细胞生物化学实验课件Exp-3-Determination-of-Km-Lei-Zhang-upd

细胞生物化学实验课件Exp3-DeterminationofKm-LeiZhang-updatedOverviewObjectivePrincipleProcedureResultsandanalysisNotesThoughtquestionsObjectiveTounderstandthesignificanceofKmandlearnhowtodeterminetheKmvalueofAKP.TobeabletocalculatetheKmofenzymeusingthestandardcurve.PrinciplesThestagesofanenzyme-catalysedreactionaresummarisedasE+S<——>ES<——>EP<——>E+PMichaelis-MentenEquationV=initailvelocityofthereactionVmax=maximumvelocityofthereaction[S]=substrateconcentrationKm=MichaelisconstantMichaelisConstant(Km)TheKmvalueforanenzymedependsonparticularsubstrateandontheenvironmentalconditions,suchastemperature,pH,andionicstrength,regardlessofenzymeconcentration.TheKmvalueisacharacteristicconstantofenzymes.PlotofMichaelis-MentenEquationWhen[S]=Km,V=Vmax/2Then,atV=Vmax/2,Km=[S]Thus,theunitofKmismol/L,justas[S]HyperbolaAtlowconcentrationsofthesubstrate[S],velocity(V)isproportionalto[S],isausualfeaturesofafirstorderreaction.Withtheincreaseofsubstrateconcentration,Vdoesnotincreaseproportionallyto[S].AtsubstrateconcentrationsfarhigherthanKm,([S]>>Km),thecurveapproachestoaplateau.Thereactionbecomesnearlyindependentofthesubstrateconcentrationandshowszero-orderkinetics.Whentheenzymeissaturatedbysubstrate,almostallenzymemoleculesarepresentasenzyme-substratecomplexandthereactionisnolongerlimitedbysubstrateavailabilitybutbytheamountandtheturnovernumberoftheenzyme.Lineweaver-BurkDouble-reciprocalPlotWhenusedfordeterminingthetypeofenzymeinhibition,theLineweaver–Burkplotcandistinguishcompetitiveandnon-competitiveinhibitorsofenzyme.AlkalinePhosphatase(AKP)Alkalinephosphatases(AKP)arethephosphatehydrolasesthathaveamaximumactivityatarelativelyhighpH(>7.0).AKPiswidespreadandoccursinbotheukaryoticandprokaryoticcells.SomedifferentsubstratescanbecatalyzedbyAKPwithdifferentKm.Inthisexperiment,asubstratecalleddisodiumphenylphosphateisusedtomeasuretheactivityofAKP.DisodiumphenylphosphatecanbehydrolyzedbyAKPandproducephenolandphosphates.ThehigheractivityofAKPthemorephenolisproduced.SotheconcentrationofphenolvariesinproportionwiththeactivityofAKP.Phenoland4-aminoantipyrinecanbeoxidizedtoquinonederivatives.Themorephenolactiveassubstrate,themorequinonederivativesareproduced,whichareredcompoundswithmaximumabsorptionpeakat510nm.ProcedureTheimpactofsubstrate’sconcentrationonvelocityofenzymereaction(1)Take6testtubesandlabelthemas1-6.(withoutKH2PO4)(2)mixtesttubes1-6wellandincubateat37℃for15minutes.(3)add1.1mlofalkalinesolutionintoeachtubetoterminatereaction.(4)add1.0mlof0.3%4-aminoantipyrineand2.0mlof0.5%potassiumferricyanideintoeachtubes,mixwellthenplacethematRTfor10minutes.(5)#6and6’tubesareasblank.MeasuretheA510ofeachsamplesusingthespectrophotometerandtheblanktubeisusedforthezerosetting.(6)Plot1/A510against1/[S]andcalculateKm.PlotastandardcurveofphenolcontentTake6testtubesandlabelthemass1-s6，#s1tubeisusedasblank.(2)mixwellandincubateatRTfor15minutes.MeasuretheA510ofsamplesandtheblanktubeisusedforthezerosetting.(3)A510isplottedagainstphenolconcentration.Resultsandanalysis1.Figureofreactionkinetics(1/A510vs.1/[S])2.ValueofKm3.EnzymeactivityOneunitofenzymeactivityisdefinedastheamountthatcatalyzestheformationofonemilligramofproduct(phenol)in15minutesat37℃.Calculatetheenzymeactivityaccordingtothestandardcurveofphenolcontent.NotesPipettingthereagentsshouldbeac