MECHANISMandKineticsofEnzymesummaryEnzymesdotwoimportantthings:theyrecognizeveryspecificsubstrates,andtheyperformspecificchemicalreactionsonthematfantasticspeeds.Enzymesarehighlyeffectivecatalysts,commonlyenhancingreactionratesbyafactorof105to1016Thesubstancesuponwhichanenzymeactsaretraditionallycalledsubstrates.Theselectivequalitiesofanenzymearecollectivelyrecognizedasitsspecificity:Lowspecificity,Intermediatespecificity,andAbsoluteornearabsolutespecificity.Theholoenzyme=

apoenzyme+Cofactors(coenzyme+prostheticgroup,i.e.,metalionsororganicmolecules)EnzymesAreClassifiedbytheReactionsTheyCatalyzeOxidoreductases:catalyzingoxidation-reductionreactions.Transferases:catalyzingthetransferofamoleculargroupfromonemoleculetoanother.Hydrolases:catalyzingthecleavagebytheintroductionofwater.Lyases:catalyzingreactionsinvolvingremovalofagrouptoformadoublebondoradditionofgroupstodoublebonds.Isomerases:catalyzingreactionsinvolvingintramolecularrearrangements.Ligases(synthetases):catalyzingreactionsjoiningtogethertwomolecules.HowEnzymesWorkThespecificsiteontheenzymewheresubstratebindsandcatalysisoccursiscalledtheactivesite.Theactivesiteofanenzymeisgenerallyapocketorcleftthatisspecializedtorecognizespecificsubstratesandcatalyzechemicaltransformations.Itisformedinthethree-dimensionalstructurebyacollectionofdifferentaminoacidsthatmayormaynotbeadjacentintheprimarysequence.Theinteractionsbetweentheactivesiteandthesubstrateoccurviathesameforcesthatstabilizeproteinstructure:hydrophobicinteractions,electrostaticinteractions,hydrogenbonding,andvanderWaalsinteractions.TRANSITIONSTATE:Thetransitionstateisthehighest-energyarrangementofatomsthatisintermediateinstructurebetweenthestructureofthereactantsandthestructureoftheproducts.Anyreactionmayhaveseveralsteps,involvingtheformationanddecayoftransientchemicalspeciescalledreactionintermediates.Whenseveralstepsoccurinareaction,theoverallrateisdeterminedbythestep(orsteps)withthehighestactivationenergy;thisiscalledtherate-limitingstep.Thedifferenceinfreeenergybetweenthetransitionstateandthereactant(s)iscalledthefreeenergyofactivation,G?.AFewPrinciplesExplaintheCatalyticPower

andSpecificityofEnzymesThisactivationenergyprovidesabarriertothereaction—thehigherthebarrier,theslowerthereaction.CATALYSISThereactionhappensatafasterrate.Thecatalystisregenerated.enzymesworkbyloweringtheactivationenergyofthereactiontheycatalyze.how?EnzymesAffectReactionRates,NotEquilibria.enzymespecificityLOCKANDKEYSpecificitymodel—thecorrectsubstratefitsintotheactivesiteoftheenzymelikeakeyintoalock.Onlytherightkeyfits.INDUCEDFITThebindingofthecorrectsubstratetriggersachangeinthestructureoftheenzymethatbringscatalyticgroupsintoexactlytherightpositiontofacilitatethereaction.Asignificantpartoftheenergyusedforenzymaticrateenhancementsisderivedfromweakinteractions(hydrogenbondsandhydrophobicandionicinteractions)betweensubstrateandenzyme.Theenzymeactivesiteisstructuredsothatsomeoftheseweakinteractionsoccurpreferentiallyinthereactiontransitionstate,thusstabilizingthetransitionstate.Theneedformultipleinteractionsisonereasonforthelargesizeofenzymes.Thebindingenergy,G

B,canbeusedtolowersubstrateentropyortocauseaconformationalchangeintheenzyme(inducedfit).Bindingenergyalsoaccountsfortheexquisitespecificityofenzymesfortheirsubstrates.Additionalcatalyticmechanismsemployedbyenzymesincludegeneralacid-basecatalysis,covalentcatalysis,andmetalioncatalysis.Catalysisofteninvolvestransientcovalentinteractionsbetweenthesubstrateandtheenzyme,orgrouptransferstoandfromtheenzyme,soastoprovideanew,lower-energyreactionpath.EnzymeKineticsasanApproachtoUnderstandingMechanismMichaelis-Mentenequationreflectsthekineticbehaviorofmanyenzymes.Thesaturationeffectisadistinguishingcharacteristicofenzymaticcatalysts.Themaximuminitialrateofthecatalyzedreaction(Vmax)isobserved,

when[S]issufficientlyhighthatessentiallyallthefreeenzymehasbeenconvertedtotheESform,undertheseconditionsfurtherincreasesin[S]havenoeffectonrate.Hyperbolickinetics,saturationkinetics[S]<<Km,Vo=Vmax[S]/Km[S]>>Km,Vo=Vmax[S]=Km,Vo=Vmax/2When[S]=Km,V0=Vmax/2,

Km

isdefinedbythesubstrateconcentrationthatgivesavelocityequaltoone-halfthemaximalvelocity.Aplotof1/Voversus1/[S]yieldsastraightline,ThedoublereciprocalplotiscalledtheLineweaver-Burkplot.

EnzymeUnits

Enzymeactivity(活力)iscommonlyexpressedbytheinitialrate(Vo).Theactualmolaramountoftheenzymecanbeexpressedintermsoftheactivityobserved.TheInternationalCommissiononEnzymesdefinesOneInternationalUnitofastheamountthatcatalyzestheformationofonemicromoleofproductinoneminuteat25℃underoptimalconditionsofmeasurement.1U=1molmin-1Anotherdefinitionforunitsofenzymeactivity(酶活力單位)isthekatal.Onekatalisthatamountofenzymecatalyzingtheconversionofonemoleofsubstratetoproductinonesecond.1kat=1molsec-1Thus,1kat=6×107U.Thetermactivityrefers

tothetotalunitsofenzymeinasolution.TurnoverNumber（轉(zhuǎn)化系數(shù)）

Theturnovernumberofanenzyme,kcat,isameasureofitsmaximalcatalyticactivity.kcatisdefinedasthenumberofsubstratemoleculesconvertedintoproductperenzymemoleculeperunittimewhentheenzymeissaturatedwithsubstrate.Theturnovernumberisalsoreferredtoasthemolecularactivityoftheenzyme.Thespecificactivity(比活力)isthenumberofenzymeunitspermilligramoftotalprotein.Thespecificactivityisameasureofenzymepurity:itincreasesduringpurificationofanenzymeandbecomesmaximalandconstantwhentheenzymeispure.EveryenzymehasanoptimumpH(orpHrange)atwhichithasmaximalactivity.Enzymeinhibition

Irreversibleinhibition

ReversibleinhibitionInhibitioncanbeasourceofinsightintothemechanismofenzymeaction:residuescriticalforcatalysiscanoftenbeidentifiedbyusingspecific(irreversible)inhibitors.Inirreversibleinhibition,aninhibitorbindspermanentlytoanactivesitebyformingacovalentbondoraverystablenoncovalentinteraction.ReversibleinhibitorsbindtoenzymenoncovalentlyReversibleinhibitioncanusuallybedividedintothreetypes:Competitiveinhibitorscompetewithsubstratebybindingreversiblytotheactivesite,buttheyarenottransformedbytheenzyme.Whichareoftenresemblethesubstrates.UncompetitiveinhibitorsbindonlytotheEScomplex,atasitedistinctfromtheactivesite.MixedinhibitorsbindtoeitherEorES,againatasitedistinctfromtheactivesite.Competitive:Slopeeffect(affecttheKm,nottheVmax)Uncompetitive:IntercepteffectMixed:Slopeandintercepteffect.whenα=α′,themixedinhibitorhasbeendefinedasnoncompetitiveinhibition.(affecttheVmax,nottheKm)

AllostericenzymeFeedbackregulationReversiblecovalentmodificationProteolyticactivationRegulationofenzymesynthesisandbreakdownRegulationofenzymeactivityAllostericenzymes(similartohemoglobin)areregulatedbyreversible,noncovalentbindingofmodulators(oftenbeingmetabolites).Theenzymecatalyzingthefirststepofasyntheticpathwayisoftenanallostericenzyme.Forexample,aspartatetranscarbamoylase(ATCase,thebestunderstoodallostericenzyme)inthepyrimidinenucleotidesynthesispathway.multisubunitproteins,Allostericeffect,sigmoidalcurve.ALLOSTERISMANDCOOPERATIVITY

Cooperativity:Observedwhenthereactionofonesubstratemoleculewithaproteinhasaneffectonthereactionofasecondmoleculeofthesubstratewithanotheractivesiteoftheprotein.Positive:Thebindingofthefirstsubstratemakesthereactionofthenextsubstrateeasier.Negative:Thebindingofthefirstsubstratemakesthereactionofthenextsubstrateharder.Allosterism:ThebindingofaneffectormoleculetoaseparatesiteontheenzymeaffectstheKmorVmax

oftheenzyme.FeedbackregulationInbiologicalsystemstheratesofmanyenzymesarealteredbythepresenceofeffectors(inhibitorsoractivators).Buildingupofapathway’sendproductultimatelyslowstheentirepathway.Thisiscalledfeedbackinhibitionandoftentakesplaceatthecommittedstepinthepathway.Thecommittedstepisthefirststeptoproduceanintermediatewhichisuniquetothepathway.Asmanymetabolicpathwaysarebranched,feedbackinhibitionmustallowthesynthesisofoneproductofabranchedpathwaytoproceedevenwhenanotherispresentinexcess.SequentialfeedbackinhibitionConcertedfeedbackinhibitionCumulativefeedbackinhibition

ReversiblecovalentmodificationsTheactivityofmanyenzymesareregulatedbyreversiblecovalentmodifications.Phosphorylation,themostcommonreversiblecovalentmodification,isahighlyeffectivemeansofswitchingtheactivityoftargetenzymes.

ProteinkinasescatalyzethetransferofaphosphategroupfromanATPmoleculetothesidechains