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1、,Chapter8IntroductiontoEnzymology,Outline,DefinitionofenzymeChemicalnatureofenzymesCatalyticpropertiesofenzymesFeaturessharedbyenzymesandnon-enzymaticcatalystsUniquefeaturesofenzymesClassificationandnomenclatureofenzymes,Enzymesarebiologicalcatalyststhatacceleratetheratesofchemicalreactions.,Snailwi
2、thoutenzymecatalyst,Snailwithenzymecatalyst,WhatareEnzymes?,Enzymescatalyzetherateatwhichsubstratesareconvertedtoproduct,Substrates,Product,Substrates,products,andenzymes,Enzymescatalyzetheconversionofsubstratesintoproducts,Whatisasubstrate?Asubstrateisthecompoundthatisconvertedintotheproductinanenz
3、ymecatalyzedreaction.Forthereactioncatalyzedbyaldolase,fructose1,6-phosphateisthesubstrate.,AllenzymesareproteinsexceptsomeRNAsandnotallproteinsareenzymesItwasassumedthatallenzymesareproteinsuntil1982whenThomasCechandSydneyAltmandiscoveredcatalyticRNAs(NobelprizeinChemistry,1989)CatalyticRNA,orriboz
4、ymes,satisfyseveralenzymaticcriteria:substratespecificity,enhancereactionrate,andemergefromreactionunchanged,Chemicalnatureofenzymes,Thedifferencebetweenenzymecatalyzed&uncatalyzedchemicalreactions,Enzymecatalyzedreactionsaremuchfasterthanuncatalyzedreactions.Enzymecatalyzedreactionsdisplaysaturatio
5、nkineticswithrespecttosubstrateconcentration.EnzymecatalyzedreactionsareoptimizedforspecifictemperatureandpHvalues.,Enzymecatalyzedreactionsaremuchfasterthanuncatalyzedreactions,.,Time,Product,Enzyme,catalyzed,reaction,Uncatalyzed,reaction,0,0,ReactionRate=,Enzymecatalyzedreactionsdisplaysaturationk
6、ineticswithrespecttoreactantconcentration,Importantthingstorememberaboutenzymesjustlikeothercatalysts,1.Enzymesarenotconsumedoralteredbythereactiontheycatalyze.,Justasaconstructionworkercantakeapileoflumberandbuildahomewithoutbeingphysicallychangedbytheprocess,anenzymetakessubstratesandconvertsthemi
7、ntoproductswithoutbeingphysicallychangedorconsumed.,Thisisanimportantpoint.Anenzymedoesnotdeterminewhichdirectionthereactiongoes,itonlyincreasestherateatwhichthereactionapproachesequilibrium.,2.Enzymescatalyzeboththeforwardandthereversereaction.,Enzymescatalyzeboththereactionsinboththeforwardandreve
8、rsedirection,LDH,LDH,3.Enzymesdonotaltertheequilibrium(orequilibriumconstant)betweensubstratesandproducts.,Atequilibrium,theratioofsubstratestoproductsisthesameregardlessofwhetheranenzymecatalystispresent.Althoughthefinalequilibriumratioofreactantstoproductsisnotalteredbyanenzyme,therateatequilibriu
9、misachievedisincreased.,GeneralPropertiesofEnzymes,1.Higherreactionrates:Theratesofenzyme-mediatedprocessesareacceleratedbyafactorof108-1010.Example1:carbonicanhydrasecatalyzes:CO2+H2OH2CO3nonenzymaticrateconstant=1.3x10-1s-1enzymaticrateconstant=1x106s-1(x7.7x106)Example2:Staphylococcalnucleasecata
10、lyzesnucleicacidhydrolysisnonenzymaticrateconstant=1.7x10-13s-1enzymaticrateconstant=95s-1(x5.6x1014),Reactantsarerequiredtoreachahigh-energy(unstable)statereferredtoasthetransitionstate.Residencemaylastonly10-13to10-14sOnlyalimitednumberofmoleculeswillpossesssufficientenergytoreachthistransitionsta
11、te.Increasingtemperatureorotherconditionsmayfacilitatereactantsreachingthetransitionstate,TransitionStateTheory,Thetransitionstateofareactionisanunstable,highenergyspeciesthatthesubstratesofthereactionmustgothroughinordertoformproduct.Byloweringtheenergyofthetransitionstate,enzymesincreasetherateoft
12、hereaction.,Whatisthetransitionstateofareaction?,Thetransitionstateisthemostunstablespeciesonthereactioncoordinate(i.e.thespecieswiththehighestenergy),Whatisthetransitionstate?,Thetransitionstateisnotanintermediatespecies,Thetransitionstatecannotbetrappedorisolated.Intermediatescanbetrappedorisolate
13、d.,.,.,UnderstandthedifferencebetweenGandGTheoverallfreeenergychangeforareaction(G)isrelatedtotheequilibriumratioofSandPThefreeenergyofactivationforareaction(G)isrelatedtothereactionrateItisextremelyimportanttoappreciatethisdistinction!,TheTransitionState,EnzymeslowerGbutdontaffectGforthereaction,Th
14、eactivesiteofanenzymeistheplacewherealloftheactionoccurs.Itcontainsthefunctionalgroups(aminoacidsidechains)thatbindthesubstrate(s)andcatalyzeitsconversiontoproduct(s).,2.Enzymesbindsubstratestotheiractivesiteandstabilizethetransitionstateofthereaction.,GeneralPropertiesofEnzymes,Activesiteofchymotry
15、psin,Theactivesitebindsthesubstratesandpositionsthemintheproperorientationforthereactiontooccur.Theactivesitecontainschemicalgroupsthatstabilizethetransitionstateofthereaction.Theactivesitedeterminesthespecificityoftheenzyme(i.e.itdetermineswhetheraparticularsubstrateisboundandwhetheraparticularprod
16、uctismade).,Whatdoestheactivesitedo?,TheactivesitetakesupasmallpartofthetotalvolumeoftheenzymeTheactivesiteis3-dimensionalandisgenerallyfoundinacreviceorcleftontheenzymeTheactivesitedisplayshighlyspecificsubstratebindingTheactivesiteisresponsibleforwhetherthereisorderedorrandombindingofsubstratesand
17、releaseofproducts,Characteristicsofactivesites,3.Greaterreactionspecificity:Enzymescandistinguishbetweenstereoisomericsubstrates,evenamongenantiomers.Reactionsarehighlyspecific,rarelyproducingsideproducts.-Absolutespecific-Relativespecific-Stereo-specific,GeneralPropertiesofEnzymes,Alaninesynthesisb
18、yanorganicchemist,EnzymespreventunwantedsidereactionsAlaninesynthesisinsideanorganicchemist,Threemodelshavebeenproposedforsubstratesubstratebinding:LockandkeymodelInducedfitmodelThree-pointattachmentmodel,EmilFicher:(1894)TheLock&Keyhypothesis-explainssubstratespecificity-saysnothingaboutwhycatalysi
19、soccursDanKoshland:(1958)InducedFithypothesis:-enzymesprefertobindtoadistortionofthesubstratethatresemblesthetransitionstate-bothenzymeandsubstratemustadjusttooneanother-inreality,enzymeisnotdistortedbuthasevolvedtobindinacertainwayandsometimesundergoconformationalchanges,“Lockandkeymodel&“theinduce
20、dfit”model,“LockandKey”model,“InducedFit”model,Inducedconformationalchangeinhexokinase,AnExample:,Thiswidely-usedmodeltoexplainthestereo-specificityofenzymeswassuggestedbyA.G.Ogston.Togetahighdegreeofstereo-specificity,substratemustbeheldfirmlyinthreedimensionalspace.Asaconsequence,theremustbeatleas
21、tthreedifferentpointsofattachmentofthesubstrateontotheactivesite.,Three-pointattachmentmodel,aconitase,AnExample-Aconitase,4.Milderreactionconditions:ChemicalcatalysisoftenrequireshightemperaturesandpressuresandextremesofpH5.Greatercapacityforregulation6.Enzymecatalyzedreactionsareoptimizedforspecif
22、icvaluesoftemperatureandpH,GeneralPropertiesofEnzymes,7.ManyEnzymesRequireCofactors,GeneralPropertiesofEnzymes,Coenzyme=smallorganicorinorganicmoleculeswhichareboundtotheapoenzymeandarerequiredfortheenzymetocatalyzethechemicalreaction.Prostheticgroup=similartoacoenzyme,butistightlyboundtotheapoenzym
23、e.Hemeisaprostheticgroupincytochromecandhemoglobin.Apoenzyme=theproteinpartofanenzymewithoutcoenzymesorprostheticgroupsthatarerequiredfortheenzymetohaveactivity.Holoenzyme=theapoenzymewiththecoenzymeorprostheticgroupboundtoit.,Somedefinitionsrelatedtocofactors,Understandinghowenzymesworkiscrucialfor
24、understandingboththephysiologicalbasisofmostdiseasesandthemechanismofactionofdrugsusedtotreatthesediseases,Whystudyenzymes?,Manydiseasesarecausedbytheabsence,malfunction,orinappropriateexpressionofaparticularenzymeEnzymesserveastargetsforavarietyofdrugsEnzymesaresometimesadministeredinthetreatmentof
25、diseaseThepresenceorabsenceofspecificenzymescanbeusedtodiagnosespecificdiseases,MedicalRelevance,Howdoserumenzymelevelshelpinthediagnosisofhumandisease?,Diseasedtissuesoftenmis-expressspecificenzymesanddiseasedordamagedtissuesoftensecreteor“leak”tissuespecificenzymesintothebloodsystem.Assayingserumf
26、orthepresenceoftissuespecificenzymesaidsindiagnosisofdiseasessuchasmyocardialinfarctionandliverdamage.Inaddition,sincemitochondrialenzymesaregenerallyreleasedaftercytoplasmicenzymeshavebeenreleased,assayingforthepresenceofserumenzymescangiveinformationonthetimeofonsetofthedisease.,Serumenzymesarecom
27、monlyusedindiagnostictestsforavarietyofdiseases,MyocardialInfarction:Lactatedehydrogenase(H4isozyme),Aspartateaminotransferase,CreatinekinaseViralhepatitis:AlanineaminotransferaseAcutepancreatitis:Amylase,LipaseLiverdisease:Alkalinephosphatase,Lactatedehydrogenase(M4isozyme),Oxidoreductases(ECClass1
28、)Transferelectrons(RedOxreactions)Transferases(ECClass2)TransferfunctionalgroupsbetweenmoleculesHydrolases(ECClass3)BreakbondsbyaddingH2OLyases(ECClass4)EliminationreactionstoformdoublebondsIsomerases(ECClass5)IntramolecularrearangementsSynthetaseorLigases(ECClass6)Joinmoleculeswithnewbonds,EnzymeNo
29、menclature,Oxidoreductasescatalyzethetransferofhydrogenatomsandelectrons,Example-LactateDehydrogenase,O,O,-,C,C,C,H,3,O,+NAD,H,+,H,+,O,O,-,C,H,C,C,H,3,H,O,+NAD,+,pyruvate,L-lactate,lactate,dehydrogenase,Transferasescatalyzethetransferoffunctionalgroupsfromdonorstoacceptors,Example-Alanineaminotransf
30、erase,pyruvate,O,O,-,C,H,C,C,H,2,H,2,N,C,H,2,O,O,-,C,glutamate,O,O,-,C,H,C,C,H,3,H,2,N,L-alanine,O,O,-,C,C,C,H,2,C,H,2,O,O,-,C,O,a,-ketoglutarate,+,+,alanine,aminotransferase,Hydrolasescatalyzethecleavageofbondsbytheadditionofwater(hydrolysis),Example-Trypsin,N,H,C,N,H,2,(,C,H,2,),4,H,O,C,O,H,H,C,N,
31、H,N,H,C,C,H,3,H,O,C,O,H,C,N,H,C,C,C,H,(,C,H,3,),2,N,H,C,N,H,2,(,C,H,2,),4,H,O,C,O,H,H,C,N,H,O,-,C,N,H,C,C,H,3,H,O,C,O,C,H,(,C,H,3,),2,H,C,H,3,N,+,C,+,H,2,O,trypsin,+,G,l,y,-,L,y,s,-,V,a,l,-,A,l,a,V,a,l,-,A,l,a,G,l,y,-,L,y,s,Lyasescatalyzetheadditionofgroupstodoublebondsorformationofdoublebondsbyremovalofgroups,Example-ATP-citratelyase,Isomerasescatalyzethetransferoffunctionalgroupswithinthesamemolecule,Example-Phosphoglucoseisomerase,LigasesuseATPtocatalyzetheformationofnewcovalentbonds,Example-DNAligase,