生物化學(xué)英文版課件：Chapter 6 Enzyme catalysis

Chapter6

EnzymecatalysisBiochemistryLecture(forOct18and23,2012)Extremeefficiencyofcatalysis;Highspecificityonsubstrateandproduct;Regulationofactivityinresponsetochanges.Whatareenzymes?Enzymes:Biologicalcatalyststhatpromoteandspeedupchemicalreactions(usuallyby105to1017fold)withoutthemselvesbeingaltered(consumed)intheprocess.Theydeterminethepatternsoftransformationsforchemicals,aswellasformsofenergyinthelivingorganisms;Playkeyrolesinmetabolism!Enzymesaffectreactionrates,notequilibria!Enzymesspeedupthethermodynamicallyfavorablereactionstoreachequilibriabyloweringtheactivationenergy(ΔG?),usingthebindingenergy

(ΔGB).Enzymescatalyzetheforwardandbackwardreactionsequally.

(aprostheticgroup)2H2O2

→2H2O+O2

200,000catalyticevents/second/subunit(nearthediffusion-controlledlimit).Thereactionisspedupbyabillionfold!(tetramers)Fe3+→1000foldHemoglobin→1,000,000foldCatalase→1,000,000,000foldRateenhancementActivesiteH2O2-inducedoxidativedamagewasfoundtobeakeyelementinsenilehairgraying(2009)!Enzymescanbesmallorlarge4-oxalocrotonatetautomeraseFattyacidsynthase(human)Monomer:62residuesMonomer:2504residuesThediscoveryofenzymesasthebiocatalysts(1)Earlystudies:Understandingthenatureofanimaldigestion(meatbystomachsecretionandtheconversionofstarchtosugarsbyplantextractsandsaliva)

andfermentation(fromsugartoalcohol).(19thcentury)Diastase(lateramylase

淀粉酶):Firstenzymediscovered(in1833)fromgeminatingbarleyandsaliva,liquefiesstarchpasteandconverteditintosugar.Thediscoveryofenzymesasthebiocatalysts(2)Pepsin:first

discoveredin1834astheactiveprincipleintheacidextractofgastricmucosacausingthedissolutionofcoagulatedeggwhite.Other“solubleferments”.“Enzyme(somethinginyeast)”wasfirstcoinedforsuch“unorganizednon-livingferments”byKühnein1877.KühneTwoexplanationswereprovidedtoexplainthebiologicalfermentationofsugar(19thcentury)JustusvonLiebig(Germany):fermentationcausedbytransmissionofvibrationfromparticlesoffermenttoparticlesofthefermentingmaterial.LouisPasteur(France):Fermentationapropertyinseparablefromlivingcells.Liebig(1803-1873)PasteurSucrosewasfoundtoprotectinvertaseactivity(1890)O’SULLIVAN,C.andTOMPSON,F.W.(1890)“Invertase:acontributiontothehistoryofanenzymeorunorganizedferment”J.Chem.Soc.57:834–931.Theactivityofinvertaseinthepresenceofsucrosesurvivesatemperaturethatcompletelydestroysitifsucroseisabsent.Enzymecombinationwithsucrosewassuggested.Earlyobservationsontherateoffermentationbyyeast(1892)BROWN,A.J.(1892)“Influenceofoxygenandconcentrationonalcoholfermentation”J.Chem.Soc.61,369–385.BROWN,A.J.(1902)“Enzymeaction”J.Chem.Soc.81,373–386.Therateoffermentationofsucroseinthepresenceofyeastseemedtobeindependentoftheamountofsucrosepresent,butontheamountoftheenzyme.Invertasemoleculespresentintheyeastwasproposedtoformadditioncomplexeswithsucrose.GrapesfermentingC6H12O6→2C2H5OH+2CO2

Thelock-and-keymetaphorwasproposedtoexplaintheexquisitespecificityofenzymes(1894)FISCHER,E.(1894)“EinflussderConfigurationaufdieWirkungdenEnzyme”Ber.Dtsch.Chem.Ges.27:2985–2993.Invertinonlyhydrolyzesa-methylglucoside.Emulsinhydrolyzesonlyb-methylglucoside.Thesetwoenzymesmustconsistof“asymmetricallybuiltmolecules”.EmilFischer(1852-1919)a-methylglucosideCell-freeextractsofyeastwasreportedtocarryoutsugarfermentation(1897)Fermentationisachemicalprocess,nota“vitalprocess”.A“zymase”wasproposedtobepresentinyeastcellsandresponsibleforfermentation.Amathematicalequationestablishedtodescriberelationshipbetweenrateandsubstrateconcentration(1913)Seekinganintegratedformoftherateequationsbystudyinginvertase.Initialrate(v)ofinvertase-catalyzedreactionmonitoredatseveralsucroseconcentrations.Theory:Invertaseformsacomplexwithsucrosethatisverylabileanddecaystofreeenzyme,glucoseandfructose.Theory:Ratemustbeproportionaltoconcentrationofsucrose-enzymecomplex.DatafittingunveiledaconstanttobecalculatedatthedifferentsubstrateconcentrationsC/KsTheconstant(C/Ks)calculatedbeingVmax/Km,thespecificityconstanttimestheenzymeconcentration(kcat/Km×E0)!CΦ=Vmax,Φisthetotalenzymeconcentration,andk=KS,thedissociationconstantofthesucrose-enzymecomplex.Forthefirsttime,revealedapictureofthemagnitudeoftheaffinityofanenzymeforitssubstrate.AplottedversionoftheMichaelis-MentondataJohnsonandGoody,(2011)TheOriginalMichaelisConstant:Translationofthe1913Michaelis?MentenPaper,Biochemistry,50:8264-8269

Theproductsofthereactionwereinhibitory.CurrentversionoftheMichaelis-Mentenequation(Briggs&Haldanein1925,usingthesteadystateapproximation)ESisinasteady-stateandbreakdownofESisthesloweststep.LeonorMichaelis(1875-1949)LeonoraMenten(1879-1960)Km=(k2+k-1)/k1Vmax

=kcat[E]0J.B.S.Haldane(1892-1964)TheactualmeaningofKmdependsonthereactionmechanismForIfk2israte-limiting,k2<<k-1, Km=k-1/k1Kmequalstothedissociationconstant(Kd)oftheEScomplex;KmrepresentameasureofaffinityoftheenzymeforitssubstrateintheEScomplex.Ifk2>>k-1,thenKm=k2/k1.Ifk2andk-1arecomparable,Kmisacomplexfunctionofallthreerateconstants.k-1k1DoublereciprocalplotsfordeterminationofKm(Ks)introducedLineweaver&Burk(1934)TheDeterminationofEnzymeDissociationConstants,J.Ame.Chem.Soc.56:658–666.LineweavernotedthesimilaritybetweentheMMequationandtheLangmuirequation.(Ks-enzymedissociationconstant)ThemostcitedpaperofJACS,withmorethan11000citations.

HansLineweaver(1907-2009)DeanBurk(1904-1988)

Note:Thisequationcanbeusedtodescribeavarietyofbio-interactions,e.g,Mb-O2,Ab-Ag,DNA-DNA,protein-protein,etc.Morereliablenonlinearregressionmethodsareusednowadays!Kmisaconstantforeachsubstrateofanenzyme(Km:

substrateconcentrationathalfVmax)Vmaxisdeterminedbykcat,therateconstantoftherate-limitingstepVmax=kcat[Et]kcat

equalstok2ork3oracomplexfunctionofboth,dependingonwhichistherate-limitingstep.kcatisalsocalledtheturnovernumber:thenumberofsubstratemoleculesconvertedtoproductinagivenunitoftimeperenzymemoleculewhentheenzymeissaturatedwithsubstrate.40,000,000moleculesofH2O2areconvertedtoH2OandO2byonecatalasemoleculewithinonesecond!ThekineticparameterskcatandKmareoftenstudiedandcomparedfordifferentenzymesKmoftenreflectsthenormalsubstrateconcentrationpresentinvivoforacertainenzyme.Thecatalyticefficiencyofdifferentenzymesisoftencomparedbycomparingtheirkcat/Kmratios(thespecificityconstant).

when[S]<<Kmkcat/Kmisanapparentsecond-orderrateconstant(withunitsofM-1S-1),relatingthereactionratetotheconcentrationsoffreeenzymeandsubstrate.Thevalueofkcat/Kmhasanupperlimit(fortheperfectedenzymes)Itcanbenogreaterthank1.ThedecompositionofEStoE+PcanoccurnomorefrequentlythatEandScometogethertoformES.Themostefficientenzymeshavekcat/Kmvaluesnearthediffusion-controlledlimitof108to109M-1S-1.Catalyticperfection(rateofreactionbeingdiffusion-controlled)canbeachievedbyacombinationofdifferentvaluesofkcatandKm.Rateenhancement

isoftenusedtodescribetheefficiencyofanenzymeRateenhancement:ratiooftheratesofconversionbeingcatalyzedandtheuncatalyzedreactions.

kunkcatuncatalyzedcatalyzedNonenzymatichalf-lifeUncatalyzedrate(kun,s-1)

Catalyzedrate(kcat,s-1)

Rateenhancement(kcat/kun)EnzymeRateenhancementofselectedenzymesTheunstableandelusiveenzymesfoundtobeproteins(1920s)Sumner,J.B.(1926)“Theisolationandcrystallizationoftheenzymeurease”J.Biol.Chem.69:435-441.Northrop,J.H.(1930)“Crystallinepepsin,1:Isolationandtestsofpurity”J.Gen.Physiol.13:739-766.Thecrystalsarepurelyproteinasshownbyallchemicaltests.

Theproteinsaretheenzymes:reactivation;chemicaltests.OctahedralcrystalsofjackbeanureasePepsincrystalsNobelPrizein1946"forhisdiscoverythatenzymescanbecrystallized"

Northrop,J.H.Sumner,J.B.Smallheatstablechemicalswerefoundtobeneededforenzymeaction(1906)HardenA,YoungWJ(1906).TheAlcoholicFermentofYeast-Juice,PartII:theCofermentofyeastjuice,Proc.Roy.Soc.B:Biol.Sci.78:369–75.

Fermentationofglucosebyyeastjuiceisdependentonadialysablesubstancewhichisnotdestroyedbyheat.ThisheatstablefactorwaslateridentifiedasNAD.thefirstorganiccofactordiscovered.ArthurHarden(1865-1940)HansvonEuler-Chelpin(1873-1964)NobelPrize1929“fortheirinvestigationsonthefermentationofsugarandfermentativeenzymes”Non-proteinchemicalcompounds(organicorinorganic)foundtobeenzymehelpersProstheticgroups:tightlyboundorganiccofactors;Coenzymes:looselyboundorganiccofactors(co-substrates).Butthereisnosharpdivisionbetweenthem!!!Someorganiccofactorsarevitaminsorarederivedfromvitamins.Manyinorganicionsfoundtobecofactorsofenzymes

Beingtheessentialtraceelementsinnutrition.Enzymescategorizedinto6classesbasedonreactionscatalyzed(thenomenclaturecommittee,IUBMB,1961)Problem:uncontrollednamingoftherapidlyincreasingnumberofknownenzymes.Someofthenamesinuseweredefinitelymisleading;othersconveyedlittleornothingaboutthenatureofthereactioncatalysed(e.g.catalase)http://www.chem.qmul.ac.uk/iubmb/enzyme/Thesixclassesofenzymes1.Oxidoreductases:transferelectronsfromonesubstance(donor)toanother(acceptor),e.g.,dehydrogenases,oxidases,oxygenases,reductases,peroxidases,andhydroxylases.2.Transferases:Transfergroups(amino,carboxyl,carbonyl,methyl,phosphoryl,andacyl)fromonemoleculetoanother,e.g.,transaminases,transcarboxylases,andtransmethylases.22subclasses9subclassesThesixclassesofenzymes3.Hydrolases:Cleavageofbondsbyaddingwater,e.g.,lipases,phosphatases,andpeptidases.4.Lyases:breakingofchemicalbondsbymeansotherthanhydrolysisandoxidation,oftenforminganewdoublebond,e.g.,decaboxylases,hydratases,dehydratases,deaminasesandsynthases.7subclasses13subclassesThesixclassesofenzymes5.Isomeases:structuralrearrangementof

isomers,e.g.,racemases,epimerases,mutasesandisomerases.6.Ligases:JointogethertwomoleculesbysynthesisofnewC-O,C-S,C-NorC-CbondswithsimultaneousbreakdownofATP,e.g.

synthetases,

carboyxlasesandligases.6subclasses6subclassesThenumberofenzymesrevealedhavebeenincreasingAsreportedbytheEnzymeCommisionorEnzymeNomenclaturecommitteeofIUBMB:Yearnumberofenzymes1961712196487519721770197821221984247719923196Eachenzymepossessaconventionalname,asystematicname,andaECnumber(assignedbyEnzymeCommissionofIUBMBLactatedehydrogenase(lactate:NAD+oxidoreductase)Lactate+NAD+pyruvate+NADH+H+

1Typeofelectrondonor(OH)TypeofelectronAcceptor(NAD)

EnzymescanbereversiblysuppressedbynoncovalentbindingofinhibitorsCompetitive

Non-competitive

Substrate&inhibitorcompeteforaccesstotheenzyme'sactivesite;Sufficientlyhighconcentrationsofsubstrateout-competetheinhibitor!Kmappincreasesasittakeshigherconcentrationofsubstratetoreach1/2Vmax

Substrateandinhibitorbindindependentlybutinhibitorbindingcompletelypreventcatalysis.BindstoE,butnottoESIdenticalaffinitiesforEandESSubstrateandproductInhibitionmightoccur.Manydrugmoleculesarereversibleinhibitors

ofkeyenzymesRitonavir(Norvir)isaninhibitoroftheHIVprotease.(fortreatingAIDS)Highspecificityandpotencymeansfewsideeffectsandlowtoxicity.

Sildenafil(Viagra)isaninhibitorofcGMP-specificphosphodiesterasetype5

(fortreatingmaleerectiledysfunction)Methotrexate,aninhibitorofdihydrofolatereductase,inhibitsthesynthesisofthymidineandpurines.(fortreatingcancer)ChemicalreactionshypothesizedtooccurviatransitionstatesTransitionstate:Ahighenergyconfigurationofthereactantsalongthereactioncoordinate.(EyringandPolanyi,1935).Beingnotachemicalspeciesofanysignificantstability,butafleetingmolecularmomentinwhichbondsarebothbrokenandformed.HenryEyringMichaelPolanyiLifetimes~10-13secondsTheactivesiteofenzymeswasproposedtocomplementthestrainedconfigurationofthesubstrates(1946)Pauling,L.(1946)MolecularArchitectureandBiologicalReactions,Chem.Eng.News,24:1375-1377.Pauling,L.(1948)Natureofforcesbetweenlargemoleculesofbiologicalinterest,

Nature161:707-709.ActiveregionofsurfaceofenzymehasaconfigurationNOT

complementarytothesubstrateinitsnormalconfiguration,butratherinthestrainedconfiguration,or“activatedcomplex”,ofthereactioncatalyzed,thusdecreasingtheactivationenergyandincreasetherateofthereaction.Ifenzymecompletelycomplementaryinstructuretosubstratethennoothermoleculewouldcompetesuccessfullywiththesubstrateincombiningwiththeenzyme,similarinbehaviortoantibodies;butanenzymecomplementarytoastrainedsubstratemoleculewouldattractmorestronglytoitselfamoleculeresemblingthestrainedsubstratemoleculethanitwouldthesubstratemolecule.Pauling(1901-1994)Enzymeactivesitehastocomplementthetransitionstate,NOTthesubstrateActivationenergywillbeIncreased,insteadofdecreased!whichwilldecreasingtherate!ONLYthiswillwork!!!Transitionstateanalogsfoundtotightlybindtoenzymes(1972)Secemskietal.(1972)Atransitionstateanalogforlysozyme.JBiolChem.247:4740-8.Transitionstateanalogsfoundtobindtoenzymes102to106timesmoretightlythansubstrates.Substratesoftenparticipateseveralenzymereactions,whereasthetransitionstatetendstobecharacteristicofoneparticularenzyme.

LactoneAlkoxylcarboniumAssumedtransitionstateforthelysozyme-catalyzedreactionTransitionstateanalogdesignedCatalyticantibodieswerepredictedbyWilliamJencks(1969)"Ifcomplementaritybetweentheactivesiteandthetransitionstatecontributessignificantlytoenzymaticcatalysis,itshouldbepossibletosynthesizeanenzymebyconstructingsuchanactivesite.Onewaytodothisistoprepareanantibodytoahaptenicgroupwhichresemblesthetransitionstateofagivenreaction.Thecombiningsitesofsuchantibodiesshouldbecomplementarytothetransitionstateandshouldcauseanaccelerationbyforcingboundsubstratestoresemblethetransitionstate."

Williamp.Jencks,CatalysisinChemistryandEnzymology,1969,p.288Catalyticantibodies(abzyme)wereproved(1986)bySchultz’slabTramontanoetal.(1986)Catalyticantibodies,Science,234:1566.Pollacketal.(1986)Selectivechemicalcatalysisbyanantibody,Science,234:1570.Postulatedtransitionstate(metallopeptidase)Transitionstateanalog(ashapten)SubstrateoftheCatalyticantibodyPeterSchultzIrreversibleinhibitorswereusedtoidentifykeyresiduesintheactivesiteofchymotrypsin(1955,1964)TurbaandGundlach,(1955)[Aminoacidsequenceintheareaofthereactiveserinegroupofthechymotrypsinmolecule.].BiochemZ.327:186-8.Ongetal.(1964)Theidentificationofthehistidineresidueattheactivecenterofchymotrypsin.JBiolChem.240:694-8.Modificationfollowedbysequencing:specificSer-195andHis-57residuesassumedtobeintheactivesitesduetotheirhighreactivity.Theinhibitor,TPCK,is14C-labeled.Onlyoneofthe2Hisresidues,oneoutofthe25Serresiduesbeinglabeled!Diisopropylphosphofluoridate(DIPF)Ser-195ChymotrypsinProteolyticenzymesfoundoftenexistasinactivezymogenprecursors(1933)Kunitz&NorthropJH.(1933)Isolationofacrystallineproteinfrompancreasanditsconversionintoanewcrystllineproteolyticenzymebytrypsin.Science.78:558-9.KunitzM,NorthropJH.(1934)Theisolationofcrystallinetrypsinogenanditsconversionintocrystallinetrypsin.Science.80:505-6Azymogenrequiresabiochemicalchange(suchasahydrolysisreactionrevealingtheactivesite,orchangingtheconfigurationtorevealtheactivesite)forittobecomeanactiveenzyme.Thetertiarystructureofthefirstenzyme(lysozyme)determined(1965)Blakeetal.(1965)Structureofhenegg-whitelysozyme.Athree-dimensionalFouriersynthesisat2Aresolution.Nature.206:757-61.JohnsonLNandPhillipsDC.(1965).Structureofsomecrystallinelysozyme-inhibitorcomplexesdeterminedbyX-rayanalysisat6Aresolution.Nature.206:761-3.

Actionmechanismcanbeproposedbasedontheenzyme-inhibitorcomplexstructure.Lysozymecleavesthepolysaccharideofthebacterialcellwall.StructureofNAD-dependentlactatedehydrogenasedetermined(1970)Adamsetal.(1970)Structureoflactatedehydrogenaseat2.8Aresolution.Nature.227:1098-103.Rossmannetal.(1974)Chemicalandbiologicalevolutionofnucleotide-bindingprotein.Nature.250:194-9.

Acommonnucleotidebindingmotif(“Rossmannfold”)wasfoundinallNAD-andFAD-dependentenzymes.RossmannfoldMichaelRossmannPyruvatelactateChymotrypsincatalysiswasfoundtooccurintwostages(1954)

HartleyandKilby,(1954)TheReactionofp-NitrophenylEsterswithChymotrypsinandInsulin,Biochem.J.,56:288-297.Esterwasusedassubstratetomakethereactionslower.Thecatalysisappearstooccurintwostages:arapidacetylation,releasingonep-nitrophenolperenzyme,followedbyaslowstep.Theslowlinearhydrolysisprecededbyarapidinitialreaction.Theextrapolatedlinearhydrolysisplotdidnotpassthroughtheoriginatzerotime:Km=20mMKcat=77s-1ColorlesssubstrateYellowproductThisreactionisfarslowerthanthehydrolysisofpeptides!“burst”(fast)phase

(rapidacylationofallEnzymesleadingtoreleaseofp-nitrophenol)Slowphase

(enzymeswillbeabletoactagainonlyafteraslowdeacylationstep)Thecatalysisofchymotrypsinisbiphasicasrevealedbypre-steadystatekinetics(burstkinetics)MillisecondsaftermixingReflectingasingleturnoveroftheenzymeChymotrypsinoperatesthrougha

ping-pongmechanismRatesofindividualstepsforanenzyme-catalyzedreactionmaybeobtainedbypre-steadystatekineticsTheenzyme(oflargeamount)isusedinsubstratequantitiesandtheeventsontheenzymearedirectlyobserved.Ratesofmanyreactionstepsmaybemeasuredindependently.Veryrapidmixingandsamplingtechniquesarerequired(theenzymeandsubstratehavetobebroughttogetherinmillisecondsandmeasurementsalsobemadewithinshortperiodoftime).“Rapidkinetics”or“pre-steady-statekinetics”isappliedtotheobservationofratesofsystemsthatoccurinveryshorttimeintervals(usuallymsorsub-msscale

)andverylowproductconcentrations.Thisperiodcoversthetimefromtheenzymeencounteringitstarget(eitherasubstrate,inhibitororsomeotherligands)tothepointofsystemsettlingtoequilibrium.

TheconcentrationofESwillrisefromzerotoitssteady-statevalue.

(msorsub-ms)Stopped-flowapparatusforpre-steadystatekinetics(since1940s)

Solutionsareforcedtogetherveryrapidly.RapidmixingRapidsamplingQuenchflowapparatusforrapidkinetics

RapidmixingRapidstoppingDeterminationofthecrystalstructureofchymotrypsin(1967)revealedacatalytictriad:Ser195,His57,Asp102.

Matthewsetal.(1967)Three-dimensionalstructureoftosyl-alpha-chymotrypsin.Nature.214:652-6.

Blowetal.(1969)Roleofaburiedacidgroupinthemechanismofactionofchymotrypsin.Nature.221:337-40.

Chymotrypsin:threepolypeptidechainslinkedbymultipledisulfidebonds;acatalytictriad.His57Asp102Ser195CleftforbindingextendedsubstratesTrypsin,sharinga40%identitywithchymotrypsin,hasaverysimilarstructure.ActivesiteAcatalytictriadhasbeenfoundinallserineproteases:theSeristhusconvertedintoapotentnucleophile(subtilisinhasnohomologywithotherSerproteasemembers,buthasthetriad)ThePeptideBondhaspartial(40%)doublebondcharacterasaresultofresonanceofelectronsbetweentheOandNThehydrolysisofapeptidebondatneutralpHwithoutcatalysiswilltake~10-1000years!Chymotrypsin(andotherserineproteases)actsviaamixtureofcovalentandgeneralacid-basecatalysistocleave(notadirectattackofwateronthepeptidebond!)1stsubstrate1stproduct2ndsubstrate2ndproductEESAcyl-EE’S2EP2AcylationphaseDeacylationphaseTheproposedcompletecatalyticcycleofchymotrypsin(rateenhancement:109)APing-PongMechanismThespecificityofserineproteasesisdeterminedbythestructuralfeaturesofasubstratebindingpocketValValAdynamicprocessforchymotrypsincatalysis:APingPangmechanism.Importanceoftheresidueswasexminedbysite-directedmutagenesis:TheSerandHisresiduesarefarmoreimportantthantheAspresidue!ManybookswerewrittenonenzymecatalysisEnzymescanfunctioninfarmorecomplexmechanismsDNApolymeraseRNApolymeraseATPsynthaseDifferentformsofglycogenphosphorylasefound(1943)Cori&Green(1943).CrystallinemusclephosphorylaseIIprostheticgroup,J.Biol.Chem.151:21–29.Glycogenphosphorylaseinmuscleexistintwoforms:formahas70%activity

withoutadditionofadenylicacid,formb

isinactivewithoutaddedAMP.Incubationofform

awithanenzymeof

muscleconvertsittoform

b.Glycogenphosphorylasefoundtobephosphorylatedascatalyzedbyaspecifickinase(1958)KREBSEG,KENTAB,FISCHEREH.(1958)Themusclephosphorylasebkinasereaction.JBiolChem.231:73-83.(NobelPrize1992)EdwinKrebs(1918-2009)EdmondFische(1920,Shanghai)Activityofglycogenphosphorylaseisregulatedviabothallostericregulatorsandreversiblephosphorylation.SitesofmodificationcanbefarawayfromtheactivesitesGlycogenphosphorylaseaPhosphorylatedsiteActivesitesAMPbindingsiteGlycogenphosphorylaseandmanyothers(for1/3to?ofalleukaryoticproteins)kinasesphosphatasesDinitrogenasereductaseRNApolymeraseGlutaminesynthetaseAspartatetranscarbamoylase(ATCase)foundtobeallostericallyregulatedGerhartandPardee(1962)Theenzymologyofcontrolbyfeedbackinhibition.JBiolChem.237:891-6.Gerh