生物化學(xué)-酶(講稿)

§6.1Introduction§6.2HowDoEnzymesPlaySpecificity§6.3HowDoEnzymesAccelerateReaction§6.4Enzymeexamples§6.5EnzymeKinetics§6.6EnzymeRegulation§6EnzymeBriefHistoryClasses&NameCompositionGeneralFeatures1In1810,JosephGay-Lussac(約瑟夫·蓋·呂薩克，1778.12-1850.5，法國化學(xué)家和物理學(xué)家)foundthatethanolandCO2weretheprincipalproductsofsugardecompositionbyyeast.BriefHistory2BriefHistory(cntd)In1835,JacobBerzelius(雅各布·貝采利烏斯,1779.8-1848.8，瑞典化學(xué)家)pointedoutthatanextractofmalt(knownasdiastase)catalyzes

thehydrolysisofstarchmoreefficientlythandoessulfuricacid.3BriefHistory(cntd)Inthemid-19thcentury,LouisPasteur(1822-1895,法國微生物學(xué)家)proposedthattheprocessesoffermentationcouldonlyoccurinlivingcells(vitalforce);Others,however,notablyJustusvonLiebig(尤斯蒂斯·馮·李比希,

1803-1873,德國化學(xué)家),arguedthatbiologicalprocessesarecausedbytheactionofchemicalsubstancesthatwerethenknownas“ferments.”4BriefHistory(cntd)In1878,WilhelmFriedrichKühne(威廉·弗里德里?！で鼉?nèi),1837.3－1900.6，德國生理學(xué)家)coinedtheword

“enzyme”(Greek:en,in+zyme,yeast)toemphasizethatthereissomethinginyeast,asopposedtotheyeastitself,thatcatalyzesthereactionsoffermentation.51941～現(xiàn)代生物化學(xué)1891～1940酶學(xué)時代1840～1890生理化學(xué)1800～1839有機(jī)化學(xué)1772~1799化學(xué)革命生物化學(xué)酶學(xué)哲學(xué)生理學(xué)化學(xué)生理化學(xué)有機(jī)化學(xué)物理學(xué)現(xiàn)代生物化學(xué)生物化學(xué)發(fā)展的脈絡(luò)BriefHistory(cntd)6BriefHistory(cntd)EduardBuchner(畢希納,1860.5-1917.8):德國化學(xué)家,1894年，與其兄弟一起發(fā)現(xiàn)酵母細(xì)胞的萃取液即可致發(fā)酵作用。1987年從這些萃取液分離出有效的成份，稱為zymase，從而導(dǎo)致對酵素的了解，并因此曾獲1907年諾貝爾化學(xué)獎7BriefHistory(cntd)In1894,EmilFischer’s(費(fèi)歇爾,1852.10-1919.7，德國化學(xué)家,

1902年諾貝爾化學(xué)獎)discoverythatglycolyticenzymescandistinguishbetweenstereoisomericsugarsledtotheformulationofhislock-and-keyhypothesis.8BriefHistory(cntd)In1926,JamesSumner(詹姆斯·薩姆納,887.11-1955.8,美國化學(xué)家,1946年諾貝爾化學(xué)獎)

crystallizedthefirstenzyme,jackbean（洋刀豆）

urease,andthesecrystalsconsistofprotein.9BriefHistory(cntd)SinceSumner’spreparationsweresomewhatimpure,however,theproteinnatureofenzymeswasnotgenerallyaccepteduntilthemid-1930s,whenJohnNorthrop(1891.7-1987.5美國生化學(xué)家

)showedthatthereisadirectcorrelationbetweentheenzymaticactivitiesofcrystallinepepsin,trypsin,andchymotrypsinandtheamountsofproteinpresent.10BriefHistory(cntd)1963年，牛胰核糖核酸酶成為第一個被完全確定一級結(jié)構(gòu)的酶:SmythDG,SteinWH,MooreS.Thesequenceofaminoacidresiduesinbovinepancreaticribonuclease:revisionsandconfirmations.JBiolChem.1963,238:227-2341965年,卵清蛋白溶菌酶成為第一解析三維空間結(jié)構(gòu)的酶：BlakeCC,KoenigDF,MairGAetal.StructureofHenEgg-WhiteLysozyme:AThree-dimensionalFourierSynthesisat2?Resolution,Nature,1965,206:757-761…11Classes&NameNo.ClassTypeofreactioncatalyzed1Oxidoreductases(氧化還原酶)Transferofelectrons(hydrideionsorHatoms)2Transferases(轉(zhuǎn)移酶)Grouptransferreactions3Hydrolases(水解酶)Hydrolysisreactions(transferoffunctionalgroupstowater)4Lyases(裂合酶)Additionofgroupstodoublebonds,orformationofdoublebondsbyremovalofgroups5Isomerases(異構(gòu)酶)Transferofgroupswithinmoleculestoyieldisomericforms6Ligases(連接酶)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輔因子依賴性酶(Cofactor-dependent~)全酶(holo~)=脫輔基酶蛋白(apo~)+輔因子(cofactor)非輔因子依賴性酶(Cofactor-independent~)酶輔因子(cofactor)無機(jī)~(inorganic~):如金屬離子有機(jī)~(organnic~)輔基(prostheticgroup)：與酶結(jié)合緊密（共價或非共價)輔酶(coenzyme)：與酶可逆結(jié)合，常為維生素的衍生物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（枯草桿菌蛋白酶）

plasmin（血纖維蛋白溶酶）

……SerineproteasefamilyAcatalytictriadhasbeenfoundinallserineproteases:theSeristhusconvertedintoapotentnucleophile(subtilisinhasnohomologywithotherSerproteasemembers,buthasthetriad)5253ChymotrypsinElastaseTrypsin54555657Thespecificityofserineproteasesisdeterminedbythestructuralfeaturesofasubstratebindingpocket58AspartylProtease:Renin（血管緊張肽原酶）AspOtherproteinhydrolases(1)59Otherproteinhydrolases(2)60MetalloproteaseThermolysin（嗜熱菌蛋白酶）Otherproteinhydrolases(3)61§6.1Introduction§6.2HowDoEnzymesPlaySpecificity§6.3HowDoEnzymesAccelerateReaction§6.4Enzymeexamples§6.5EnzymeKinetics§6.6EnzymeRegulation§6EnzymeSteadystate&Pre-steadystateOnesubstratekineticsTwosubstrateskinetics62SteadystatekineticsPre-steadystatekinetics63Onesubstratekinetics:Michaelis-Mentenequation合理簡化Kcat代表“暗箱”的表觀速率常數(shù)，是各正向速率常數(shù)的函數(shù).如果“暗箱”中只有一個限速步驟，則kcat

近似于該步的正向速率常數(shù)。ES代表“暗箱”中從ES1到EPm各種復(fù)合物；穩(wěn)態(tài)的開始階段,[P]很低,形成復(fù)合物的逆反應(yīng)可忽略64

65在底物過量的情況下，酶被底物所飽和，[ES]=[E]ThisistheMichaelis-MentenEquation66When[S]<<KmWhen[S]>>KmKmisequivalenttothesubstrateconcentrationatwhichV0

isVmax/2Mostenzymes(excepttheregulatoryenzymes)havebeenfoundtofollowtheMichaelis-Mentenkinetics67對kcat、Km、kcat/Km的說明表觀速率常數(shù)Kcat：又稱酶的轉(zhuǎn)換數(shù)(turnnumber)，反映了一個酶的催化效率.

kcat/Km：反映酶與底物受擴(kuò)散速率限制的匹配程度，絕大多數(shù)高效酶的kcat/Km比值為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.磷酸丙糖異構(gòu)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(天冬氨酸轉(zhuǎn)氨甲酰酶,ATCase)isaclassic(alsobeststudied)allostericenzyme:ItisnegativelyregulatedbyCTPbutpositivelyregulatedbyATP.100Allostericenzymesareoftenoligomeric:

ATCaseconsistsoftwocatalytictrimersandthree