生物化学-酶(讲稿)

§6.1Introduction§6.2HowDoEnzymesPlaySpecificity§6.3HowDoEnzymesAccelerateReaction§6.4Enzymeexamples§6.5EnzymeKinetics§6.6EnzymeRegulation§6EnzymeBriefHistoryClasses&NameCompositionGeneralFeatures1In1810,JosephGay-Lussac(约瑟夫·盖·吕萨克，1778.12-1850.5，法国化学家和物理学家)foundthatethanolandCO2weretheprincipalproductsofsugardecompositionbyyeast.BriefHistory2BriefHistory(cntd)In1835,JacobBerzelius(雅各布·贝采利乌斯,1779.8-1848.8，瑞典化学家)pointedoutthatanextractofmalt(knownasdiastase)catalyzes

thehydrolysisofstarchmoreefficientlythandoessulfuricacid.3BriefHistory(cntd)Inthemid-19thcentury,LouisPasteur(1822-1895,法国微生物学家)proposedthattheprocessesoffermentationcouldonlyoccurinlivingcells(vitalforce);Others,however,notablyJustusvonLiebig(尤斯蒂斯·冯·李比希,

1803-1873,德国化学家),arguedthatbiologicalprocessesarecausedbytheactionofchemicalsubstancesthatwerethenknownas“ferments.”4BriefHistory(cntd)In1878,WilhelmFriedrichKühne(威廉·弗里德里希·屈内,1837.3－1900.6，德国生理学家)coinedtheword

“enzyme”(Greek:en,in+zyme,yeast)toemphasizethatthereissomethinginyeast,asopposedtotheyeastitself,thatcatalyzesthereactionsoffermentation.51941～现代生物化学1891～1940酶学时代1840～1890生理化学1800～1839有机化学1772~1799化学革命生物化学酶学哲学生理学化学生理化学有机化学物理学现代生物化学生物化学发展的脉络BriefHistory(cntd)6BriefHistory(cntd)EduardBuchner(毕希纳,1860.5-1917.8):德国化学家,1894年，与其兄弟一起发现酵母细胞的萃取液即可致发酵作用。1987年从这些萃取液分离出有效的成份，称为zymase，从而导致对酵素的了解，并因此曾获1907年诺贝尔化学奖7BriefHistory(cntd)In1894,EmilFischer’s(费歇尔,1852.10-1919.7，德国化学家,

1902年诺贝尔化学奖)discoverythatglycolyticenzymescandistinguishbetweenstereoisomericsugarsledtotheformulationofhislock-and-keyhypothesis.8BriefHistory(cntd)In1926,JamesSumner(詹姆斯·萨姆纳,887.11-1955.8,美国化学家,1946年诺贝尔化学奖)

crystallizedthefirstenzyme,jackbean（洋刀豆）

urease,andthesecrystalsconsistofprotein.9BriefHistory(cntd)SinceSumner’spreparationsweresomewhatimpure,however,theproteinnatureofenzymeswasnotgenerallyaccepteduntilthemid-1930s,whenJohnNorthrop(1891.7-1987.5美国生化学家

)showedthatthereisadirectcorrelationbetweentheenzymaticactivitiesofcrystallinepepsin,trypsin,andchymotrypsinandtheamountsofproteinpresent.10BriefHistory(cntd)1963年，牛胰核糖核酸酶成为第一个被完全确定一级结构的酶:SmythDG,SteinWH,MooreS.Thesequenceofaminoacidresiduesinbovinepancreaticribonuclease:revisionsandconfirmations.JBiolChem.1963,238:227-2341965年,卵清蛋白溶菌酶成为第一解析三维空间结构的酶：BlakeCC,KoenigDF,MairGAetal.StructureofHenEgg-WhiteLysozyme:AThree-dimensionalFourierSynthesisat2ÅResolution,Nature,1965,206:757-761…11Classes&NameNo.ClassTypeofreactioncatalyzed1Oxidoreductases(氧化还原酶)Transferofelectrons(hydrideionsorHatoms)2Transferases(转移酶)Grouptransferreactions3Hydrolases(水解酶)Hydrolysisreactions(transferoffunctionalgroupstowater)4Lyases(裂合酶)Additionofgroupstodoublebonds,orformationofdoublebondsbyremovalofgroups5Isomerases(异构酶)Transferofgroupswithinmoleculestoyieldisomericforms6Ligases(连接酶)FormationofC-C,C-S,C-O,andC-NbondsbycondensationreactionscoupledtoATPcleavage12Trivial(common)name：addingthesuffix“-ase”tothenameoftheirsubstrateortoaword/phrasedescribingtheiractivityUrease(hydrolysisofurea).Transaminase(transferaminogroup).RNApolymeraseButmanyenzymesarenamedbeforethisrulewasestablished(e.g.,pepsin,trypsin).Classes&Name(cntd)13Lactatedehydrogenase(lactate:NAD+oxidoreductase)E.C.1.1.1.27Class:OxidoreductaseEnzyme

CommissionSub-Class:Actingontheprimary&secondaryalcoholsSub-Sub-Class:NAD+aselectronacceptorSpecificenzymewithinsub-sub-classSystemicname14辅因子依赖性酶(Cofactor-dependent~)全酶(holo~)=脱辅基酶蛋白(apo~)+辅因子(cofactor)非辅因子依赖性酶(Cofactor-independent~)酶辅因子(cofactor)无机~(inorganic~):如金属离子有机~(organnic~)辅基(prostheticgroup)：与酶结合紧密（共价或非共价)辅酶(coenzyme)：与酶可逆结合，常为维生素的衍生物Composition151617GeneralFeaturesEnzyme(biocatalyst)contrastwithchemicalcatalystHigherreactionratesMilderreactionconditionsGreaterreactionspecificityCapacityforcontrol1819§6.1Introduction§6.2HowDoEnzymesPlaySpecificity§6.3HowDoEnzymesAccelerateReaction§6.4Enzymeexamples§6.5EnzymeKinetics§6.6EnzymeRegulation§6EnzymeSpecificityinshapeSpecificityinchirality20Specificitybetweenproteinsandligands21SpecificityinshapeTheactivesiteofanenzymeiscompatibleinbothshapeandinteractionforcewithitssubstrate.Afewenzymes:

onlyonesubstrate.Mostenzymes:

asmallrange

ofrelatedsubstrates.Someenzymes,

particularlydigestiveenzymes:

alargerange

ofrelatedsubstrates.Hydrophobicgroups22SpecificityinchiralityAnenzymecatalyticreactionisachiralreactionbecausetheactivesiteisachiralenviromentItiseasytounderstandachiralreactionifthesubstrateisachiralmoleculeHowever，therearealotofachiralmoleculesasenzymesubstratesincell,andtheproductsarechiralmolecules.Why?Prochirality!23Whatisprochirality?Ifamoleculecanbeconvertedfromachiraltochiralinasinglestep,themoleculeisprochiralorhasprochirality.R/Ssystem24ProchiraldifferentiationofenzymeThespecificattachmentofaprochiralcentertoanenzymebindingsitepermitstheenzymetodifferentiatebetweenprochiralgroupsEthanol’stwomethyleneHatomsmaybedistinguishedifthemoleculeisheldinsomesortofasymmetricjig.Thesubstrate-bindingsitesofenzymesare,ofcourse,justsuchjigsbecausetheyimmobilizethereactinggroupsofthesubstrateontheenzymesurface.25§6.1Introduction§6.2HowDoEnzymesPlaySpecificity§6.3HowDoEnzymesAccelerateReaction§6.4Enzymeexamples§6.5EnzymeKinetics§6.6EnzymeRegulation§6EnzymeAmacroscopicview

—EnzymesdecreasetheactivationenergyAmicroscopicview—Howdoenzymesdecreasetheactivationenergy?26

Amacroscopicview—Thermodynamicsdefinesthereactionrates&equilibriaFromthermodynamicsBoltzmannconstantPlanck'sconstantRateconstant27Amacroscopicview—Enzymesdecreasetheactivationenergy28EScomplexE-transitionstatecomplexnon-covalentinteractionsbetweenenzymeandsubstrateareoptimizedinthetransitionstateTransitiontheory29(1)Bindingenergy(2)SpecificCatalyticGroupsGeneralAcid-BaseCatalysisCovalentCatalysisMetalioncatalysisAmicroscopicview—

Howdoenzymesdecreasetheactivationenergy?30Inducedconformationalchange—underthiscondition,thefollowingactionsoccur.DesolvationEntropyreductionElectornredistributionBindingenergycanbeusedtoovercomethesebarriersProximityorientation31InducedfitinhexokinaseAnexampleforthetransitiontheoryofenzymes32Desolvation33Rateenhancementbyentropyreduction34Whenprotontransfer

toorfromH2Ois

fasterthanthe

rateofbreakdown

ofintermediates,

thepresenceof

otherprotondonors

oracceptorsdoesnot

increasetherateof

thereaction.SpecificCatalyticGroups/GeneralAcid-BaseCatalysis+OH-H2OH+HOH-H2OB:AHBH+A-WhenprotontransfertoorfromH2Oisslowerthantherateofbreakdownofintermediates,onlyafractionoftheintermediatesformedarestabilized.Thepresenceofalternativeprotondonors(HA)oracceptors(B:)increasestherateofthereaction.35Aminoacidsingeneralacid-basecatalysis36SpecificCatalyticGroups37Covalentandacid-basecatalysisworktogether

38SpecificCatalyticGroups/metalioncatalysis

39§6.1Introduction§6.2HowDoEnzymesPlaySpecificity§6.3HowDoEnzymesAccelerateReaction§6.4Enzymeexamples§6.5EnzymeKinetics§6.6EnzymeRegulation§6EnzymeChymotrypsinSerineproteasefamilyOtherproteinhydrolases40His57Asp102Ser195Crystalstructure(1967)revealedacatalytictriad:Ser195,His57,Asp102Chymotrypsin41HNphenylNO42Asp102functionsonlytoorientHis57.43444546

47

48

49AnimationofChymotrypsincatalyticMechanism50pHdependenceofchymotrypsin-catalyzedreactionsWhy?Effectontheacid/basecatalysisofHis57EffectontheconformationofPhenylpocket51SerineProteaseisalargefamilyofenzymeswhosecatalyticmechanismisbasedonanactive-siteserineresidue，including：

chymotrypsin

trypsinelastasethrombin（凝血酶）

subtilisin（枯草杆菌蛋白酶）

plasmin（血纤维蛋白溶酶）

……SerineproteasefamilyAcatalytictriadhasbeenfoundinallserineproteases:theSeristhusconvertedintoapotentnucleophile(subtilisinhasnohomologywithotherSerproteasemembers,buthasthetriad)5253ChymotrypsinElastaseTrypsin54555657Thespecificityofserineproteasesisdeterminedbythestructuralfeaturesofasubstratebindingpocket58AspartylProtease:Renin（血管紧张肽原酶）AspOtherproteinhydrolases(1)59Otherproteinhydrolases(2)60MetalloproteaseThermolysin（嗜热菌蛋白酶）Otherproteinhydrolases(3)61§6.1Introduction§6.2HowDoEnzymesPlaySpecificity§6.3HowDoEnzymesAccelerateReaction§6.4Enzymeexamples§6.5EnzymeKinetics§6.6EnzymeRegulation§6EnzymeSteadystate&Pre-steadystateOnesubstratekineticsTwosubstrateskinetics62SteadystatekineticsPre-steadystatekinetics63Onesubstratekinetics:Michaelis-Mentenequation合理简化Kcat代表“暗箱”的表观速率常数，是各正向速率常数的函数.如果“暗箱”中只有一个限速步骤，则kcat

近似于该步的正向速率常数。ES代表“暗箱”中从ES1到EPm各种复合物；稳态的开始阶段,[P]很低,形成复合物的逆反应可忽略64

65在底物过量的情况下，酶被底物所饱和，[ES]=[E]ThisistheMichaelis-MentenEquation66When[S]<<KmWhen[S]>>KmKmisequivalenttothesubstrateconcentrationatwhichV0

isVmax/2Mostenzymes(excepttheregulatoryenzymes)havebeenfoundtofollowtheMichaelis-Mentenkinetics67对kcat、Km、kcat/Km的说明表观速率常数Kcat：又称酶的转换数(turnnumber)，反映了一个酶的催化效率.

kcat/Km：反映酶与底物受扩散速率限制的匹配程度，绝大多数高效酶的kcat/Km比值为108-109M-1S-1.6869ThetransitionstatetheoryofenzymecatalysishasstrongsupportingevidencesCertainmodificationsonthesubstrateofchymotrypsinwerefoundtohaveminimaleffectonthe

Km,butmajoreffectonthekcat.Transition-stateanalogsbindtoenzymes102to106timesmoretightlythannormalsubstrates.Theideaofcatalyticantibodieswasalsosuggestedbythistheory(Jencks,1969)andapprovedtobecorrect(LernerandSchultz,1980s).70Smallstructuralchangesonthesubstrateofchymotrypsinhaveamajoreffectonthekcat,butminimalontheKmvalues.71Transition-stateanalogscanbedesignedaccordingtotheproposedreactionmechanismandusedformakingcatalyticantibodies.72Doublereciprocalplot

(i.e.,theLineweaver-BurkPlot).如何求Km和kcat73EachsubstratewillhaveonecharacteristicKmvalue.Ternarycomplexmayormaynotbeformedforthebisubstratereactionsdependingonthemechanism.Steady-statekineticscanoftenhelpdistinguishthesetwomechanisms.Twosubstrateskinetics7475Keeping【S2】constant,thedoublereciprocalplotsmadebyvarying【S1】Goto9676Ping-Pong(ordoubledisplacement)mechanism77Keeping【S2】constant,thedoublereciprocalplotsmadebyvarying【S1】Goto9478§6.1Introduction§6.2HowDoEnzymesPlaySpecificity§6.3HowDoEnzymesAccelerateReaction§6.4Enzymeexamples§6.5EnzymeKinetics§6.6EnzymeRegulation§6EnzymeArtificialinhibitionPhysiologicalregulation79ArtificialinhibitionSuchinhibitorsareimportantpharmaceuticalagentsandusefulinunderstandingtheactionmechanismofenzymesInhibitionIrreversibleReversibleCompetitiveUncompetitiveMixed

Group-specificAffinitySuicideIrreversibleinhibitors(alsocalledinactivator)chemicallymodifyorformtightnoncovalentinteractionswithfunctionalgroupsintheactivesiteofenzymes.80Group-specificinactivatorreactswithspecificRgroupsofAAsthataffectenzymeactivity.81DIPFirreversiblyinactivatechymotrypsin(andotherserineproteases)andreactsonlywithSer195(outofthe25Serresidues).82Affinityinactivatorsaremoleculesthatarestructurallysimilartothesubstratefortheenzymethatcovalentlymodifyactivesiteresidues.Theyarethusmorespecificfortheenzymeactivesitethanaregroup-specificreagents.磷酸丙糖异构酶83TPCKalkylatesHis57ofchymotrypsin(Itdoesnotoccurwhenchymotrypsinisdenaturedinurea)84Antibioticmechanismofpenicillin(1)Structureofpenicilline.g.85Antibioticmechanismofpenicillin(2)L-alaD-gluL-lys

D-alaD-alaGly-Gly-Gly-Gly-GlyPeptidoglycaninSaureus(葡萄球菌)cellwall

e.g.86Antibioticmechanismofpenicillin(3)Formationofcross-linksTranspeptidationreactiontranspeptidasee.g.87Antibioticmechanismofpenicillin(4)e.g.88Suicideinactivatorisdesignedtocarryoutthefirstfewchemicalstepsofthenormalenzymereaction.Insteadofbeingtransformedintothenormalproduct,however,theinactivatorisconvertedtoaveryreactivecompoundthatcombinesirreversiblywiththeenzyme(alsocalledmechanism-basedinactivator)

Monoamineoxidaserequiresthecofactorflavinprostheticgroup(FAD).N,N-DimethylpropargylamineinhibitsmonoamineoxidasebycovalentlymodifyingtheFADonlyaftertheinhibitorisfirstoxidized8990

91CompetitiveinhibitorsalterstheKmbutnottheVmaxofenzymes92InhibitoronlybindstotheEScomplex

93UncompetitiveinhibitorsalterboththeKmandtheVmaxofanenzymeGoto7894Mixedinhibition

95MixedinhibitorsalterboththeKmandtheVmaxofanenzymeGoto7696Noncompetitiveinhibitionisaspecialmixedinhibitionwhena=a'NoncompetitiveinhibitorsalterstheVmaxbutnottheKmofenzymes97PhysiologicalregulationAllostericregulation(noncovalentmodifications,reversible);Covalentmodifications(reversible);Proteolyticcleavage(irreversible).(Generegulation:changingtheamountofspecificenzymes).98AllostericregulationThebindingofallostericmodulators(oftensmallmetabolitesorcofactors)atallostericsites(distinctfromtheactivesite)triggersconformationalchangesthataretransmittedtotheactivesite(intramolecularsignaltransduction).99AllostericmodulatorscanbeeitherinhibitoryorstimulatoryAspartatetranscarbamoylase(天冬氨酸转氨甲酰酶,ATCase)isaclassic(alsobeststudied)allostericenzyme:ItisnegativelyregulatedbyCTPbutpositivelyregulatedbyATP.100Allostericenzymesareoftenoligomeric:

ATCaseconsistsoftwocatalytictrimersandthree