. 104,000), UwB (M.78,000),and UwC (M.68,000).The term "excinuclease"is used to describethe unique capacity of this enzyrnecomplex to catalyzetwo speciflc endonucleolyticcleavages,distinguishingthis activity A complexof the from that of standardendonucleases.

AP endonuclease

F"l

@

25-25 DNA repair by the base-excisionrepair pathway. FIGURE glycosylaserecognizesa damagedbaseand cleavesbe,l oNn @ in the backbone. tweenthe baseand deoxyribose @ An AP endonubackboneneartheAP site O DNA the phosphodiester cleasecleaves from the free3' hydroxylat the polymerase repairsynthesis I initiates activity)and replacinga nick,removing(with its 5'-;3' exonuclease afterDNA polystrand. portionof thedamaged @The nickremaining is sealedby DNA ligase meraseI hasdissociated

UwA and UwB proteins (A2B) scansthe DNA and binds to the site of a lesion.The UwA dimer then dissociates, leaving a tight UwB-DNA complex. UwC protein then binds to UwB, and UwB makes an incision at the fifth

DNA Metaborism

[trt]

DNA lesion

3',

5',

('xcrnucleasc

a) o

-u*-----"n*-.*-*@>=*=

,--r*-r=--\.s*:-r..=--m-ry-r

;-=r-=i*,L"L,

"L*t*l*L^i*r-+*i*;-";-*g-*.L=l*i*.

DNA helicas

DNA helicas T.1

G) -\**

DNA polvnierase I

*1.--L, J.i

*r--i*L

,oH

6

@

*:"-,L;

.-i_"T*=i=,*]*.,F.

FfGURE 25-26 Nucleotide-excision repair in E. coti and humans. The generalpathwayof nucleotide-excision repairis similarin all organisms. bindsto DNA at the siteof a bulky @ An excinuclease lesionand cleavesthe damagedDNA strandon eitherside of the

(13 mer)or 29 nulesion.@ The DNA segment-of 13 nucleotides cleotides(29 mer)-is removedwith the aid of a helicase.@The gap is filled in by DNA polymerase, and @ the remainingnick is sealedwith DNA ligase.

phosphodiester bond on the 3' sideof the lesion.This is followed by a UvrC-mediatedincision at the eighth phosphodiester bond on the 5' side.The resulting l2 to 13 nucleotide fragment is removed by UvrD helicase.The short gap thus created is filled in by DNA polymerase I and DNA ligase. This pathway (Fig. 25-26,left) is a primary repair route for many types of lesions,including cyclobutanepyrimidine dimers, 6-4 photoproducts(seeFig. 8-31), and severalother types of baseadductsincludingbenzo[o]pyrene-guanine, which is formed in DNA by exposure to cigarette smoke. The nucleolytic activity of the ABC excinucleaseis novel in the sensethat two cuts are made in the DNA. The mechanismof eukaryoticexcinucleasesis quite similar to that of the bacterial enzyrne,although 16 polypeptideswith no similarity to the E cola excinuclease subunits are required for the dual incision. As describedin Chapter26, someof the nucleotide-excision repair and base-excisionrepair in eukaryotesis closely tied to transcription. Geneticdeflcienciesin nucleotide-

excisionrepair in humansgive rise to a variety of serious diseases(see Box 25-1). Direct Repair Several types of damage are repaired without removhg a baseor nucleotide.The bestcharacterizedexample is direct photoreactivationof cyclobutanepyrimidine dimers, a reaction promoted by DNA photolyases. Py'rimidinedimers result from a UV-induced reaction, and photolyases use energy derived from absorbedlight to reverse the damage (Fig.25-27). Photolyasesgenerallycontain two cofactors that serve as light-absorbing agents, or chromophores.One of the chromophoresis alwaysFADH-. In E. coli, and yeast, the other chromophoreis a folate. The reaction mechanismentails the generation of free radicals.DNA photolyasesare not presentin the cells of placentalmammals(which include humans). Additional examples can be seen in the repair of nucleotides with alkylation damage. The modified nucleotide O6-methylguanineforms in the presenceof allcylatingagents and is a corunon and higlrly mutagenic

r-

--'l

ReoairI 999| 25.2DNA 'Ll

*MTHFpolyGlu

MTHFpolyGIu

Cyclobutanepyrimidine dimer

o

o

;A w

r\o

"/.-,o/ Monomeric pyrimidines in repaired DNA

ME(HAlllSMFIGURE 25-27 Repairof pyrimidine dimers with photolyase.Energyderivedfrom absorbedlight is usedto reversethe photoreactionthat causedthe lesion.The two chromophoresin E. coli photolyase(M, 54,OOO),Ns,Nr o-methenyltetrahyd rofolylpolyglutafunctions. mate(MTHFpolyClu)and FADH-, performcomplementary

lesion (p. 292).lt tends to pair with thymine rather than cytosine durir€ replication, and therefore causesG:C to A:T mutations (Fig. 25-28). Direct repair of 06methylguanine is carried out by O"-methylguanine-DNA methyltransferase,a protein that catalyzestransfer of the methyl group of O6-methylguanineto one of its own Cys residues.This methyltmnsferaseis not strictly an enz),rne,becausea singlemethyl transfer event perrnanently methylates the protein, making it inactive in this pathway. The consumption of an entfe protein molecule to correct a sing[edamagedbaseis another vivid illustration of the priority given to maintaining the integrity of cellular DNA.

to absorbbluelightphotons. MTHFpolyclufunctionsasa photoantenna The excitation energy passesto FADH-, and the excited flavin (*FADH-) donatesan electronto the pyrimidinedimer,leadingto the as snown. rearranSement

O6-Methylguanine nucleotide

Guanine nucleotide

Metabotism It ooo DNA

(a)

H

I

Guanine R

I

H I J

methylation md replicatron replication

Ala (f) His-+Glu (c) Ala+Thr (g) Pro-+Ser (d) Phe--+Lys 10. Basis of the Sickle-Cell Mutation Sickle-cellhemoglobin has a Val residueat position 6 of the B-globinchain,instead of the Glu residuefound in normal hemo$obin A. Canyou predict what changetook placein the DNA codonfor glutamateto accormtfor replacementof the Glu residueby Val? 11. Proofreading by Aminoacyl-tRNA Synthetases The isoleucyl-tRNA slmthetasehas a proofreading function that ensures the fldelity of the aminoacylationreaction, but the histidyl-tRNA syrrthetaselacks such a proofreadingfunction. Explain. 12. Importance of the "Second Genetic Code" Some amiroacyl-tRNA synthetasesdo not recognizeand bind the anticodonof their cognatetRNAs but insteaduse other structural features of the tRNAs to impart binding speci_flcity. The tRNAs for alanineapparentlyfalt into this category. (a) What featuresof IRNAAUare recognizedby Ala-tRNA synthetase? (b) Describethe consequences of a C-+ G mutationin the third position of the anticodonof tRNAa'. (c) What other kinds of mutations might have similar effects? (d) Mutations of these types are never found in natural populations of organisms.Why? (Hint: Considerwhat might happen both to individual proteins and to the organismas a whole ) 13. Maintaining the Fidelity of Protein Synthesis The chemicalmechanismsusedto avoid errors in protein sy'nthesis are dilferent from those used during DNA replication. DNA poly'rnerases use a 3'-+5' exonucleaseproofreadingactivity to remove mispaired nucleotides incorrectly inserted into a growingDNA strand There is no analogousproofreadingfunction on ribosomesand, in fact, the identity of an aminoacid attached to an incoming tRNA and added to the growing polypeptide is never checked. A proofreading step that hydrolyzed the previously formed peptide bond after an incorrect amino acid had been inserted into a growing polypeptide (analogousto the proofreading step of DNA poly.rnerases) would be impractical. Why? (Hint: Considerhow the link between the growing polypeptide and the mRNA is maintained during elongation;see Figs 27-29 and 27-30.) 14. Predicting the Cellular Location of a Protein The gene for a eukaryoticpolypeptide300 amino acid residues Iongis altered so that a signalsequencerecognizedby SRPoccurs at the po\peptide's amino terminus and a nuclear localization signal (NLS) occurs internally, beginning at residue 150.Whereis the protein likely to be found in the cell?

15. Requirements for Protein Ttanslocation across a Membrane The secretedbacterial protein OmpA has a precursor, ProOmpA, which has the amino-terminal signal sequence required for secretion. If purified ProOmpA is denaturedwith 8 M urea and the urea is then removed (such as by runnhg the protein solution rapidly through a gel flltration column) the protein can be translocatedacrossisolated bacterialirner membranesin vitro. However,translocationbecomesimpossibleif ProOmpAis first allowedto incubatefor a few hours in the absenceof urea. Furthermore, the capacity for translocationis maintained for an extended period if ProOmpAis first incubatedin the presenceof another bacterial protein calledtrigger factor.Describethe probablefunction of this factor. 16. Protein-Coding Capacity of a Viral DNA The 5,386bp genomeof bacteriophageQXl74 includesgenesfor 10 proteins, designatedA to K, with sizesgiven in the table below. How much DNA would be required to encode these l0 proteins? How can you reconcile the size of the @X174genome with its protein-codingcapacity?

Protcin

Numberof amino acidresidues

A455F BI?OG C86H DT52J E91

Protein

Numberof arnino acidresidues

K

427 175 328 38 56

DataAnalysis Problem 17. Designing Proteins by Using Randomly Generated Genes Studies of the amino acid sequence and corresponding three-dimensional structure of wild-t1pe or mutant proteins have led to signiflcant insights into the prhciples that govern protein folding. Ar important test of this understanding would be to desi,gn a protein based on these principles and see whether it folds as expected. Kamtekar and colleagues (1993) used aspects of the genetic code to generate random protein sequences with defined patterns of hydrophilic and hydrophobic residues Their clever approach combined knowledge about protein structure, amino acid properties, and the genetic code to explore the factors that influence protein structure. They set out to generate a set of proteins with the simple four-helix bundle structure shown at the top of page 1113 (right), with a helices (shown as cylinders) connected by segments of random coil (pink). Each a helix is amphipathicthe R groups on one side of the helix are exclusively hydrophobic (yellow) and those on the other side are exclusively hydrophilic (blue). A protein consisting of four of these helices separated by short segments of random coil would be expected to fold so that the hydrophilic sides of the helices face the solvent

P r o b l e m1s1 1 3 1 !l

coo-

'

oo-

An amphipathic a helix

Four-helix bundle

(a) What forces or interactions hold the four a helices together in this bundled structure? Figure 4-4a shows a segment of a helix consisting of 10 amino acid residues With the gray central rod as a divider, four of the R groups (purple spheres) extend from the left side of the helix and six extend from the right. (b) Number the R groups in Figure 4-4a, from top (amino terminus; 1) to bottom (carboxyl terminus; 10). Which R groups extend from the left side and which from the right? (c) Suppose you wanted to design this 10 amino acid segment to be an amphipathic helix, with the left side hydrophilic and the right side hydrophobic Give a sequence of 10 amino acids that could potentially fold into such a structure There are many possible correct answers here (d) Give one possible double-stranded DNA sequence that could encode the amino acid sequence you chose for (c). (It is an internai portion of a protein, so you do not need to include start or stop codons.) Rather than designing proteins with specific sequences, Kamtekar and coileagues designed proteins with partialiy random sequences, with hydrophilic and hydrophobic amino acid residues placed in a controlled pattern. They did this by taking advantage of some interesting features of the genetic code to construct a library of synthetic DNA molecules with partially random sequences arranged in a particular pattern. To design a DNA sequence that would encode random hydrophobic amino acid sequences,the researchers began with

the degeneratecodonNTN,whereN canbe A, G, C, or T. They fllled eachN position by inciuding an equimolarmixture of A, reaction to generatea mixG, C, and T in the DNA s5,rrthesis ture of DNA moleculeswith different nucleotidesat that position (see Fig. 8-35). Similarly,to encoderandom polar amino acid sequences,they beganwith the degeneratecodon NAN and usedan equimolarmixture of A, G, and C (but in this case, no T) to flli the N positions. (e) Which amino acids can be encoded by the NTN triplet? Are all amino acids in this set hydrophobic?Does the set include all the hydrophobicamino acids? (f) Which amino acids can be encoded by the NAN triplet? Are all of these polar?Doesthe set include all the polar amino acids? (g) In creating the NAN codons,why was it necessaryto leaveT out of the reaction mixture? Kamtekar and coworkers cloned this library of random DNA sequencesinto plasmids,selected48 that producedthe correct patterning of hydrophilic and hydrophobic amino acids,and expressedthesein.8. co\i,.The next challengewas to determine whether the proteins folded as expected.It would be very time-consumingto express each protein, crystallize it, and determine its complete three-dimensional structure. Instead,the investigatorsused the E. coli' proteinprocessingmachineryto screen out sequencesthat led to highly defective proteins. In this initial screening,they kept only those clones that resulted in a band of protein with the expected molecular weight on SDS polyacrylamide gel (seeFig. 3-18). electrophoresis (h) Why would a grosslymisfoldedprotein fail to produce a band of the expectedmolecularweight on electrophoresis? Severalproteins passedthis initial test, and further exploration showedthat they had the expectedfour-helix structure. (i) Why didn't all of the random-sequenceproteins that passedthe initial screeningtest produce four-helix structures? Itel'eren pK. of the a-carboxyl group and pI < pK. of the a-amino group, so both groups are charged (ionized) (b) I n 2 19 x 107 The pl ofalanine is 6 01 From Table 3-1 arLd the Henderson-Hasselbalch equation, 1/4,680 carboxyl groups and 1/4,680 amiro groups are uncharged The fraction of alanine molecules with both groups rurcharged is the product of these fractions 4. (a)-(c)

H+

3

CHt FuIIy deprotonated

29. (a) Blood pH is controlled by the carbon dioxide-bicarbonate buffer system, CO2 + H2O + H+ + HCOt Dtrtnghypouenttlatzon, [COr] increases in the lungs and arteria.lblood, driving the equilibrium to the right, raising [H-] and lowering pH (b) Dunng hyperuenti\ati,on, ICOz] decreases in the lungs and arterial blood, reducing [H-] and increasi4g pH above the normal 7 4 value. (c) Lactate is a moderately strong acid, completely dissociatfirg urder physiological conditions and thus lowenng the pH of blood and muscle tissue Hlperventilation removes H-, raisng the pH of blood and tissues in anticioation of the acid builduo

cH-Nn,-4{11-

X.",-

I HN-\ i\N xcHr-CH-NH2

pH

Structure

1 4 8 12

I 2 3 4

Migrates toward

Net charge

Cathode Cathode Does not migrate Anode

T' TI

0 -1

5. (a) Asp (b) Met (c) Glu (d) Gly (e) Ser 6. (a) 2 (b) a (c) :+

+E

HrN--C--H

H3N--C..H

H--C'-NHs

I

HrC--g..11

H-Q-CHa

CH,

I

H--Q-.CH3

ii

9H, ll

coo

coo

coo

coo-

H--C'-NHs

I H3C-C..H

9H,

CH,

CHo

CH,

CHt

CHt

t-

I

at pH 7: 7. (a) Structure

oo ll

+.

ItN-CH-C-N-

,ll

CH-C-N-CH

CH,HCH,HCH, pKr:8.03

P K r: 3 . 3 9

(b) Electrostatic interaction between the carboxylate anion and the protonated amino group of the alanine zwitterion favorably affects the ionization ofthe carboxyl group This favorable electrostatic interaction decreases as the length ofthe poly(A-la) increases, resulting in an increase in p& (c) Ionization of the protonated amino group destroys the favorable electrostatic interaction noted in (b). With increasing distance between the chargecl groups, removal of the proton from the amino group in poly(Ala) becomes easier and thus pKz is lower The intramolecular effects of the amide (peptide bond) linkages keep pKu values lower than they would be for an allTl-substituted amine. 8. 75,000 9. (a) 32,000 The elements of water are lost when a peptide bond forms, so the molecular weight of a Tfp residue is not the same as the molecular weight of free tryptophan (b) 2 10. The protein has four subunits, with molecular masses of 160, 90, 90, and 60 kDa The two 90 kDa subunits (possibly identical) are tinked bv one or more disulfide bonds -1 (b) pl : 7.8 11. (a) at pH 3, +2;atpH 8, 0; at pH 11,

COOH t

|

HN-\

oK.=\.82

L2. pI:1;

,)-cHr-cH-NH3j-J-----l_ \NH H I

13. Lys, His, Arg; negatively charged phosphate groups in DNA interact with positively charged side groups in histones L4. (a) (Glu)2s (b) (Lys Ala)3 (c) (Asn-Ser-His)s (d) (Asn-Ser-His)u

coo |

HN-\

0(,-60

_)-cHr-cH-NH3--5l_ \NH H

,

carboxylate groups; Asp and Glu

15. (a) Specif,c activity after step 1 is 200 units/mg; step 2, 600 units/mg; step 3, 250 units/mg; step 4, 4,000 units/mg; step 5' 15,000units/mg; step 6, 15,000unlts/mg (b) Step 4 (c) Step 3 (d) Yes. Speciflc activity did not increase in step 6; SDS polyacrylamide ge1electroPhoresis

f-l

!AS-41

Abbreviate 5d o l u t i o nt so p r o b l e m s

16. (a) [NaCl]: 0 5 mn (b) [NaCl]: 0 05 mu 17. C elutesf,rst,B second,A last 18. Tlr-Gly-Gly-Phe-Leu l9' ---al,eu1 Orn Phe

/

\

Val

Pro

Pro \

Val

,t

t

Phe

\L"'1

Orri

The arrows correspond to the orientation of the peptide bonds, CO --+ NH-. 2O. 88o/o, 970/o The percentage (r) of correct amino acid residues released in cycle n is r,,fr All residues released in the first cycle are correct, even though the efficiency of cleavage is not perfect 2L. (a) Y (1), F (7), and R (9) (b) Posirrons4 and 9; K (Lys) is more comrnon at 4, R (Arg) is invariant at g (c) positions b and 10; E (Glu) is more common at both positions (d) Position 2; S (Ser) 22. (a) The protein to be rsolated (citrate synthase, CS) is a relatively small fraction of the total cellular protein Cold temperatures reduce protein degradation; sucrose provides an isotonic enuronment that preserves the rntegrity of organelles during homogenization. (b) This step separates organelles on the basis of relative size (c) The first addrtion of ammonium sulfate removes some unwanted proteins from the homogenate Additionat ammonium sulfate precipitates 0S. (d) To resuspend (solubilize) CS, ammonium sulfate must be removecl under conditions of pH and ionic strength that support the native conformation (e) CS molecules are larger than the pore size of the chromatographic gel Protein is detectable at 280 nm because of absorption at this wavelength by Tj,r and T?p residues (f) CS has a posrtive charge and thus binds to the negatrvely charged catron-exchange column After the neutral and negatively charged proteins pass through, CS is displaced from the column using the washing solution of higher pH, which alters the charge on CS (g) Different proteins can have the same pI The SDS gel confirmed that only a single protein lras purrfied SDS is difficult to remove completely from a protein, and its presence distorts the acid-base Dropertrtes of the protein, including pl 23. (a) Any linear polypeptide chain has only two kinds of free amino groups: a single a-amino group at the amino terminus, and an e-amino group on each Lys residue present. These amino groups react with FDNB to form a DNp-amino acicl derivative Insulin gave two different a-amino-DNp derivatives, suggesting that it has two amino termini and thus two polypep_ tide chains-one with an amino-terminal Gly and the other with an amlno-terminal Phe Because the DNp-lysine product is e-DNPJysine, the Lys is not at an amino terminus (b) yes The A chain has amino-terminal Gly; the B chain has amino-terminal Phe; and (nonterminal) residue 29 in the B chain is Lys (c) Phe Val-Asp-Glu- Peptide 81 shows that the amino-terminal resrdue is Phe Peptide 82 also includes Val, but since no DNp_ Val is formed, Val is not at the amino terminus; it must be on the carboxyl side of Phe Thus the sequence of 82 is DNpPhe Val Similarly, the sequence of 83 must be DNp-phe Val_Asp, and the sequence of the A chain must begin phe-Val Asp-Glu_ (d) No The known amrno-terminal sequence of the A chain is Phe-Val-Asn-GlnThe Asn and Gln appear in Sanger's analysis as Asp and Glu because the vigorous hydrotysis in step (! hydrolyzed the amide bonds in Asn and Gln (as well as the peptide bonds), formrng Asp and Glu Sanger et al could not dlstinguish Asp from Asn or Gju from Gln at this stage in their analysis (e) The sequence exactly matches that rn Fig S_24 Each peptide in the table gives speciflc information about whlch Asx residues are Asn or Asp and which Glx resiclues are Glu or Gln

AcJ: residues 20-21 Thisis the onlyCys-Asxsequence in theA chain;thereis -1 amidogroupin thispeptide,soit mustbe Cys-Asn: N-Gly-Ile -Val-Glx-Glx-Cys-Cys-Ala-Ser-Val-Cys-Ser1510 Leu-Tyr-Glx-Leu-Glx-Asx-Tyr-Cys-Asn-C 15 20 ,4p15:residues14-15-16Thlsis the onlyTP-Glx-Leuin theA chain;thereis -1 amidogroup,sothepeptidemustbe Tlr-Gln-Leu: N-Gly-IIe -Val-Glx-Glx-Cys-Cys-Ala-Ser-Val-Cys-Ser1510 Leu-Tyr-Gln-Leu-Glx-Asx-Tyr.-Cys-Asn- C 15 20 Ap14:residues 14-15-16-17 . It has- 1 amidogroup,andwealreadyknowthat residue15is Gln,soresidue17mustbe Glu: N-Gly-Ile -Val-Glx-GIx-Cys-Cys-Ala-Ser-Val-Cys-Ser1510 Leu-Tyr'-Gln-Leu-Glu-Asx-T1r-Cys-Asn-C 15 20 ,4p3:residues18-19-20-2IIt has-2 amidogroups,andwe knowthatresidue 21is Asn,soresidue18mustbeAsn: N-Gly-Ile -Val-Glx-Glx-Cys-Cys-AIa-Ser-Val-Cys-Ser1510 Leu-Tyr-Gln-Leu-Glu-Asn-Tyr-Cys-Asn-C 15 20 ,4p1:residues l7-IU19-20-21,whichis consistent with residues 18and21beingAsn Ap5pal: residues1-2-3-4 It has-0 amidogroup,soresidue4 mustbe Glu: Leu-Tyr-Gln-Leu-Glu-Asn-Tyr-Cys-Asn-C 15 20 Leu-Tyr-Gln-Leu-Glu-Asn-Tyr-Cys-Asn-C 15 20 ,4p5 residuesI through13 It has-1 amidogroup,andweknow that residue14is Glu,soresidue5 mustbe Gln: N-Gly-Ile -Val-Glu-Gln-Cys-Cys-Ala-Ser-Val-Cys-Ser1510 Leu-Tyr'-Gln-Leu-GIu-Asn-Tyr-Cys-Asn15 20

C

(hapter 4 l. (a) Shorter bonds have a higher bond order (are multiple rather than single) and are stronger. The peptide C-N bond is stronger than a single bond and is midway between a single and a double bond in character (b) Rotation about the peptide bond is difficult at physiological temperatures because of its partial doublebond character. 2. (a) The principal structural units in the wool fiber polypeptide (a-keratin) are successive turns of the a helix, at b 4 A intervals; coiled coiis produce the 5.2 A spacing Steaming and stretching the fiber yields an extended polypeptide chain with the B conformation, with a distance between adjacent R groups of about Z 0 A As the polypeptide reassumes an a-helical structure, the fiber shortens (b) Processed wool shrinks when polypeptide chains are converted from an extended p conformation to the native a-helical conformation in the presence of moist heat The structure of silk-B sheets, with their small, closely packed amino acid side chains-is more stable than that of wool. 3. -42 peptide bonds per second 4. At pH > 6, the carboxyl groups of poly(Glu) are deprotonated; repulsion among negatively charged carboxylate groups leads to

s f] A b b r e v i aS t eodl u t i otnosP r o b l e m[ns

unfolding. Similarly, at pH 7, the amino groups of poly(Lys) are protonated; repulsion among these positively charged groups also leads to unfolding 5. (a) Disulfide bonds are covalent bonds, which are rnuch stronger than the noncovalent interactions that stabiljze most proteins They crossJink protein chains, increasing their stiffness, mechanical strength, and hardness (b) Cystine residues (disulflde bonds) prevent the complete unfolding of the protein 6. (a) Bends are most likely at residues 7 and 19; Pro residues in the cis configuration accommodate turns well (b) The Cys residues at positions 13 and 24 can form disulfide bonds. (c) External surface: polar and charged residues (Asp, Gln, Lys); interior: nonpolar and aliphatic residues (Ala, Ile); Thr, though polar, has a hydropathy index near zero and thus can be found either on the extemal surface or in the interior of the orotein 7. 30 amino acid residuesl 0 87 8. Myoglobin is all three The folded structure, the "globin fold," is a motif found in all globins The polypeptide folds into a single domain, which for this protein represents the entrre threedimensional structure, 9. The bacterial enzlnne is a collagenase; it destroys the connectivetissue barrier of the host, allowing the bacterium to invade the trssues Bacteria do not contain collagen f0. (a) The number of moles of DNP-valine formed per mole of protein equals the number of amino termini and thus the number of polypeptide chains (b) 4 (c) Different chains would probably run as discrete bands on an SDS polyacrylamide gel lf . (a);rt has more amino acid residues that favor a-helical structure (see Table 4-1) 12, (a) Aromatic residues seem to play an important role in stabilizing amyloid fibrils Thus, molecules with aromatic substituents may inhibit amyloid formation by interfering with the stacking or association of the aromatic srde chains (b) Amyloid is formed in the pancreas in association with type 2 diabetes, as it is in the brain in Alzheimer's disease. Although the amyloid flbrils in the two diseases involve different proteins, the fundamental structure of the amylord is similar and similarly stabilized in both, and thus they are potential targets for similar drugs designed to disrupt this structure 13. (a) NFrB transcription factor, also called RelA transforming factor (b) No You will obtain similar results, but with additional related proteins listed (c) The protein has two subunits There are multiple variants of the subunits, with the best-characterized being 50, 52, or 65 kDa These pair with each other to form a variety of homodimers and heterodimers The structures of a number of different variants can be found in the PDB (d) The NFrts transcription factor is a dimeric protein that binds specific DNA sequences,enhancing transcnption ofnearby genes One such gene is the immunoglobulin r light chain, from which the lranscription factor gets its name 14. (a) Aba is a suitable replacement because Aba and Cys have approximately the same sized side chain and are similarly hydrophobic However, Aba cannot form disulfide bonds so it will not be a suitable replacement ifthese are required (b) There are many important differences between the synthesized protein and HIV protease produced by a human cell, any of which could result in an rnactive synthetic enz),'Ine: (l) Although Aba and Cys have similar size and hydrophobiclty, Aba may not be similar enough for the protein to fold properly (2) HIV protease may requrre disulfide bonds for proper functioning (3) Many proteins s)'nthesized by ribosomes fold as they are produced; the protein in this study folded only after the chain was complete (4) Proteins synthesized by ribosomes may interact with the ribosomes as thev fold; this is not possible for the protein in the study (5) Cltosol is a more complex solution than the buffer used in the study; some proteins may require specific, unknown proteins for proper folding (6) Proteins slrrthesized in cells often reqtdre

chaperones for proper foiding; these are not present in the study buffer. (7) In cells, HIV protease is slmthesized as part of a larger chain that is then proteoll'tically processed; the protein in the study was slnthesized as a single molecule (c) Because the enzt ne zs finctional with Aba substituted for Cys, disulflde bonds do not play an important role in the structure of HIV protease (d) Moclel, 7 it would fold like the L-protease Argument Jor: the covalent structure is the same (except for chirality), so it should fold Like the l-protease. Argument agai'nst: chirality is not a trivial detail; three-dimensional shape is a key feature of biological molecules The synthetic enzgne rvill not fold like the L-protease Model 2: it would fold to the mirror image of the l-protease Ior: because the individual components are mrrror images of those in the biological protein, it will fold in the mirrorimage shape Agaznst the interactions involved in protein folding are very complex, so the sSmthetic protein will most likely fold in another form Mode\ 3: it would fold to something else. For: the interactions involved in protein folding are very complex, so the slnLthetic protein will most likely fold in another form Agai,nst: because the individual components are mlrror images of those in the biological protein, it will fold in the mirrorimage shape (e) Model 1. The enz5.nneis active, but with the enantiomeric form of the biological substrate, and it is inhibited by the enantiomeric form of the biological hlLibitor This is consistent with the D-protease being the mirror image of the L-protease (f) Evans blue is achiral; it binds to both forms of the enzyrne (g) No. Because proteases contain only r,-amino acids and recognize only t -peptides, ch5nnotrypsin would not digest the D-protease (h) Not necessarily. Depending on the individual enzSane,any of the problems listed in (b) could result in an inactive enzyme.

5 Chapter 1. Protein B has a higher affinity for ligand X; it will be halfsaturated at a much lower concentration of X than will protein A 1 Protein A has K. : 106u-1; protein B has K. = 10e lt 2. (a), (b), and (c) all have "H < 1 0. Apparent negative cooperativity in ligand binding can be caused by the presence of two or more tJ,?es of ligand-binding sites with different affinities for the ligand on the same or different proteins in the same solution. Apparent negative cooperativity is also commonly observed in heterogeneous protein preparations There are few welldocumented cases of tme negative cooperativity. 3. (a) decreases(b) increases (c) decreases(d) decreases 5s I 4 . k . 1: 8 9 x 1 0 5. The cooperative behavior ofhemoglobin arises from subunit interacttons 6. (a) The observation that hemoglobin A (HbA; maternal) is about 6070 saturated when the pO2 is 4 kPa, whereas hemoglobin F (HbF; fetat) is more than 90% saturated under the same physiological conditions, indicates that HbF has a higher 02 affinity than HbA (b) The higher 02 affinity of HbF ensures that oxygen will flow from maternal blood to fetal blood in the placenta Fetal blood approaches full saturation where the 02 affinity of HbA is low. (c) The obserwation that the 02 -saturation curue of HbA undergoes a larger shift on BPG binding than that of HbF suggests that HbA binds BPG more tightly than does HbF. Differential binding of BPG to the two hemoglobins may determine the difference in their 02 affinities 7. (a) Hb Memphis (b) HbS, Hb Milwaukee, Hb Providence, possibly Hb Oowtonn (c) Hb Providence 8. More tightly An inability to form tetramers would limit the cooperativity of these variants, and the binding curve would become more hyperbolic. Also, the BPG-binding site would be clisrupted. Oxygen binding would probably be tighter, because the default state in the absence of bound BPG is the tightbindine R state

b'.u-iA b b r e v i aSt eodl u t i otnosP r o b l e m s 9. (a) 1 x 10 Er,r(b)5 x t0 8iu(c)8 x 10-8r,r (d) 2 X 10 7 M Note that a rearrangement of Eqn 5-8 gives [L): eK,r/(l - 0) 10. The epitope is likely to be a structure that is buried when G-actin pol5.rnerizes to F-actin 11. Many pathogens, including HIV, have evolved mechanisms by which they can repeatedly alter the surface proteins to which immune system components initially bind. Thus the host organism regularly faces new antigens and requires time to mount an immune response to each one As the immune system responds to one variant, new variants are created 12. Binding of ATP to myosin triggers drssociation of myosin from the actin thin fi.lament In the absence of ATp, actin and mvosirr bind tightly to each other

13.

(a)

(b)

(c)

(d)

f4. (a) Chain L is the light chain and chain H is the heary chain of the Fab fragment of this antibody molecule Chaln y is lysozyme (b) B structures are predominant in the variable and constant regions ofthe fragment (c) Fab heary-chain fragment,218 amino acid residues; Iight-chain fragment, 214; lysozvme, l2g Less than l5% of the lysozyme molecule is in contact with the Fab fragment (d) In the H chain, residues that seem to be in contact with lysozl'rne include Gly31,Tyr'r2,Argetr,Asp1o0,and Tyrl0t. In the L chain the residues that seem to be in contact wrth lysozlme include Tlr32, Tyron, Ty.on, and Ttp92. In lysozyme, r e s i d u e sA s n r u ,G l y 2 2 T , y r 2 3 ,s e r 2 a ,L y s r 1 6 G , l y 1 1 7T, h r I r 8 , A s p r r s ) , Glnl2r, antl Arg'25 seem to be situated at the antigen-antibody rnterlace Not all these resrdues are adjacent in the primary structure Folding of the polypeptide chain into higher levels of structure brings the nonconsecutive resrdues toAether to form the antigen-binding site 15. (a) The protein with a K,1 of 5 pu wil have the highest affinity for ligand L When the Ka is 10 pu, doubling [L] from 0 2 to 0 4 pu (vahres well below the K,1) will nearly double d (the actual increase factor is 1.96) This is a property of the hyperbolic curve; at low ligand concentrations, d is an almost linear function of [L] On the other hand, doubling [L] from 40 to 80 g,u (wetl above the K4, where the binding curves is approaching its aslmptotic limit) wrll ircrease d by a factor of only I I The increase factors are identical for the curves generated from Eqn 5 ll (b) 0: 0 ggg (c) A variety of answers will be obtained depending on the values entered for the different Darameters 16. (a)

would block actin binding and prevent movement An antibody that bound to actin would also prevent actin-myosin interaction and thus movement (d) There are lwo possible explanations: (1) T\psin cleaves only at Lys and Arg residues (see Table 3-7) so would not cleave at many sites in the protein (2) Not all Arg or Lys residues are equally accessible to trypsin; the most-exposed sites would be cleaved first (e) The 51 model The hinge model predrcts that bead-antibody-HMM complexes (wth the hinge) would move, but bead-antibody-SHMM complexes (no hinge) would not The 51 model predicts that because both complexes include Sl, both would move The finding that the beads move with SHMM (no hinge) is consistent only with the 51 model (f) With fewer myosin molecules bound, the beads could temporarily fall off the actin as a myosin let go of it. The beads would then move more slowly, as time is requrred for a second myosin to bind. At higher myosin density, as one myosin lets go another qulckly binds, leading to faster motion (g) Above a certain density, what limits the rate of movement is the intrinsic speed with which myosrn molecules move the beads. The myosin molecules are moving at a maximum rate and adding more will not increase speed (h) Because the force is produced in the St head, damaging the 51 head would probably inactivate the resulting molecule, and SHMM would be incapable of producing movement (i) The 51 head must be held together by noncovalent interactions that are strong enough to retain the active shape of the molecule

(hapter 6 l. The activity of the enz),me that converts sugar to starch is destroyed bv heat denaturation 2.24xI0

"M

3.95X108years 4. The enzl'rne-substrate complex is more stable than the enzvme alone 5. (a) 190 A (b) Three-dimensional folding of the enzyme brings the amino acid residues into proximitv. 6. The reaction rate can be measured by following the decrease in absorption by NADH (at 340 nm) as the reaction proceeds Determrne the K^ value; using substrate concentrations well above the K*,, measure initial rate (rate of NADH disappearance with time, measured spectrophotometrically) at several known enzyrne concentrations, and make a plot of initial rate versus concentration of enzyme. The plot should be linear, with a slope that provides a rneasure of LDH concentration.

7. (b),(e),(s) 8. (a) 17 x 10-3M(b0 ) 3 3 ;0 6 7 ;0 9 1( c ) T h e u p p e r c u rcvoer r e sponds to enzlrne B ([X] > K. for this enzyme); the lower curve, enzyme A 9. (a) 400 s t (b) 10 pu (c) a - 2, a': 3 (d) Mixed inhibitor 10. (a) 24 nv (b) 4 4a (tr/6is exactly hatf 7-*, so IAI : KJ k) 40 p.rrt (fr is exactly half Z^*, so [A] = 10 times K.. in the presence of inhibitor) (d) No. k"orlK^:0.33/(4 x 10 6 M-r s 1) = 8 25 x 104M-t s-t, v/ell below the diffusion-controlled limit. I l. I/*"* - 140 prdmin; K", - I < l0-5 r,r f 2. (a) 7^u* : 51.5 mu/min, K.. : 0.59 mv ( b ) C o m p e t i ti v e i n h i b i t i o n 13, 1l^ : 2 2 mxt; V^u*: 0 50 g.moVmin 14. Curve A 15.k.^t:20x107min-l 16. The basic assumptions of the Michaelis-Menten equation still hold. The reaction is at steady state, and the rate is determined by Vo: k2 [ES] The equations needed to solve for [ES] are

The drawing is nol to scale; any given cell would have marlv rnore myosin molecules on its surface (b) ATp is needed to pronde the chemical energy to drive the motion (see Chapter 18) (c) An antibody that bound to the myosin tail, the actin-bindrng srre,

tE,l : tEl + tESl + tEIl and tEIl :

tE:ltll AI

[E] can be obtainedby rearuangingEqn 6-19 The rest followsthe pattern of the Michaelis-Mentenequationderivationin the text

5d o l u t i o nt so P r o b l e m s A S - 7 | Abbreviate

17. MinimumM,:

29,000

18. Activity of the prostate enzyrne equals total phosphatase activity in a blood sample minus phosphatase activity in the presence of enough tartrate to completely inhibit the prostate enzyme 19. The inhibition is mixed Because K* seems not to change appreciably, this could be the special case of mixed inhibition called nnnnnmnotitirro

20. The [S] at which Vs: V^ *l2a' is obtained when all terms except 7*,o* on the right slde of Eqn 6-30-that is, [S]/(aK* + a' Begin with [S]/(aK,, + a'[S]) : ] a'and a'[S])-equali solve for [S]. 21. The optimum activity occurs when Glu35 is protonatecl and Asp52 ic rrnnrntnnatpd

22. (a) Increase factor : 7 96;V0: 50 g.u s-r; increase factor : 1 048 (b) When a -- 2 0, the curve is shifted to the right as the 1{.. is increased by a factor of 2. When o' : 3 0, the asymptote of the curve (the I/.u*) declines by a factor of3 When a : 2 0 and o' : 3 0, the curve briefly rises above the cuwe where both a and o' : 1.0, due to a decline in K^. However, the asymptote is lower, because 7-.* declines by a factor of 3 (c) When o : 2 0, the r intercept moves to the right When a : 2 0 and a' : 3.0,

(f) The mutant enzymerejectspyruvatebecausepyruvate's hydrophobicmethyl group will not interact with the highly hydrophilic guanidiniumgroup of Argr02.The mutant binds becauseof the strong ionic interaction betweenthe oxa.loacetate Arg102side chain and the carboxylof oxaloacetate(g) The protein must be flexible enoughto accommodatethe addedbulk of the side chaln and the larger substrate

7 Chapter l With reduction of the carbonyloxygento a hydroxyl group' the chemistryat C-1 and C-3 is the same;the glycerolmoleculeis not chiral. 2. Epimersdiffer by the confulurationabout only one carbon. (a) l-altrose (C-2),o-glucose(C-3),o-gulose(C-4) (b) o-idose (C-2), (C-a) (c) D-arabinose (C-2),l-galactose(C-3),D-al1ose o-xylose(C-3) 3. Osazoneformation destroysthe configurationaround C-2 of aldoses,so aldosesdiffering oniy at the C-2 configurationgive the samederivative,with the samemelting point. 4.(a)

the l' intercept moves to the left. 23. (a) In the wiid-type enzyme, the substrate is held in place by a hydrogen bond and an ion-dipole interaction between the charged side chain of Argroe and the polar carbonyl of pyruvate During catalysis, the charged Arg1oe side chain also stabilizes the polarized carbonyl transition state In the mutant, the binding is reduced to just a hydrogen bond, substrate binding is weaker, and ionic stabihzation of the transition state is lost, reducing catal5.'ticactivity (b) Because Lys arLdArg are roughly the same size and have a simrlar positive charge, they probably have very similar properties Furthermore, because plruvate binds to Argt?l by (presumably) an ioruc interaction, an Arg to Lys mutation would probably have little effect on substrate binding (c) The "forked" arrangement aligns two positively charged groups of Arg residues with the negatively charged oxygens of pyruvate and facrlitates two combined hydrogen-bond and iondipole interactions When Lys is present, only one such combined hydrogen-bond and ion-dipole interaction is possible, thus reducing the strength ofthe rnteraction The positionng ofthe substrate is less precise (d) I1e250interacts hydrophobically with the ring of NADH This type of interaction is not possible with the hydrophilic side chain of Gln (e) The structure is

HOH B-l-Glucose

HOH a-l-Glucose

(b) A fresh solution of a-l-glucose, or of B-o-glucose,undergoes mutarotation to an equilibrium mixture of the a and B forms (c) 36% a form; 64% B form 5, To convert d-D-glucose to p-o-glucose, the bond between C-l and the hydroxyl on C-5 (as in Fig 7-6) To convert D-glucose to D-mannose, either the -H or the -OH on C-2 Conversion between chair conformations does not require bond breakage; thls is the critical distinction between confu;uration and conformation 6. No; glucose and galactose differ at C-4 ?. (a) Both are poll'mers of o-glucose, but they differ in the glycosidic linkage: (Fl-+4) for cellulose, (a1->4) for glycogen (b) Both are hexoses, but glucose is an aldohexose, fructose a ketohexose. (c) Both are disaccharides, but maltose has two (a1+4)Jinked D-glucoseunits; sucrose has (a1e2p)Jinked D-glucose and o-fructose

shown below 8. Arg10z

l

NH

li

H

OH reducing sugar

HH 9. A hemiacetal is formed when an aldose or ketose condenses with an alcohol; a glycoside is formed when a hemiacetal condenses with an alcohol (see Fig 7-5, P. 238) 10. Fructose cyclizes to either the pyranose or the furanose structure Increasing the temperature shifts the equilibrium in the direction of I he furanose, the less sweeLforrn ll. Maltose; sucrose has no reducing (oxidizable) group, as the anomeric carbons of both monosaccharides are involved in the glycosidic bond.

AS-B

A b b r e v i aS t eodl u t i otnosP r o b l e m s

12. The rate of mutarotation is sufliclently high that, as the enzyme consumes p-o-glucose, more a-n-glucose is converted to the B form and, eventually, all the glucose is oxidizecl Glucose oxidase is specific for glucose and does not detect other reducing sugars (such as galactose) that react wrth Fehling's reagent

27. q

Oligosaccharide chains:

q L

0 L

13. (a) Measure the change in optical rotation with time (b) The optical rotatron of the mixture is negatlve (invertect) relative to that of the sucrose solution. (c) -2 0. 14. Prepare a slurry of sucrose and water for the core; add a small amount of sucrase (rnvertase); immediately coat with chocolate 15. Sucrose has no free anomeric carbon to undergo mutarotation 16.

z c

o 28. Oligosaccharides; their subunits can be combined in lLore ways than the amino acid subunits of otigopeptides Each hydroxyl group can participate in glycosidic bonds, and the configuration of each glycosidic bond can be elther a or B. The pol5.'rnercan be linear or branched 29. (a) Branch-point residues yield 2,3-di-O-methylglucose;the unbranched residues yield 2,3,6-tri-O-methylglucose (b) B.Tb\o

H

30. Chains of (1-+6)Jinked l-glucose residues with occasional (1-+3)-linked branches, wrth about one branch every 20 residues

OH HOH

Yes;yes 17. N-Acetyl-B-D-glucosamine is a reducing sugar; its C-l can be oxidized (see Fig. 7-10,p 241) o-Gluconate is not a reducing sugar; its C-1 is already at the oxidatron state of a carboxylic acid GlcN(a1 glyceraldehyde 3-phosphate + NAD* + HpO?(c) Unchanged; pyruvate -| H+ --; acetaldehyde f CO2 (d) Oddized; pyuvate + NAD+ -+ acetate + CO2 + NADH + H+ (e) Reduced; oxaloacetate + NADH * H+ --+ malate + NAD+ (f) Unchanged; acetoacetate * H+ --+ acetone * CO, 6, TPP: thiazolium ring adds to a carbon of pyruvate, then stabilizes the resulting carbanion by acting as an electron stnk Li,poic acid: oxidizes pyruvate to level of acetate (acetyl-CoA), and activates acetate as a thioester. CoA-SH: activates acetate as thioester FAD: oxldizes lipoic acld. N,4D+: oxidizes FAD. 7. Lack of TPP inhibits pyruvate dehydrogenase; pyruvate accumulates 8. Oxidative decarboxylation; NAD+or NADP+; o-ketogiutarate dehydrogenase

The optimum value of g" (i e , at maximum/) is 13. In nature, g" varies from 12 to 74, which corresponds to;fvalues very close to the optimum If you choose another value for t, the numbers will differ but the optrmal9" will still be 13

(hapter 16 r. (a) @Citrate sgnthase: Acetyl-CoA * oxaloacetate+ H2O-+ citrate * CoA @Aconi,tase: Citrate + isocltrate

9. Oxygen consumption is a measure of the activity of the flrst two stages of cellular respiration: glycolysis and the citric acid cycle. The addition of oxaloacetate or malate stimuiates the citric acid cycle and thus stimulates respiration The added oxaloacetate or malate serves a cata\4ic role, because it is regenerated in the latter part of the citric acid cycle 10. (a) 5 6 x 10-6 (b) 1.1 x 10-8 u (c) 28 molecules ll. ADP (or GDP), P,, CoA-SH, TPP, NAD+; notlipoic acid, which is covalently attached to the isolated enzyrnes that use it 12. The flavin nucleotides, FMN and FAD, would not be s),nthesized. Because FAD is required in the citric acid cycle, flavin deficiencv would strongly inhibit the cycle.

toProblems Solutions Abbreviated ins-td

13. Oxaloacetate might be withdrawn for aspartate synthesis or for gluconeogenesis Oxaloacetate is replenished by the anaplerotic reactions catalyzed by PEP carboxykinase, PEP carboxylase, malic enzyme, or pyruvate carboxylase (see Fig 16-15, p 632). 14. The terminal phosphoryl group in GTP can be transferred to ADP in a reaction catalyzed by nucleoside diphosphate kinase, with an equilibrium constant of 1.0: GTP + ADP + GDP + ATP (succinate) (b) Malonate is a 15, (a) OOC-CHr-CH2-COO competitive inhibitor of succinate dehydrogenase. (c) A block in the citric acid cycle stops NADH formation, which stops electron transfer, which stops respiration (d) A large excess of succinate (substrate) overcomes the competitive inhibition 16. (a) Add uniformly labeled [taC]glucose ancl check for the release laCor. (b) of Equally distributed in C-2 and C-3 of oxaloacetate; an infinite number 17. Oxaloacetate equilibrates with succinate, in which C-1 and C-4 are equivalent. Oxaloacetate derived from succinate is labeled at C-l and C-4, and the PEP derived from it has label at C-i, which gives rise to C-3 and C-4 ofglucose. 18. (a) C-1 (b) C-3 (c) C-3 (d) C-2 (methyl group) (e) C-a (f) C-a (g) Equally distributed in C-2 and C-3 19. Thiamine is required for the synthesis of TPP, a prosthetic group in the pyruvate dehydrogenase and a-ketoglutarate dehydrogenase complexes A thiamine deflciency reduces the activity of these enz;ane complexes and causes the observed accumulation of precursors 20. No For every two carbons that enter as acetate, two leave the cycle as CO2; thus there is no net synthesis of oxaloacetate Net s5mthesis of oxaloacetate occurs by the carboxylation of pyruvate, an anaplerotic reaction 21. Yes, the citric acid cycle would be inhibited Oxaloacetate is present at relatively low concentrations in mitochondria, and removinpl it for gluconeogenesis would tend to shift the equilibrium for the citrate s),nthase reaction toward oxaloacetate. 22, (a) Inhibition of aconitase (b) Fiuorocitrate; competes with citrate; by a large excess of citrate (c) Citrate and fluorocitrate are inhibitors of PFK-1 (d) All catabolic processesnecessary for ATP production are shut down 23. Glycolysi,s

r e s t r i c t e d ,a s i n a m e d i u m l o w i n F e 3 - . A c o n i t a s er e q u i r e s Fe3*, so an Fe3+-restricted medium restricts the s1'nthesis of aconltase (b) Sucrose * H2O -+ glucose + fructose Glucose + 2Pi + 2ADP + 2NAD- ---> 2 pyruvate + 2ATP + 2NADH + Fructose + 2P, + 2ADP + 2NAD- --> 2 py'ruvate + 2ATP + 2NADH + 2 Pwuvate + 2NAD* * 2CoA + 2 acetyl-CoA + 2NADH + 2 Pr,mvate + 2CO2 + 2ATP + 2H2O + 2 oxaloacetate + 2ADP 2 Acetyl-CoA * 2 oxaloacetate t 2H2O --+ 2 citrate

2H+ + 2H2O 2H+ + 2H2O 2H- + 2CO2 + 2Pi+ 4H+ + 2CoA

The overall reaction is Sucrose + H2O + 2Pi + 2ADP + 6NAD- --+ 2 citrate + 2ATP + 6NADH + 10H+ (c) The overall reaction consumes NAD+. Because the cellular pool of this oxidized coenzlme is limited, it must be recycled by the electron-transfer chain with consumption of 02 Consequently, the overall conversion of sucrose to citric acid is an aerobic process and requires molecular oxygen. 28. Succinvl-CoA is an intermediate ofthe citric acid cycle; its accumulation signals reduced flux through the cycle, calling for reduced entry of acetyl-CoA into the cycle Citrate synthase, by reguiating the primary oxidative pathway of the cell, regulates the supply of NADH and thus the flow of electrons from NADH to 02 29. Fatty acid catabolism increases Iacetyl-CoA], which stimulates pyruvate carboxylase. The resulting increase in Ioxaloacetate] stimulates acetyl-CoA consumption by the citric acid cycle, and [citrate] rises, inhibiting glycolysis at the level of PFK-1. In addition, increased Iacetyl-CoAl inhibits the p]Tuvate dehydrogenase complex, slowing the utilization of pyruvate from glycolysis. 30. Oxygen is needed to recycle NAD* from the NADH produced by the oxidative reactions of the citric acid cycle. Reoxidation of NADH occurs during mitochondrial oxidative phosphorylation 31. Increased [NADH]/[NAD+] inhibits the citric acid cycle by mass action at the three NAD*-reducing steps; high [NADH] shifts equilibrium toward NAD-

Glucose + zPi + 2ADP + 2NAD- --+ 2 pyruvate + 2ATP + 2NADH + 2H+ + 2H2O

32. Toward citrate; AG for the citrate synthase reaction under these conditions is about -8 kJ/mol

P lJnn ate carb org las e re acti,on :

33. Steps @ and @ are essential in the reoxidation of the enzyrne's reduced lipoamide cofactor.

2 P;'ruvate + 2CO2 + 2ATP 1- 2H2O --> 2 oxaloacctate + 2ADP + 2P, + 4Hj M alate de hA dro g enas e re act'ton : 2 Oxaloacetate + 2NADH 1- 2H+ + 2l-malate

+ 2NAD*

This recycles nicotinamide coenzlirnes under anaerobic conditions. The overall reaction is Glucose + 2CO2+ 2 r,-malate f 4HThis produces four H* per glucose, increasing the acidity and thus the tartness of the wine. 24. Net reaction: 2 Plruvate + ATP + 2NAD* + HzO -) a-ketoglutarate + CO2 + ADP + Pi + 2NADH + 3H* 25. The cycle participates in catabolic and anabolic processes For example, it generates ATP by substrate oxidation, but also provides precursors for amino acid s1'nthesis (see Fig. 16-15,

p 632) 26. (a) Decreases(b) Increases (c) Decreases 27. (a) Citrate is produced through the action of citrate slmthase on oxaloacetate and acetyl-CoA Citrate synthase can be used for net synthesis of citrate when (1) there is a continuous influx of new oxaloacetate and acetyl-CoA and (2) isocitrate s5mthesis is

34. The citric acid cycle is so central to metabolism that a serious defect in any cycle enzyrne would probably be letha.l to the embryo. 35. The first enzlme in each path is under reciprocal allosteric regulation Inhibition of one path shunts isocitrate into the other path 36, (a) The only reaction in muscle tissue that consumes signiflcant amounts of oxygen is cellular respiration, so 02 consumption is a good proxy for respiration. (b) Freshly prepared muscle tissue contains some residual glucose; 02 consumption is due to oxidation ofthis glucose (c) Yes.Because the amount of02 consumed increased when citrate or l-phosphoglycerol was added, both can serve as substrate for cellular respiration in this system (d) Erperimenf I citrate is causing much more 02 consumption than would be expected from its complete oxidation. Each molecule of citrate seems to be acting as though it were more than one molecule. The only possible explanation is that each molecule of citrate functions more than once in the reaction-which is how a catalyst operates Erperi'ment II: the key is to calculate the excess 02 consumed by each sample compared with the control (samPle 1)

E'-'d

A b b r e v i aS t eodl u t i otnosP r o b l e m s

Substrate(s) Sample added I

No extra

2

03mL0.2u

3 4

1-phosphoglycerol 0 15 mL 0.02u citrate 0.3mL02u I -phosphoglycerol + 0 15 mL 0 02 lr citrate

ttL Oz absorbed

Excess pL 02 consumed

342 757

0 415

431

89

1,385

1,043

If both citrate and 1-phosphoglycerol were simply substrates for the reaction, you would expect the excess 02 consumption by sample 4 to be the sum of the individual excess consumptions by samples 2 and 3 (415 pL + 89 pL: 504 pL) However, the ex_ cess consumption when both substrates are present is rougNy twice this amount (1,043 p.L) Thus citrate increases the ability of the tissue to metabolize l-phosphoglycerol This behavior is typical of a catalyst Both experiments (I and II) are required to make ttus case convincing Based on experiment i only, citrate is somehow accelerating the reaction, but it is not clear whether it acts by helping substrate metabolism or by some other mechanism Based on experiment II only, it is not clear which molecule is the catalysl, citrate or 1-phosphoglycerol Together, the experiments show that citrate is acting as a ,,catalyst,,for the oxidation of l-phosphoglycerol (e) Given that the pathway can consume citrate (see sample 3), if citrate is to act as a catalyst it must be regenerated If the set of reactions first consumes then regener_ ates citrate, it must be a circular rather than a linear pathway. (f) When the pathway is blocked at a-ketoglutarate dehydrogenase, citrate is converted to a-ketoglutarate but the pathway goes no further Oxygen is consumed by reoxidation ofthe NADH produced by isocitrate dehydrogenase.

7. 4 acetyl-CoA and 1 propionyl-CoA 8. Yes Some of the tritium is removed from palmitate during the dehydrogenation reactions of p oxidation. The removed tritium appears as tritiated water 9. Fatty acyl groups condensed with CoA in the cltosol are first transferred to carnitine, releasing CoA, then transported into the mitochondrion, where they are again condensed with CoA. The cytosolic and mitochondrial pools of CoA are thus kept separate, and no radioactive CoA from the cytosolic pool enters the mitochondrion 10. (a) In the pigeon, p oxidation predominates; in the pheasant, anaerobic glycolysis ofglycogen predominates. (b) Pigeon muscle wotlld consume more 02 (c) Fat contains more eners/ per grarn than $ycogen does in addition, the anaerobic breakdown of glycogen is limited by the tissue's tolerance to lactate buildup. Thus the pigeon, operating on the oxidative catabolism of fats, is the jongdistance flyer. (d) These enzyrnes are the regulatory erzlmes of their respective pathways and thus limit AIP production rates ll.

Malonyl-CoA would no longer inhibit fatty acid entry into the mitochondrion and p oxidation, so there might be a futile cycle of simultaneous fatty acid sytrthesis in the cytosot and fatty acid breakdown in mitochondria

f2. (a) The carnitine-mediated entry of fatty acids into mitochondria is the rate-limiting step in fatty acid oxidation. Carnitine deficiency slows fatty acrd oxidation; added carnitine increases the rate (b) A1l increase the metabolic need for fatty acid oxidation (c) Camitine deficiency might result from a deficrency of lysine, its precursor, or from a defect in one of the enzS.mesin the biosynthesis of carnitine. 13, Oxidation offats releasesmetabolic water; 1 4 L ofwater per kg of tripalmitoylglycerol (ignores the small contribution of glycerol to the mass) 14. The bacteria can be used to completely oxidize hydrocarbons to CO2 and H2O However, contact between hydrocarbons and bacterial enz5.rnesmay be difflcult to achieve Bacterial nutrients such as nitrogen and phosphorus may be limiting and inhibit growth. 15. (a) M,136; phenyiacetic acid (b) Even 16. Because the mitochondrial pool of CoA is small, CoA must be recycled from acetyl-CoA via the formation of ketone bodies This allows the operation of the B-oxidation pathway, necessary for energy production 17. (a) Glucose yields pytuvate via glycolysis, and pyr.uvate is the main source of oxaloacetate. Without glucose in the diet,

This djffers from Fig 16-Z in that it does not include czs-acorutate and isocitrate (between citrate and a-ketoglutarate), or succrnyl_ CoA, or acetyl-CoA (h) Establishing a quantitative conversron was essential to rule out a branched or other, more complex pathway.

(hapter 17 1. The fatty acid portion; the carbons in fatty acids are more re_ duced than those in glycerol. 2. @) a 0 x 105kJ (9 6 x 104kcal) (b) 48 days (c) 0.48 lb/day 3. The first step in fatty acid oxiclation is analogous to the conver_ sion of succinate to fumarate; the second step, to the converslon of fumarate to malate; the third step, to the conversion of malate to oxaloacetate 4. 7 cycles; the last releases 2 acetyl-CoA 5. (a) R-COO + ATp -+ acyl-AMp + pp1 Acyl-AMP + CoA --> acyl-CoA + AMp (b) Irreversible hydrolysis of pp, to 2p1 by cellular inorgaruc py'rophosphatase 6. czs-A3-dodecanoyl-CoA; it is converted to czs-A2-dodecanovlCoA, then p-hydroxydodecanoyl-CoA

[oxaloacetate] drops and the citric acid cycle slows. (b) Oddnumbered; propionate conversion to succinyl-CoA provides intermediates for the citric acid cycle and four-carbon precursors for gluconeogenesis 18. For the odd-chain heptanoic acid, B oxidation produces propionylCoA, which can be converted in several steps to oxaloacetate, a starting material for gluconeogenesis. The even-chain fatty acid cannot support gluconeogenesis, because it is entirelv oxidized to acetyl-CoA 19. B Oxidation of afluorooleate forms fluoroacetyl-CoA, which enters the citric acid cycle and produces fluorocitrate, a powerful inhibitor of aconitase. Inhibition of aconitase shuts down the citric acid cycle. Mthout reducing equivalents from the citric acid cycle, oxidative phosphorylation (ATP s}'nthesis) is fatally slowed 20. Ser to AIa: blocks p oxidation in mitochondria fatty acid synthesis, stimulates p oxidation.

Ser to Asp: blocks

21. Response to glucagon or epinephrine would be prolonged, giving a greater mobilization of fatty acids in adipocytes 22. Erz-FATt, having a more positive standard reduction potentral, is a better electron acceptor than NAD+, and the reaction is driven in the direction of fatty acyl-CoA oxidation This more favorable equilibrium is obtained at the cost of 1 ATP; only 1 b ATp are produced per FADH, oxidized in the respiratory chain (vs 2 5 per NADH) 23. 9 turns; arachidic acid, a 20-carbon saturated fatty acid, vields 10 molecules of acetyl-CoA, the last two formed in the ninth turn

t0Problems Solutions Abbreviated [ns-zil LJ

2. This is a coupled-reaction assay.The product of the slow transamination (pyruvate) is rapidly consumed in the subsequent "indicator reaction" catalyzed by iactate dehydrogenase, which consumes NADH Thus the rate of disappearance of NADH is a measure of the rate of the aminotransferase reaction. The indicator reaction is monitored by observing the decrease in absorption of NADH at 340 nm with a

24. See Fig 17-11 [3-'"C]Succinyl-CoA is formed, which gives rise to oxaloacetate labeled at C-2 and C-3 25. Phytanic acid -+ pnstanic acid -+ propionyl-CoA -+ -+ -+ succinylCoA -+ succinate -+ fumarate + malate All malate carbons would be labeled, but C-l and C-4 would have only half as much label as C-2 and C-3 26. ATP hydrolysis in the energy-requiring reactions of a cell takes up water in the reaction ATP + HzO + ADP + Pi; thus, in the steady state, there is no ze, production of H2O.

spectrophotometer 3. Alanine and glutamine play special roles in the transpod of amino groups from muscle and from other nonhepatic tissues,

27. Methylmaionyl-CoA mutase requires the cobalt-containing cofactor formed from vitamin B12

respectively, to the liver. 4. No The nitrogen in alanine can be transferred to oxaloacetate via transa[unation, to form aspartate.

28. Mass lost per day is about 0 66 kg, or about 140 kg in 7 months Ketosis could be avoided by degradation of non-essential body proteins to supply amino acid skeletons for gluconeogenesis

5 . l S m o l o f A l P p e r m o l e o f l a c t a t e ;1 3 m o l o f A T P p e r m o l e o f aianine, when nitrogen removal is included

29. (a) Fatty acids are converted to their CoA derivatives by enz)Tnes in the cytoplasm; the acyl-CoAs are then imported into mitochondria for oxidation Given that the researchers were using isolated mitochondria, they had to use CoA derivatives (b) Stearoyl-CoA was rapidly converted to 9 acetyl-CoA by the B-oxidation pathway AII intermediates reacted rapidly and none were detectable at signi-flcant levels. (c) T\vo rounds. Each round removes two carbon atoms, thus two rounds convert an 18-carbon to a l4-carbon fatty acid and 2 acetyl-CoA. (d) The K- is higher for the trans lsomer than for the cis, so a higher concentration of trans isomer is required for the same rate of breakdown Roughly speaking, the trans isomer binds less well than the cis, probably because differences in shape, even though not at the target site for the enzyme, affect substrate binding to the enzlme (e) The substrate for LCAD/\'LCAD builds up differently, dependi4g on the particular substrate; this is expected for the rate-Iimiting step in a pathway. (f) The kinetic parameters show that the trans isomer is a poorer

6. (a) Fasting resulted in low blood glucose; subsequent administration of the experimental diet led to rapid catabolism of glucogenic amino acids (b) Oxidative deamination caused the rise in NH3 levels; the absence of arginine (an intermediate in the urea cycle) prevented conversion of NH3 to urea; arginine is not slrlthesized in sufflcient quantities in the cat to meet the needs imposed by the stress of the experiment This suggests that arginine is an essential amino acid in the cat's diet (c) Ornithine is converted to arginine by the urea cycle. 7. H2O + glutamate + NAD* -+ a-ketoglutarate + NHI + NADH + H+ NHi + zATP + H2O + CO2 -+ carbamoyl phosphate + 2ADP + Pi + 3H* Carbamoyl phosphate 1- ornithine -+ citrulline + Pi + H+ Citrulline * aspartate + ATP -+ argininosuccinate + AMP + PP, + Hr Argininosuccinate + arginine + fumarate Fumarate t H2O -+ malate Malate t NAD+ + oxa-loacetate + NADH + H+ Oxaloacetate + glutamate -+ aspartate * a-ketoglutarate

substrate than the cis for LCAD, but there is little difference for VLCAD Because it is a poorer substrate, the trans isomet accumulates to higher levels than the cis (g) One possible pathway is shown below (indicating "inside" and "outside" mitochondria)

Arginine + H2O -+ urea * ornithine Elaidoyl-CoA (outside)

L , r , L r L r r ., r, . , l t r . r r L - t i rr . I L

elaidoyl-carnitils (inside)

elaidoyl-carnitine (outside)

L . L r r r r r r r L , , , , i l r r , r - ' r r - , el l l,a i d o y l - C o A 2 r \ ' L r r r i r ' . 1 r r " \ 1 1 : r r r ' r i > (inside)

5-t ran s -tetr adecenoyl-CoA (inside)

i-trans-tetradecanoic acid (inside)

r'

,

5-trans-tefi adecanoic acid (outside) (h) It is correct insofar as trans fats are broken down less efficiently than cis fats, and thus trans fats may "leak" out of mitochondria It is incorrect to say that trans fats are not broken down by cells; they are broken down, but at a slower rate than cis fats.

(hapter 18 1.O (a)

tl -OOC-CH2-C-COO-

Oxaloacetate

o (b)

ooc-cH2-cH2-c-coo-

a-Ketoglutarate

U

(c)

tl cH3-c-coo

o ll /-\ (d) ( )-cHr-c-coo\-./

Pyruvate

PhenYlPl.n-rvate

2 Glutamate+ CO2+ 4H2O+ 2NAD+ + 3ATP -+ 2 d-ketoglutarate+ 2NADH + ?H+ + urea * 2ADP * AMP + PPi + 2Pi Additional reactionsthat need to be considered: AMP + ATP -+ 2ADP 02 + 8H+ + 2NADH + 6ADP * 6P1-r 2NAD'+6ATP+8H2O H2O + PPi -+ 2P1* H+ Summingequations(1) through (4),

(1)

(2) (3) (4)

2 Glutamate+ COz+ 02 + ZADP + zPi-) 2 a-ketoglutarate+ urea + 3H2O+ 2ATP 8. The secondaminogroup introduced into urea is transferred from aspartate,which is generatedduring the transamination of glutamateto oxaloacetate,a reaction catalyzedby aspartate aminotransferaseApproximatelyone-halfof all the amino groups excretedas urea must passthrough the aspartate aminotransferasereaction,making this the most highly active aminotransferase. 9. (a) A personon a diet consistingonly of protein must use amino acidsas the principal sourceofmetabolic fuel Becausethe catabolismof amino acidsrequiresthe removalof nitrogen as urea' the processconsumesabnormallylarge quantitiesofwater to dilute and excrete the urea in the urine Furthermore,electrolltes in the "liquid protein" must be diluted with water and excreted If the daily water lossthrough the kidney is not balancedby a sufficientwater intake, a net lossof body water results (b) When considenngthe nutritional benefitsof protein, one must keep in mind the total amount of amino acidsneededfor protein sl,nthesisand the distribution of amino acidsin the dietary protein Gelatincontainsa nutritionally unbalanceddistribution of amino acids As large amountsof geiatin are ingested

-

-_t

S0lutions toproblems BS-2?l Abbreviated and the excess amino acids are catabolized, the capacity of the urea cycle may be exceeded, leading to ammonia toxicity. This is further complicated by the dehydration that may result from excretion oflarge quantities ofurea A combinatlon ofthese two factors could produce coma and death

18. A likely mechanism is:

10. Lysine and leucine fl.

(a) Phenylalanine hydroxylase; a low-phenylalanine diet (b) The normal route of phenylalanine metabolism via hydroxylation to tyrosine is blocked, and phenyalanine accumulates (c) Phenylalanine is transformed to phenylpyruvate by transamination, and then to phenyllactate by reduction The transamination reaction has an equilibrium constant of I 0, and phenylpytuvate is formed in significant amounts when phenylalanine accumulates (d) Because of the deficiency in production of tyrosine, which is a precursor of melanin, the pigment normally present in hair

12. Catabolism of the carbon skeletons of valine, methionine, and isoleucine is inpaired because a functronal methylmalonyl-CoA mutase (a coenz).rne B12 enzyme) is absenl The physiological effects of loss of this enzyme are described in Table l8-2 and Box 18-2

HOH

tl -ooc_c_c_H *NH3

H

Serine PLP

,,H n., e_{GD) -----7 T l,t -ooc_c_c_H lul *NH H

13. The vegan dret lacks vitamrn B,,, leading to the increase in homocysteine and methylmalonate (reflecting the deficiencres in methionine synthase and methylmalomc acid muLase, respectively) in individuais on the diet for several years Dairy products provide some vitamin Br2 in the lactovegetarian

CH

I

diet 14. The genetrc foms of pernicious anemia generally anse as a result of defects in the pathway ihat mediates absorption of dietary utamin Br, (see Box 17-2, p 6b8). Because dietary supplements are not absorbed in the rntestine, these conditions are treated by injecting supplementary B12 directty into the bloodstreant 15. The mechanism is identical to that for serine dehydrarase (see Fig. 18-20a, p 693) except that the extra methyl group of threonine is retained, yeelding a-ketobutvrate instead of pyruvate 16. (a) 15NH2-CO-'5NH2

(b) -ootac-cH2-cH2-rncoo

H

I -ooc-c-H I *NH CH HO

Glycine H

I -ooc-c-H * NIH t

H

"ri-T\',

tsNH (c) R-NH-C-15NH2

o ( d ) R-NH-c-15NH2

(e) No label tuNHo

(f)

t-

oo14c-c-cHo-14coo

t-

H

cc@@@@

17. (a)Isoleucine------+ II + IV + I + V + III + acetyl-CoA * propionyl-OoA (b) Step @ transaminatron, no analogous reaction, PLP; @ oxidative decarboxylation, analogous

thiolase reaction, CoA

PLP The formaldehyde (HCHO) produced in the second step reacts rapidly with tetrahydrofolate at the enz1me active site to produce M, N10-methylenetetrahydrofolate (see Fig 18 1Z) f9. (a) Transamination; no analogies; PLP (b) Oxidative decarboxylation; analogous to oxidative decarboxylatron of pyruvate to acetyl-CoA prior to entry into the citric acid cycle, and of aketoglutarate to succinyl-CoA in the citric acid cycle; NAD+, FAD, lipoate, and TPP (c) Dehydrogenation (oxidation); analogous to dehydrogenation of succinate to fumarate in the citric acid cycle, and of fatty acyl-CoA to enoyl-CoA in p oxidation; FAD (d) Carboxylation; no analogies in citric acid cvcle or p oxidation; ATP and biotin (e) Hydration; analogous to hydration of fumarate to malate in the citric acid cycle, and of enoylCoA to 3-hydroxyacyl-CoA in 6 oxidation; no cofactors (f) Reverse aldol reaction; analogous to reverse of crtrate synthase reactron in the citric acid cycle; no cofactors 20. (a) Leucine; valine; isoleucine (b) Cysteine (derived from cystine) Ifcysteine were decarboxylated as showr in Fig 18-6, it would yield H3N*-CH2-CH2-SH, whlch could be oxidized to taurine (c) The January 1957 blood shows significantly elevated levels of isoleucine, leucine, methionine, and valine; the January 1957 urine, signiicantly elevated isoleucine, leucine, taurine, and valine (d) All patients had high levels ofisoleucine, leucine, and

toProblems Abbreviated 5olutions [nS-rl

valine in bolh blood and urine, suggesting a defect in the breakdown ofthese amino acids Given that the unne also contained high levels of the keto forms of these three amino acids, the block in the pathway must occur after deamtnation but before dehydrogenation (as shown in Fig 18 28) (e) The model does not explain the high levels of methionine in biood and taurine in urine The lugh taurine levels may be due to the death ofbrain cells during the end stage ofthe disease However, the reason for high levels of methionine in blood are unclear; the pathway of methionine degradation is not linked with the degradation of branched-chain amino acids Increased methionine could be a secondary effect of buildup of the other amino acids It is important to keep in mind that the January 1957 samples were from an individual who was dying, so comparing blood and urine results with those of a healthy individual may not be appropriate (f) The followrng information is needed (and was eventually obtained by other workers): (1) The dehydrogenase activity is significantly reduced or missing in individuals wrth maple syrup urine disease (2) The disease is inherited as a single-gene defect (3) The defect occurs in a gene encoding all or part of the dehydrogenase (4) The genetic defect leads to production of inactive enzyrne

Chapter 19 l. Reacti,on (1) (a), (d) NADH; (b.), (e.) E-FMN; (c) NAD+/NADH and E-FMN/FMNH2

which causes the rate of electron transfer to increase This results in an increase in the H* gradient, 02 consumption, and amount of heat released 8. (a) The formation of ATP is inhibited. (b) The formation of ATP is tightly coupled to electron transfer: 2,4-dinitrophenol is an uncoupler of oxidative phosphorylation. (c) Oligomycin 9. Cltosolic malate dehydrogenase plays a key role in the transport of reducing equivalents across the inner mitochondrial membrane via the malate-aspartate shuttle f0. (a) Glycolysls becomes anaerobic (b) Oxygen consumption ceases (c) Lactate formation increases. (d) ATP synthesis decreases to 2 ATP/glucose 11. The steady-state concentration of P1in the cell is much higher than that of ADP. The P1released by ATP hydrolysis changes total [Pi] very little 12. The response to (a) increased [ADP] is faster because the response to (b) reduced pO2 requires protein slmthesis 13. (a) NADH is reoxidized via electron transfer instead of lactic acid fermentation. (b) Oxidative phosphorylation is more efficient (c) The high mass-action ratio of the ATP system inhibits phosphofructokinase- 1 14. Fermentation to ethanol could be accomplished in the presence of 02, which ls an advantage because strict anaerobic conditions are difficult to maintain The Pasteur effect is not observed, since the citric acid cycle and electron-transfer chain are inactive.

Reaction (2): (a), (d) E-FMNHz; (b), (e) Fe3*; (c) E-FMNIFMNH2

15. More-efficient electron transfer between complexes.

and Fe3*/Fe2*

16. (a) External medium: 4 0 x 10-B u; matrix: 2 0 x 10-8 u (b) tH*l gradient contributes 1 7 kJ/mol toward ATP synthesis (c) 21 (d) No (e) From the overali transmembrane potential f7. (a) 0.91 p.moVs' g (b) 5 5 s; to provrde a constant level ofATP,

Reaction(3):

(a), (d) fe2*; (b), (e) Q; (c) Fe3*/Fe2* and

Q/QHz 2. The side chain makes ubiquinone soluble in lipids and allows it to diffuse in the semifluid membrane 3. From the difference in standard reduction potential (AE'') for each pair of half-reactions, one can calculate AG'', The oxidation of succinate by FAD is favored by the negative standard freeenergy change (AG" 3 7 kJ/mol) Oxidation by NAD- would requrre a large, positive, standard free-energy change (AG'" 68 kJ/mol) 4. (a) Ail carriers reduced; CN- blocks the reduction of02 catalyzed by cytochrome oxidase (b) All carriers reduced; rn the absence of 02, the reduced carriers are not reoxidized (c) A11 carriers oxidized (d) Early carriers more reduced; later carriers more oxidized 5. (a) Inhibition ofNADH dehydrogenaseby rotenone decreases the rate of electron flow through the respiratory chain, which in turn decreasesthe rate ofATP production Ifthis reduced rate is unable to meet the organism's ATP requirements, the organism dies (tr) Antimycin A strongly inhibits the oxidation of Q in the respiratory chain, reducing the rate of electron transfer and leading to the consequences described in (a) (c) Because antrmycin A blocks alJ electron flow to oxygen, it is a more potent poison than rotenone, which blocks electron flow from NADH but not from FADH2 6. (a) The rate of electron transfer necessary to meet the ATP demand increases, and thus the P/O ratio decreases. (b) High concentrations ofuncoupler produce P/O ratios near zero The P/O ratio decreases, and more fuel must be oxidized to generate the same amount of ATP The extra heat released by this oxidation raises the body temperature (c) Increased activity of the respiratory chain in the presence of an uncoupler requires the degradation of additional fuel By oxidizing more fuel (including fat reserves) to produce the same amount of ATP, the body loses weight. When the P/O ratio approaches zero, the lack of ATP results in death 7. Valinomycin acts as an uncoupler Il combines with K- to form a complex that passes through the inner mitochondrial membrane, dissipating the membrane potential ATP synthesis decreases,

regulation of ATP production must be tight and rapid 18. 53 pmoVs ' g With a steady state [ATP] of 7 0 pmoVg, this is equivalent to 10 turnovers of the ATP pool per second; the reservoir would iast about 0 13 s 19. Reactive oxygen species react with macromolecules, including DNA If a mitochondrial defect leads to increased production of ROS, the nuclear genes that encode proto-oncogenes (pp 473, 474) can be damaged, producing oncogenes and leading to unregulated cell division and cancer 20. Different extents of heteroplasmy for the defective gene produce different degrees of defective mrtochondrial function. 21. The inner mitochondrial membrane is impermeable to NADH, but the reducing equivalents of NADH are transferred (shuttled) through the membrane indirectly: they are transferred to oxaloacetate in the cl,'tosol, the resulting malate is transported into the matrix, and mitochondrial NAD+ is reduced to NADH. 22. The citric acid cycle is stalled for lack of an acceptor of electrons from NADH Pyruvate produced by glycolysis cannot enter the cycle as acetyl-CoA; accumulated pyruvate is transaminated to alanine and exported to the liver. 23. Pyruvate dehydrogenase is located in mitochondria; glyceraldehyde 3-phosphate dehydrogenase in the c5,'tosol.The NAD pools are separated by the inner mitochondrial membrane. 24. Complete lack of glucokinase (two defective alleles) makes it impossible to carry out glycolysis at a sufficient rate to raise [ATP] to the threshold required for insulin secretion 25. Defects in Complex II result in increased production of ROS, damage to DNA, and mutations that lead to unreguiated cell division (cancer). It is not ciear why the cancer tends to occur in the midgut. 26. For the maximum photosynthetic rate, PSI (which absorbs light of 700 nm) and PSII (which absorbs light of 680 nm) must be operating simultaneouslY. 27 . The extra weight comes from the water consumed in the overall react10n.

Ies-z+lA b b r e v i aSt eodl u t i otnosP r o b l e m s 28. Purple sulfur bacteria use H2S as the hydrogen donor in photosynthesis. No 02 is evolved, because the single photosystem lacks the manganese-containing water-splitting complex 29.0.44 30. (a) Stops (b) Slows; some electron flow continues by the cyclic pathway. 31. During illumination, a proton gradtent rs established When ADP and P1are added, ATP slrrthesis is driven by the gradient, which becomes exhausted in the absence of Light. 32. DCMU blocks electron transfer between PSII and the first site of ATP production 33. In the presence of venturicidin, proton movement through the CF.CFI complex is blocked, and electron flow (oxygen evolution) continues only until the free energy cost of pumping protons against the risrng proton gradient equals the free energy available in a photon DNP, by dissipating the proton gradient, re-

sumed (2) In the presenceoflight, glucoseis producedand is metabolizedby cellular respirationto produceATq with changes in the levelsof phosphorylatedintermediates.(3) In the presenceof light, ATP is producedand other phosphorylatedintermediatesare consumed (e) Light energyis used to produce ATP (as in the Emersonmodel) ond is usedto produce reducing power (as in the Rabinowitchmodel) (f) The approximatestoichiometryfor photophosphorylation(Chapter 19) is that 8 photons yield 2 NADPH and about 3 ATP TWoNADPHand 3 ATP are required to reduce I CO2(Chapter20). Thus, at a minimum, 8 photonsare required per CO2moleculereduced This is in good agreementwith Rabinowitch'svalue. (g) Becausethe energz of light is usedto produceboth NIP and NADPH,eachphoton absorbedcontdbutesmore than just 1 ATP for photosynthesis.The processof energyextraction from light is more efficient than Rabinowitchsupposed,and plenty of energy is availablefor this process-even with red light.

stores electron flow and oxygen evolution. 34. (a) 56 kJ/mol (b) 0 29 V

Chapter 20

35. From the difference ir reduction potentials, one can calculate that AG'' = 15 kJ/mol for the redox reaction. Figure 19-46 shows that the energy of photons in any region of the visrble spectrum is more than sufficient to dnve this endergonic reactron

l. Within subcellular organelles, concentrations of speciflc enzyrnes and metabolites are elevated; separate pools of cofactors and intermediates are maintained; regulatory mechanisms affect only one set of enz)tnes and pools.

36. 1 35 x 10-77; the reaction is highly unfavorable! In ch-loroplasts, the input of light energy overcomes this barrier 37. -920 kJ/mol

2. This observation suggests that ATP and NADPH are generated ln the light and are essential for CO2 f,xation; conversion stops as the suppiy ofATP and NADPH becomes exhausted Furthermore, some enzynes are switched off in the dark

38. No. The electrons from H2O flowto the artiflcial electron acceptor Fe3*, not to NADP+ 3 9 . A b o u l o n c e e v e r y 0 . 1 s ; I i n 1 0 si s e x c i t e c l 40. Light of 700 nm excites PSI but not PSII; electrons flow from P700 to NADP*, but no electrons flow from P680 to replace them When light of680 nrn excites PSII, electrons tend to flow to PSI, but the electron carriers between the two photosystems quickly become completely reduced 41. No The excited electron from P700 returns to reflll the electron "hole" created by illumination PSII is not needed to supply electrons, and no 02 is evolved from H2O NADPH is not formed, because the excited electron retums to P700. 42. (a) (1) The presence of Mg2* supports the hypothesis that chlorophyll is directly involved in catalysis of the phosphorylation reaction: ADP + P, -+ AfP (2) Many enzyrnes (or other proteins) that contain Mg2+ are not phosphorylating enzyrnes, so the presence of Mg2+ in chlorophyll does not prove its role in phosphorylation reactions (3) The presence of Mg2+ is essential to chlorophyll's photochemical properties: light absorptron and electron transfer (b) (1) EnzS.mescatalyze reversible reactions, so an isolated enzyme that can, under certain laboratorv conditions, catalyze removal of a phosphoryt group could probably, under different conditions (such as in celts), catalyze addition of a phosphoryl group. So it is plausible that chlorophyll could be involved in the phosphorylation of ADP (2) There are two possible explanations: the chlorophyll protein is a phosphatase only and does not catalyze ADP phosphorvlation under cellular conclitions, or the crude preparation contains a contaminating phosphatase activity that is unconnected to the photosynthetic reactions (3) It is likely that the preparation was contaminated with a nonphotos)'nthetic phosphatase activity. (c) (l) This llght inhibition is what one would expect if the chlorophyll protein catalyzed the reaction ADP + Pi + light --) ATp Without light, the reverse reaction, a dephosphorylation, would be favored. In the presence of light, energy is provided and the equilibrinm would shift to the right, reducing the phosphatase activity. (2) This inhibition must be an artifact of the isolation or assay methods (3) It is unlikely that the crude preparation methods in use at the time preserved intact chloroplast membranes, so the inhibition must be an artifact (d) (1) In the presence oflight, ATp rs synthesized and other phosphorylated intermediates are con-

3. X is 3-phosphoglycerate;Y is ribulose 1,5-bisphosphate. 4. Ribulose 5-phosphate kinase, fructose 1,6-bisphosphatase, sedoheptulose 1,7-bisphosphatase,and glyceraldehyde 3-phosphate dehydrogenase; all are activated by reduction of a critical disulfide bond to a pafu of sulfhydryls; iodoacetate reacts irreversibly with free sulfhydryls. 5, To carry out the disulfide exchange reaction that activates the Calvin cycle enzlmes, thioredoxin needs both of its sulfhydryl groups 6. Reductive pentose phosphate pathway regenerates ribulose 1, 5-bisphosphate from triose phosphates produced during photosyrrthesis. Oxidative pentose phosphate pathway provides NADPH for reductive blosynthesis and pentose phosphates for nucleotide synthesis 7. Both types of "respiration" occur in plants, consume 02, and produce CO2. (Mitochondrial respiration also occurs in animals ) Mitochondrial respiration occurs continuously; electrons derived from various fuels are passed through a chain of carriers in the inner mitochondrial membrane to 02 Photorespiration occurs in chloroplasts, peroxisomes, and mitochondria Photorespiration occurs during the daytime, when photosynthetic carbon fixation is occurring; mitochondrial respiration occurs prrmarily at night, or during cloudy days. The path of electron flow in photorespiration is shown in Fig 20-21; that for mitochondrial respiration, in Fig 19-16 8. This hypothesis assumes directed evolution, or evolution with a purpose-ideas not generally accepted by evolutionary biologists Other processes, such as burning fossil fuels and global deforestation, affect the global atmospheric composition Ca plants, by fixing CO2 under conditions when rubisco prefers 02 as substrate, also contdbutes to setting atmospheric CO2/O2ratios. 9. (a) Without production of NADPH by the pentose phosphate pathway, cells wor,rld be unable to synthesize lipids and other reduced products (b) Without generation ofribulose 1,5-bisphosphate, the Calvin cycle is effectivelv blocked 10. In maize, CO2 is flxed by the Ca pathway elucidated by Hatch and Slack, in which PEP is carboxylated rapidly to oxaloacetate (some of which undergoes transamination to aspartate) and reduced to malate Only after subsequent decarboxylation does the CO2 enter lhe Calvin cycle

Solutions toProblems Abbreviated Es-zil

11. Measure the rate of fixation of raC carbon dioxide in the }ight (daytime) and the dark. Greater fixation in the dark identifies the CAM plant One could also determine the titratable acidity; acids stored in the vacuole during the night can be quantified in thls way. 12. Isocitrate dehydrogenase reaction 13. Storage consurnes I mol of ATP per mole of glucose 6-phosphate; this represents 3 3% of the total ATP available from glucose 6-phosphate metabolism (i e , the efflciency of storage is 96.7%). 14. [PP,] is high in the cytosol because the cl4osol iacks inorganic pytophosphatase. f5. (a) Low [P1]in the cltosol and high [triose phosphate] in the chloroplast (b) High [triose phosphate] in the cy4osol 16. 3-Phosphoglycerate is the primary product of photosynthesis; [P,] rises when light-driven s}'nthesis of ATP from ADP and Pi slows f7. (a) Sucrose + (glucose)z -+ (glucose),a1 + fructose (b) Fructose generated in the synthesis of dextran is readily imported and metabolized by the bacteria. 18. Species 1 is Ca; species 2, C3. 19. (a) In peripheral chloroplasts (b) and (c) In central sphere 20, (a) By analogy to the oxygenic photos5.nthesis carried out by plarLts (H2O + CO2 -+ glucose + O), the reaction wouid be HrS + 02 + CO2 -+ glucose + H2O + S This is the sum of the reduction of CO2 by HzS (HzS + CO2 -+ giucose + S) and the energy input (H2S + Oz -r S + H2O) (b) The H2S and CO2 are produced chemically in deep-sea sediments, but the 02, like the vast majority of 02 on Earth, is produced by photos},nthesis, which is driven by light energy. (c) In Robinson et al.'s assay, 3H labels the C-l of ribulose 1,5-bisphosphate, so reaction wrth COz yields one molecule of l3H]3-phosphoglycerate and one molecule of unlabeled 3-phosphoglycerate; reaction with 02 produces one molecule of [3H]2-phosphoglycolateand one molecule of unlabeled 3-phosphoglycerate. Thus the ratio of [3H]3phosphoglycerate to [3H]2-phosphoglycolate equals the ratio of carboxylation to oxygenation. (d) If the 3H labeled C-5, both oxygenation and carboxylation would yield [3H13-phosphoglycerate and it would be impossible to distinguish which reaction had produced the labeled product; the reaction could not be used to measure O.

lco'zl o ooo38 : 0.0019 (e) SubstitutinC inLo toi YcarboxyJation : Yoxygenation Ycarboxylation

O-

lCO"l*

divpc

tu2l

: (8.6X0.0019) : 0.016

Yoxygenation

Therefore, the rate of oxygenation would be roughly 60 times the rate of carboxylationl (f) If terrestrial plants had O : 8 6, carboxylation would occur at a much lower rate than oxygenation. This would be extremely inefficient, so one would expect the rubisco of lerrestrial plants to have an O substantially higher than 8.6. In fact, O values for land piants vary between 10 and 250 Even with these values, the expected rate of the oxygenation reaction is still very high (g) The rubisco reaction occurs with 13CO2 CO2 as a gas. At the same temperature, molecules diffuse 12CO2 13CO2 more slowly tharL the lighter molecules, and thus will enter the active site (and become lncorporated into subt'COr. strate) more slowly than thl For the relationship to be truly slmbiotic, the tube worms must be getting a substantial amount of their carbon from the bacteria. The presence of rubisco in the endosl'rnbionts simply shows that they afe capable of chemosynthesis, not that they are supplyirg the host with a significant fraction of its carbon. On the other hand, showirg that the r3C:r2C ratio in the host is more smilar to that in the endosr.rynbiont than that in other marine ar-rimalsstrongly suggests that the tube worms are gettiru the majority of their carbon from the bacteria.

Chapter 21 l. (a) The 16 carbonsof palmitateare derivedfrom 8 acetylgroups l4clabeled acetyl-CoAgivesrise of 8 acetyl-CoAmoleculesThe to malonyl-CoAlabeledat C-l and C-2. (b) The metabolicpool of malonyi-CoA,the sourceof aJIpalmitate carbonsexcept C-16 raCand C-15,doesnot becomelabeledwith small amountsof palmitateis formed. Hence,orily [15,16-raC] labeledacetyl-CoA. 2. Both glucoseand fructoseare degradedto plruvate in glycolysis Py.ruvateis convertedto acetyl-CoAby the pyruvate dehydrogenasecomplex.Someof this acetyl-CoAentersthe citric acid cycle, which producesreducing equivaients(NADH and NADPH). Mitochondrialelectrontransfer to 02 yields ATP 3. 8 Acetyl-CoA + 15ATP+ 14NADPH+ 9H2O-+ palmitate + 8CoA + 15ADP+ 15Pi + 14NADP- + 2H4. (a) 3 deuteriumsper palmitate;all locatedon C-16;all other two-carbonunits are derivedfrom unlabeledmalonyl-CoA (b) 7 deuteriumsper palmitate;locatedon all eerera-numbered carbonsexceptC-16. 5. By using the three-carbonunit malonyl-CoA,the activatedform of acetyl-CoA(recall that malonyl-CoAsy'nthesisrequiresATP), metabolismis driven in the direction of fatty acid synthesisby the exergonicreleaseof CO2 is carboxylationof 6. The rate-limiting step in fatty acid s5mthesis acetyl-CoA,catalyzedby acetyi-CoAcarboxylaseHigh [citrate] and [isocitrate]indicate that conditionsare favorablefor fatty an active citric acid cycle is providing a plentifuJ acid s5'rLthesis: supply of ATP,reducedpyridine nucleotides,and acetyl-CoA. Citrate stimulates(increasesthe 7^* of) acetyl-CoAcarboxylase(a) Becausecitrate binds more tightly to the fllamentous form of the enzyme(the activeform), high [citrate] drivesthe protomer # flIamentequilibriumin the direction of the activeform (b) In contrast,palmitoyl-CoA(the end product of fatty acid synthesis)drives the equilibriumin the direction of the inactive (protomer) form Hence,when the end product accumulates,the biosyntheticpath of fatty acid sS,rrthesis s1ows. + ADP + ?. (a) Acetyl-CoA66s-l-AIP f CoA1"ytl-+ acetyl-CoA1.r1) (b) 1 ATP per acetyl group (c) Yes P1f CoA6mit; 8. The doublebond in palmitoleateis introduced by an oxidation catalyzedby fatty acyl-CoAdesaturase,a mixed-functionoxidasethat requires02 as a cosubstrate 9. 3 Palmitate + glycerol + TATP+ 4H2O-) tripalmitin + TADP + 7Pi + 7H10. in adult rats, storedtriacyl$ycerols are maintainedat a steadystate level through a balanceof the rates of degradationand biosyrrthesis.Hence,the triacylglycerolsof adipose(fat) tissue are constantlyturned over,which explainsthe incorporationof tnOlabel from dietary glucose. 11. Net reaction: Dihydroxyacetonephosphate+ NADH * palmitate + oleate + SATP+ CTP + choline + 4H2O-+ r NAD* + 2AMP + ADP + H+ + CMP+ 5Pi phosphatidylcholine 7 ATP per moleculeof phosphatidylcholine 12. Methioninedeflciencyreducesthe Ievelof adoMet,which is required for the de novo slrrthesisof phosphatidylcholine.The salvagepathwaydoesnot employadoMet,but usesavailable choline.Thus phosphatidyicholinecan be synthesizedeven when the diet is deflcientin methionine,as long as cholineis available 13. 14Clabel appearsin three placesin the activatedisoprene:

'NCII, \tc-tncHo-cHo-

,NCrt"

5otutions toprobtems bt-rql Abbreviated 14. (a) ATP (b) UDP-glucose (c) CDP-ethanolamine (d) UDPgalactose (e) Fatty acyl-CoA (f) S-Adenosylmethionine (g) Malonyl-CoA (h) A3-isopentenyl pyrophosphate Lycopene (C-40)

15. Linoleate is required in the synthesis of prostaglandils Animals are unable to transform oleate to linoleate, so linoleate is an essential fatty acid Plants can transform oleate to linoleate, and they pror,rde ammals with the required l_rnoleate(see Fig 21-12)

t,, bend ends around for cyclization J

16. The rate-determinng step in the biosynthesis of cholesterol is the symthesisof mevalonate, catalyzed by hydroxlnnethylglutaryl-CoA reductase This enzl'rne is allosterically regulated by mevalonate and cholesterol derivatives High intracellular [cholesterol] also reduces transcription of the gene encoding HMG-CoA reductase

J

17. When cholesterol levels decline because of treatment with a statin, cells attempt to compensate by increasing expression of the gene encoding HMG-CoA reductase The statins are good competitive inhibitors of HMG-CoA reductase activitv nonetheless and reduce overall production of cholesterol 18. Note: There are several plausible alternatives that a student might propose in the absence of a detailed knowledge of the literature on this enz5rme Thzo\ase reaction begins with nucleophilic attack of an active-site Cys residue on the f,rst acetyl-CoA substrate, displacing -S-CoA and forming a covalent thioester lnk between Cys and the acetyl group A base on the erzyrne then extracts a proton from the methyl group of the second acetyl-CoA, leaving a carbanion that attacks the carbonyl carbon of the thioester formed in the first step The sulfhydryl of the Cys residue is displaced, creating the product acetoacetyl-CoA HMG-CoA sAnthase reaction: begins in lhe same way, with a covalent thioester link formed between the enzyme's Cys residue and the acetyl group of acetyl-CoA, with displacement of the -S-CoA. The -S-CoA dissociatesas CoA-SH, and acetoacetylCoA binds to the enzlrne A proton is abstracted from the methyt group of the enz)..rnelinked acetyl, forming a carbamon that attacks the ketone carbonyl of the acetoacetyl-CoA substrate The carbonyl is converted to a hydroxyl ion in this reaction, and this is protonated to create -OH The thioester link with the enzyme is then cleaved hydroly'tically to generate the HMG-CoA product HMG-CoA reductLrse reacti,on two successive hydride rons derived from NADPH first displace the -S-CoA, and then reduce the aldehyde to a hydroxyl group 19. Statins inhibit HMG-CoA reductase, an enzyme in the pathway to the s5'nthesis of activated isoprenes, whrch are precursors of cholesterol and a wide range of isoprenoids, including coenz),Tne Q (ubiquinone) Hence, statins might reduce the levels of coenz5.meQ available for mitochondrial respiration Llbiquinone is obtained rn the diet as well as by direct biosynthesis, but it is not yet clear how much is required and how well dietary sources can substitute for reduced synthesis Recluctions in the levels of particular isoprenoids may account for some side effects of statins 20. (a)

Astuanthin

cvclize

F-Carotene (C-40)

(e) StepsC through@. The enzymecan convert IPP and DMAP to geranylgeranylplrophosphate,but catalyzesno further reactionsrn the pathway,as confirmedby resultswith the other subshates (f) Strains 1 through 4lack crtE and havemuch lower astaxanthinproduction than strains5 through 8, all of which overexpresscl"tE Thus, overexpressionof crfE leadsto a substantialincreasein astaxanthinproduction Wild-typeE colz has somestep @ activity,but this conversionof farnesylpyrophosphateto geranylgeranylpyrophosphatels stronglyratelimiting. (g) IPP isomerase.Comparingstrains5 and 6 shows that addingzsp, , which cata\yzessteps@ and @, has little effect on astaxanthinproduction,so thesestepsare not rate-timiting. However,comparingstrains5 and 7 showsthat addingzde substantiallyincreasesastaxanthinproduction, so IPP isomerase must be the rate-limiting step when crfE is overexpressed. (h) A low (+) level, comparableto that of strains5, 6, and 9 Without overexpressionof zdz,production of astaxanthinis limited by low IPP isomeraseactivity and the resulting limited supply of IPP

Chapter 22 l. In their symbiotic relationship with the plant, bacteria supply amrnonium ion by reducing atmospheric nitrogen, which requires large quantities ofATP 2. The transfer of nitrogen from NH3 to carbon skeletons can be catalyzed by (1) glutamine sj4rthetase and (2) glutamate dehydrogenase The latter enzyrne produces glutamate, the amino group donor in all transamination reactions, necessary to the formation of amino acids for protein syrrthesis. 3. A link between en4nne-bound PLP and the phosphohomoserine substrate is fust formed, with rearrangement to generate the ketimine at the a carbon ofthe substrate This activates the B carbon for proton abstraction, leading to displacement of the phosphate and formation of a double bond between the B and 7 carbons A rearrangement (beginning with proton abstraction at the pyridoxal carbon adjacent to the substrate amino nitrogen) moves the double bond between the c and B carbons, and converts the ketimine to the aldimine form of PLP Attack of water at the B carbon is then facilitated by the linked pye'idoxal, followed by hydrolysis of the imine link between PLP and the product, to generate threonine.

(b) Head-to-head There are two ways to look at this First, the "tail" of geranylgeranyl py'rophosphate has a branched climethyl structure, as do both ends of phytoene, Second, no free -OH is forrned by the release of PP1,inclicating that the two -O-@-@ "heads" are Linked to forrn phytoene (c) Four rounds of dehy-

4. In the mammalian route, toxic ammonium ions are lransformed to glutamine, reducing toxic effects on the braln 5, Glucose + 2CO2 + 2NH3 -+ 2 aspartate + 2H+ + 2H2O

drogenation convert four single bonds to double boncls. (d) No A count of single and double bonds in the reaction below shows that one double bond is replaced by two single bonds-so, there i s n o n e t o x i d a t i o no r r e d u c t i o n

6. The amino-terminal glutaminase domain is quite simtlar in all glutamine amidotransferases A drug that targeted this active site would probably inhibit many enzymes and thus be prone to producing many more side effects than a more specific

solutions to Problems Abbreviated Et-rl inhibitor targeting the unique carboxyl-terminal synthetase active site. 7. Ifphenylalaninehydroxylaseis defective,the biosS,rLthetic route to tyrosine is blocked and tyrosine must be obtainedfrom the diet 8. In adoMets5'nthesis,triphosphateis releasedfrom ATP Hydrolysis of the triphosphaterendersthe reactionthermod5'namicaily more favorabie. 9. If the inhibition of glutaminesynthase\.,rerenot concerted,saturating concentrationsof histidine would shut down the enz;,me and cut off production of glutamine,which the bacteriumneeds to synthesizeother products. 10. Folic acid is a precursor of tetrahydrofolate(Fig 18-16), required in the biosynthesisofglycine (Fig 22-12), a precursorof porphyrins.A folic acid deflciencythereforeimpairs hemoglobin s5'nthesis. ll. For glgci,neau,rotrophs: adenineand guanine;/or gLutami,ne auaotrophs: adenine,guanine,and c)'tosine;/or aspa?-tate auaotrophs: adenine,guanine,cltosine, and uridine. f2. (a) SeeFigure 18-6, step @, for the reactionmechanismof amino acid racemization.The F atom of fluoroalanineis an excellent leavinggroup. Fluoroalaninecausesirreversible(covalent) hhibition of alanineracemase.One plausiblemechanismis (Nuc denotesany nucieophilicamino acid side chain in the enzlme activesite):

-OO14C_ 14CH2_14CH2 _ 14COOSuccinate

Y

-oo14c \,/

H

14c:14c

,/\ H

14coo-

Fumarate

II

J

OHH -oo14c - 14c-14c - 14cooHH Malate

II

J

OH l -oo14c-14cl l-14c-14coo-

il

r-,8-@

H

HB

l-r -ooc-c:cH" t-

Ng.

Oxaloacetate I Jtransamination

-ooc

N

N ll CH

CH

HO

fr",

+

HO CH, N H

-OO14C-14C -14CH2 - 14COOI H Aspartate

I

J

o

@

H Nu'c

-ooc-c-cH" tN

H I

CH

I

HO

o CH, (b) Azaserine(seeFig. 2248) is an analogof glutamine.The diazoacetylgroup is higNy reactiveand forms covalentbonds with nucleophilesat the active site of a glutamine amidotransferase. 13. (a) As shownin Figure 18-16,p-aminobenzoateis a component of AP,lflO-methylenetetrahydrofolate, the cofactorinvolvedin the transfer of one-carbonunits. (b) In the presenceof sulfanilamide,a structural ana.logofp-aminobenzoate,bacteriaare unable to synthesizetetrahydrofolate,a cofactornecessaryfor convertingAICAR to FAICAR;thus AICAR accumulates.(c) The competitive hlLibition by sulfanilamide of the enz5'rneinvolved in tetrahydrofolatebiosynthesisis overcomeby the addition of excesssubstrate(p-aminobenzoate). 14. The t'C-labeledorotate arisesfrom the foilowing pathway (the first three stepsare part of the citric acid cycle):

H

II

J

o --*,-u._fi/

H14AH

l , r l l -'rl

o

ozv\N,/"\14COO-l H Orotate

Er-rqlA b b r e v i aSt eodl u t i otnosP r o b l e m s 15. OrgarLisms do not storenucleotidesto be usedas fuel, and they do not completelydegradethem, but rather hydrolyzethem to releasethe bases,which can be recoveredin salvagepathways.The iow C:Nratio of nucieotidesmakesthem poor sourcesof energy. 16. Theatmentwith allopurinolhastwo consequences. (1) It inhibits conversionof hypoxanthine to uric acid, causingaccumulationof hypoxanthine,which is more solubleand more readilyexcreted; this alleviatesthe clinical problemsassociatedwith AMP degradation (2) It inhibits conversionof guanineto uric acid, causingaccumulation of xanthine, which is less solublethan uric acid; this is the sourceof xanthinestones.Becausethe amountof GMPdegradation is low relative to AMP degradation,the kidney damagecaused by xanthinestonesis lessthan that causedby untreatedgout 17. 5-Phosphoribosyl-l-pyrophosphate; this is the first NH3acceptor in the purine biosyntheticpathway. f8. (a) The a-carboxylgroup is removedand an -OH is addedto the y carbon (b) BtrI has sequencehomologywith acyl carrier proteins.The molecularweight of BtrI increaseswhen incubated under conditionsin which CoA could be addedto the protein. Adding CoA to a Ser residuewould replacean -OH (formula weight (FW) 17) with a 4'-phosphopantetheinegroup (see Fig.21-5, p. 809).This grouphasthe formulaClrH2rN2OzPS (FW356).Thus,11,182- 17 + 356 :12,151,whlchisvery closeto the observedM.of 12,153(c) The thioestercouldform with the o-carboxylgroup. (d) In the most commonreactionfor removingthe a-carboxylgroup of an amino acid (seeFig. l&-6c, p 679), the carboxylgroup must be free. Furthermore,it is difficult to imaginea decarboxylationreaction starting with a carboxyl group in its thioesterform. (e) 12,240- 12,281:41, close to lhe M, of CO2(44) Giventhat BtrK is probablya decarboxylase,its most likely structure is the decarboxvlatedform: +r) H"N S t BtrI (t) 12,370- 12,240: 130 Glutamicacid (C5HeNO4tM,I4T), minus the -OH (FW 17) removedin the glutamylationreaction, leavesa glutamyl group of FW 130;thus, 7-glutamylatingthe moleculeabovewould add 130to its M. BtrJ is capableof 7-glutamylatingother substrates,so it may 7-glutamylatethe structure above.The most likely site for this is the free aminogroup, giving the following structure:

o

o

(e)

ft' -o-"]--.^.fo d

d+BtrI

frn,

-o->,,\---zo , "tt | Arp tlp 6 io, Glutamate

coo

/ BtrK ,

NADH Antibiotic

Glutamate + BtrI OH d1q,r-t "l

a-tZO

o-A,,tibioti.

Chapter 23 l. They are recognizedby two different receptors,typically found in different cell types, and are coupledto different downstream effects. 2. Steady-statelevelsof ATP are maintainedby phosphorylgroup transfer to ADP from phosphocreatine.1-Fluoro-2,4-dinitrobenzeneinlLibitscreatinekinase. 3. Ammoniais very toxic to nervoustissue,especiallythe brain ExcessNH3is removedby transformationof glutamateto glutamine, which travelsto the liver and is subsequentlytransformed to urea. The additionalglutaminearisesfrom the transformation of glucoseto d-ketoglutarate,transaminationof a-ketoglutarate to $utamate, and conversionof glutamateto glutamlne. 4. Glucogenicamino acidsare used to make $ucose for the brain; others are oxidizedin mitochondriavia the citric acid cycle. 5. From glucose,by the followingroute: Glucose-+ dihydroxyacetone phosphate(in glycolysis);dihydroxyacetonephosphate* NADH + H+ -+ glycerol 3-phosphate+ NAD+ (glycerol 3phosphatedehydrogenasereaction) 6, (a) Increasedmuscularactivity increasesthe demandfor ATP, which is met by increased02 consumption.(b) After the sprint, lactate producedby anaerobicglycolysisis convertedto glucose and glycogen,which requiresATP and therefore02. 7. Glucoseis the primarv r'-.:' ,, iire brain. TPP-dependentoxidative decarboxr'lruonof pytuvate to acetyl-CoAis essentialto nor..pleteL Juosemetabolism. 8. 190m 9. (a) Inactivationprovidesa rapid meansto changehormoneconcentrations.(b) Insulin level is maintainedby equalrates of synthesisand degradation.(c) Changesin the rate of releasefrom storage,rate of transport, and rate of conversionfrom prohormone to activehormone 10, Water-solublehormonesbind to receptorson the outer surface of the cell, triggeringthe formation of a secondmessenger (e.g.,cAMP) inside the cell. Lipid-solublehormonescan pass through the plasmamembraneto act on target moleculesor receptorsdirectly. ff. (a) Heart and skeleta"lmuscleiack glucose6-phosphatase. Any glucose6-phosphateproducedenters the glycolytic pathway, and under O2-deficientconditionsis convertedto lactatevia p,'mvate. (b) In a "fuht or flight" situation,the concentrationof glyco\'tic precursorsmust be high in preparationfor muscular activity.Phosphorylatedintermediatescannotescapefrom the cell, becausethe membraneis not permeableto chargedspecies, and glucose6-phosphateis not exported on the glucose

A b b r e v i aS t eodl u t i otnosP r o b l e m sA S - 2 9

transporter The liver, by contrast, must release the glucose necessary to maintain blood glucose level; glucose is formed from glucose 6-phosphate and enters the bloodstream 12. (a) Excessive uptake and use of blood glucose by the liver, leading to hypoglycemia; shutdown of amino acid and fatty acid catabolism (b) Little circulating fuel is available for ATP requirements. Brain damage results because glucose is the main source of fuel for the brain. 13. Thyroxine acts as an uncoupler of oxidative phosphorylation Uncouplers lower the P/O ratio, and the trssue must increase respiration to meet the normal ATP demands Thermogenesis could also be due to the increased rate of ATP utilization bv the thyroid-stimulated tissue, as increased ATP demands are met by increased oxidative phosphorylation and thus respiration 14. Because prohormones are inactive, they can be stored in quantity in secretory granules. Rapid activation is achieved by enzymatic cleavage in response to an appropriate signal. 15. In animals, glucose can be synthesized from many precursors (see Fig 14-15) In humans, the principal precursors are glycerol from triacylglycerols and glucogenic amino acids from protein. 16. The ob/ob monse, which is initially obese, will iose weight The OB/OB mouse will retain its norrnal body weight 17. BMI : 39 3 For BMI of 25, weight must be 75 kg; must lose 43 kg - 95 lbs 18. Reduced lnsulin secretion Valinomycin has the same effect as opening the K- channel, allowing K- exit and consequent hyperpolarization 19. The liver does not receive the insulin message and therefore continues to have high levels of glucose 6-phosphatase and gluconeogenesis, increasing blood glucose both during a fast and after a glucose-containing meal The elevaled blood glucose triggers insulin release from pancreatic B cells, hence the high level of insulin in the blood. 20. Some things to consider: What is the frequency of heart attack attributable to the drug? How does this frequency compare wrth the number of individuals spared the long-term consequences of type 2 diabetes? Are olher, equally effective treatment options, with fewer adverse effects, available? 21. Mthout intestinal glucosidase activity, absorption of glucose from dietary glycogen and starch is reduced, blunting the usual rise in blood glucose after the meal The undigested oligosaccharides are fermented by bacteria in the large intestine, and lhe gases released cause intestinal discomfort 22. (a) Closing the ATP-gated K- channel would depolarize the membrane, leading to increased insulin release (b) T\rpe 2 diabetes results from decreased sensitilety to insulin, not a deficit of insulin production; increasing circulating insulin levels wrll reduce the slrnptoms associated wrth this disease (c) Individuals with type I diabetes have deficient pancreatic B cells, so glyburide will have no beneficial effect (d) Iodine, llke chlonne (the atom it replaces in the labeled glyburide), is a halogen, but it is a larger atom and has slightly different chemical properties It is possible that the iodinated glyburide would not bind to SUR If it bound to another molecule instead, the experiment would result in cloning of the gene for this other, incorrect protein (e) Although a protein has been "purified," the "purified" preparation might be a mixture of several proteins that co-purify under those experimental conditions In this case, lhe amino acid sequence could be that of a protein that co-purifies with SUR. Using antibody binding to shoru that the peptide sequences are present in SUR excludes this possibility (f) Although the clonecl gene does encode the 25 amino acid sequence found in SUR, it could be a gene that, coincidentally, encodes the same sequence in another protein In thrs case, this other gene would most likely

be expressed in different cells than the St/fi gene The mRNA hybridization results are consistent with the putative SU/? cDNA actualiy encoding SUR. (g) The excess unlabeled glyburide competes with labeled glyburide for the binding site on SUR. As a result, there is signiflcantly less binding of labeled glyburide, so lttle or no radioactivity is detected in the 140 kDa protein (h) In the absence of excess unlabeled glyburide, Iabeled 140 kDa protein is found only in the presence of the putative SUR cDNA. Excess unlabeled glyburide competes with the labeled glyburide, and no l25llabeled 140 kDa protein is detected. This shows that the cDNA produces a glyburide-binding protein of the same molecular weight as SUR-strong evidence that the cloned gene encodes the SUR protein (i) Several additional steps are possible, such as: (1) Express the putative SI/,? cDNA in CHO (Chlnese hamster ovary) cells and show that the transformed cells have ATP-gated K+ channel activity. (2) Show that HIT cells with mutations in the putative SUfi gene lack ATPgated K+ channel activity. (3) Show that experimental animals or human patients wrth mutations in the putative SUfi gene are unable to secrete insulin

24 Chapter 1. 6.1 x 104nm; 290 times longer than the T2 phage head 2. The number of A residues does not equal the number of T residues, nor does the number of G equal the number of C, so the DNA is not a base-paired double helix; the M13 DNA is single-stranded. 55,200L ; k : 51,900 : 576 bp. x acids 192 amino 4. The exons contain 3 bp/amino acid The remairung 864 bp are in introns, possibly in a leader or signal sequence, and/or in other noncoding DNA 3. M,:3

8 x 1 0 8 ;l e n g t h = 2 0 0 , p t mL; k s :

5. 5,000 bp (a) Doesn't chartge;Lk cannot change without breaking and re-forming the covalent backbone of the DNA (b) Becomes undefined; a circular DNA with a break in one strand has, by definition, no .Lk (c) Decreases; in the presence of ATP, gyrase underwinds DNA (d) Doesn't change; this assumes that neither of the DNA strands is broken in the heating process 6. For Lk to remain unchanged, the topoisomerase must introduce the same number of positive and negative supercoils 7. o : -0 067; >70o/o ProbabiLitY 8. (a) Undefined; the strands of a nicked DNA could be separated ancl thus have no Lk (b) 476 (c) 476; the DNA is already relaxed, so the topoisomerase does not cause a net change. (d) 460; gyrase plus ATP reduces the L/c in increments of 2 (e) 464; eukaryotlc type I topoisomerases increase the.Lk of underwound or negatively supercoiled DNA in increments of 1. (D a60; nucleosome binding does not break any DNA strands and thus cannot change .Lk 9. A fundamenta.l structural unit in chromatin repeats about every 200 bp; the DNA is accessible to the nuclease only at 200 bp interuals The brief treatment was insufflcient to cleave the DNA at every accessible point, so a ladder of DNA bands is created in which the DNA fragments are multiples of 200 bp. The thickness of the DNA bands suggests that the distance between cleavage sites varies somewhat For instance, not all the fragments in the iowest band are exactly 200 bp long. 10. A right-handed helix has a positive -Lk; a left-handed helix (such as Z-DNA) has a negative,Lk. Decreasing the Lk of a closed circular B-DNA by underwinding it facilitates formation of regions of Z-DNA within certain sequences. (See Chapter 8, p 281, for a description of sequences that permit the formation of Z-DNA ) f1. (a) Both strands must be covalently closed, and the molecule must be either circular or constrained at both ends (b) Formation of cruciforms, left-handed Z-DNA, plectonemic or soienoidal supercoils, and unwinding of the DNA are favored (c) E. coli,

5otutions toprobtems Lp-rql Abbreviated DNA topoisomerase II or DNA gyrase (d) It brnds the DNA at a point where it crosses on itself, cleaves both strands of one of the crossing segmenls, passesthe other segment through the break, then reseals the break The result is a change inLk of -2 12, Oentromere, telomeres, and an autonomous replicating sequence or replication origin 13. The bacterial nucleoid is organized into domains approximately 10,000 bp long Cleavageby a restnction enzlme relaxes the DNA within a domain, but not outside the domain Any gene in the cleaved domain for which expression is affected by DNA topology will be affected by the cleavage; genes outside the domain wrll not. 14. (a) When DNA ends are sealed to create a relaxed, closed circle, some DNA species are completely relaxed but others are trapped in slightly under- or overwound states Thrs gives rise to a distribution of topoisomers centered on the most relaxed species (b) Positively supercoiled (c) The DNA that is relaxed despite the addition of dve is DNA wrth one or both strands broken DNA isolation procedures inevitably introduce small numbers of strand breaks in some of the closed-circular molecules (d) Approxrmately -0 05 This is determined by simply comparing native DNA with samples of known a In both gels, the native DNA migrates most closely wrth the sample of o - 0 049 'f5. (a) In nondisjunction, one daughter cell and all of its descendants get two copies of the svnthetic chromosome and are white; the other daughter cell and all of its descendants get no copies of the svnthetic chromosome and are red This gives rise to a halfwhite, half-red colony (b) In chromosome loss, one daughter cell and all of its descendants get one copy of the slmthetic chromosome and are pink; the olher daughter and all its descendants gel no copies of the sr,nthetic chromosome and are red This gives rise to a half-pink, half-red colony (c) The minimum functional centromere mLlst be smaller than 0 63 kbp, since all fragntents of this size or larger confer relative mitotic stability. (d) Telomeres are required to fully replicate only linear DNA; a clrcular molecule can rephcate without them. (e) The larger the chromosome, the more faithfully it is segregated The data show neither a minimum size below which the slrnthetic chromosome is completely unstable, nor a maximum size above which stabilitv no longer changes (f)

2. In this extension of the Meselson-Stahl experiment, after three generations the molar ratio of r5N-14N DNA to l4N-14N DNA is 2/6:033 3. @) a 42 x 705 turns; (b) 40 min. In cells dividing every 20 min, a repiicative cycle is initiated every 20 min, each cycle beginning before the prior one is complete (c) 2,000 to 5,000 Okazaki fragments The fragments are 1,000 to 2,000 nucleotides long and are f,rmly bound to the template strand by base pairing. Each fragment is quickly joined to the lagging strand, thus preserving the correct order ofthe fragments 4. A 28 7o/o; G 27.3o/o; C 21 30/o ; T 28 7o/o The DNA strand made from the template strand: A 32 7o/o;G 78 5o/o;C 24 \o/o;T 24 7o/o; the DNA strand made from the complementary template strand: A 2 4 7 o / o ; G 2 4 l o / o ; C7 8 5 o / o ; " 13 2 . 7 o / o . lits a s s u m e dt h a t t h e t w o template strands are replicated completely. 5. (a) No Incorporation of 32Pinto DNA results from the sy'nthesis of new DNA, which requires the presence of aLLJour n;;cleotide precursors. (b) Yes Although all four nucleotide precursors must be present for DNA synthesis, only one of them has to be radioactive in order for radioactivity to appear in the new DNA. (c) No Radioactivity is incorporated only if the 32Plabel is in the a phosphate; DNA pollmerase cleaves off pS,rophosphate-i.e , the p and 7 phosphate groups 6. Mechani,sm 1; 3'-OH of an incoming dNTP attacks the a phosphate of the triphosphate at the 5' end of the growing DNA strand, displacing pyrophosphate This mechanism uses nonnal dNTPs, and the growing end of the DNA always has a triphosphate on the 5' end

5'end

@-@-@-9t).o,,

Pe

)

OH I

,-o-l:o o I

@-@

r02

o\

C}fz

H

H

t0 c chromosomes

1 10 1

OH

I o-P:o I

\\_r_r\_r_

E I

lo2

b!

10-3

fu,-o \ -;'

Actual chromo.o-I."

10-4 10-5

0

OH

50

100 150 200 Length (kbp)

250

300

As shownin the graph, evenif the sJ,ntheticchromosomeswere as long as the normal yeast chromosomes,they wouid not be as stable This suggestsother, as yet undiscovered,elementsare requiredfor srability.

(hapter 25 1. In random, disperslve replication, in the second generation, all the DNAs would have the same density and would appear as a single band, not the lv/o bands observed in the Meselson-Stahl expenment.

I o-P:o I o

I -o-P:o I o 3'end I

3'end l

Mechanzsm 2: Thrs uses a ne\.,rb,?e of precursor, nucleotide 3, triphosphates. The growing end ofthe DNA strand has a 5'-OH, which attacks the a phosphate of an incoming deoxynucleotide 3'-triphosphate, displacing py'rophosphate. Note that this mechanism would require the evolution of new metabolic pathways to supply the needed deoxynucleotide 3'-triphosphates

Solutions toProblems Abbreviated lot ti]

5'end

HO_CH, HO-CH,

I

o I

CH,

I

o I

CHz

OH

I -o- P:O I o

3'end

I -o-P:o I o I

CH,

OH I -O-P:O

DNA containsthe many fragmentscausedby the cleavage,and the averagemolecularweight is lowered,Thesefragmentsof single-strandedDNA are absentfrom the XPG samples,as indicatedby the unchangedaveragemolecularweight (b) The absenceof fragmentsin the single-strandedDNA from the XPG cells after irradiation suggeststhe specialexcinucleaseis defective or mrssing. 14. During homologousgeneticrecombination,a Holliday intermediwithin the two paired,hoate may be formed almostan1'rvhere mologouschromosomes;the branch polnt of the intermediate can move extensivelyby branch migration In site-specificrecombination,the Hollidayintermediateis formed betweentwo specificsites,and branch migrationrs generallyrestricted by heterologoussequenceson either side of the recombinationsites. 15. Oncereplicationhas proceededfrom the origin to a point where one recombinationsite hasbeen replicatedbut the other has not, site-speciflcrecombinationnot only inverts the DNA between the recombinationsitesbut also changesthe direction of one replicationfork relative to the other. The forks will chaseeach other aroundthe DNA circle, generatingmany tandem copiesof the plasmid.The multimeric circle can be resolvedto monomers by additionalsite-specificrecombinationevents.

I o

7. Lead'ing strand: Precursors:dATP,dGTR dCTP,dTTP (also needsa template DNA strand and DNA primer); enzymesand other proteins:DNA gyrase,helicase,single-strandedDNAbindingprotein,DNA polymeraseIII, topoisomerases, and pyt'ophosphatase. Lagging strand: Precursors:ATR GTR CTq UTe dAIP, dGTR dCTR dTTP (alsoneedsan RNA primer); enzlrnesand other proteins: DNA gyrase,helicase,singlestrandedDNA-binding protein, primase,DNA poll'rneraseIII, DNA polyrneraseI, DNA ligase,topoisomerases, and py'rophosphatase.NAD+ is alsorequired as a cofactorfor DNA ligase 8. Mutants with defectiveDNA ligaseproducea DNA duplex in which one of the strandsremainsin pieces(as Okazakifragments). When this duplex is denatured,sedimentationresults in one fraction containingthe intact singlestrand (the high molecularweight band) and one fraction containingthe unspiicedfragments(the low molecularweight band) 9, Watson-Cnckbasepairing betweentempiate and leadingstrand; proofreadingand removalof wrongly insertednucleolidesby the 3'-exonucleaseactivity of DNA pol5.,rnerase III. Yes-perhaps Becausethe factors ensuringfidelity of replicationare operative in both the leadingand the laggingstrands,the laggingstrand would probablybe madewith the samefidelity However,the greaternumber of distinct chemicaloperationsinvolvedin making the laggingstrand might provide a greater opportunity for errorsto anse 10. -1,200 bp (600in eachdirection) 11. A smallfraclion(13 of l0e celJs)of the histidine-requiring mutants spontaneouslyundergoback-mutationand regaintheir capacityto sl,nthesizehistidine 2-Aminoanthraceneincreases the rate ofback-mutationsabout 1800-foldand is therefore mutagenic.Sincemost carcinogensare mutagenic, 2-aminoanthracene is probablycarcinogenic (seep. 289) pro12. Spontaneous deaminationof 5-methylcytosine ducesthymine, and thus a G-T mismatchedpair Theseare amongthe most commonmismatchesin the DNA of eukaryotes. The specializedrepair systemrestoresthe G=C pair. f3. (a) Ultravioletirradiation producespyrimidine dimers;in normal flbroblastslhese are excisedby cleavageof the damagedstrand by a specialexcinuclease.Thus the denaturedsingle-stranded

recombination at sites I and 2 (inversion)

initiation of

3'end i

replication ->

Plasmid with recombination sites 1 and 2

Partially replicated plasmid

Origin

Plasmid with reoriented replication forks

Multimeric plasmid

f6. (a) Even in the absenceof an addedmutagen,backgroundmutations occur due to radiation,cellular chemicalreactions,and so forth. (b) If the DNA is sufflcientlydamaged,a substantialfraction of geneproducts are nonfunctionaland the cell is nonviable (c) Cellswrth reducedDNA repair capabilityare more sensitive to mutagens.Becausethey lessreadily repair lesionscausedby R7000,uvr- bacteriahavean increasedmutation rate and increasedchanceof lethal effects.(d) in the uw* strain, the excision-repairsystemremovesDNA baseswith attached the 3Hin thesecellsovertime In the decreasing [3H]R7000, 3H uw strain, the DNA is not repairedand level increasesas [''H]R7000continuesto react with the DNA. (e) All mutations Iisted in the table exceptA:T to G:C show signiflcantincreases over background.Each type of mutation results from a different type of interaction betweenR7000and DNA. Becausedifferent types of interactionsare not equallylikely (due to differencesin reactivity,steric constraints,etc.), the resultingmutations occur

f-'l

AS-32

Abbreviate Sdo l u t i o nt os P r o b l e m s

with different frequencies.(f) No. Only those that start with a G:C basepair are explainedby this model.Thus A:T to C:G and A:T to T:A must be due to R7000attachingto an A or a T. (g) R7000-Gpairs with A. First, R7000addsto G:C to give R7000-G:C. (Comparethis with what happenswith the CH3-G in Fig. 25-28b,p 1000.)Ifthis is not repaired,one strandis replicatedas R7000-G:A, which is repairedto T:A. The other strand is wild-type. If the replicationproducesR7000-G:T, a similar pathwayleadsto an A:T basepair. (h) No Compare data in the two tables,and keep in mind that different mutations occurat differentfrequencies. A:T to C-G: moderatein both strains;but better repair in the uw- strain G-C to A:T: moderatein both; no real difference G:C G-C A:T A:T

to C:G: higher in uw*; certainly lessrepair! to T:A: high in both; no real difference to T:A: high in both; no real difference to G:C: low in both; no real difference Certainadductsmay be more readily recognizedby the repair apparatusthan others and thus are repairedmore rapidly and result in fewer mutations.

Chapter 26 l. (a) 60 to 100s; (b) 500to 900nucleotides 2. A singlebaseerror in DNA replication,if not corrected,would causeone of the two daughtercells,and all its progeny,to have a mutated chromosome.A singlebaseerror in RNA transcription would not affect the chromosome;it would lead to formation of somedefectivecopiesof one protein, but becausemRNAsturn over rapidly,most copiesof the protein would not be defective. The progenyof this cell would be normal. 3. Norma"lposttranscriptionalprocessingat the 3' end (cleavage and polyadenylation)would be inhibited or blocked. 4. Becausethe template-strandRNA doesnot encodethe enzymesneeded to initiate viral infection, it would probably be inert or simply degradedby cellular ribonucleases Replication of the template-strand RNA and propagation of the virus could occur only if intact RNA replicase (RNA-dependentRNA polymerase)were introducedinto the cell alongwith the template strand. 5. (1) Use of a template strand of nucleic acid; (2) synthesisin the 5'-+3' direction; (3) use of nucleosidetriphosphatesubstrates, with formation of a phosphodiesterbond and displacementof

PPt. Polynucleotidephosphorylaseforms phosphodiesterbonds, but differs in all other listed properties. 6. Generallytwo: one to cleavethe phosphodiesterbond at one intron-exonjunction; the other to link the resuiting free exon end to the exon at the other end ofthe intron Ifthe nucleophile in the fust step were water, this step would be a hydrolytic event and only one transesterificationstep would be required to complete the splicingprocess. 7. Many snoRNAs,required for rRNA modi-flcationreactions,are encodedin introns. If splicingdoesnot occur,snoRNAsare not produced. 8. Theseenzyrnes lack a 3'-+5'proofreadingexonuclease and have a high error rate; the likelihood of a replication error that would inactivatethe virus is much lessin a smallgenomethan in a large one (c) For the "unnaturalselec9. (a) 432= 1.8 x 10re(b) 0 006%o tion" step,use a chromatographicresin with a bound molecule that is a transition-stateanalogof the ester hydrolysisreaction (e.9.,an appropriatephosphonatecompound;seeBox 6-3). 10. ThoughRNA synthesisis quickly halted by a-amanitintoxin, it takes severa"l daysfor the critical mRNAsand the proteins in the liver to degrade,caus.ingliver dysfunctionand death lf. (a) After lysis of the cells and partial puriflcation of the contents, the protein extract could be subjectedto isoelectricfocusing. The B subunit could be detectedby an antibody-basedassay. The dilferencein amino acid residuesbetweenthe normal B subunit and the mutated form (i.e., the different chargeson the amino acids) would alter the electrophoreticmobility of the mutant protein in an isoelectricfocusinggel, relative to the protein from a nonresistantstrain (b) Direct DNA sequencing(by the Sanoer

methnd-\

12. (a) 384nucleotidepairs(b) 1,620nucleotidepairs(c) Mostof the nucleotidesare untranslatedregionsat the 3' and 5' endsof the mRNA.Also, most mRNAscode for a signalsequence(Chapter 27) in their protein products,which is eventuallycleavedoff to producethe mature and functional protein. 13. (a) cDNA is producedby reversetranscription of mRNA; thus, the mRNA sequenceis probablyCGG.Becausethe genomicDNA transcribedto make the mRNA has the sequenceCAG,the primary transcript most likely has CAG,which is posttranscriptionally modifiedto CGG.(b) The unedited mRNA sequenceis the sarneas that ofthe DNA (except for U replacingT) Unedited mRNA has the sequence(* indicatessite of editing)

(5')...GUCUCUGGUUUUCCUUGGGUGCCUUUAUGC;.GCAAGGAUGCGAUAUUUCGCCAAG...(3') In step1,primer1 amealsasshown:

(5')...GUCUCUGGUUWCCUUGGGUGCCUUUAUOC.I,CCN.q.CCEUGCGAUAUUUCGCCAAG...(3')

||t|||||t||||l

Primer 1: (3')-CGTTCCTACGCTATAAAGCGGTTC-(5') cDNA (underlined) is synthesizedfrom right to left:

(5')...GUCUCUGGUUWCCUUGGGUGCCUWAUGCAGCAAGGAUGCGAUAUUUCGCCAAG...(3')

| |||!r

|||||||||!||||||||

|||tl

(3')...CAGAGACCAAAAGGAACCCACGGAAATACGTC GTTCCTAC GCTATAAAGCGGTTC-(5' ) Then step 2 yieldsjust the cDNA:

'* (3')...CAGAGACCAAAAGGAACCCACGGAAATACGTCGTTCCTACGCTATAAAGCGGTTC-(s' ) In step3, primer2 anneals to the cDNA: (5')-CCTTcccTGCCTTTA-(3') ||l|||||ll (3')...CAGAGACCAAAAGGAACCCACGGAAATACGTCGTTCCTACGCTATAAAGCGGTTC-(5' ) Primer2:

toProblems Abbreviated 5olutions Et-rd

DNA pol5.rnerase adds nucleotrdes to the 3' end of this primer Moving from left to dght, it inserts T, G, C, and A However, because the A from ddATP lacks the 3' -OH needed to attach the next nucleotide, the chain is not elongated past this point This A is shown in zlolzic; the new DNA is underlined: Primer2:

(5')-CCTTGGGTGCCTTTATGCA

||||||||l (3')...CAGAGACCAAAAGGAACCCACGGAAATACGTCGTTCCTACGCTATAAAGCGGTTC-(5' ) This yields a 19 nucleotidefragmentfor the unedited transcript. In the edited transcript, the +A is changedto G; in the cDNA this correspondsto C. At the start of step 3: Primer 2:

(5')-CCTTGGGTGCCTTTA-(3')

It|||||l

(3')...CAGAGACCAAAAGGAACCCACGGAAATACGCCGTTCCTACGCTATAAAGCGGTTC-(5') In this case,DNA pol5anerase can elongatepast the edited baseand will stop at the next T in the cDNA. The dideoxyA is i,taL'ic;the new DNA is underlined. Primer 2:

(5'FCCTTGGGTGCCTTTATGCGGCA

||t|||||||||t

(3')...CAGAGACCAAAAGGAACC CACGGAAATACGCCGTTCCTACGCTATAAAGCGGTTC-(s' ) This gives the 22 nucleotide product (c) Tfeatments (proteases, heat) known to disrupt protein function inhibit the editing activity, whereas treatments (nuclease) that do not affect proteins have little or no effect on editing A key weakness of this argument is that the protein-disrupting treatments do not completely abolish editing. There could be some background editing or degradation of the mRNA even without the enzyme, or some of the enzyme might survive the treatments (d) Only the a phosphate of NTPs is incorporated into polynucleotides If the researchers had used the other t5.pesof 132P1NTPs, none ofthe products would have been labeled (e) Because only an A is being edited, only the fate of any A in the sequence is of interest (f) Given that only AIP r,vaslabeled, if the entire nucleotide were removed all radioactivity would have been removed from the mRNA, so only unmodified [3zP]AMP would be present on the chromatography plate (g) Ifthe base were removed and replaced, one would expect to see only [3H]AMP The presence of [3H]IMP indicates that the A to I change occurs without removal of H at positions 2 and 8. The most likelv mechanism is chemical modification of A to I by hydrolytic deamination (see Fig 22-34,p 885). (h) CAG is changed to CIG This codon is read as CGG.

Chapter 27 1. (a) Gly-Gln-Ser-Leu-Leu-I1e (c) His-Asp-Ala-Cys-Cys-Tlr fMel-Asp Glu in bacteria

rior positionsin a pot5,peptideOnly fMet-tRNArM"tis recognized by the initiation factor IF-2 and is alignedwith the initiating AUG positionedat the ribosomalP site in the initiation complex AUG codonsin the interior of the nRNA can bind and incorporate only Met-tRNAM"t 6. Allow polynucleotidephosphorylaseto act on a mixture of UDP and CDPin which UDP has,say,five times the concentrationof CDP The result would be a s5,ntheticRNA pol;rner with many UUU triplets (codingfor Phe), a smallernumber of UUC (Phe), UCU (Ser), and CUU (Leu), a much smallernumber of UCC (Ser), CUC (Leu), and CCU (Pro), and the smallestnumber of CCC(Pro). 7. A minimum of 583 ATP equivalents(basedon 4 per amino acid residueadded,except that there are only 145translocation steps) Correctionof eacherror requires2 ATP equivalents For glycogensynthesis,292 NIP equivalents The extra energycost reflectsthe cost of the information content for B-globins5mthesis of the protein. At least 20 activatingenzlrnes,70 ribosomalproteins, 4 rRNAs,32 or more tRNAs,an mRNA,and 10 or more auxiliary enzyrnesmust be madeby the eukaryoticcell in order of an to s)'nthesizea protein from amino acids The sS,rrthesis (a1-+4) chain ofglycogenfrom glucoserequiresonly 4 or 5 enz)flres(Chapter 15). 8.

(b) Leu-Asp-Ala-Pro (d) Met-Asp-Glu in eukaryotes;

2. UUAAUGUAU, UUGAUGUAU, CUUAUGUAU, CUCAUGUAU, CUAAUGUAU, CUGAUGUAU, UUAAUGUAC, UUGAUGUAC, CUUAUGUAC, CUCAUGUAC, CUAAUGUAC, CUGAUGUAC 3. No. Because nearly all the amino acids have more than one codon (e g , Leu has six), any given po\peptide can be coded for by a number of different base sequences However, some amrno acids are encoded by only one codon and those with multiple codons often share the same nucleotide at two of the three positions, so certain parts of the mRNA sequence encoding a protein of known amino acid sequence can be predicted with high certainty. 4. (a) (5') CGACGGCGCGAAGUCAGGGGUGUUAAG(3') (b) Arg-Arg-Arg-Glu Val-Arg-Gly-Val-Lys (c) No The complementary antiparallel strands in double-helical DNA do not have the same base sequence in the 5'-+3' direction RNA is transcribed from only one specific strand of duplex DNA The RNA pol:rmerase must therefore recognize and bind to the correct strand. 5. There are two tRNAs for methionine: tRNArMur,which is the initiating tRNA, and tRNAMet, which can insert a Met residue in inte-

Glycine codons

(5',)GGU (5',)GGC (5',)GGA (5',)GGG

Anticodons (5')ACC,GCC,ICC (5')GCC,ICC (5')UCC,ICC (5',)CCC,UCC

(a) The 3' and middle position (b) Pairingswith anticodons (5')GCC,ICC,and UCC(c) Pairingswith anticodons(5')ACC and CCC 9. (a), (c), (e), and (g) only; (b), (d), and (f) cannotbe the result of single-basemutations:(b) and (f) would require substitutions of two bases,and (d) would require substitutionsof all three bases. 10. The two DNA codonsfor GIu are GAA and GAG,and the four DNA codonsfor Val are GTT,GTC,GTA,and GTG A singlebase changein GAA to form GTA or in GAGto form GTGcould account for the GIu -+ Val replacementin sickle-cellhemoglobin. Much lesslikely are two-basechanges,from GAA to GTG,GTT, or GTC;and from GAGto GTA,GTT, or GTC 11. Isoleucineis similarin structure to severalother amino acids, particularlyvaline.Distinguishingbetweenvaline and isoleucine

Et-rolA b b r e v i aSt eodl u t i otnosP r o b l e m s in the aminoacylation process requires the second fi-lter of a proofreading function Histidine has a structure unlike that of any other amino acid, and this structure provides opportunities for binding specificity adequate to ensure accurate aminoacylalion of the cognate tRNA. 12. (a) The AIa-tRNA syrrthetase recognizes the G3-U70 base pair in the amino acid arm of tRNAao (b) The mutant tRNAau would inseft Ala residues at codons encoding Pro (c) A mutation that mrght have similar effects is an alteration in TRNAP'othat allowed it to be recognized and aminoacylated by Ala-tRNA synthetase (d) Most of the proteins in the cetl would be inactivated, so these would be lethal mutations and hence never observed This represents a powerful selective pressure for maintaining the genetic code 13. The amino acid most recentlv added to a growrng polypeptide chain is the only one covalently attached to a tRNA and thus is the only link between the polypeptide and the nRNA encoding it A proofreading activity would sever this link, halting syrrthesis of the polypeptide and releasing it from the mRNA 14. The protein would be directed into the ER, and from there the targeting would depend on additional signals SRP binds the amino-terminal signal early in protein synthesis and directs the nascent polypeptide and ribosome to receptors in the ER Because the protein is translocated into the lumen of the ER as it is synthesized, the NLS is never accessrbleto the proteins involved in nuclear targeting 15. Trigger factor is a molecular chaperone that stabilizes an unfolded and translocation-competent conformation of ProOmpA 16. DNA with a mrnimum of 5,784 bp; some of the coding sequences must be nestedor ovcrlapping 17. (a) The helces assoclatethrough hydrophobic and van der Waals interactions (b) R groups 3, 6, 7, and 10 extend to the left; 1, 2, 4, 5, 8, and 9 extend to the right. (c) One possible sequence ls 12345678910

N-Phe-Ile -Glu-Val-Met-Asn- Ser-Ala-Phe-Gln-C (d) One possible DNA sequence for the amino acid sequence in (c) ls

Nontemplate

strand

(5')_TTTATTGAAGTAATGAATAGTGCATTCC AG_(3') |||||||l||t|ll||l|| (3')_AAATAACTTCAT (5') TACTTATCACGTAAGGTCTemplatestrand (e) Phe, Leu, Ile, Met, and Val All are hydrophobic, but the set does not include a,ll the hydrophobic amino acids; Tfp, Pro, Ala, and Gly are missing (f) IVr, His, Gln, Asn, Lys, Asp, and Glu. A11of these are hydrophrlic, although Tlr is less hydrophilic than the others The set does not include all the hydrophilic amino acids; Ser, Thr, and Arg are missing (g) Omitting T from the mixture excludes codons starting or ending with T-thus excluding Tlr, which is not very hydrophilic, and, more importantly, excluding the two possible stop codons (TAA and TAG) No other amino acids in the NAN set are excluded by omitting T (h) Misfolded proteins are often degraded in the cell Therefore, if a synthetic gene has produced a protein that forms a band on the SDS gel, it is likeiy that this protein is folded properly (i) Protein folding depends on more than hydrophobic and van der Waals interactions There are many reasons why a synthesized random-sequence protein mrght not fold into the four-helix structure. For example, hydrogen bonds between hydrophilic side chains could disrupt the structure Also, not all sequences have an equal propensity to form an a helix

Chapter 28 1. (a) Tiyptophan synthase levels remain high in spite of the presence oftryptophan (b) Levels again remain high. (c) Levels rapidly decrease, preventing wasteful synthesis of tryptophan 2. (a) Constitutive, lowJevel expresslon of the operon; most mutations in the operator would make the repressor less likely to bind (b) Either constitutive expression, as in (a), or constant repression, if the mutation destroyed the capability to bind to lactose and related compounds and hence the response to inducers. (c) Either increased or decreased expression of the operon (under conditions in which it is induced), depending on whether the mutation made the promoter more or less similar, respectively, lo the consensus E coli promoter 3. 7,000 copies s 4. 8 x 10 u, aboul 105times greater than the dissociation constant With 10 copies of active repressor in the cell, the operator srte is always bound by the repressor molecule 5. (a)-(e)

Each condition decreases expressron of lac operon genes.

6. (a) Less attenuation of transcription. The ribosome completing the translatlon of sequence 1 would no longer overlap and block sequence 2; sequence 2 would always be available lo pat with sequence 3, preventing formation of the attenuator structure (b) More attenuation oftranscription Sequence 2 would pair less efficiently wrth sequence 3; the attenuator structure would be formed more often, even when sequence 2 was not blocked by a ribosome (c) No attenuation oftranscription The only regulation would be that afforded by the Trp repressor (d) Attenuatron loses its sensitivrty to Trp IRNA It might become sensitive to His IRNA (e) Attenuation would rarely, if ever, occur Sequences 2 and 3 always block formation of the attenuator (f) Constant attenuation of transcription Attenuator always forms, regardless of the avarlability of try-ptophan 7. Induction of the SOS response could not occur, making the cells more sensitive to high levels of DNA damage. 8. Each Salmonella cell would have ffagella made up of both types of flagellar protein, and the cell would be r,ulnerable to antibodi e s g e n e r a t e di n r e s p o n s et o e i t h e r p r o t e i n 9. A drssociable factor necessary for activity (e g , a speciicity factor simrlar to the o subunit of the -O.colz enz5.nne)may have been lost during purification of the polymerase. 10. Gal4 protein

Gal4 DNA-binding domain

I

Cata activator domain

Lac repressor DNA-binding domain I

Cat+ activator domain

Engineered protein

The engineered protein cannot bind to the Ga14binding site in the GAL gene (UASc), because it lacks the Gal4 DNA-binding domain Modify the Gal4p DNA binding slte to give it the nucleotrde sequence to which the Lac repressor normally binds (using methods described in Chapter 9). 11. Methylamine. The reaction proceeds with attack of water on the guamdinium carbon of the modified arginine 12. The bcd mRNA needed for development ls contdbuted to the egg by the mother. The egg develops normally even if its genotype ts bcd-,/bcd-, as long as the mother has one normal bcd gene and the bcd- allele is recessive. However, the adult bcd /bcd female will be sterile because she has no normal bcd mRNA to contribute to her own eggs 13, (a) Hydrogen bonds form between the protein and DNA backbone at 4.106,A110, ,A118,T119, and A122, and between the

t0 Pr0blems 5olutions Abbreviated [ns-ttl

proteinand DNA basesat 4106,T107,A118,and T119.The latter four nucleotidescontribute directly to DNA sequence recognition(b) ,N,4 backbone:A106-Arg2e0, A110-Sel12, A1l&Argles, Ti l9-Arg20a,AI22-Ser303 . DNA basas:A106Asn253, T107-Asn253, A1l8-Asnt63,T11g-Asn163. Asn,Gln,GIu, Lys, and Arg are commonlyfound hydrogen-bondedto basesin DNA. The majority of residuesin the TATA-bindingprotein that are involvedin hydrogenbonds are Arg and Asn. (c) TATATATA(residues103to 110) ATATATAT(residues 122 Lo ll5) proteinrecognizes The TATA-binding A106,T107lI119,A118 (d) The hydrophobicinteractionsare numerous Many binding interactionsof this type involvethe burying of large amountsof hydrophobicsurface. 14. (a) For 10%expression(90%repression),10%ofthe repressor has bound inducer and 90% is free and availableto bind the operator.The calculationusesEqn 5-8 (p. 156), with 0 : 0 I and Ka : 10-an ,_ 01:

[IP/lc] _ tIPfrGl IIPTGI+ Kd [IPTG]+ 10-4M --lryIql so0.9[IPTG]: 10-5or tIPTGl= 1.1x 10-5M [PTG]+ 10 'u

For 90%o expression,90% of the repressorhas bound inducer, so 0 : 0.9 Entering the valuesfor 0 and K6 in Eqn 5-8 gives IIPTGI = 9 x 10-a rrl.Thus,gene expressionvaries l0-fold over a roughly 10-fo1d[IPTG]range.(b) Youwould expect the protein levelsto be low before induction, rise during induction, and then decayas syrrthesisstopsand the proteins are degraded (c) As

shownin (a), the lac operonhas more levelsof expressionthan just on or off; thus it doesnot have characteristicA. As shownin (b), expressionof lhe lac operon subsidesonce the inducer is removed;thus it lacks characteristicB. (d) GFP-on: tept" and GFP are expressedat high levels;rep'" repressesOP1,so no LacI protein is produced.GFP-off:LacI is expressedat a high level; LacI repressesOP66,so rept'and GFParenot produced.(e) IPTG treatment switchesthe systemfrom GFP-offto GFP-on. IPTG has an effect onJywhen LacI is present,so alfects onJythe GFP-offstate.AddrngIPTG relievesthe repressionof OP6"aIlowing highJevelexpressionof rept",which turns off expression of Lacl, and high-levelexpressionof GFP.(f) Heat treatment switchesthe systemfrom GFP-onto GFP-off Heat has an effect only when repc"is present,so affectsonly the GFP-onstate.Heat inactivatesrep" and relievesthe repressionof OP1,allowing highJevelexpressionofLacl LacI then acts at OP6"to repress synthesisofrept" and GFP (g) Chnracteristic,4; the systemis not stablein the intermediatestate.At somepoint, one repressor will act more stronglythan the other due to chancefluctuations in expression;this shuts off expressionof the other repressor and locks the systemin one state. Characteristi'cB: once one repressoris expressed,it preventsthe synthesisof the other; thus the systemremainsin one state evenafter the switching stimulushas been removed.(h) At no time doesany cell express an intermediatelevel of GFP-this is a con-firmationof characteristic A. At the intermediateconcentration(X) of inducer,some cells haveswitchedto GFP-onwhile othershave not yet made the switch and remain in the GFP-offstate;none are in between. The bimodaldistribution of expressionlevelsat [IPTG] = a it causedby the mixed populationof GFP-onand GFP-offcells.

Key: b : boxedmaterial;f = figures;s = structural formulas;t = tables;boldfa.ce = boldfacedterms tn text

a A bands,176, I77f A (aminoacyl)binding site, ribosomal,1089 A khase anchoringproreins (AIGPs), a3t-a32 AAA+ ATPase,985 abasicsite, in base-excisionrepair, 996-997, 997f ABC exinuclease,997-998 ABC1protein, 840,843-844 ABC transporter,400, 400f,840 abioticslnthesis,30-31,31f absolutecon-figuration, 74 absolutetemperature,units of, 491t absorbance(,4), 76b absorption of fat, in small intestine,648-649,648f othght,744-749 See alsoltg!|, absorptionof absorptionspectra ofcltochromeq 71I, 71lf of nucleotides,276, 276f of opsins,465, 465f ACAT (acyl-CoA-cholesterol acyl transferase), 836,841 acceptorcontrol, 733 acceptorcontrol ratio, ?33 accessorypigments,747 in light absorption,745f,747 acetaldehyde,513s acetals,238, 238f acetate,505s activated,in citric acid cycle,616-617,616f, 617f,619f in cholesterolslrrthesis,831-832,832f in fatty acid synthesis,813-814,813f oxidation of, 631 as sourceof phosphoenolpyruvate, 638 transportof, 813-814,813f aceticacid,505s,513s pK" of, 80, 80f titration curve for, 58, 58f aceticacid-acetatebuffer system,60, 60f acetoacetate,666, 929s acetoacetatedecarboxylase,666 acetoacetyl-ACP, 809, 810f, 811f acetoacetyl-CoA, 298 acetone,513s,666, 929s in diabeticketoacidosis,929 acetylgroups in fatty acid snthesis, 813-814,813f transportof, 813-814,813f N-acetyl-B-o-glucosamine, 240s acetylcholinereceptor,410 defective,4l2t open/closedconformationof, 453,454t in signaling,453,454t structure of, 453, 454f s)'napticaggregationof, 384 acetyl-CoA, 13s,607s aminoacid degradationto, 695-696,695f, 696f AMPK and, 934 F oxidationyielding,653f, 654-655 in cholesterolsynthesis,832-836,832f decarboxylationofplruvate to, 619, 619f in fatty acid sl4:rthesis, 805-806,806f, 807f,811, 8r2,922 in glucosemetabolism,922, 923f,927f,928 hepaticmetabolismof, 915-916 hydrolysisof, 505, 505s,505t oxidationof, in citric acid cycle,655,656t oxidationof pl'ruvate to, 61G617, 6f6f

in plant $uconeogenesis,798 productionof, 813-814 in citric acid cycle,616-620,616f, 619f,631f by ppuvate dehydrogenase complex,636f, ODUTDI

acetyl-CoAcarboxylase in bacteria,814 in fatty acid synthesis,806-806, 806f, 807f, 814,814f in plants, 814 810f, 8llf acetyl-CoA-ACPtransacetylase, acetyl-coenzyneA Seaacetyl-CoA acetylene, 513s N-acetylgalactosamine, in $ycosaminoglycans, 249,250t (GlcNAc), 240, 240f N-acetylglucosamine in bacterialcell walls,249 in glycosaminoglycans, 249, 250f in peptidoglycarsJmthesis, 796, 797t N-acetylglutamate,regulatorof urea cycle,686 N-acetylglutamate synthase, 686 N-acetylrnuramicacid (Mur2Ac), 240,240t in peptidoglycansynthesis,796, 797t N-acetylneuramhicacid (NeubAc),240,240s, 25U259, 2581,259s,354s in gangliosides,354, 354s acetylsalicylate(aspirin), 818 acetylsalicylicacid (aspirin), 818s achrralmolecules,16f o,cid,e g , aceticacid acid(s) Saealso specxrt,c qminn

qnirlq ac

R1

as buffers,59-63, 6lf definition of, 57 dissociationconstant(K^) of, 57f-59f, 68-59 fatty ,Seefatty acid(s) relative strengthof. SeepKu strong,57-58 titration curve for, 58-59, 58f, 59f Henderson-Hasselbalch equationfor, 60-61, 80 weak,57-59 acid anhydride,standaxdfree-energzchangesof, 493t acid dissociationconstant(I{J, 57f-59f, 68-59, 68-59 oairl-hoco

nofolwcic

general,192, 193, 193f specific,192-193 acid-basepairs, conjugate,67, 57f as buffer systems,59-63 acid-basetitration, 58-59, 58f, 59f, 79-8I, 79f acidemra arginmosuccinic,694t methylmalonic,694t, 700b acidicactivationdomain,l142-1 143 qnidin

crroqrc

24Oc

acidosis, 57, 64b,667 diabetic,930 acivicin,894,894f aconitase,622,625b aconitase,moonJightilg,624 cis-aconitate,622, 622s isocitrate f ormati on iq 622-623, 623f aconitatehydratase,622 ACP (acyl carrier protein), 810f acridine,1033,f0$g actin,176-178,178f in ATP hydrolysis,176,178-179,178f in musclecontraction,178-179,178f qfnfnturc

al

177-17R

in thin filaments,177-1?8,178-179,l78t actin fllaments,9, 9f d-actinin, 177 actin-myosincomplex ATP in, 609-510 phosphorylationof, 472

actin-myosminteractions,178-179,178t actnomycin D, 1093, 1033f action spechum,for photosynthesis,747 activationbarrier. 25. 25f activationenergy(AG+),26, 25f, 187 of enzymaticreactions,187 in membranetransport,390-391,390f rate constantand, 188 activators.1117-1I 18.1118f activesite, 164, 186, 186f transition-statecomplementariwand, 210b-21lb,21lf activetransport,391 ATP in, 509-510,730-73I primary transportersin, 391 secondarytransportersin, 391 transportersin, 391 activetransport,ATP and, 509-510,730-731 Actos,808s,824,936 acyclovir,992 acyl carrier protein (ACP), 808, 809f, 810f acyl phosphate,536 acyl-carnitine/carnitine transporter, 661 acyl-CoA,fatty, 660 conversionoffatty acidsinto, 650-651,651f 664 acyl-CoAacetyltransferase, 664, 716 acyl-CoAdehydrogenase, medium-chain,661 acyl-CoAsynthetases,660 in triacylglycerolslmthesis,820, 820f acyl transferase(ACAT), acyl-CoA-cholesterol 836,841 acyl-eruyme intermediate, in chymotrlpsin mechanism,205-207,206t, 208t, 209 N-acylsphmganine, E29, 83lf 829, 831f N-acylsphrngosine, adaptorh'?othesis, 1066,1066f adaptorproteins,in signaling,481' 446449 ADARs,1074 adenine,9s,272,272t,502s,517sSeealso purine bases deamination of. 289, 290t evolutionarysigniflcanceof, f057, 1057f adeninenucleotidetranslocase,731 adeninenucleotides biosj,nthesisof, regulatorymechanismsin, 885,886f ceUularconcentrations,502t in metabolic regulation, 575-57 7 adenosine,2T3s anti form of. 280-281.280f as enzJ'rnecofactor,257-298, 297f evolutionary signifi canceof , 297-298 methylationof, 292 sl'n form of, 280-28I,280t adenosine2'-monophosphate,274s adenosme2',3'-cyclic monophosphale,274s adenosine3',5'-cyclic monophosphate.SeecAMP (adenosine3', 5'-cyclic monophosphate) adenosinedeaminase,892 adenosinedeaminasedeflciency,893 genetherapy for, 335-336 adenosinediphosphate(ADP) SaaADP (adenosinediphosphate) adenosine3'-monophosphate,274s adenosine5'-monophosphate,2T4sSeealso AMP (adenosinemonophosphate) adenosinemonophosphate(AMP) SeeAMP (adenosinemonophosphate) 893 adenosinephosphoribosyltransferase, adenosinetriphosphate (ATP) See ATP (adenosinetriphosphate) 690 ,S-adenosylhomocysteine, as mutagen,291f adenosylmethionine,

l-1

,l i

l-2

lndex

S-adenosylmethionine, 689, 689s as mutagen,292 sSmthesis of, 69lf adenylate,272I,273s adenylatekinase,510, 676, 888 adenylyl cyclase activation of, 424f, 431 in B-adrenergicpalhway, 426 adenylylgroup, ATP and, 508 adenylyltransfer,508f adenylylation,508 adenylylation,enzyrte,223,223f adenylyltransferase, 858 adhesionreceptors,in signaling, 422,422t adipocrtes,346,346t,916, 9l6f NADPHsynthesisin, 812,812f adipokines,930-931 adiponectin,9341-936,935,935f adiposetissue brown,736, 917,917t heat generatedby, in oxidativephosphorylation regulation,736, 736f mitochondriain, 736 endocrinefunctionsof, 909f,916-917 in fastrng/starvation, 927f , 928f fatty acidreleasefrom,821-822,822f,823, 916-9i7 fatty acid sJ,nthesis in, 916-917 SeeaLsofally acid synthesis glucagonand, 926,927r glyceroneogenesis in, 822-823, 822f, 823f leptin slnthesisin, 930-931,931f metabolicfunctionsof, 909f,916-917 triacylglycerolmobilizationin, 648f, 650f triacylglycerolrecyclingin, 822, 822f w h i t e ,9 1 6 , 9 1 6 f A - D N A ,2 8 1 , 2 8 1 f adoMet Seeadenosylmethionine ADP (adenosinediphosphate) ATP stabilizedrelativeto, on F1 component,726f in fatty acidsynthesis, 810f,811 phosphoryltransfer to from 1,3-bisphosphoglycerate, 536-537 from phosphoenolp),Tuvate, 538 in photosynthesis,783 synthesisof, 24 ADP-glucose, 791 in glycogensJ,'nthesis, 791-792,791f in starchsynthesis,79).-T92, 79tf ADP-glucosepyrophosphorylase, in starch synthesis,793,794f. ADP-ribosylation,enzyrre,223,223t adrenal,909f adrenalineSaaepinephrine B-adrenergicreceptor desensitization of, 430-431,430f in rafts, 449 B-adrenergicreceptor kinase(BARK), 430f, €f adrenergicreceptors,423-431 adrenocofticalhormones,906t, 908 adrenoleukodystrophy, X-linked,662 adsorptionchromatography,364-365,364f AE (anion exchange)protein, 395 ApquorpoL)icloria.fluorescent proteinsh, 434b-436b aerobicmetabolism,of smallveftebrates,548b aerobicorganisms,evolutionof, 32-33, 33f affinity, receptor-Iigand, 4lg-420, 420f affinity chromatography,87f, 88, 88t, 313-314 tagsin, 313-314,313r African sleepingsickness,880b-881b agar,249 agarose,2 49, 249f, 25It agarosegels,in electrophoresis,249 aggrecan,254 agurg,mitochondrialDNA darnageand, 239 agonists,receptor,423 Agre, Peter, 404,404t agriculture Seeolso plant(s) genetic engineeringin, 330-334 Agrobacteri,umtumeJaciens,in plant cloning, 330-332,330f,331f

AIDS Saehumanimmunodeflciencyvirus infection AKAPs (A kinaseanchoringproteins), 431-432 alanine,9s, l5f, 15s,75, 75s,692, 865 degradationof to pyruvate,692,692t propertiesof,73t, 75 stereoisomersof, 72-73, 72f structure of, 15, I5f in transport of amrnoniato liver, 681 alanineaminotransferase(ALT), 681 measurement of,678b alanylglutamylglycyllysine, 83s Alberts,A]fred, 843b,843f albinism,694t albumin,serum,649-650, 916 alcohol(s),513s,556s fermentationof, 528, 530f, 547-549,547f hemiacetalsand, 238, 238f hemiketalsand, 238, 238f. in lipid extraction,363-364,364f alcoholdehydrogenase, 518t, 547, 664 reactionmechanismof, 547f aldehyde(s),l3 hemiacetalsand, 238, 238f hemiketalsand, 238, 238f aldehydedehydrogenase, 664 aldohexoses, 237,237f aldol condensation,497, 497s aldolase, 575r,783 in Calvincycle,779 in glycolysis,533 aldolasereactionmechanism,classI, 534f aldonicacids,240 aldopentoses, 236 structure of, 2361,239,2391 a l d o s e s2,3 6 , 2 3 6 f n isomersof, 237 L isomersof, 237,237f structure of, 237-238, 237f aldosterone,359, 359s algae,cell walls of, heteropolysaccharides in, 249,249t alkalinephosphatase,305t alkalosis,57, 64b alkaptonuria,6941,698 AlkB protein, in DNA repair, 1000,1000f alkene,5l3s alkylatingagents,as mutagens,291t,292 allantoin,892 alleles,168 alligator,anaerobicmetabolismand movementof,548b allopurinol,S94 xanthine oxidaseinhbition by, 894, 894f allose,237s allostericeffectors,220 allostericenzyrnes,220-222, 221f, 222t conformationalchangesin, 220-221,22If feedbackinhibitionby,221-222,22Lt k r n e t i co sf,222,222t allostericmodulators,220 allostericproteins,162 Iigandbinding of, 162,I62t ^gee.rlso proteinligandinteractions allostericregulation of acetyl-CoAproduction,635-636,636f of aminoacid biosynthesis,870f, 872-873, 872f of aspartatetranscarbamoylase, 887,887f of carbohydratemetabolism,606-608,608f lipid metabolicintegrationwith, 608 of fat metabolism,608 of glutaminesylthetase,857-858 of glycogenphosphorylase,603-604,603f of phosphofructokhase-1,585-587,586f ofplruvate kinase,579-580,589f all-trarzs-rethal,463 Alper, Tikvah, 147 o cells,pancreatic,924,924f a chains,in collagen,I25, I25f a helix, 117-120,118f,120f,I2I-122, 122t, r23t in aminoacids,119,119t in keratin, 123-124, I24f

in membraneproteins,376, 377t,378 in myoglobin,130 rn polysaccharide s, 2 48,249f in protein folding, 135,148 in smallglobularprotens, 131t a helix-tJ,pecharnels,39t, 392t a oxidation,664-665 Saaolso oxidation in peroxisomes,664-665,665t o-actin,1764 c-amanitin, 1033 c-amiro groups,transfer of to d-ketog)utarate,677, 677f,679f alB baruel,136-136,l36f a-A-glucose l-phosphate,391,595-596,595f d-oxoglutarateSeeo-ketoglutarate ALT See alanine aminotransferase(ALT) Aitman, Sidney,1048 altrose,237s Alzheimer'sdisease amyloiddepositsin, l47b apolipoproteinin, 839 Ames,Bruce,993 Amestest,993,993f amrde,standardfree-energrchangesof, 493t annnes,13 as products of aminoacid decarboxylation, 878-879,879f,880f aminoacid(s), 9f,72-81 Seaolso protein(s); specif,c amino acids in a helix, II7-119,118f,1i9t, 122f abbreviationsfor, 73t acid-basepropertiesof, 81 activationof, in proten synthesis,I081-1084, 1082f-1084f addition of, in protein synthesisSaaprotein slrlthesis ammoniumassimilationinto, 857 as ampholytes,79 aromatic,as precursorsof plant substarces, 878,878f in p sheet,121,121f,I22t in B tums, 121,121.f biosy,nthesis of, 860-873,861f a-ketoglutaratein, 861+64, 862f, 863f allostericregulationof, 870f, 872-873,872t chorismatein, 865-869,868f-870f histidine in, 869-872,87lt inrerlockngregulatorymechanisms in, 872-473,872f metabolicprecursorsin, 861, 861t oxaloacetateand pyeuvatein, 865 3-phosphoglycerate in, 863-864,863f, 864f reactionsin, 859,861f branched-chain,not degradedin liver, 701, 70If as buffers,80, 80f carbondesignationsfor, 73-74 codonsfor, 1066-1074 Saaalso codons conversionof to glucoseand ketonebodies,688-689,688f to d-ketoglutarate,698-699,698f to succinyl-CoA,699-700,699f degradationof to acetyl-CoA,695-696,695f, 696f to pJ,a'uvate, 692-694,692f,693f, 694t discoveryof, 73 electric chargeof, 80-81 essential,686, 686t, 860 biosl'nthesisof, 865,866f-867f in expandedgeneticcode, 1085b-1087b found in bacteria,877-878 in generalacid-basecatalysis,193, 193f glucogenic,667, 5571,688 hepaticmetabolismof, 91rt-915,915f ketogenic,6E8 Iight absorptionby, 76b, 76t molecularweight of, 84 moleculesderivedfrom, 873-882 nonessential,686t, 860 biosynthesisof, 865 number of, 831,84, 84t in peptide s),nthesis,100-102,101f pK. of, 79-80, 79f-81f

in plant gluconeogenesis, 798 polarity of, 75 pol)lners of Seepeptide(s);protein(s) as precursorsof creatineand glutathione, 876-877,877t R groups of, 78, 73t, 74-77 relativeamountsof, 84, 84t stereoisomerism in, 72.73.721 structureof,72-74,72f,72s,79f s)rynbols for, 73t titration curvesof, 79-81, 79f, 81f uncommon,TT zwitterionicform of, 79-80, 79f aminoacid arm, of IRNA, 1081 673-682,675f aminoacid cataboLism, acetyl-CoArn, 695 696, 695f,696f a-keto$utarate in, 698f ammoniain, 681-682 aladne transport of, 681,681f glutamatereleaseof,677 680,680f glutaminetransport of, 680-681,680f asparaginein, 70I -7 02, 702f aspartaten, 701-702, 702f enzJ.(ne cofactorsin, 689-692,689f, 690f,69lf enzlme degradationof protein in, 674 677,693f geneticdefectsrn, 694t, 696-698,697f glucoseand ketonebodiesin, 688 689,688f a-ketoglutaratein, 698-699 overuiewof,687 688, 688f oxaloacel ate n, 701-T 02,7021 pathwaysof, 673-674,674f,687-702,913t pyruvatein, 692-694,692f,693f,694t succinyl-CoAin, 699-700,699f transfer of a-aminogroupsto c-ketoglutaratein, 677,677f, 679t aminoacid metabolism,570f,9l3t amho acid oxidation,673-702 fates of aminogroupsin, 673 682 nitrogen excretionand urea cyclein, 682487 pathwaysof degradationin, 687-702 aminoacidresidues,72,77 , 78s amino-teminal, 82 carboxyl-terminal,82 in consensussequences,225-226,226r farnesylationof, 1097,1097f isoprenylationof, 1097,1097f numberof, 83,83t,84t phosphorylationot, 2231,22+226 sequenceot,92-93,92t aminoacid sequences , 28f, 29, 92-93, 92t, 102 a heiix and, 117-119,1\8f, ).20f,121.-122 determinationof, 93-100 Seeo,lsoamino acid sequencirg evolutionarysignificanceof, 102-106 in geneticcode,1067-1068Seealso codons 104-105,104f,105f homologous, hydrophobicityof, 73t, 378 identificationof, in probe design,311, 31lf membraneprotein topologyand, 376f-379f, 378-379 nucleic acid sequencesand, 97f, 98-I00,948,948f protein function and, 93 proteh structure and, 102 signature,105, 105f amho acid sequencing,93-100 bond cleavagein, 95-96, 96f, 96t bond locationin, 97-98 104-106 computerized, DNA sequencingand, 97f, 98-100 Edmandegradationin, 95, 94f in evolutionaryanalysis,102-106,l04f-106f historicalperspectiveon, 93-94 for homologs,104-105,104f,l05f for largepob?eptides,95-96 massspectrometryin, 98-100,98b-100b, 99f-100f,100f peptide cleavagein, 95-96, 96f, 961,97f reagentsfor, 94-95, 95s for smallpollpeptides, 9tt-95 stepsin, 94f

amrnoacid substitutions,104 in homologs,104,105f aminogroups,transfer of, to a-ketoglutarate,677, 677t,679t ^-r-^ dluru

^,.d^-^ DuSdrr,

o/A^ a*uJ

aminoacyl(A) binding site, ribosomal,1089 aminoacyl-tRNA,1066 bindingof, 1091,1092f sitesof, 1089, 1089f formationof, 1081-1084,1082f-1084f amuioacyl-tRNAsynthetases,1066 rn proteurslmthesis,108i-1084,1082f-1084f reactionmechanismof, 1082f y-aminobuty'ricacid (GABA), receptorfor, as ion channel,410 6-aminolel'ulinate,biosl'nthesisof, 874f aminopeptidase,676 aminopterin,894-895 amino-terminalresidues,82 protein half-life and, 1107,tl09t aminotransferase ,677. Seealso transaminase PLP as prostheticgroup of, 677,695t aITImOrua in aminoacid catabolism alaninetransportof, 681,68lf glutamatereleaseof, 677-680,680f glutaminetransportof,680 681,680f from aminoacld metabolism,914-915 in nitrogenmetabolism,857 reduction of nitrogen to, 852-85,1 solubilityof in water,47,47l toxicity of, 681-682 urea productionby, enzymaticstepsin, 682-684, 683f,684f ammoniumcyanide,adeninesynthesisfrom, 1056-1057,1057f amnonotelic species,disposalof, 682 amodcillh,218 AMP (adenosinemonophosphate) allostericenz)'memodification by, 227 concentrationof, 575-576 relativechangesin, 576t variant forms of, 274, 274s AMP-dependentprotein kinase(AMPK), 576, 576f adiponectinand, 934-936, 935,935f Ieptin and, 934, 934 amphibolicpathway,631 amphipathiccompourds,46t solubilityof in water, 46t, 48-49 amphitropicproteins,375 amphol,'tes,amlno acidsas, 79 amphotericsubstances,aminoacidsas, 79 AMPK SeeAMP-dependentprotein kinase(ADPK) amplification,signal,42O,420f, 429 amylase,246,543 amylo (1+4) to (1--+6)transglycosylase, 601,60tf amyloid,145 amyloidoses,145-147 amylopectin,245-246,245f, 251t, 792 Seealso siarch amyloplast,774-775, 775f amylose,245-246,245t, 248,248f, 251t Seea\so slatch structure of, 245f, 248,248f, 249f anabolicpathways,773,9l3t energycarriersin, 487f in glycogenmetabolism,595-596 anabolism,26,26f, 487, 487f citric acidcyclein, 631,632f anaerobicbacteria,incompletecitric acid cyclein, 631,631f anaerobicmetabolism,of coelacanths,548b anal).tes,98 anammox,8521852f anafimoxosome,854, 854f anandamide,442s anapleroticreaction,in citric acid cycle,631 633, 632f,632t 599t Andersen's disease, androgens,908 synthesisof, 844-815,844f

anemla megaloblastic,691 pernicious,role ofvitamin Blr, 659' 691 Anfinsen,Christian,i41 anginapectoris,nitrovasodilatorsfor, 446 angstrom(AJ, II / anion exchanger,90. Seealso ion-exchange chromatography anion-exchange(AE) protein, 395 ankyrin, 384f anneahng,of DNA, 287-288, 287f annotatedgenome,34 annularlipids, 376, 377f anomedccarbon,239 anomers,239 anorexigenicneurons,933 antagonists,receptor,423 anterua chlorophylls,in light-drivenelectronflow, 752,753f,75+755 antennamolecule,747-T 48 dntelxnapedia, 1).51 anterior pituitary, 909, 909f antibiotics mechanismof action of, 216-219, 404,404f,896, 960b-961b protein glycosylationinhibition by, I 102 resistanceto ABC transportersand, 400 plasmidsin, 949 topoisomeraseinhibitor, 960b-961b transcriptioninhibition by, 1033 translationrnhibitionby, 1098-1099 antibodies,l7O. Seealso immunoglobulin(s) in analltic techniqtes, 173-174, 774f antigensand, 170-175,l7lt-173t Seea|so antigen-antibodyinteractions binding sites on,17I-173, l72f binding speciflcityof, 170, 173,173f catalltic,2ll diversityof, 170 recombinationand, 1014 monoclonal,173 polyclonal,173 anticodon(s),1070-1072,1071f wobblebaseof, 1072 anticodonarm, of tRNA, 1081 antidiuretichomone (ADH), 9lls antigen(s),171 epitopeof, 171 T-cell binding of, 170 antigen-antibodyinteractions,770-175, rTrf-173t binding affinity in, 173,l73f bhding sitesfor, 171-173,l72f conformationalchangesin, 173, 173f haptensin, 171 inducedflt in, 164,173, I73f specificrfof, 170,173 strengthof, 173 antigenicdeterminant,171 antrgenicvariation,I 135t antiporter(s),395' 395f Na*K*-ATPase,398-399,398f,403,403f in membrane polarization, 450, 450f in membranetransport,in neurons,920 in neurons,920 in retina, 463 for triose phosphates,783-784,7841 antiviral agents,mechanismof action of, 218-279 997 AP endonucleases, AP site, in base-excisionrepaA, 996' 997,997f Apaf-1 (apoptosisproteaseactivatingfactor-l), 738 ,4PCmutation, 477, 477f 625b apo-aconitase, apoB-48,genefor, RNA editing in, 1074 837f,838,840-841 apoB-100, genefor, RNA editing in, 1074 APOBEC,1074 APOE, n Nzheimer'sdisease,839b apoE,LDL receptor binding to, 841 apoenzlme,184

f

!-4

Index

apolipoproteins,649, 836-837, 837f in Alzhermer'sdisease,839b genefor, RNA editing in, 1074 apoprotein,184 apoptosis,47747 8, 478t, 73? cytochromeP-450n, 737-7 38, 737t mitochondriain, 737-7 38, 737t apoptosisproteaseactivatingfactor-l (Apaf-l), 738 apoptosome,738 appetite,hormonalcontrol of, 930-937 App(NH)p (B,7-imidoadenosine 5'lriphosphate), 727s aptamers,1068b aquaporins(AQPs),404-406, 405f, 406t classiflcationof, 406t distribution of, 406t microscopyof, 385b permeabilityof, 406t aqueousenvironments,adaptationto, 6b-66 aqueoussolutions Seeako water amplupathiccompoundsin, 46f,4Ug buffered,59-63, 61f coliigativepropertiesof, 61, 5lf hydrophilic compoundsin, 4648, 47f hydrophobiccompoundsin, 10, 46,4U9, 4Tt,48t hlpertonic, 62, 52f hy?otonic,52,52f ionic interactionsn, 4647 isotonic,62, 52f osmolarityof, 62, 52f pH of, 56-57,56f,56t weak acids,/bases in, 57-63 weak interactionsin, 43-54 Seeal,sowater Arabidopsi,sthaliana aquaporhsin,404 celluloseslnthesis n, 79fF796,795t signaliruin, 458-460,459t arabinose,237s,238s arachidicacid,344t arachidonate,815f, 817, 8l8f See aLso arachidonicacid arachidonicacid, 344t, 358,358s Seealso arachidonate Arber, Wemer, 304 archaea,4-5, 4f, 5f membranelipidsof, 352,353f arginase,684 arginine,75s,77, 699,861 biosynthesisof, 861-863,862f conversionof, to a-ketoglutarate,698-699,698f in nitric oxide biosy'nthesis, 882,882f propertiesof,73t,77 argininemia,694t argininosuccinate,684 argininosuccinatesynthetase,684 reactionmechanismof, 684f argininosuccinicacidemia,694t Arnon, Daniel,759, 759t aromaticaminoacids Seealso aminoacid(s) as precursorsof plant substances,828,878f ailestin 1, 466 anestin 2, 430431,43I B-arrestin(Barr), 430f, 431 ARS (autonomously replicaringsequences), 991 artiicial chromosomes,310f, 953 bacterial,309,310 human,953 yeast,310, 310f,953 ascorbate, 127s ascorbicacid Seavitamin C (ascorbicacid) asparaginase, 701 asparagine, 75s,77,7OL, 866 degradationof, to oxaloacetate,Z0l-702, T02f propertiesof, 73t,77 aspartame,S3s stereoisomersof, 19f aspartate, 9s,75s,77,7O2,866 in C4pathway,789-790,789t degradationof, to oxaloacetate,T0\-702, 702t propertiesoI, 73t,77 pyrimidine nucleotidesynthesisfrom, 886-887,886f

aspartateaminotransferase(AST), 678b aspartatetranscarbamoylase, 220-22I, 22It, 887 aspaxtate-axgininosuccinate shunt, 684 Aspergi,ILtLs nige4 citric acid productionby, 633b aspidn, 818,818s.Seeolso acetylsalicylate; acetylsalicylicacid associationconstant(i'"), 156 in Scatchardanalysis,421b Astbury, William, 117 aslrnrnetry,molecular,16, 16f atherosclerosis,842-843 trans fatty acidsand, 348 atom(s) electronegativityof, 208 hydrogen,electrontransferard, 514 atomic force microscopy,385b atomic massuut, 14b ATP (adenoshetriphosphate),23s in activetransport,509-510 p oxidationyielding,654-655,656t in Calvin cycle, 782-7 83, 782f-T 85f cAMP synthesisfrom, 426427,428f , 429f concentrationof, 575 energyby group transferand, 506-507,506f,507f in glycolysis,528-533 balancesheetfor, 538-539 payoff phase,535-538,536f, 537f preparatoryphase,531-535 m preparatoryphase,534f,535f in heart muscle,919 hydrolysisof SeeATP hydrolysis h hlpoxia, 733 trtrbition of plruvate kinaseby, 579-580,589f magnesiumcomplexesof, 502,502f in metabolism,25, 26f in musclecontraction,509-510,918-919,9l9f nucleophilicdisplacementreactionof, 508,508f in nitrogenfixation, 856 phosphorylgroup transfersand, 501-511 in photos5,Trthesis, 782-786 synthesisof, 782-783, 784, 785t transport ot, 783-784, 784f in proteolysis,I 106f, 1l08f regulationof, oxidativephosphorylationin, 732-735,734t,735f relative changesIn, 576t in replicationinitiation, 985-986,985f supply of, 575 slrlthesis,SeeATP synthesis yield of, from oxidationof glucose,733t ATP hydrolysis,296-297,296f actinin, l7&179, l78f biochemtcalequationfor, 511 chemicalequationfor, 5l I equilibriumconstantfor, 495 free energyof, within cells,503 free-energychangeassociatedwith, chemical basisfor, 502f free-energychangefor, 50I-503, 502t,504t during ischemia,hhibitory proteins in, 733, 733f in membranetranspoft, 397-400,398f in musclecontraction,178-t79,178f, 918-gl9 myosinin, 17U179,778t as two-stepprocess,506, 506i ATP synthase(s),723, 725 B subunitsof, conformationsot,725t-726t, 72U728 binding-changemodetfor, 729f of chloroplasts,760-767, 760t functionaldomainsof, 725 in membranetransport,399 ATP synthasome,731 ATP synthesis,723-732 ATP synthasein conformationsof B subunitsot, 725f-726f, 726-728 functionaldomainsof, 725 binding-changemechanismfor, rotational catalysis in, 728-7 29, 729f, 730t chemiosmotictheory of, 707, 707-708,723, 723-724,723f couplingof electrontransfer and, 723-724,TZgf

cl,tosolicNADH oxidationin, shuttle systems conveyanceof, 731-732,731t,732f equationfor, 723 haloplulicbacterian, 762-764 O2consumptionand, nonintegralstoichiometdes of,729-730 by photophosphorylation,759-761,759f, 760t proton gradientin, 724,725t, 726,726t proton-motiveforce and activetransport in, 730-731,730f stabilizationof, 725-726, 726t standa"rd free-energSz changefor, 725-726 stoichiometryof, 630t ATPase(s) AAA+,986 F-type, 399, 399f Seeolso ATP synthase(s) in membranetransport,397J99, 397f-399f, 403,403t Na'K--, 398-399,398f,403,403f in membranepolarization,450, 450f in membranetransport,in neu_rons, 920 in retina, 463 P-type,396-397 V-t1pe,399 ATP-bindingcasette(ABC) transporter,400, 400f,840 ATP-gatedK' charurels,924 ATP-producingpathways,regulationof, 734,735f atrial natriuretic factor (ANF), 444 attractants,457 autocrinehormones,906 autonomouslyreplicatingsequences(ARSs),991 autophosphorylation,439. See also phosphorylation of insulin-receptortyTosinekinase,439, 440t in receptor enzlme activation,439,440f in signalug,439 in bacteria,457458, 458f in plants,460, 460f autotrophs,4f, 6 auxins,33lf,459s,878 Avandia,808s,824,936 A v e r y ,O s w a l d T , 2 7 8 Avery-MacLeod-McCarty expenment,278 aviansarcomavirus, 1052,1052f avidin,633-634 Avogadro'snumber (N), 491t azaserine, 894,894f Azotobacteruinelandii, nltrogenfixation by, 856 AZT, 1053b

b BACs @acterialartificial chromosomes),309,309f bacteria,4-5,4f SeeaLsoEscherickia coli, aminoacidsfound in, 877-878 anaerobic,incompletecitric acid cycle in, 631,631f antibiotic-resistant,277-218, 278f ABC transportersand, 400 plasmidsin, 949 cell structure of, 5-7, 5f cell walls of heteropolysaccharides in, 249 s}'nthesisof, 797f cellulosesynthesisin, 796 DNA replicationin ,977-991 endoslrnbiotic,33, 34f chloroplastsevolvedfrom, 761 in eukaryoticevolution,33, 34f mitochondriaevolvedfrom, 739,762f evolutionof, 32-33, 33f, 76l-7 64 fatty acid synthesisin, 806-811,815 generegulationin, 1126-1136 genesin, 952 geneticmap of, 976f glycogenslnthesis in, 792 gram-negative,5f gram-positive,5f greensulfur, photochemicalreactioncenter n,749,750f halophilic,in ATP synthesis,762-764,763f

lectins and, 260, 260f, 262t lipopolysaccharides of, 256-257,257f nitrogen-fixing, symbiotic relationship with legunmous plants,856-857,856f nucleoidsin, 970, 970f peptidoglycanslnthesis in, 796, 797f photochemicalreactioncentersin, 749-751, 750t,7'rt potassiumchannelin, 408-409,408f purple photochemicalreactioncenter in, 749-751, 750f,7'rf. photoreactioncenter of, 751f photoslnthetic reactioncenter of, 376,377f signalingin, 457458, 458f,459t structue of, 5-7, 5f bacterialartr.flcialchromosomes(BACs), 309,309f bacterialDNA, 949,950f, 950t packagingof, 970 topoisomerases and, 959 bacterialgenes,952 mappingof, 976f namingconventionsfor, 976 bacterialgenome,949, 950t sequencingof, 322, 323t bacteriophagel vector, 305t, 307,308, 309f, 101.2,10r2f bacteriorhodopsin,376, 762 Iight-drivenproton pumpingby, 762-764,763f mrcroscopyof, 385b structureof,376,377f ball-and-stickmodel, 15, 15f Ballard,Jotm,822 Baltimore,David, 1050,1051f Banting,FrederickG, 903b basaltranscriptionfactors,1139t base(s) aminoacidsas,8l in buffer systems,5943,62t nucleotide/nucleicacid,27I-27 7, 272f-274f, 272s-274s,272t,273s,274s alkylated,repair of, 999, 1000f anti form of, 280-281,280f Chargaffsnrles for, 278 chemicalpropertiesof, 276-277 in codons,1067-1068^9eeolso codons deamination of, 289-290, 290f n DNA, 276-277, 277t, 278-279, 279t, 280f estimation of via denaturation, 287-288, 287t functionalgroupsof, 276-277 in geneticcode, 1067-1068Seealso geneticcode hydrogenbondsot, 277, 277t, 278-279,279f, 280f,285,286f methylationof, 292 mnor,274,274f. nitrous acid and, 291,291f pairingof, 277,277t,278-280,279f,280fSee cr,lsobase pairs/pairing h replication,979f, 980f in RNA, 276, 284,285,286t structure of, 9s, 27l-27 4, 272-274, 272f-274t, 272t,276-277 syn form of, 280-281,280f. tautomericforms ot, 276, 276f. variant forms ot, 276, 280-281, 280f weak interactionsof, 277,277f , 279, 280f wobble,1072 relativestrength of SeepK" weak,57 basepairs/pairing,277 in DNA, 278-280,279f SeeaJsoDNA structure in replication,980,98if in DNA-proteinbinding,l121-1122,1121f in RNA, 284t-285,285t, 286f wobblein, 1072 base stackin4,27G277 in DNA, 27&279, 279f in replication,979f, 980f in RNA,276,284 base-excisionrepair, 994t, 996-997, 997f basichelix-loop-hehx,1124, ll25 B - D N A 2, 8 1 , 2 8 1 f

Beadle,GeorgeW , 624b,947,948t beads-on-a-string formation,963t, 964,964t beer brewing,fermentationin, 549 beeswax,349,349f benzoate,687s for hyperammonemia,686 benzoyl-CoA,686, 687s Berg,Paul,303,303f Bergstrom,Sune,358f, 359 Berson,Solomon,902 Best,Charles,903b termination B adrenergicresponse,in signaLing, of, 430 B barrel, 13lf, 379, 379t in membranetransport,379,379f B cells,170, 170t,921 pancreatic,923-924,924f recombirationin, 1015-1016,1016f B conJormation,l2O, \20f-123f in smallglobularproteins, 131t structural correlatesof, 123t B conformationsheet SeeB sheet B oxidation,647-649 Seaolso oxidation in bears,655b enzyrnesof, 663-664,663f in peroxisomes, 662-663, 662t plarts and, 662-663,663f stepsin, 653-655,653f yieldng acetyl-CoAand ATP, 653f, 654-655 B sheet,120-121, I20f-123f in largeglobularproteins, 135,135f in protein foldurg,135-136,135f,148 structural correlatesof, 123t twisted, 135, 135f B slidingclamp,983-984,984f, 988,988f B subunits,of ATP synthase,different conformationsof, 725t-726f, 726-728 B tum, 121,l22f ,123, I23t B-c-Bloop,131, 131f,l36f B-adrenergicreceptor. 42943 I etrflnf,rra ^f

4r4

4r.4t

B-adrenergicreceptor kinase(BARK), 431 p-adrenergicsignalng pathway,423431, 424t, 428f430f 361,361s,745s,747 B-carotene, B-lactamantibiotics,resistanceto, 217-218,218f 217 -218, 2l8f B-lactamases, bicarbonate as buffer, 57f, 59-63, 6t-63 formationof, 165-166 bicoid, 1748-1749 bilayer,lipid, 372,374, 374f formationof,374,374f gel phaseof, 381 Iiquid-disordered(fluid) state of, 381 liquid-orderedstate of, 381 bile acids,357,357s,836 bile pigment,heme as source of,875-876,876f bilirubin,875 breakdownproducts of, 876f binary switches,G protein(s) as, 425b427b bindrng,cooperativityin, 160-165,16if, 162f binding energr (AGB),189-192 in eru;'rnaticreactions,189-192 enz)'mespecificityand, 191-192 binding sites, 16A, rc2 SeeaLsospecifi'ctgpes antibody,171-L73, 172f characteristicsof, 170 binding specificity, antibody,\7 0-77I, 173, 173t biochemicalreactions bond cleavagein, 496-497 commont}?es of, 495-501 electrophilic,496, 496t eliminationsin, 497-498 free-radical,498 group transfer,499-500,499f internal rearrangementsin, 497-498 isomerizationin, 497-498 nucleophilic,496,496f oxidation-reductionSeeoxidation-reduction reactions vs chemicalreactions,500-501

biochemicalstandardfree-energychange(^G''), 186, 187f biochemistry fundamentalprinciplesof, 2 whole cell, 98-100 bioenergetics,22-24, 490495 Seealso energy oxidation-reductionreactionsand, 512-521 phosphorylationand, 501-511 thermod)'namicsand. 490-495 biohformatics, 103 biologicwaxes,348,349, 349f biologicalenergytrarsformation, in thermodl'namcs, 490491,491i bioluminescencecycle,of fuefly, 509b biomolecnles, L See also 1t'nder molec'alat amphipathic,46t asFrunetric,16, 16f averagebehaviorof,28 chirality of, 16, 16f conformationof, 18, 18f derivedfrom aminoacids,873-882 functionalgroupsof, 12f, 13, l3f interactionsbetween,10-12 lrght absorptionby, 76b, 76t macromolecules,14 molecularmassof, 14b molecular weight of, 14 nonpolar,45,46t origin of, 30-31, 31f Saeo,so evolution polar,45,46t sizeof, 10-11,10f small,10-11 of, 15' 18, 18f, 19f stereospeci.flcity in supramolecularcomplexes,29 bioorganisms classificationof, 4-5, 4t, 5f distinguishingfeaturesof, 1-2, 2f biosignaling See signaling biotin,664,633 deflciencyof, 633-634 in acetyl-CoAcarboxylasereaction,806f in phosphoenolplruvate slrtthesis from plruvate, 554,554t in pynuvatecarboxylasereaction,554,554f, 633, 634f,635f Bishop,Michael,1052 (BPG), 504s,636 1,3-bisphosphoglycerate hydrolysisof, 504, 540s phosphoryltransfer from, to ADP, 536-537 synthesisof, 728t, 778-T80, 779f, 782-783 (BPG), in hemoglobin2,3-bisphosphoglycerate orygen binding, 167,168f bisulfltes,as mutagens,291 Blobel,Giinter,1100,1100f Bloch, Koruad,835,835f blood bufferingof, 57f, 61-63 compositionof, 92I, 921t electrolltes of, 921 glutarune transport of ammoniain, 680-681,680f metabolic functio^s of , 920-922 normalvolume of, 921 pH of, 62-63 transportfunctionsof, 920-921 blood clotting integrinsin, 456 proteolysisin, 227 blood groups,sphingolipidsin, 354, 355f blood plasma,g2l,92lf blue fluorescentprotein (BFP), 434b-436b blunt ends,306,307 boat conformation,239f body mass.Seealso fat, bodY regulationof, 8 -a22,930-937 teptin in, 930-931' 931f-934f body massindex, 930 Bohr, Christian,166 Botu effect, 166,166

f

l-6

Index

Bohr,Niels,166 boilingpoint of corunon solvents,44t of water, 43, 44t Boltzmannconstant(k), 49lt bond(s) Seealso weak interactions carbon,11-13,12f carbon-carbonSeecarbon-carbonbond bond(s) carbon-hydrogen,cleavageot, 496 497, 496f covalent,10 in enzlmatic reactions,189 heterol''tic cleavageof, 496,496f homolltic cleavageof, 496,496t of phosphorus, 499-500,499f disruption of in aminoacid sequencing,95, 96f energrfor, l14 glycosidic,243 phosphorolysisvs hydrolysisreactionsof, 595 596 hydrogen Seehydrogenbonds N-p-glycosyl,272 hydrolysisof, 290, 290f noncovalent, 10,50-51 Seenlso weak interactions O-glycosidic,243 peptide Seepeptide bonds phosphate,high-energr,506 phosphorus-oxygen, 499-500,499f bond dissociationenergy,44 bovineFr-ATPase, structureof, ?33,Z33f bovinespongiformencephalopathy,147-148 Boyer,Herbert,303,303f Boyer,Paul,728,729f. BPG (2,3-bisphosphoglycerate), in hemoglobinoxygenbindhg, 167,168t brain glucosesupply for, 926-928,927f metabolismin, 920,920f branchmigration, 1005-1006,1006f,1008f brassinoLide, 360s,459s brassinosteroids, 459,459s,460 BRCAL/2,breastcancerand, 1003b breast cancer,1003b brown adiposetissue(BAT), 736, 9IT, gITf heat generatedby, in oxidativephosphorylation regulation,736,736f mitochondriain, 736 Brom, Michael,841,841f Buctmer,Eduard, 184, l84f , 528,577 buffers,59-63,59-66,61f bulges,in RNA, 285,285f bungarotoxin,4ll-412 trorzs-l2-butenoyl-AcP,809, 810f, 81lf butlryl-ACP,809, 810f,811f b),?assreaction,in gluconeogenesis fructose 1,6-bisphosphate to fructose 6-phosphateconversionas,553t, 556 glucose6-phosphateto glucoseconversionas, 553r,556 pyruvate to phosphoenolppuvateconversionas, 553-557,553t, 554f, 555f

c C See catbonentries C2cycle,789 Capathway,789 C3plants,776 photosJ,'nthesis in, 776, 789, 790 Caplants,photosynthesisin, 789-790 Ca"- channels defective,diseasescausedby, 4l2t in glucosemetabolism,924,924f in signaling,451 452,451f, 4534b4 Ca'- concentration,in cttosol vs. extracellular fluid,451t oscillationof, 437, 438t Ca2+ion channel,406,409+10 Ca" pump,39?-398,39Zf Ca2+/calmodulin-dependent protein kinases,4i|2, 43Zf

Cairns,John, 978,982 calcitonin,stnthesis of, 1040,1041f calcitoningene,altemativeprocessingof, 1040,1041f peptide.synthesisol', calciloninEene-1p121"6 1 0 4 0 i,0 4 1 f calcrtriol,360,360s,906t,908 calcium blood levelsof, 921 IP3ard, 432-433, 433f.,447 in musclecontraction,178 regulationof, 908 in vision, 464, 464f, 465 calnodulm (CaM), 436, 437t cahexur,261 Calvincycle,774 ATP in, 782-786,782f-785f 1,3-bisphosphoglycerate in, 778, 779f in Caplants, 789-790,789f in CAMplants, 790 carbon-fixationreactionin, 776-778, 775t 2-carboxryarabinitol 1-phosphatein, 778 glyceraldehyde3-phosphatesynthesisin, 773, 776f,777f,778 NADPHin, 773,775f, 778,782-786,782f-785f 3-phosphoglycerate in conversionof to glyceraldehyde3-phosphate, 775,778 slrrthesisoI, 773,776 778 rubiscoin, 7787 78, 776f-778f regenerationof, 778-780, 780f,781f stoichiometryof, 782f triose phosphatesh 1,5-bisphosphate regenerationfrom, 778-780 regenerationof rubiscofrom, 77U780,780f,78lf synthesisof, 779f Calvh, Melvin,774, 774f CaMkhases,437 CAM plants, 790 cAMP (adenosine3',5'-cyclic monophosphate), 274,298,298s degradationof, by cyclic nucleotidephosphodiesterase,424f, 43O hormonalregulationof, 431-432 measurementof, by FRET,434b 436b in protein kinaseA activation,424t,428,428t as secondmessenger, 298,424f,43I-432,431l rn p-adrenergicpaLhway,426427, 428f, 430f structure of, 298s,424f sl,nthesisof, adenylylcyclasen,426427 , 428t,429f cAMPreceptor protein (CRP), 1029 in generegulation,\126-1127 cAMPresponseetement(CRE),1144 cAMP responseelementbinding protein(CREB),431 protein kinase Seeprotein cAMP-dependent kinaseA (PKA) camptothecin,961 cancer citric acid cyclemutationsin, 637-638 detectionby PET scanning,541 DNA repair in, 1003b as genetherapy complication,336 genetherapy for, 336 glucosecatabolismin, 539, 540-541 integrinsin, 456 mutationsin, 47447 5, 477f , 637,993, 1003b oncogenes a:,d.47347 4, 477f retroviruses and,1051-1052,1052f selectinsand, 259 treatmentof chemotherapeuticagentsin, 328, 894-896, 960b-961b microarraysin, 328 protein kinasesn, 475b47 6b targetingenzl.mesin nucleotidebiosynthesis in, 894-896,894f, 895f topoisomeraseirhibitors in, 960b-961b transcriptionalprofllesir, 328 genesand,474475,477t tumor suppressor cannabinoids,442, 442s CAP SeacAMP receptorprotein (CRP)

cap-bindingcomplex,1035,1066f capsid,viral, icosahedralsyrnrnetryof, 139-140,140f carbaminohemoglobin, I 66s formation of, 166 carbamoylglutamate,687s carbamoylphosphate,pyrimidine nucleotide s],nthesisfrom, 886-887,887f carbamoylphosphateslnthetaseI, 684 activationof, 685, 686f deficiencyof, 6941 reactionmechanismof, 684f carbamoylphosphatesynthetaseII, 886 carbamylphosphate ,9aecarbamoylphosphate carbadon, 496,782f carbocation,496 carbohydrate(s),235-265 analysisof, 263-265, 264f chemical slalthesis ot, 263-265 classificationof, 235 disaccharide,236, 243-244 dolicholsand, 362 Fischerprojectionformulasfor, 236r 236f glycoconjugatesof, 235 intermediatesof, 240f, 241, lectin binding of, 258-261, 258t, 259f, 261f, 262f monosaccharide, 285, 235-241 Seeolso monosaccharides nomenclature of , 235-236, 236-238, 240-24I oligosaccharideSeeoligosaccharides overuiewof, 235 oxidationof, 241, 243-244 polysaccharide,235, 244-252 See also polysaccharide(s) sizeclassesof, 235 carbohydrateanabolism,pathwaysof, 913t carbohydratecatabolism in cellularrespiration,615,616f pathwaysof, 913t carbohydratemetabolism,570f anabolic,913t catabolic,9l3t in diabetesmellitus, 542f enzlmesof,574,575t geneexpressionh, insulin and, 606 hepatic,914,915f metaboliccontrol analysisappliedto, 581-582 pathwaysof, 9l3t in plants, 773-786,797-800,798f Seealso Calvincycle regulationof allostericand hormonal,606-608,608,608f Lipidmetabolicintegrationwith, 608 in Liver,606-608 in liver and muscle,608f in muscle,608 xylulose5-phosphatein, 588 carbohydrateresponseelementbindng protein (chREBP),5gr-592, 591f carbohydrateslnthesis, 552, 552f, 773-800 Seealso gluconeogenesis C. pathwayin, 789-790 celluloseslnthesis and, 795-796,797f glycolatepathwayn, 787-7 89, 787f integratedprocessesin, 797-800,798f,799t pentosephosphatepathwayin, 775, 785-786,798 peptidoglycans)'nthesisand, 796-797,797f photorespirationin, 786-790 photosJ,nthetic, 773-T86 See also photosynthesis starch slaithesisin , 791-792,793 sucrosesJ.nthesis n,792 794 carbon anomeric,239 asyrnrnetric,16, 16f oxidationof, 496f, 500,5l3f carbonbonds,11-13,l2f carbondioxide,5l3s cyclingof, 486,486f hemoglobintransport of, 166 in hemoglobin-orf/genbinding, 166 oxidationof a-ketoglutarateto, 623 oxidationof glucoseto, 516

oxidationof isocitrateto, 623,623f oxidationof pyruvateto, 616-617,616f partial pressureof, 62 in photos5nthesisSeeCalvincycle solubility of in water, 47, 47t carbondioxide assimilation,774, 774f, 775-780 Seeolso Calvincycle in C3plants,789,790 in C4plants,789 790,789t in CAM plants, 790 carbonfixation, 664, 774f, 775-T 78, 775f See also Calvh cycle in Caplants, 789-790 carbonmonoxide,513s hemoglobin bindingof, 155,158, 1 6 2 ,1 6 8 physiologicaleffectsof, 168 carbonmonoxidepoisonmg,168 reactions,742, 743, carbon-assimilation 785-786,785f in Cj plants, 789-790,789f in C4plants, 789 790, 789f carbonates,hydrolysisof, transition state in, 2).t,zltf carbon-carbonbond cleavageof, 496,496f reactionsof,496 497,504f carbon carbonbonds,cleavageof, 496-497,497f carbon-furationreactions,742 carbon-hydrogenbond, cleavageof, 496497,196f carbonicanhydrase,166 carbonylgroups, 497, 4971 2 carboxyarabinitol1-phosphate,778s in Calvincycle,778 634s carboxybiotinyl-enz),rne, 77, 78s 7-carboxyglutamate, carboxyhemoglobin,168 physiologicaleffectsof, 168 carboxylicacids,13,5l3s carboxyl-termiralresidues,82 posttranslationalmodificationof, 1096 A, 676 carboxypeptidase carborl,peptidaseB, 676 cardiacmuscle,919,919f cardiolipin,35lf, 370f,372 stnthesis of,8261,827 caretakergenes,477 carnaubawax, 349 carnithe,651 carnitineacyltransferaseI/ll, 651 camitineshuttle,650 carotenoid,747 carriers,391, 391f Seearsotransporter(s) Caruthers,Mawin, 294 carvone,stereoisomersof, 19f caseinkinaseII, 605 CASPcompetition,143 in apoptosis, 478 caspase, 738 caspases, catabolicpathways,773, 913f energydeliveryin, 487f in glycogenmetabolism,595-596 catabolism,25,26f, 487, 487f aminoacid, 673-674,687-702 Seealso amino acid catabolism glucose,in cmceroustissue,539,540 541 protein, fat, and carbohydrate,in cellular respiration,6l6f purinenucleotide,892,893f pyrimrdine,892,893f 1126 cataboliterepression, catalase, 662 25,26f,186 194 Seealso catalysis, 6hr\rm.fi^

ra,.fi^na

acid-base general, 192, 193f specific, 192-193 covalent,193 in membrane lipid diffusion, 382t metal ion, 193 194, 21.3,2l3f regulalion of,220-227

rotational,T2S 729 vs specificity,l9l catalJ,ticantibodies,2ll catalj,ticsite, 154 catalj,tictdad, 207 catecholamines, 906t,907,907 as hormones,902,906t,907 as neurotransmitters,902 catenanes, 991 cation-exchangechromatography,86, 87f, 88t Sae o.isoion-exchangechromatography caudal, I 149-I 150,ll50f caveolae,386, 386f caveolh,386,386f,449 CB1receptor,442 C-Cbond Seecarbon-carbonbond ccA(3'), 1044-1045 CCAAT-bindingtranscriptionfactor I (CTF1), proline-richactivationdomainsof, 1143 cDKg, 1032 cDNA (complementaryDNA), 316 cloningof, 1060-i061 289,290,1051 in hybridization, cDNAlibrary,316, 316f.1060-1061 CDP (cltidine diphosphate),824 in lipid biosynthesis,824-827, CDP-diacylglycerol, 825f,826f Cech,Thomas,1037,1037f cell(s),2-1I b a c t e r i a l , 57 , 5 f death of Seeapoptosis energyneedsof, in oxidativephosphorylation regulation,733 fat cellsin, 346,346f free-energyof ATP hydrolysiswithin, 503 metabolictransformationsin, 495-496 origin of,30-31 Seealso evolution sizeof, 3 sourcesof free energyfor, 491 structure of in bacteria,5-7, 5f in eukaryotes,6f, 7-9 10-11,10f hierarchical, cell cycle chromosomechangesin, 962,963f meiosisin, 1004-1005,1004f 1004f 1004-1005, in eukaryotes, 1006f in, 1004-1005, recombination regulationof cyclin-dependentprotein kinasesin, 4701472f, 470f-473t cltokrnes in, 47If,472 growth factorsin , 47lf, 472 replicationin SeeDNA replication retinoblastomaproten in, 472, 473f stagesof, 469,469f cell death,programmed,4TT-478,478f See also apoptosis cell envelope,5f, 6 cell fractionation,8-9, 8f, 52 cell membrane Seemembrane(s) slnthesis of cell wall polysaccharides, in bacteria,795-T96, 797f in plants,795-796 388-389 cell-cellinteractions/adhesion, cadherinsin, 388 ntegrins in, 254, 2551,388' 455-456, 455f lectins in, 260,262f in, 252-255, proteoglycars/oligosaccharides 257-262 selectrnsin, 388 cell-ecllsignalingSppsignaling cellulardifferentiation,571 cellularfunctions,of proteins,324,325-329 cellularimrnunesystem,170. Seeolso imune system cellularrespiration,615 stagesin, 615,616f 247,247f cellulase, cellulose,246 247,2511Seealso polysaccharide(s) conformationsof, 248f function of. 246, 248,25lr

structure of, 246, 246f, 248,248f, 25)i, 794,794t synthesisof,795 796 tensilestrength of, 248 celluloseslnthase, 795, 795 centraldogma,945,945f,1050 centrifugation,differential,8-9 953, 953f centromere, ceramide,353f,354 in cell regulation,358 350f,353f,354' 355f, cerebrosides, 829,831f slmthesisof, 829 ceruloplasmin,258-259 CFTR,401,410,840 cGMP(guanosine3',5'-cycLicmonophosphate), 446446 273s,298,298s, as secondmessenger,445446,445f in signaling,445-446 stmcture of, 298s as secondmessenger,445446 in vision, 463-464,464f, 465 cGMPPDE,446 protein kinase(protein cGMP-dependent kinaseG), 445-446 C-H bond ,Seecarbon-hydrogenbond chair conformation,239f Changelu,Jean-Pierre,165 chamels Seeion charmels 143 145,144f,145f,1104,1106f chaperones, chaperonins,144,1451 Chargaff,Erwin,278 Chargaffsmles, 278 charge of aminoacids,80-81,81f p H a n d ,8 0 - 8 1 , 8 1 f Chase,Maftha, 278 chemicalelements essential,11f trace,1lf, 13 chemicalmodels 15-16,15f ball-and-stick, I5, 15f space-filling, chemicalpotential energy,of proton-motive force,720 chemicalreaction(s), 19-26 Seealso under reaction activationenergyin, 25' 25f directionof,22,24 driving force of, 25 dynamicsteadystate and, 20 22,24,261 endergonic, energ/ consenationin, 20, 20f energysourcesfor, 22 energy-coupled,22-24, 23t enzymatic, 183-227 See a\so catalysis; enz5,rne(s) 25, 26f, 186-794 Seealso enzl'rne-catalyzed, enzFnaticreactions at equilibrium,2S equilibriumconstantfor, 24 exergonic,22, 24, 25, 26f in citric acid cycle,636-637 in conversionof p;mrvateto phosphoenolpyruvate, 553-557,553r,554f,555t coupledwith endergonicreactions, 22-24,23f freeenergyin, 22,23-24,186-187,187f ground state ln, 186 intermediatesin, 209 oxidation-reduction,22 rate constantfor, 188 rate equationfor, 188 rate-iimitingstepsin, 187 reactionintermediatesin, 187 sequential,494-495 standardfree-energychangesof, 492, 492r at pH 7 0 and 25 "C, 493t steady-state,21 transition statein, 25, 18?, 189-1I 1, 207,209, 2r0b-21Lb chemicalreactionmechanisms,208,208f

T Ll-8

Index

chemicalreactions,vs biochemicalreactions, 500-501 chemicalsynthesis,263-265 of DNA,294,295f phosphoramiditemethod in, 294,29bt chenxcaluncouplers,of oxidativephosphorylation, 724,724f chemicals,mdustrial, fermentation yieldirg, 549,549t chemiosmoticcoupLing,of 02 consumptionand ATP synthesis,729-730 chemiosmotictheory,7 07, 723-T24 ofATP synthesis,723 prediction of, 724,725f chemistry,prebiotic,30-31 chemoheterotrophs,32 chemotaxis,two-componentsignalingin, 457458,458f chemotherapeuticagents targetingeMlmes in nucleotidebioslnthesis, 894-896,894f,895f transcriptionalprofilesfor, 328 chemotherapy, topoisomerase hhibitors ur, 960b-96rb,961s chemotrophs,4f, 5 chiral center, 16, I6f,72 chiral molecules,16, ),6t,72-73 opticalactivity of, 17, 73 chitin,247, 247f,251t chloramphenicol,1098 ctrloride,blood levelsof, 921 chlorideion channels ur cysticfibrosis,401,401f,410 in signaling,453-454 chlorideion concentration,in cytosolvs extracellularfluid, 45lt chloride-bicarbonate exchanger,384f, 895, 395f chloroform,rn lipid extraction,363-364,364f cNorophyll, 745 antenna,in hght-drivenelectronflow, 252, 754-755,754f in light absorption,745-T47, T4bt,T46f excitationtransfer and,747 749, T48t chlorophylla, 745f chlorophyllb, 745f chloroplast(s),773, 774, 775f ATP sJ,arthase of, 760-761,760f ATP slnthesis ur,707-708 Seealso ATP synthesis evolutionof, 761-762 fatty acid slmthesisin, 812, 8l2f integrationof photosystemsI andII n, Tb2-T54 lipid metabolism in. 812,8t2I membranelipidsot. 352,352f NADPH slmthesisin, 812 photosgrthesisin, 743-T44,743t electronflow in, 743-744 protein targetingto, 1104 starchsynthesisin ,79If,792 chloroplastDNA (cpDNA), 951 cholecalciferol(vitamin Ds), 360, 360f,360s cholecystokinin,675 choleratoxin, 258t, 260, 426427 cholesterol,355, 355f Seealso sterols esterificationof, 836, 836f excessproduction of, 843 fate of, 836 in isoprenoidslrthesis, 84b,84bf membrane,372t microdomainsof, 384-386, B86f receptor-mediatedendocJ,tosis of, 840f, g4l in steroidhormoneslnthesis,359, 359s, 844-845,844t structure of, 355-357,355f,839f slnthesis of, 832-836,832f-836f regulationof, 847-842,841t, 842t trans fatty acidsand, 348 transport of, 836-841,838f, 839f cholesterolintemediates, slnthesis of, 844t, 84b cholesterol-loweringdrugs,842b 843b,843 cholesterylesters,836 receptor-mediatedendocrtosisoI, 840f, 842 transpoft of, 836-841,g39f

chondroitin sulfate,250,250s,253f chorismate,in amino acid biosJ,nthesis, 865-869, 868f-870f ChREBP(carbohydrateresponseelementbinding p r o t e i n ) , 5 9 15 9 2 , 5 9 1 f chromatids, sister,962,1004f,1005,1005f chromatin,962,965f in 30 nm fiber, 966, 967f acetylation/deacetylation of, 1137 1138 assemblyof, 965f beads-on-astring appearanceof, 963f, 964f condensed, 1136 euchromatin,l136 heterochromatin,ll36 histonesand,963, 963f,966b-967b,1137 1138 Saaolso histone(s) nuclearscaffoldsand, 968, 968f nucleosomesin, 964f, 965f,967f remodelingof, 1137-1138,11371 transcriptionallyactivevs inactive, 1136-1137 transcnption-associated changesin, 1136-1138,1137t chromatography,173 adsorption,364-365,364f affility, 86, 87f,88, 88t, 313-314,313t column,85-88,86,86f gasliquid,364f,365 high-performanceIiquid, 88 ion-exchange,86, 87f, 88t in lipid analysis,364-365 size-exclusion, 87, 87f thinlayer, 364-365, 364f chromatophore,759 chromosome(s),947953 artiflcial,953 bacterialartificial,309, 309f cell-cyclechangesin, 962-963,963f condensationof, 962, 963f,969, 970f contiguousdrmeric,repauof, 1013,1013f daughter,469 definition of, 962 elementsof,947-953 eukaryotic,949-951,951f genesin, 947,948 Seealso gene(s) paftitioning of, in bacteria,991 repLicationof, 962, 963f structure of,962-971 yeastartificial,3f0, 310f, 953 chylomicrons,649, 837f,837t, 838, 838f molecularstructure of, 649f chlmotrypsin,676 catalltic activity ot, 194,204f,205-207, 207t 209t transition-statecomplementarityand, 202, 2r0b-2r1b,2r0t reactionmechanismof, 208f-209f,499 structureof, 113f,205,206f slmthesisof. proteoll.4ic cleavage in, 226-227,227f ch)Tnotr'?srnogen,225,227t, 676f cimetidine,879 ciprofloxacin,960b,960s circular dichromism(CD) spectroscopy, 122, r22t cis configuation, ofpeptide bonds, l2I, IZlf cis-transisomers,l5 citrate, 622, 622s asynmetric reactionof, 629b,629t in fatty acids''nthesis,813-814,813f,814,814f formation of, in citric acid cycle,622,622f, 623f citrate lyase,813 citrate s),nthase,622, 622s,633b,8f 3 reactionmechanismof, 623f structure of, 622f citratetransporter,813, 813f citric acid,AspergilLusniger productionof, 6B3b citric acid cycle,615, 615-638 activatedacetatein, 616-620,616f, 617f,618f,634f anapleroticreactionreplenishing,631-633, 632t,632r citrate formationin, 622, 622f, 623f componentsof, 631, 632f

conversionof succinyl-CoAto succmaten, 626,626t energyof oxidationsin, 629f,630t in glucosemetabolism, 914-915,914t,9151 927f,928 glyoxylatecycleand, 639-640, 639f, 640f in hepaticmetabolism, 914-915,915f, 927f,928 rncomplete,anaerobicbacteriain, 631, 63lf intermediate anapleroticreactionin, 631-633,632f,632t in gluconeogenesis, 557,927f, 928 isocitrate fomati on n, 622-623, 623f in Lipidmetabolism,915f, 916 oncogenicmutationsin, 637-638 oxidationof acetatein, 631,63lf oxidationof acetyl-CoAin, 655, 6561 oxidationof isocitrateto a-ketoglutarateand Co2trq623,624f oxidationof malateto oxaloacetatein, 628-630 oxidationof succinateto fumarateur, 628 of oxidationsin, 630-631 products of, 630f p1'ruvatecarboxylasereactionh, biotin and, 633, 634f,635f reactionsof, 620-635,636f regulationof, 635-638 allostericand covalentmeehanisms h, 635-636,636f exergonicstepsin,636 637 glyoxylatecycle in, 639 640, 639f,640f multienzj,mecomplexesin, 637,637f role of, in anabolism,631,632f stepsin, 622-630 urea cyclelinks to, 684-685,685f citrulline, 77, 78s Cl- Seechloride Claisenester condensation,497, 497s clathrates,48 clathrin,1106, 1107f Clausius, Rudolf,2lb clamlanic acid, 217-218 clonalselection,170 clone(s),173 definition of, 304 in DNA libraries,315f,316f,317 clonedgenes,expressiorvalteration of, 312,3r2f clonhg, 304,304-314 in animals,332-334 in bacteria,304-312 blmt endsin, 306 cDNAin, 290f,1051,1060-1061 DNA cleavagein, 304-307, 304f,305t, 306f DNA fragmentsizein, 306-307 DNA hybddizationin, 310-311,311f DNA ligasesin, 304, 304f,306f, 307,351f electroporationin, 307 enzymesin, 305t in eukaryoticcells,315-316 fusionprotehs in, 313 gene expressionand, 312,372f lmkers/polylinkersin, 306f,307 nucleicacidprobesin, 311,311f oligonucleotide-directed mutagenesisand, 3 1 2 ,3 1 3 f in plants,330-332,330f-333f pollmerase chain reactionln, 3l?, 318f proceduresusedin, 304,304f restriction endonucleases in, 3041-307,304f, 305t,306f site-dhectedmutagenesisand, 312 stepsin, 304, 304f stickf/endsin, 306, 306f,307 transformationin, 307 in vitro packagurgin, 308 cloningvectors,304,305t,307-310,307f 3lOf bacterialartificial chromosome,309,309f in DNA library creation,316 expression,312 1,309f l phage,305t,307,308,1012,1012t for plmt s. 330-332,330I-333f plasmid,307, 308f,309f

shuttle,3l0 Ti plasmid,33G331 viral,333,334f yeastartificial chromosome,309,3f0 closedsystem,in thermodynamics,20 closed-circularDNA, 955, 955f linkmg number for, 956-958, 956f, 957f, 965 Clostl"LdiurnacetobutgrLcurryin fermentation,549 Clostrndt um botulinum" 388 clotting nr^fa^lr,cia

in

997

selectinsh, 388 CLVI peptide,460 CMP (cltidine 5'-monophosphate),273s,824 CO2 Seecarbondiodde A), 297t,298, 607s Seealso CoA (coer-rzyrne specxncWpe, e g, succinyl-CoA coactivators,I 139-1 140,1139f coagulation proteolysisrn, 227 selectinsin, 388 coatedpits, 1106, 1107f cobalamrn,Seevitamin B12(cobalamin) cobrotoxin,4l I codingstrand,in transcription,LO24,I024f CODISdatabase,320, 320r codons,31lf, 1066-1074,1066f of, 1068-1069,1069f assignment variationsin, 1070b-I071b basecompositionof, discoveryof, 1067 basesequencesin, di.scoveryof, 1067-1069 dictionaryof, 1069,1069f initiation, 1069, 1069f 1090f in proteinslnthesis,1088-1091, readingframe for, 1038f,tO67, 1067f,1072 terrunation,1069, 1069f,1070b-1071b coelacanth,anaerobicmetabolismof, 548b coenzl'rne(s),3, 184, 185t ,Seeolso erulme(s) examplesof, 184t flavin nucleotide,520t NAD' or NADP' as,sl,ereospecificity of dehydrogenase employing,518t prosthetic-group,184 rn pyruvatedehydrogenasecomplex,617,6l7f as universalelectroncarrier, 516 A (CoA),297f,298,607sSeea\so coenzl.rne specific tg72e,e g, succuryl-CoA coenzlrneB12,65?, 658b-659b,658f, 659f coenzyrne Q, 362,710, 7l0f coenzlme reduction,stoichiometryof, 630t cofactors enzl.rne,184, 194t of nitrogenasecomplex,855f Cohen,Stanley,303, 303f cohesins,969 coiled coils collagenas, 124f,125 d-keratin as, 123-124,I24f 1014 cointegrate, collagen aminoacidsn, 124f,125,126-127, I28 ascorbicacidand, 126-127 disordersof, 126-128 proteoglycansand, 254 structure of, L22f, 1231,124-128,124f, I25f triple helix of, 122t, 723r, 124-125, I24f , 126-127 typesof, 125 colligativeproperties,solute concentrationand, 61,51f Collins,Francis,317,322f Collip,JB,903b coloncancer,1003b mutationsin, 475,477f color vision, 465,465f colurnnchromatography,86+8 See ^L-^-^+^d--^L., 4PrrJ rru urlaLUSr

comparativegenomics,35-36, 326, 326, 328,329f competitiveinhibitor, 2Ol-203, 202f, 203t,203t complementaryDNA SeacDNA (complementary DNA) Complexi: NADH to ubiquinone flow of electronsand protonsthrough, 719f in oxidativephosphorylation,7 l2-7 | 4, 7I 4f

ComplexII: succinateto ubiquinone flow of electronsand protonsthrough, 719f ur oxidativephosphorylation,715, 7l5t ComplexIII: ubiqurnoneto cltochrome q 7J.6f,7r7f flow of electronsand protonsthrough, 7l9f in oxidativephosphorylation,7I5-7 16,7I6t c to 02,716-T18,7l8f Complex[V: c]'tochrome flow of electronsand protons through, 719f path of electronsthrough, 7I8f complextransposons,1013 concentrationgradient,389-390, 389f,390f h membranepolarization,450,450f concertedinhibition, 872 concertedmodel, of protein-ligard binding, 165,165f condensation,65f 963f chromosomal,962, in peptide bond formation,82, 82f condensationreacton,66 condensins,969 cone cells, 462465, 462t in color vision, 465,465f configuration,16 isomeric,15-18,15f,16f vs conformation,239 conformation boat,239f chair, 239f molecular,18, 18f native,29, 114 proteh,1l3-114 vs configuration,239 conjugateacid-basepairs,67, 57f as buffer systems,60, 60f, 61-62 conjugateredox pair, 512 electrontransferof, 514-515,514f conjugatedproteins,84, 85t consensussequences,102 oriC, 985f,986-987 of promoters,1025, I025f, 1117f of protein kinases, 225-226, 226r consensustree of life, l06f conseruationof energy,20, 20f constitutivegeneexpression,1116 contig,315 repairof. contiguousdimericchromosome. 1 0 1 3 1, 0 1 3 f contractileproteins, 175-179,176t-I79t binding,160-165,161f,162f cooperativity, copperions,in ComplexIV, 716-718,718f Corey,Robert,115f,117,124,130 Cori, Carl F , 548b,598,598f Coricycle,548b,552,919,9l9f Cori, Gerty T , 548b,598,598f Cori'sdisease,599t Cornforth,John,835,835f coronaryartery disease hyperlipidemiaand, 842-843 trans fatty acidsand, 348 corrin ring system,668 corticosteroids,359, 359f,359s,906t, 908 Seealso under sLeroid hormone(s) synthesisot, 844-a45,844f cortisol,359,359s,823s,911,929 in glucosemetabolism,823 cotransportsystems,396, 395f couplurg,operationaldefinition of, 723 covalentbonds,10 in eruymaticreactions,189 heterolfiic cleavageof, 496,496f homolltic cleavageof, 496' 496f of phosphorus,499-500,499f covalentcatalysis,193 covalentmodification,of regulatoryenzlmes,220' 223-227,223f,226t,226t,227f 817' 818 COX(cyclooxygenase), inhibitors of, 818,818f cox4,734,734f DNA),951 cpDNA(chJoroplast C-protein,177 Crassulaceae,photosFthesis in, 790

creatine,505s,876-877, 918s amino acids as precursors of, 87G877 biosynthesisof, 877f in musclecontraction,918, 918f creatinekinase,610, 678 Creutzfeldt-Jakobdisease,147-148,147f Crick, FYancis,93, I23, 277, 277t, 278-279, I 45, 977,1056,1056f,1066 crocodile,movementof, 548b crossingover, i004f, 1005,1005f CRP SeecAMPreceptor protein (CRP) cruciform DNA, 282,282t,958, 958f crude extract, 86 cryptoctuome,620 crystalstructures,bound water moleculesin, 50-51,51f culture, tissue,332 cyan fluorescentprotein (CFP), 434H36b cyanobacteria,5f evolutionof, 33, 34f nitrogenfixation by, 852 phosphorylationin, 762, 762t 658 cyanocobalamin, cyclic AMP SaacAMP (adenosine3',5'-cyclic monophosphate) cyclic GMP Sea cGMP(guanosine3',S'-cyclic monophosphate) cyclic nucleotidephosphodiesterase, 424t,430 cyclic symmetry,199' 139f cyclin,469,469f degradationof, 471472, 471f, I 109 synthesisof, 472 kinasecomplex,469-473, cyclin-cyclin-dependent 470f,47lt cyclin-dependentkinase9, 1032 cyclin-dependentprotein kinases,469473, 47Of in cell cycle regulation,469473, 470f inhibition of, 472-473 oscillatinglevelsot, 437,438t phosphorylationof,470473, 470t, 47Lf,473t regulationof, 47047 3, 470f472f synthesisof, 472 cycloheximide,1098, 1098s cyclooxygenase(COX), EI?' 818 cycloorygenase1/2 inhibitors,817,818, 8l8f cycloserine,8TS cystathionined-slrlthase,864 cystathionineTJyase,864 cysteine,9s,75s,77,77s,692,864 bioslmthesisof, 864,864f degradationof, to pyruvate, 692,692t propertiesof, 73t, 77 cystic fibrosis defectiveion channelsin, 401,410 protein misfolding n, 146, 147 cystine,?7, 77s crtidine, 273s cytidine diphosphate(CDP), 824,825f cytidne 5'-monophosphate(CMP), 273s,824 cltidylate, 272t,273s cltidylate synthetase,E87 cltochome(s), 7lO-711 prostheticgroupsof, 7l0-7 II, 710f cltochromeb5,815 cltochrome b5reductase,815 cl'tochromebq, 7 16-7 17, 7I6f cytochromebcr complex,7161776.Seealso ComplexIII cytochromeb6J dual roles of, 762f cytochromeb6lcomplex, photosystemsI and II Iinkage by, 755-756, 7551 cltochrome c absorptionspectraof, 711, 711f in apoptosis,737-7 38, 737t structure of, 13lt cltochromej water chain in, 50-51, 51f c)'tochromeoxidase,716' Saaorso Complex[V subunitsof, 717f cytochromeP-450,7A6-737,816' 916 mitochondriaand, 736 in xenobioticmetabolism,736-737

l-10 ]

Index

cytokines,443 in cell cycle regulation,47 If, 472 cytokinins,331f cltoplasm, 3, 3f fllamentsin, 9-10, 9f cltosine,9s,272, 272t,273f Seeolso pyrimidine bases deaminationof, 289, 290f methylationof, 292 cltoskeleton,9-10, 9f cltosol,3, 3f cellulosesS.nthesis in, 795-796 llpid syrthesisul, 8\2,812t sucrosesynthesisn, 772, 827f cltotoxic T cells(T,, cells),170, 170t

d D arm,ofIRNA, 1080f,1081 dabsylchloride,94,95s Dalgarno,L1rrn,i088 daltons,1.lb Dam,Henrik,361,36lf dam methylase,986,986t in mismatchrepair, 994 dAMP(deoxyadelosine 5'-monophosphate), 2Z3s dansylchloride.94,95s databases genomic,323 for proteinstructure,136-138,l36f l3Zf daughterchromosomes,469 Dayhoff,MargaretOakley,72, 72f dCMP (deo4'cltidine 5'-monophosphate),273s DDI, 1053b De Duve,Christian,599 de novo pathrvay,882 deamination of nucleotidebases,289 290,290f oxidative,679 debranchingenz1.me, in glycogenmetabolism, 644,596 decarboxylation of aminoacids,878-879,879f, 880f ofB-ketoacid,497s free radical-rnitiated,498,499f oxidative,616 degeneracy, of geneticcode,1069 dehydration,of 2-phosphoglycerate to phosphoenolplruvate,538 dehydroascorbate, 127s 7-dehydrocholesterol, 360,360s dehydrogenase, 513 stereospecificityof, employingNAD* or NADp+ as coenzyrnes, 516-519,b18t dehydrogenation,513 oxidationsinvolving,513-514,bl3f dehydrohydroxylsinonorleucine, 128s deletionmutations,993 y'C (free-energychange) Sec free-energy change(zlG) dS (entropy change),22, 22 denaturation of DNA,287-288,287t,288f of proteins, l4O-142, 141f of RNA-DNAhybrids, 288 denaturationmapping,952 dendrotoxin,411 denitrification,862 deoxy sugars,240s deoxyadenosure, 273s deoxyadenosine 5'-monophosphate(dAMp), 2Z3s 5'-deoxyadenosylgroup, 658 5'-deoxyadenosylcobalamin, 65?, 6b8 deoxyadenylate,273s 2-deoxy-c-l-ribose,9s deoxycltidine, 273s deoxycltidine 5'-monophosphate(dCMp), 2ZBs p-2'-Deory-o-ribofurmose, 274f deoxy-o-ribose, 274 2-deoxy-n-ribose, 236s,272 deoxygumosine,273s

deoxyguanosine5'-monophosphate(dGMP),273s deoxyguanylate,273s deoxyhemoglobin,164 deoxlnucleosidetriphosphate,regulationof ribonucleotidereductaseby, 890, 909f deoxJ,Tibonucleotides, 272t, 273-274, 273f See also nucleotides ribonucleotidesas precursorsof, 888-890,888f, 889f, 909f deorrythycltidylate,273s deoxlth)'midine,273s deorythyrrudine5' -monophosphate (dTMP),273s deoxythJ,Tnidylate, 273s depurination,290,290t dermatansulfate,250 desaturases, 815-817,8f Zf desensitization,receptor,42O, 420f, 42If , 430-431,430f desmin,177 desmosine, 77, 78s desolvation,in enzlrnatic reactions,192, 192f development generegulationh. genesiJencing h, l145-1146,1146f pattern-regulatinggenesand, ll47-1152 dexamethasone, 823s in glucosemetabolism,823 dextran,246, 248f, 25It Seealso polysaccharide(s) synthetic,246 dextrose,235,236s Seealso glucose 4G+ (activation enersr) Seeactivation eners/ (y'G+) DGDG(digalactosyldiacylglycerol), 352, 352f dGMP (deoxyguanosine5'-monophosphate),273s diabetesinsipidus,394b diabetesmellitus, 929-930 acidosisin, 63-64 carbohydratemetabolismi:r, 542f defectiveglucoseand water transport in, 394b diagnosis of,24lb, 930 early studiesof, 903b fat metabolismin, 542f fatty acid synthesisin, 821-822 glucosemetabolismin, 927f,929-930 glucosetestingin , 24I, 24|b-242b ketosis/ketoacidosis in, 667,929-930 mature onsetof the young (MODY), 593b-594b mitochondrialmutationsand, 741 pathophysiologyof, 929-930 sulfonylureadrugs for, 925 treatment of, 808,824, 936 tlpe 1, 929 type 2, 929, 938-940 drug therapyfor, 808, 824 insulin insensitivityin, 929, 935-936,938-940 managementof, 839-840 diabeticketoacidosis,63-64, 667,929 diacylglycerol,357, 432, 433,433f in triacylglycerolsynthesis,82lf diacylglycerol3-phosphate,in triacylglycerol slrnthesis,821,821f dialysis,protein,8S diastereomers, 16, 16f dichromats,465,466 dideoxymethod,for DNA sequencing,292,294, 293t,294t dideoxyinosine(DDI), 1053b dideorf,nucleotides,in DNA sequencng,292-294, 293f,294f dielectric constant,46 2,4-dienoyl-CoAreductase,65? differentialcentrifugation,8-9, 8f diffusion facilitated,390-391, 390f Seealso transporter(s) hop,384 of membranelipids, 380-833,382f-384f simple,389f,390,390f of solutes,389 391,389f,390f SeeaLso membranetransport

dihedralangles in secondarystruclves, I2l-I22, 122f secondarystructuresand, 122f dihedralsl.rnmetry,139, 139f dihydroacetonephosphate,533 dihydrobiopterinreductase,697 dihydrofolatereductase,890-891 substratebinding to, 189f dihydrogenphosphate,as buffer, 58-59, 59f dihydrolipoyldehydrogenase, 6l 8 dihydrolipoyltransacetylase, 618 dihydroxyacetone,236,236s,237s dihydroxyacetonephosphate,533s,545s catalysisof, 191 in Calvin cycle,779, 780f, 782t, 783 in glyceroneogenesis, 822f, 823 in glycolysis,530,539f P1exchangefor, 783-784, 783t, 784f. transport of, 783-T84, 784f 1,25-dihydroxycholecalciferol, 360, 360s,906t, 908 dimethylallylpyrophosphate,in cholesterol qmthesis,833, 833f, 834f dimethylnitrosarnine, as mutagen,291t,292 dimethylsulfate,as mutagen,29lf , 252 dinitrogenase,866 dinitrogenasereductase,855 Dntzis experiment,1088 Dhtzis, Howard,1088 dioxygenases, 8l6b diphtheriatoxin, 1098-1099 directtransposition, 1014,1014f disaccharides,236, 243-244 SeeaLso carbohydrates;oligosaccharides conformationsof, 247-248, 248f formationof,243,243f hydrolysisof,243-244 to monosaccharides, 543, 544f nomenclatureof, 243-244 oidalion of,243-244 reducng,243-244 structure of, 2 43-244,243t,244t dissociationconstant(acid) (KJ, 57f-59f, 68-59 dissociationconstant(KJ, 166-157, 1611 for enzgne-substratecomplex,198 in Scatchardanalysis,421b dissociationenergy,44 distal His, 168 disulfidebonds,in aminoacid sequencing,95, 96f D,Lsystemof stereochemicalnomenclature,17, 72t,74 DNA, 14, 141,27,28f,29f Seeako nucleicacids A-form,281,281f armealing of, 287 -288, 287t bacterial,949,950f, 950t packagingof, 970 topoisomerases and, 959 basepairs n, 277, 277f , 278-280,279f, 280f. See olso base(s),nucleotide/nucleicacid;base pairs/pairing B-form, 281, 287f,955 Chargaffsrules for, 278 chefiucal slnthesis of , 294, 295f cNoroplast,95l cleavageof, 30+307 ,304f blunt endsh, 306, 306f,307 restdction endonucleases in, 304-307, 304t, 305t,306f sticky endsur, 306, 306f,307 closed-circu.lar, 955, 955f,956 coding,952 compactionof, 963-968,970f complementary Sae cDNA (complementary DNA) damaged,993-1002,1000,1000f,1001-1002, 1001f ,Saealso DNA repair; mutation(s) repair of, 993-1002.SeeaLsoDNA repair SOSresponseand,lo0l, 10021, 1130-113r,1130f degradationof, 979-980 denaturationrnappingof, 978 denaturationof, 287-288,288f, 290f doublehelix of, 27U280,279t,280t See(rtso DNA structure

supercoilingand, 954-962,955f Seealso DNA, supercoilurgof in transcription,1023f underwindingof, 956-958, 955f unwhding of/rewinding of, 287-288, 287t, 288t 978-979,979f Seeako DNA replication variations of, 280-281, 281f double-strandbreaksin in recombination, 1006-1007,1015f,1016f repairof, f004-1009,1006f,1016f early studiesof, 278-280, 278f enzymaticdegradationof, 979 eukaryotic,949-951,950t evolutionarystability of, 27 folding of, 968 highly repetitive,953 histonesand, 963-968,964f human,949-951,950t hybridizationof, 288-289,290f hydrophilicbackboneof, 275, 275f, 278 junk, 1060 Iight absorptionby, 287-288 linker, in nucleosome,963f, 964-965 lhking number of, 956-958, 956f, 957f,965 topoisomerases and,958-961,959f meltrngpoint for, 288,288f methylationof, 292 microinjectionof, 333 in animalcloning,333 mitochondrial,738f,95i Seealso mitochondria agingand, 739 mutagenicchangesin from alkylatingagents,29If , 292 from nitrousacid,29I, 29If from radiation,290,29lf noncodingregionsof, 953 Saeorso introns nontemplate(coding) strand of, in transcription, 1023f,1024 nucleosomesand, 963, 964t,965f, 967f nucleotidesequencesin, ammo acid sequences and,97f,98-100 nucleotidesof, 272t, 273-274, 273f Seeal,so nucleotides packagingof, 947, 947f, 954-962,962-97I See olso DNA, supercoilingof in bacteria,970 ut eul