生物反應(yīng)工程：chap2-enzyme-1-課件

Chapter2EnzymesReducedDsbAfromE.coliLysozyme9/17/20231EnzymeChapter2EnzymesReducedDsbA§2.1IntroductionDefinitionHistoryBüchner,1897:AbreakthroughinEnzymology.Catalystsatworkinalivingorganismcouldalsofunctioncompletelyindependentlyofanylifeprocess.EffortstoisolateandpurifyindividualenzymeswereBüchner’sdiscoveries.Sumner,1926:Firstisolationofapureenzyme.Enzymesareusuallyproteinsofhighmolecularweight(15,000<MW<severalmillionDaltons)thatactascatalysts.9/17/20232Enzyme§2.1IntroductionDefinitionHisCharacteristicsThree-dimensionalstructureofthefoldedprotein,determinedbythesequenceoftheaminoacids.Fragile:mildtemperature,pressure,pH,ionstrength(ambientconditions).Lowertheactivationenergyofthereaction,butDoesn’taffectFree-energychangeEquilibriumconstant9/17/20233EnzymeCharacteristicsThree-dimension生物反應(yīng)工程：chap2_enzyme_1-課件生物反應(yīng)工程：chap2_enzyme_1-課件Enzymesarenamedbyaddingthesuffix–asetothe:EndofthesubstrateSuchasureaseThereactioncatalyzedSuchasalcoholdehydrogenaseNomenclatureEnzymesusingfamiliarnames:PepsininthedigestivetractTrypsininthedigestivetractRenninusedin

cheesemaking“Oldyellow”,whichcausedbrowningofslicedapples9/17/20236EnzymeEnzymesarenamedbyaddingthEC(EnzymeCommission)SIXclassesnumberedinFOURdigitsClassificationThefirstdigitalTheseconddigitalThethirddigitalThefourthdigitalTypeofreactioncatalyzed——mainclasses——actualsubstance9/17/20237EnzymeEC(EnzymeCommission)ClassifiOxidoreductasesFirstdigit1––theclassoxidoreductases.Seconddigit––thedonorofhydrogenatomorelectroninvolved.AlcoholAldehydeorketoneAlkene–CH=CH-PrimaryamineSecondaryamineNADH,NADPHThirddigit––hydrogenatomorelectronacceptor.NAD+,NADP+Fe3+O2OtherwiseunclassifiedFourthdigit––numberforfurtheridentification.9/17/20238EnzymeOxidoreductasesFirstdigit1–TransferasesFirstdigit2––theclasstransferases.Seconddigit––generaltypeofgroupstransferred.1-carbongroupAldehydeorketoneAcylgroup(-CO-R-)GlycosylgroupPhosphategroupSulphurcontaininggroupThirddigit––providedetailsontheexactnameofthe grouptransferred.Transferasescatalyzethefunctionalgrouptransferreactions,withageneralformgivenbelow:AX+BBX+A9/17/20239EnzymeTransferasesFirstdigit2––tHydrolasesFirstdigit3––theclasshydrolases.Seconddigit––thetypeofbondhydrolyzedEsterGlycosidicPeptideOtherC-NbondsAcidanhydridesHydrolyasescatalyzehydrolyticreactions,withageneralformgivenbelow:A-X+H2OX-OH+HA9/17/202310EnzymeHydrolasesFirstdigit3––theLyasesFirstdigit4––theclasslyases.Seconddigit––thetypeofbindsbroken.C-CC-OC-NC-SThirddigit––Thegroupremoved.CarboxylAldehydeKetoacidFourthdigit––numberforfurtheridentification.Lyasescatalyzethenon-hydrolyticremovalofgroupsfromsubstances.Oftentheproductcontainsadoublebond.9/17/202311EnzymeLyasesFirstdigit4––theclaIsomerasesFirstdigit5––theclassisomerases.Seconddigit––thetypeofreactioninvolved.RacemizationorepimerizationCis-transisomerizationIntramolecularoxidoreductasesIntramoleculartransferreactionsThirddigit––thetypeofmoleculeundergoingisomerization.AminoacidsHydroxyacidscarbohydratesFourthdigit––numberforfurtheridentification.9/17/202312EnzymeIsomerasesFirstdigit5––theLigasesFirstdigit6––theclassligases.Seconddigit––thetypeofbondsformed.C-OC-SC-NC-CLigases

catalyzethesynthesisofvarioustypesofbonds,wherethereactionsarecoupledwithbreakdownofenergy-containingmaterials,suchasATPornucleosidetriphosphates.X+Y+ATPX-Y+ADP+PiX+Y+ATPX-Y+AMP+PPi9/17/202313EnzymeLigasesFirstdigit6––theclSomeExamplesofEnzymeAlcoholDehydrogenase EC1.1.1.1GlucoseOxidase EC1.1.3.4Catalase EC1.11.1.6Tryptophan2,3-dioxygenase EC1.13.11.11PyruvateKinase EC2.7.1.40CreatineKinase EC2.7.3.2Alpha-amylase EC3.2.1.1Chitinase EC3.2.1.14OxaloacetateDecarboxylase EC4.1.1.3LactateRacemase EC5.1.2.1RiboseIsomerase EC5.3.1.20Acetate—CoALigase EC6.2.1.1GlutathioneSynthase EC6.3.2.39/17/202314EnzymeSomeExamplesofEnzymeAlcohol§2.2HowEnzymesWork?LockandKeyModelDevelopedbyEmilFischerin1895.Theenzymesandsubstratescombinebecausetheyhavecomplementarymoleculargeometries.9/17/202315Enzyme§2.2HowEnzymesWork?LockandAnExampleofLock-KeyModel9/17/202316EnzymeAnExampleofLock-KeyModel8/Induced-FitModel

Likeahandandglove.

Theenzymeisamoleculewhoseconformationcanchangeasthesubstrateapproachesandstartstobind.9/17/202317EnzymeInduced-FitModel

8/6/202317EnAnExampleofInduced-FitModel9/17/202318EnzymeAnExampleofInduced-FitMode§2.3EnzymeKineticsTwoassumptions:Reactionoccurredinwell-mixedreactor.Thatistosay,spatiallyuniform.Onlyinitialrateisused:Twomajorapproaches:Rapidequilibriumapproach;Quasi-steady-stateapproach.whichhastheunitofM/s.9/17/202319Enzyme§2.3EnzymeKineticsTwoassump§2.3.1MechanisticModelsforSimpleEnzymeKineticsSingle-substratekineticswasfirstdeveloped:V.C.R.Henriin1902L.MichaelisandM.L.Mentenin1913Asimplereactionscheme:Saturationkineticscanbeobtainedforthereactionschemeabove.9/17/202320Enzyme§2.3.1MechanisticModelsforThesamefewinitialstepsinderivingarateexpression:TherateofvariationoftheEScomplex:Eq.(2.2)Theconservationequationontheenzyme:Eq.(2.3)Therateofproductformation:Eq.(2.1)9/17/202321EnzymeThesamefewinitialstepsinRapidEquilibriumAssumption(DevelopedbyHenriandMichaelisandMenten)ASSUMPTION:Arapidequilibriumbetweentheenzymeandthesubstratecanbeachievedtoforman[ES]complex.

Thedissociationconstant:

Eq.(2.4)SubstitutingEq.(2.3)intoEq.(2.4)gives,Eq.(2.5)9/17/202322EnzymeRapidEquilibriumAssumptionASSubstitutingEq.(2.5)intoEq.(2.1)yields,Eq.(2.6)where,.Alowvalueofsuggeststhattheenzymehasahighaffinityforthesubstrate.––themaximumforwardvelocityofthereaction;iftheamountoftheenzymechanges,changes

––theMichaelis-Mentenconstant9/17/202323EnzymeSubstitutingEq.(2.5)intoEqExperimentaldatademonstratedtheconcentrationprofiles.ASSUMPTION:Initialsubstrateconcentrationgreatly

exceedstheinitialenzymeconcentration.issmall,thenTheQuasi-Steady-StateAssumption(DevelopedbyG.E.BriggsandJ.B.S.Haldane)9/17/202324EnzymeExperimentaldatademonstratedFromEq.(3.2)andtheassumption,wehave,Eq.(2.7)SubstitutingEq.(2.3)intoEq.(2.7)givesEq.(2.8)SubstitutingEq.(2.8)intoEq.(2.1)yieldsEq.(2.9)where,.9/17/202325EnzymeFromEq.(3.2)andtheassumptBothGiveSaturationKineticsSaturationkinetics,similartoLangmuir-Hinshelwoodisothermaladsorptionkinetics,whichshowsafirst-orderkineticsatthelowsubstrateconcentrations,butzero-orderkineticsathighsubstrateconcentrations.9/17/202326EnzymeBothGiveSaturationKineticsSQuestionsWhyonlyinitialratecanbeused?Whyalowvalueofsuggeststhattheenzymehasahighaffinityforthesubstrate?9/17/202