扬州大学：《生物化学 Biochemistry》课程教学课件（PPT讲稿）9 Enzymes（Part2）

Part2Enzymes

Enzymes Part2

Enzyme kinetics as an approach to understandingmechanism

Enzyme kinetics as an approach to understanding mechanism

SubstrateConcentration AffectstheRate ofEnzyme-Catalyzed Reactionspre-steadystate=initialtransientperiodduringwhichESbuildsupsteadystate=periodduring which [ES] andotherintermediatesLEremainconstant[ES]steady-statekinetics=TimethetraditionalanalysisofPre-steadySteadystatestatereactionrates

Pre-Steady State KineticsCan ProvideEvidenceforSpecificReactionStepsusedtomeasurerateconstantsofindividualstepsstopped-flowdeviceallowsmixingandsamplingonasecondormillisecondtimeAbsorbuenascaleFiowceWast

Dissociation of the Product is Rate.Limiting for Many Enzymesforanenzymewhereproductrelease,EP→E+P,israte-limiting (k,israte-limiting):k3k2kE+S=ES(6-28)EP=E+P1k_1k-2

UsingPre-SteadyStateKinetics toDetermine the Rate-Limiting Stepthe observation of aburst indicates that a rate-limitingstep(productrelease,enzymeconformationalchangeetc.)occursafterformationoftheproductbeingmonitored6050Increasing[E](wu40(wu) Ed)30011020304050101520[E](nm)Time(s)(c)(b)

EnzymesAreSubjecttoReversibleorIrreversible lnhibitionenzyme inhibitors=moleculesthat interferewithcatalysis,slowingorhaltingenzymaticreactions2classesofenzymeinhibitors:-reversible-irreversible

Reversible Inhibitiontypes of reversible inhibition:-competitiveinhibitionuncompetitive inhibition-mixedinhibition--noncompetitiveinhibition

Competitive Inhibitioncompetitiveinhibitor:competeswiththesubstrateforthe(a)E+SE+PCactive site ofan1enzymeK-typeofreversibleEIainhibitionbydefinition,theNelson&Cevalue of α'foracompetitiveinhibitoris 1

Competitive Inhibitors Affect theApparent Km, but Not the Vmax"apparent"Km=theexperimentally(b)determinedvariableaKm0.8when[S] >>[],the:0.6Vreactionexhibitsnormal0.4VmaxOKmKm30.020inthepresenceof406080100[S] (p)inhibitor, the [S] at whichehonsCoVo=V2Vmax, the apparentKm,increasesbythefactora