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Chapter10MechanismsofEnzymeCatalysisOutlineMajormethodsforstudyingenzymemechanisms“Thetransition-statestabilization”theoryMechanismsoftransitionstatestabilizationProximityandOrientationGeneralAcid-basecatalysisElectrostaticcatalysisMetalcatalysisCovalentcatalysisSubstratestrainStructureandfunctionofseveralcommonenzymesProteasesLysozyme

(self-study)MajormethodsforstudyingenzymemechanismsFindouttheconservativeresiduesbyaligninghomologousenzymesStudythe3-DstructureofenzymesPerformsite-directedmutagenesisCarryoutthekineticanalysisMakechemicalmodificationMolecularComputerMimicsEnzymesbringsubstratestogetherintheproperorientationforareactiontooccurEnzymespossessfunctionalgroupsthatstabilizethetransitionstateofthereactionEnzymeslowertheactivationGibbsenergyofareactionEnzymesdrivethermodynamicallyunfavorablereactionsbycouplingthemtothermodynamicallyfavorablereactionsHowdoenzymeswork?AnimaginarystickasedesignedtocatalyzebreakageofametalstickEvidencesofstabilizingthetransitionstateofthereactionTransitionanaloguesarepotentinhibitorsAbzymesEnzymeshaveevolvedtorecognizethetransitionstateofthereactiontheycatalyzeTodesignanenzymeinhibitor,weshouldtrytomimicthetransitionstateofthereaction,notthesubstratesorproductsDrugdesign:TransitionstateanaloguesCytidine

deaminaseisatargetforanticancerdrugsAnexample-tetrahydrouridineisatransitionstateanalogueofcytidine

deaminase

Antibodiesareimmunoglobulins.Antibodiesareelicitedinanorganizminresponsetoimmunologicalchallengebyaforeignmoleculecalledantigens;Antibodieselicitedinresponsetotransitionstateanalogshavetheabilitytostabilizethetransitionstateandthuscancatalyzeareactionbyforcingthesubstrateintothetransitionstatestructure;Examplesofabzymes:Abzymes-CatalyticAntibodiesImmunizeanimals,produceantibodyHydrolysizeScreenabzymeCocainePreparationofabzymecatalyzinghydrolysisofcocaineHowdoenzymesstabilizethetransitionstateofareactionCatalysisbyproximityandorientationGeneralAcid-basecatalysisElectrostaticcatalysisMetalcatalysisCovalentcatalysisSubstratestrainCatalysisbyproximityandorientationThisincreasestherateofthereactionasenzyme-substrateinteractionsalignreactivechemicalgroupsandholdthemclosetogether.Thisreducestheentropyofthereactantsandthusmakesreactionssuchasligationsoradditionreactionsmorefavorable,thereisareductionintheoveralllossofentropywhentworeactantsbecomeasingleproduct.Thiseffectisanalogoustoaneffectiveincreaseinconcentrationofthereagents.Thebindingofthereagentstotheenzymegivesthereactionintramolecularcharacter,whichgivesamassiverateincreaseIntramolecularandintermolecularreactionsGeneralacidcatalysisisaprocessinwhichprotontransferfromanacidlowersthefreeenergyofareaction’stransitionstate.Areactionmayalsobestimulatedbygeneralbasecatalysisifitsrateisincreasedbyprotonabstractionbyabase.Somereactionsmaybesimultaneouslysubjecttobothprocesses;theseareconcertedacid–basecatalyzedreactions.GeneralAcid-basecatalysisGeneralbasecatalysisGeneralAcidcatalysisGeneralAcid-basecatalysisMechanismsofketo–enol

tautomerization.(a)Uncatalyzed.(b)Generalacidcatalyzed.(c)Generalbasecatalyzed.DifferencesbetweenSpecificacid/basecatalysisandgeneralacid/basecatalysisTheRNaseAmechanism(1)TheRNaseAmechanism(2)GeneralAcid-BaseCatalysisLysozymeisanexampleofgeneralacidcatalysisGlu35inLysozymeissurroundedbyhydrophobicaminoacidsEnzymesusechargedaminoacidstoneutralizechargesthatdevelopduringformationofthetransitionstateofareaction.Thisisknownaselectrostaticcatalysis.ElectrostaticcatalysisConsiderthehydrolysisofapeptidebondWhydoesneutralizingthechargethatdevelopsinthetransitionstateincreasethereactionrate?LysozymeuseselectrostaticcatalysistocatalyzethecleavageofpolysaccharidesThepHprofileofpolysaccharidehydrolysisreflectsthepKavaluesofGlu35andAsp52MetalscanactasLewisacidsMetalscanstabilizechargesthatdevelopinthetransitionstateMetalscanacceptanddonateelectronsinoxidation-reductionreactionsMetalscanbeimportantforthestructureoftheenzymeMetalcatalysisALewisacidisaspeciesthatcanacceptapairofelectronsforbondingZnfunctionsasaLewisacidincarbonicanhydraseMetalscanactasLewisacidsZnfunctionsasaLewisacidincarbonicanhydraseMetalscanstabilizechargesthatdevelopinthetransitionstateMetalscanacceptanddonateelectronsinoxidation-reductionreactionsThisistheprincipaleffectofinducedfitbinding.Thisinducesstructuralrearrangementswhichstrainsubstratebondsintoapositionclosertotheconformationofthetransitionstate,soloweringtheactivationenergyandhelpingcatalyzethereaction.Inadditiontobondstraininthesubstrate,bondstrainmayalsobeinducedwithintheenzymeitselftoactivateresiduesintheactivesite.SubstratestrainLysozymeisanexampleofhowenzymescreatestraininthesubstrateGeneralacidcatalysis

Glu35donatesaprotonElectrostaticcatalysisthepositivelychargedcarboniumionisstabilizedbyAsp52StrainedsubstrateconformationthesugarboundtotheactivesiteontheenzymeisforcedtoadoptahalfchairconformationLysozymecatalyzedcleavageofpolysacharridesinvolves:Covalentcatalysisacceleratesreactionratesthroughthetransientformationofacatalyst–substratecovalentbond.Usually,thiscovalentbondisformedbythereactionofanucleophilicgrouponthecatalystwithanelectrophilicgrouponthesubstrate,andhencethisformofcatalysisisoftenalsocallednucleophiliccatalysis.Inaddition,severalcoenzymes,notablythiaminepyrophosphateandpyridoxalphosphate,functioninassociationwiththeirapoenzymesascovalentcatalysts.CovalentcatalysisIncovalentcatalysis,theenzymeformsatransientcovalentintermediatewiththesubstratesofthereaction.CovalentcatalysisBiologicallyimportantnucleophilicandelectrophilicgroupsBiologicallyimportantnucleophilicandelectrophilicgroupsThedecarboxylationofacetoacetateTheuncatalyzedreactionmechanismisatthetop,andthemechanismascatalyzedbyprimaryaminesisatthebottom-CatalyzegrouptransfertowaterUncatalyzedhydrolysisofapeptidelinkageisveryslowwithahalf-lifeatneutralpHand25oCof~300-600years

WhatareProteases?Serineproteases-inactivatedbydiisopropylfluorophosphateMetalloproteases-inactivatedbyEDTAoro-phenanthroline(example-thermolysin)Carboxylproteases-notactiveatneutralorslightlyalkalinepH(example-HIVprotease)Thiolproteases-inactivatedbyiodoacetamideoriodoacetateFourMajorClassesofProteasesGeneralCharacteristicsofProteasesTheyallgothroughatransitionstateinwhichthenormallytrigonalcarbonylcarbonofthepeptidebondbecomestetrahedral.Thistetrahedralgeometryoccursbecauseofthetemporaryadditionofanucleophile.Inthecaseoftheserineandthiolproteases,thenucleophileistheserinehydroxylorcysteinethiolattheactivesitewhereasintheothertwoclassesitisawatermolecule.Thereactionproceedsmoredirectlyinthemetalloproteasesandthecarboxylproteases,whereastheacyl-enzymeintermediatethatoccurswiththeserineandthiolproteasesmustbehydrolyzedbywaterinasecondstep.BestunderstoodoftheproteasesAllhaveapeculiarlyreactiveSerresidueTherearethreedistinctstructuralfamilies-theyhaveverysimilarmechanismsofactionbutarenotdetectablyrelated-theyprovideoneofthemoststrikingexamplesofapparentevolutionaryconvergenceOnefamilyisrepresentedbythebacterialproteasesubtilisin,anotherbyenzymesincludingchymotrypsin,trypsin,elastase,thrombin,kallikrein,etc.and,finallyafamilyrepresentedbywheatgermserinecarboxypeptidaseII

Membersinthetrypsinfamilyareinvolvedinarangeofcellularfunctionsincludingbloodclotting,complementactivation,hormoneproduction,andfertilizationSerineProteasesallshareanoxyanionholeallcontainacatalytictriad-Serine,Histidine,andAspartate:HisdirectlyabstractsaprotonfromSertherebyconvertingittoanalkoxideionTheydiffermoststrikinglyintheirpreferenceforaminoacidsidechainsontheC-sideofPeptidebondTrypsincleavesbondsonlyafterLysandArgresidues,chymotrypsinafterlargehydrophobicresidues;theotherproteasesofthisfamilyhavelessdistinctpreferencesProteasescancatalyzehydrolysisofbothamidesandestersGener

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