生物化学英文版课件：Chapter 16 the Citric Acid Cycle

Chapter16

TheCitricAcidCycle(alsoTricarboxylicAcidCycle,orKrebscycle)Biochemistrylecture,Nov.27,2012(ByProf.ChangZengyi昌增益教授）Thefinalcommonpathwayinvolvedintheoxidationofcarbohydrates,fats,andproteinsintoCO2andH2OtogenerateATPbylinkingwithoxidativephosphorylation);Italsoprovidesprecursorsforthebiosynthesisofmany

compounds(e.g.aminoacids);

O2isneededonlyindirectlytoregenerateNAD+andFAD.

OccurringinthematrixofmitochondriaineukaryotesOutlineThechemicalreactionsandenzymesofthecitricacidcycleandtheglyoxylatebypass(cycle,shunt).Theelucidationofthecitricacidcycleandtheglyoxylatebypass.SummaryC2C4(Regenerated)C6(tricarboxylicacid)C5C4ThiscycleneithergeneratesalargeamountofATPnorincludeO2asareactant.Pyruvate

dehydrogenasecomplexPyruvateisconvertedtoacetyl-CoAviaoxidativedecarboxylationascatalyzedbyamulti-enzymecomplexInyeastcytoplasmInmitochondrialmatrixThelipoamideVitaminB5Cryoelectronmicrograph(PDHfrombovinekidney)ReconstructedimageZHongZhou2001,PNAS98:14802-7.

Theflexiblelipoyl

groupcovalentlylinkedtoE2allowthethreeactivesitestoworkinconcertE2DescriptionReactantsProductsAllreactionsinthecitricacidcycle:Acetyl-CoA+3NAD++FAD+GDP+Pi+2H2O→CoA-SH+3NADH+3H++FADH2+GTP+2CO2OverallreactionsofcitricacidcycleFurtheroxidationofeachNADHprovideenoughenergyforproducingabout2.5ATPmoleculesandeachFADHabout1.5.TheenzymesofthecitricacidcycleAldolcondensationIron-sulfurproteinDehydration-rehydrationOxidativedecarboxylationOxidativedecarboxylationHydrolysisOxidationHydrationDehydrogenationThesubstrate-levelphosphorylationcatalyzedbysuccinyl-CoA

synthetaseoccursviaaphosphorylatedenzymeintermediateNosuchphosphorylatedenzymeintermediatesformedinthetwoATPformationstepsinglycolysis!Allostericeffectorsregulateactivitiesofregulatoryenzymesinthecitricacidcycle:

MoleculesindicatingalowenergylevelorrequirementofenergywillactivateenzymesTheenzymesofmetabolicpathwaysproposedtoexistasmulti-enzymecomplexes(metabolons)inlivingcellsTemporarycomplexesformedbetweensequentialenzymesofametabolicpathway.Allowingpassing(substratechannelling)intermediarymetabolicproductfromoneactivesitetoanotherofconsecutiveenzymesinthepathway.Directevidencestilllacking.BorisIKurganovThecitricacidcycle

alsoprovidesprecursorsforbiosynthesis,andisthusamphibolic.OxaloacetateisreplenishedfrompyruvateandPEPThecarboxylationofpyruvateneedstheparticipationofbiotin(covalentlylinkedtopyruvate

carboxylaseandswitchesbetweentwoactivesites)

(biotinbindstoavidinwithaKdof10-15M)Somemodernanaerobicmicroorganismsuseanincompletecitricacidcycleasasourceofbiosyntheticprecursors,notofenergy!a-ketoglutarate

dehydrogenaseisnotpresentinsuchorganisms!Ananabolicpathway:Convertingacetyl-CoAtoglucoseandotherbiomolecules,occurringinplantsandcertainmicroorganisms.Phosphoenoypyruvatemightbeconsideredastheendproductoftheglyoxylatebypass.Thepartitionofcarbonfluxbetweentheglyoxylatebypassandthecitricacidcyclehasbeenunderextensivestudies.Reversiblephosphorylation(inE.coli);Kmvaluedifferences;Malate

synthase(1956)Isocitrate

Lyase(1953)Theglyoxylatebypass(Revealedin1957)Fattyacidisconvertedtosugaringerminatingseedsviathecooperationof:glyoxylatecycle,citricacidcycleandgluconeogenesispathwayRegulationofglyoxylateandcitricacidcyclescoordinated

Allostericeffectorsinhibitingtheglyoxylatecyclewillactivatethecitricacidcyclebyexhibitingreciprocaleffectontheisocitrate

lyaseandisocitrate

dehydrogenaseEarlystudiesonbiologicaloxidationTheoxidationreactionsofcarbohydratewerenotknowndespiteofthesuccessinunderstandingthechemicalreactionsoffermentation.CellfreesystemscouldNOTbesuccessfullydeveloped.TheWieland-Warburgcontroversy:Whetherhydrogenoroxygenactivationisthekeytobiologicaloxidation.Oxidizabilityoforganicsubstancesweresystematicallyexaminedinisolatedmuscletissues:rapidoxidationofsaltsofanumberofacids(4-carbondicarboxylicacidlikesuccinate,malate,oxaloacetate,fumarate)wasobserved,butnotlinkedtotheoxidationofcarbohydrates(Thunberg,1910s-1920s).ThelinkbetweenanaerobicbreakdownofcarbohydrateandtherespiratoryutilizationofO2wassearchedinthe1930sSearchedwerecarboncompoundsthatmightserveasintermediatesforpyruvatetobeoxidizedtoCO2andH2O.Suspensionsofmincedpigeon-breastmusclewasfoundtobeagoodworkingsystemforstudyingrespiration(Szent-Gyorgyi,1934).Afew4-carbondicarboxylicacidswerefoundtoplayacatalyticroleinrespiration(1930s)Theaddedfumaratedidnotdisappear!FumarateThe“contribution”ofSzent-Gyorgyi(1930s)BothfumaricacidandvitaminCwereconsideredashydrogencarriersbetweenthefoodstaffandO2.Nosix-carboncompoundsyetrevealed.Alinear,insteadofcyclic,pathwaywasproposed.VitaminCwasproposedtobeahydrogencarrierinbiologicaloxidation!FoodstaffFoodstaffNobelPrizeinPhysiologyorMedicine1937

Forhisdiscoveriesinconnectionwiththebiologicalcombustionprocesses,withspecialreferencetovitaminCandtothecatalysisoffumaricacid.Buthelatermadeamajorcontributioninunderstandingmusclecontraction.AlbertSzent-Gyorgyi(1893-1986)Six-carbontricarboxylicacidswerealsofoundtoplaysimilarcatalyticrolesCitratewasfoundtoexhibitasimilarcatalyticeffectassuccinateonO2consumptionandpyruvateoxidation(1930s,Martius&Knoop).a-ketoglutarate

werefoundtobeaproductofcitrateoxidation(MartiusandKnoop).ThecontributionofHansKrebs(1930s)Isocitrate

andcis-aconitateareoxidizedaseffectivelyascitratebythesuspensionofmincedpigeonmuscle.Thepresenceof

malonate,aknowninhibitorofthesuccinate

dehydrogenase,causedtheaccumulationofcitrate,a-ketoglurarate,inadditiontosuccinatewhenfumaratewasadded.Additionofpyruvate(derivedfromcarbohydrate)andoxaloacetateledtoaccumulationofcitrateinthemedium.KrebsandJohnson(1937)“Theroleofcitricacidintheintermediatemetabolisminanimaltissues.Enzymologica4:148-156.

RejectedbyNatureCitricacidcycleisassumedtobethechiefpathwayfortheoxidationofcarbohydrateinpigeonmuscles(1937).UnknownsubstanceUnknownsubstanceKrebsandJohnson(1937)“Theroleofcitricacidintheintermediatemetabolisminanimaltissues.

Enzymologica4:148-156.SirHansAdolfKrebs(1990-1981)CoenzymeAnotyetfound;Radioisotopenotyetused.ThecontributionofFritzLipmannPyruvateoxidationinbacteriadependsonthepresenceofinorganicphosphate:proposedacetyl-phosphateasakeyintermediate,servingasacetylandphosphoryldonors.Activeacetylationofsulfonamidewasdetectedincell-freepigeonliverpreparations,butnotderivedfromacetyl-phosphate!Participationofanewcoenzyme:disappearedonaginganddialysis;presentinboiledextractsofallorgansandmicroorganisms;couldnotbereplacedbyanyknowncofactor.ThecontributionofFritzLipmannThenewcoenzymewaspurifiedandcharacterized(byLipmannandotherlabs):containingpantothenicacid(aknownvitamin),adenine,phosphorusandsulfur(1940s-1950s).CoenzymeAfoundtobepromotingpyruvateutilizationandcitratesynthesisStrikingparallelwasobservedbetweenCoAcontentandpyruvateutilization(mostlybyothers).CoA-dependentcitratesynthesisobservedinpigeonliverfactionandE.coliextract.PyruvateutilizationdependsoncoANobelprizeinPhysiologyormedicine1953HansKrebs(1900-1981)Forhisdiscoveryofthecitricacidcycle.FritzLipmann(1899-1986)ForhisdiscoveryofCo-enzymeAanditsimportanceforintermediarymetabolism.Krebsdiscoveredtheureacyclein1932beforeheelucidatedthecitricacidcycle!ThecitricacidcyclewasfoundtooccurinmitochondriaAlsothefattyacidoxidationactivities.NoGlycolyticactivitiesKennedyandLehninger(1949)OxidationoffattyacidsandTricarboxylicacidcycleintermediatesbyisolatedratlivermitochondria,JBC,179:957-972.O2-consumingbiologicaloxidationhasbeenassociatedwithinsolubleparticulateportionofthecell.succinooxidase&cytochrome

oxidasehavebeenfoundinmitochondriabefore.Radioisotopelabelingstudiesrevealsthestereospecificityoftheaconitase:Theapparentlysymmetricalcitrateisrecognizedbyaconitaseinanasymmetricalmanner.

Citrateisconsideredasprochiral.

Thecitricacidcyclewasoncerenamedasthetricarboxylicacid(TCA)cycle.Apparentexplanation:citrateisnotanintermediate!Characterizationofthe“condensingenzyme”Citratewassynthesizedfromacetate,ATP,oxaloacetateinthepresenceofasolubleenzymepreparationfromanimaltissuessupplementedwithE.coliextracts(providingthetransacetylaseactivity),andCoAisrequired(1949-1951,Ochoa).SeveroOchoaThe“condensingenzyme”catalyzestheconversionofpyruvatetocitrate(1951)Orthophosphateisnotneeded;acetyl-phosphateisnotanintermediate;Thiaminepyrophosphate(TPP)isrequired;Withstoichiometricproductionof–SHgroups;Acyl-mercaptidebondofacetyl-CoAisenergy-rich.TPPSeveroOchoaDiscoveryofthehugea-ketoaciddehydrogenasecomplexes(1950s)a-lipoicacidwasdiscoveredasa“pyruvateoxidationfactor”(Reed,Gunsalus,1950s).Thepyruvateanda-ketoglutarate

oxidaseswereisolatedfrommuscleandE.coli:MWbeing2-5million,eachcontainingmultiplemoleculesoflipoicacidsandFAD(1951).Successfulseparationandreconstitutionofthea-ketoaciddehydrogenationcomplexes(1961)KoikeandReed(1961)ResolutionandreconstitutionoftheE.coli

pyruvatedehydrogenationcomplex,JBC,236:PC33-34.

Elucidationoftheglyoxylatecycle:

backgroundTheintermediatesofthecitricacidcycleareobligatoryprecursorsofmanybiosynthesis,thuswillbecontinuouslyremoved.Certainreplenishmentmechanismmustexist.Isocitrate

lyasewasfirstdiscoveredin1953(Campbelletal,BBA11:594):catalyzingtheformationofglyoxylateandsuccinatewhencitrateorcis-aconitatewasaddedintobacterialextracts.Malate

synthasewasfirstrevealedin1956(WongandAjl,JACS,78:3230.):catalyzingthecondensationofacetyl-CoAandglyoxylatetoformmalate.Elucidationoftheglyoxylatecycle:

ActualoccurrenceofapathwayKornberg,H.L.;Madsen,N.B.(1957)SynthesisofC4-dicarboxylicacidsfromacetatebyaglyoxylatebypassofthetricarboxylicacidcycle,BiochimicaetBiophysica

Acta,24,651-3.

Whencell-freeextracts.ofacetate-grownPseudomonasKB1wereincubatedwithC14H3CO2Na(I),ATP,coenzymeA,glutathione,andNaglyoxylate,malatewastheonlylabeledcompoundformedintheearlystagesofincubation.Whenisocitratereplacedglyoxylate,malatewasagainthe1stlabeledcompoundformed.Intheabsenceofglyoxylateorisocitrate,nolabeledcompounds.wereformedotherthantracesofacetylCoA.ItisconcludedthatPseudomonasKB1,growingonacetateassolesourceofC,possesses,inadditiontotheenzymicreactionsofthetricarboxylicacidcycle,amechanismofferinganalternativeroutefromisocitratetomalate,i.e.,cleavageofisocitratebyisocitrataseandthecondensationofacetylCoAandglyoxylatebymalate

synthetase.Elucidationoftheglyoxylatecycle:

ActualoccuranceofapathwayKornberg,H.L.;Krebs,H.A.j(1957)SynthesisofcellconstituentfromC2unitsbyamodifiedtricarboxylicacidcycle,

Nature179,988-91.OccurrenceofametaboliccycleinmicroorganismswhichcanderivealltheirCrequirementsfrom2-Ccompds.Thecycleisavariantofthetricarboxylicacidcycle:acetate+oxaloacetate

→citrate→

cis-aconitate

→

isocitrate

→

succinate+glyoxylate;Glyoxylate+acetate→

malate,malate+1/2O2

→

oxaloacetate.AcetatereactsintheformofacetylcoenzymeA.Thestagesbetweencitrateandmalateinthetricarboxylicacidcyclearereplacedbyreactioninwhichglyoxylateisakeymetabolite,therefore,thecycleisreferredtoastheglyoxylatecycle.Maindiscoveriesleadingtotheelaborationofthecycle:(1)thefindingthatisocitrate,apartfromundergoingdehydrogenation,issplitenzymicallytoformsuccinateandglyoxylate;(2)therecognitionbyWongandAjlofanenzymesystembringingaboutthesynthesisofmalatefromglyoxylateandacetylcoenzymeA;(3)thedemo