Chapter9 Enzyme kinetics.ppt

《Chapter9 Enzyme kinetics.ppt》由会员分享，可在线阅读，更多相关《Chapter9 Enzyme kinetics.ppt（48页珍藏版）》请在淘文阁 - 分享文档赚钱的网站上搜索。

1、Chapter9EnzymeKinetics,Outline,DefinitionofenzymekineticsFactorsaffectingenzyme-catalyzedreactionsMichaelis-MentenKineticsEnzymeinhibitionkineticsAllostericenzymekinetics,Kineticsconcernswiththeratesofchemicalreaction.Enzymekineticsaddressesthebiologicalrolesofenzymeandquantifytheremarkablefunctiono

2、fenzymes;Enzymekineticsinformationcanbeexploitedtocontrolandmanipulatethecourseofmetabolicevents.Pharmaceuticalsordrugsareoftenspecialinhibitorstargetedataparticularenzyme.,LeonorMichaelis(1875-1949),MaudMenten(1879-1960),IntroductiontoEnzymeKinetics,SubstrateconcentrationEnzymeconcentrationpH,tempe

3、ratureandionicstrengthThepresenceofinhibitorsoractivatorsWhichoneisthemostimportant?,Factorsaffectingenzyme-catalyzedreactions,Howdoessubstrateconcentrationaffectthereactionrate?,AmajorityofEnzymes,AminorityofEnzymes,Michaelisenzyme,Allostericenzyme,YouneedtoknowhowthisisderivedThisisthecompletechem

4、icalformulaforanenzyme-catalyzed(E)reactionofsubstrate,Sandproduct,P;Mechaelis-Mentenequationdescribestherelationshipbetweenreactionrateandsubstrateconcentration.Itcanexplainthesaturationbehaviorincatalyzedreactionsasshowninthepreviousslide.Mechaelis-Mentenequationisderivedbasedonthefollowingthreeco

5、nditions:Statesteadyassumption;Initialvelocityassumption;Ratelaw.,TheMechaelis-MentenEquation,Inthebeginningofthereaction,thereisverylittleproduct,orPissmall.SotheamountofEScontributedbyE+Pisnegligible.Thus,theMMequationconcernsthereactionratethatismeasuredduringearlyreactionperiod.Inwhichcase,theen

6、zymecatalyzedreactioncanbemodifiedto:,K-1,InitialVelocityAssumption,Ratelawstillappliesinenzymecatalyzedreactions.Theforwardvelocity,orrate,vfis,Thereversevelocityorrate,ortherateofdisappearancevdis,Atsteadystate,thereisnoaccumulationofES,thus:,RateLawinEnzymeCatalyzedReactions,Weneedonemoreconditio

7、n,thatis,thetotalenzymeconcentration,Etisthesumofthatofenzyme-substratecomplex,ES,andthatoffreeenzyme,E:Atsteadystate,theforwardrateshouldequaltothereverserate:Rateofproductionformation(ratelaw),v=k2ES.So:,DerivationofMichaelis-MentenEquation,Therateofproductionformationcanusuallybemeasuredexperimen

8、tallybymonitoringtheprogresscurveofproductionformation.Themaximumratecanbereachedatsaturatingsubstrateconcentration,orwhenSSoMMequationcanbere-writtenas:,Enzyme-catalyzedrateissaturated,NotesontheMMEquations,UnderstandingKm,KmisaconstantderivedfromrateconstantsKmis,undertrueMichaelis-Mentencondition

9、s,anestimateofthedissociationconstantofEfromS,becauseatequilibrium,Reversiblereaction,dissociationconstantisSosmallKmmeanstightsubstratebinding;highKmmeansweaksubstratebinding.Kmequalstothesubstrateconcentrationatwhichv=1/2vmax,UnderstandingVmax,ThetheoreticalmaximalvelocityVmaxisaconstantVmaxisthet

10、heoreticalmaximalrateofthereaction-butitisNEVERachievedinrealityToreachVmaxwouldrequirethatALLenzymemoleculesaretightlyboundwithsubstrateVmaxisasymptoticallyapproachedassubstrateisincreased,Combinationof0-orderand1st-orderkineticsWhenSislow,theequationforrateis1storderinSWhenSishigh,theequationforra

11、teis0-orderinSTheMichaelis-MentenequationdescribesarectangularhyperbolicdependenceofvonS!,ThedualnatureoftheMMequation,Ameasureofcatalyticactivitykcat,theturnovernumber,isthenumberofsubstratemoleculesconvertedtoproductperenzymemoleculeperunitoftime,whenEissaturatedwithsubstrate.IftheM-Mmodelfits,k2=

12、kcat=Vmax/EtValuesofkcatrangefromlessthan1/sectomanymillionspersec,Theturnovernumber,ValuesofkcatforSomeEnzymes,Nameforkcat/Kmkcat/Kmisanapparentsecond-orderrateconstantItmeasureshowtheenzymeperformswhenSislowTheupperlimitforkcat/Kmisthediffusionlimit-therateatwhichEandSdiffusetogether,Anestimateofh

13、owperfecttheenzymeis,Beauty+wisdom,MissEnzyme,TriosePhosphateIsomerase(TIM),kcat+Km,TheValuesofkcat,Kmandkcat/Kmforsomeenzymes,AbettermethodfordeterminingthevaluesofVmaxandKm,whichwasformulatedbyHansLineweaverandDeanBurk,usesthereciprocaloftheMichaelisMentenequation,DeterminingtheVmaxandKmLineweaver

14、-Burkordouble-reciprocalplot,Enzymecanbeinhibitedbyinhibitors.Inhibitorsaretoolstoscientiststounderstandenzymes.Inhibitorsarealsoinmanycasespharmaceuticalreagentsagainstdiseases;Inhibitorsinhibitenzymefunctionbybindingwithenzymes.Thebindingreactioncanbeeitherreversibleorirreversible;Reversibleinhibi

15、torsassociatewithenzymesthroughnon-covalentinteractions.Reversibleinhibitorsincludethreekinds:Competitiveinhibitors;Non-competitiveinhibitors;Un-competitiveinhibitorsIrreversibleinhibitorsassociatewithenzymesthroughcovalentinteractions.Thustheconsequencesofirreversibleinhibitorsistodecreaseintheconc

16、entrationofactiveenzymes.,EnzymeInhibition,Thestructureissimilartosubstrateandthebindingsiteisjusttheactivesite,CompetitveInhibitor1,Thestructureisnotsimilartosubstrateandthebindingsiteisotherthantheactivesite,CompetitveInhibitor2,Aclassicexample-Succinatedehydrogenase,Anotherexample-Dihydrofolatere

17、ductase,HIVproteaseneedstobindandcleavepolypeptideduringlifecycle,OnemoreexampleHIVprotease,v,S,Vmax,Km,1/S,1/v,1/Vmax,-1/Km,Slope=Km/Vmax,KI,KmincreasesVmaxunchanged,+inhibitor,Km(1+I/KI),-1/(Km(1+I/KI),Slope=Km(1+I/KI)/Vmax,KineticsforCompetitiveInhibition,k1,k-1,k2,KI,KmunchangedVmaxdecreases,KI,

18、KineticsforNoncompetitiveInhibition,k1,k-1,k2,KmdecreasesVmaxdecreasesSlopeunchanged,+inhibitor,KI,1/S,1/v,1/Vmax,-1/Km,Slope=Km/Vmax,(1+I/KI)/Vmax,Slope=Km/Vmax,v,S,Vmax,Km,Km/(1+I/KI),Vmax/(1+I/KI),-(1+I/KI)/Km,KineticsforUncompetitiveInhibition,Combineswithordestroysanessentialfunctionalgrouponth

19、eenzyme(e.g.formscovalentbonds)InhibitenzymesirreversiblyKineticbehaviorresemblesnoncompetitiveinhibitorFourdifferenttypes:GroupSpecificReagent:-inhibitordoesnotresemblesubstrateSubstrateAnalogue:-inhibitorresemblessubstrateTransition-stateAnalogue:-inhibitorresemblestransitionstateSuicideInhibitors

20、:-inhibitorresemblessubstrate,turnsdangerousafterprocessedbyenzyme,IrreversibleInhibitor,DoesnotresemblesubstrateirreversiblyinactivatesenzymebymodifyinganessentialRgroupe.g.DIPF(potentnervegas)blocksacetylcholinesterase,whichisessentialforproperneuraltransmission,activesiteserine,GroupSpecificReage

21、nt,ResemblesubstrateBindsatenzymeactivesiteThenirreversiblymodifies(orbindsto)totheactivesitee.g.TPCKformscovalentbondwiththeHistidineattheactivesiteofChymotrypsin(aproteasesecretedbythepancreas),SubstrateAnalogue,AnexampleofSubstrateAnalogue,Transitionstateanalogs,Acompoundthatmimicsatransitionstat

22、eexploitsthesebindinginteractionsinwaysthatasubstrateanalogcannot,soitcanbeaparticularlyeffectiveinhibitor.,ResemblesubstrateBindsatenzymeactivesite(notaninhibitoryet)ProcessedbyenzymevianormalcatalyticmechanismBecomesachemicallyactiveintermediatesthatmodifiesandinactivatestheenzymeirreversiblyGoodc

23、andidatefordrugduetominimalsideeffect,SuicideInhibitor,Penicillinisasuicideinhibitor,Allostericenzyme,AllosteryandCooperativityAllostericenzymesareenzymeshavingallosteryThetermsallosteryandcooperativityhavebeenmuddled.Allosterystrictlyreferstoinfluenceofactivitybyadistantsite.Cooperativityindicatest

24、hattheoccupancyofonesiteinamultisubunitenzymeinfluencesthebindingontheothers.Thisisaformofallostery,butisonlyonemanifestationofageneralphenomena.Unfortunatelyallosteryhadbecomealmostexclusivelyassociatedwiththebehaviorofmulti-subunitenzymes.,KineticsofAllostericEnzyme,MainEntry:allostericPronunciati

25、on:a-lO-ster-ik,-stir-Function:adjectiveEtymology:all-+stericDate:1962Meaning:of,relatingto,orbeingachangeintheshapeandactivityofaprotein(asanenzyme)thatresultsfromcombinationwithanothersubstanceatapointotherthanthechemicallyactivesite-allosterically/-i-k(&-)lE/adverb-allostery/a-lO-ster-E,-stir-/no

26、un,Allostery:WebstersDefinition,GeneralPropertiesofAllostericEnzymes,Theactivitiesofallostericenzymesarechangedbymetabolicinhibitorsandactivators.Allostericmodulatorsbindnoncovalentlytotheenzymestheyregulate.Withfewexceptions,regulatedenzymesaremultisubunitproteinsAnallostericallyregulatedenzymeusua

27、llyhasatleastonesubstrateforwhichthev0versusScurveissigmoidalratherthanhyperbolicCompetitiveinhibitorscanactivateallostericenzymesataverylowconcentrationAscomparedtoMichaelisenzyme,allostericenzymeareminorities,KineticSignatureofCooperativityinAllostericEnzymes,Multisubunitenzymesthatexhibitcooperat

28、ivityshowasigmoidalinitialvelocitycurveincontrasttothehyperboliccurveforindependentsubunits.,KineticConsequencesofAllostericEffectorsonCooperativeEnzymes,Thisisthetraditionalviewoffeed-backinhibitionandregulationin“allosteric”enzymes.,Hemoglobin(notanenzyme)ThiswastheoriginoftheTandRstatesAspartatet

29、ranscarbamoylase.ExampleoffeedbackinhibitionThesearegreatexamples,buttherearemanyothers.,ClassicExamplesofAllostery,Concerted(conceptuallysimpleandofteneffective)Sequential(probablycorrectbutdifficulttoprove),ModelsforAllostericRegulation,Assumes2conformationstates:R&TBindingofsubstrateinducesallsub

30、unitstochangetoRstate.NoT-Rhybrids.Allowsfor+cooperativityonly.,+cooperativity,Tstate,Rstate,TheConcertedModel,TheSequentialModel,Thebindingofsubstrateswitchesconformationofonlythesubunittowhichitisbound.Conformationalchangeinonesubunitmayortheaffinityofothersubunitshaveforthesubstrate.Allowsfor+or-

31、cooperativity.,Boththesequentialandconcertedmodelsaccountforallostericregulationofcooperativeenzymes,AllostericactivatorsthataltertheKmofanenzymestabilizethehighaffinitystateoftheactivesite.Allostericactivatorsthatalterthekcatofanenzymestabilizethehighactivitystateoftheactivesite.,Allostericinhibito

32、rsthataltertheKmofanenzymestabilizethelowaffinitystateoftheactivesite.Allostericinhibitorsthatalterthekcatofanenzymestabilizethelowactivitystateoftheactivesite.,Thistooisagrosssimplificationofreality,butembodiestheconceptthateachbindingeventgeneratesadifferent(group)ofconformationalstates.k1etcaremi

33、croscopicrateconstants.,SimplifiedRateEquationsfortheSequentialModelforaHighlyCooperativeenzyme,SimplifiedRateEquationsfortheSequentialModelforaHighlyCooperativeenzyme,ThisassumesthattheconcentrationsofES,ES2.andEsn-1areverysmallduetothehighcooperativity.ThisissimilarinformtotheHillequation.,nisrefe

34、rredtoastheHillconstantandisameasureofthecooperativityoftheenzyme.thegreaterthevalueofn,thehigherthecooperativity.Forthecasewheren=1(nocooperativity)At1/2VmaxS0.5givesk=S0.5n.Theseparameterscanbederivedbynon-linearregressionanalysis.Thisisthesimplestkineticmodelforcooperativeenzymes.,ThevelocitydataforcooperativeenzymescanbepresentedinalinearformbyuseofEquation:,TheslopeofthebestfitlineprovidesanestimatedoftheHillcoefficientn,andtheyinterceptprovidesanestimateoflog(K).,HillPlot,