Ch395G Fall 2009: Lecture 13The Kinetic AnalysisChapter 13 and 14 Voet & VoetVoet Biochemistry 3e© 2004 John Wiley & Sons, Inc.The Kinetic Analysisof Enzyme BehaviourNomenclature of EnzymesTrivial names e.g. TrypsinBy type of substrate e.g. proteasesSystematic – Enzyme CommissionVoet Biochemistry 3e© 2004 John Wiley & Sons, Inc.eg glucose + ATP → glucose-6-P + ADPTrivial name -- hexokinaseSystematicADP-D-glucose-6-phosphotransferaseE.C. 2.7.1.1Voet Biochemistry 3e© 2004 John Wiley & Sons, Inc.Table 13-3 Enzyme Classification According to Reaction Type.Page 470Enzyme Kinetics Chapter 14Study of rates of reactionsThermodynamics tells us whether a reaction can occur spontaneously but tells nothing about rates or about pathway followed.Voet Biochemistry 3e© 2004 John Wiley & Sons, Inc.about rates or about pathway followed.Kinetics can inform about rates, how rates change with conditions, and provide a guide as to the pathway followed.Kinetics studies can provide data on the affinity of enzymes for substrate and inhibitors and on the maximum rate of a reaction.Properties of EnzymesEnzyme have some remarkable properties:a) Great catalytic power -- often accelerate reactions by more than a million fold.b) Some enzymes have incredible specificityVoet Biochemistry 3e© 2004 John Wiley & Sons, Inc.b) Some enzymes have incredible specificityEg: Can distinguish between D- and L- forms. Can distinguish beween very small structural differences.c) Almost no byproductsd) Operate efficiently at ambient temperatures and pressuresProperties of EnzymesSome drawbacks:Easily denatures by high temperatures and organic solventsMay be difficult to get exactly the catalytic properties requiredVoet Biochemistry 3e© 2004 John Wiley & Sons, Inc.properties requiredOften difficult to produce in large quantities (especially if one cannot use bacterial production)Voet Biochemistry 3e© 2004 John Wiley & Sons, Inc.Voet Biochemistry 3e© 2004 John Wiley & Sons, Inc.Voet Biochemistry 3e© 2004 John Wiley & Sons, Inc.Voet Biochemistry 3e© 2004 John Wiley & Sons, Inc.Figure 14-1 Plot of ln[A] versus time for a first-order reaction.Page 474Voet Biochemistry 3e© 2004 John Wiley & Sons, Inc.Figure 14-2 Comparison of the progress curves for first-and second-order reactions that have the same value of t1/2.Page 474Voet Biochemistry 3e© 2004 John Wiley & Sons, Inc.Voet Biochemistry 3e© 2004 John Wiley & Sons, Inc.Figure 14-4a Transition state diagrams. (a) For the H + H2reaction. This is a section taken along the a—c—d line in Fig. 14-3.Page 475Voet Biochemistry 3e© 2004 John Wiley & Sons, Inc.Figure 14-4b Transition state diagrams. (b) For a spontaneous reaction, that is, one in which the free energy decreases.Page 475Voet Biochemistry 3e© 2004 John Wiley & Sons, Inc.Figure 14-5 Transition state diagram for the two-step overall reaction A → I → P.Page 477Voet Biochemistry 3e© 2004 John Wiley & Sons, Inc.Figure 14-6 The effect of a catalyst on the transition state diagram of a reaction.Page 477Voet Biochemistry 3e© 2004 John Wiley & Sons, Inc.The Triose Phosphate Isomerase ReactionVoet Biochemistry 3e© 2004 John Wiley & Sons, Inc.Progress of the triose phosphate isomerase reaction.Voet Biochemistry 3e© 2004 John Wiley & Sons, Inc.Relationship between enzyme concentration and reaction velocity.Voet Biochemistry 3e© 2004 John Wiley & Sons, Inc.A plot of reaction velocity versus substrate concentration.Voet Biochemistry 3e© 2004 John Wiley & Sons, Inc.Changes in concentration for a simple enzyme-catalyzed reaction.Voet Biochemistry 3e© 2004 John Wiley & Sons, Inc.Voet Biochemistry 3e© 2004 John Wiley & Sons, Inc.Figure 14-7 Progress curves for the components of a simple Michaelis–Menten reaction.Page 478Voet Biochemistry 3e© 2004 John Wiley & Sons, Inc.Figure 14-8 Plot of the initial velocity voof a simple Michaelis–Menten reaction versus the substrate concentration [S].Page 479Analysis of a very simple enzyme catalyzed reactionSubstrate (S) Product (P)Involves the following simplified pathway:E + S ES E + PVoet Biochemistry 3e© 2004 John Wiley & Sons, Inc.Making a number of simplifying assumptions, Michaelis and Menton, together with later work by Briggs and Haldane were able to provide the following analysis for a single substrate enzyme ctatlyzed reactionVoet Biochemistry 3e© 2004 John Wiley & Sons, Inc.Voet Biochemistry 3e© 2004 John Wiley & Sons, Inc.Voet Biochemistry 3e© 2004 John Wiley & Sons, Inc.Chapters 14: Enzyme Kinetics -- continuedUnderstanding the Michaelis Menton EquationVoet Biochemistry 3e© 2004 John Wiley & Sons, Inc.Inhibitor AnalysisVoet Biochemistry 3e© 2004 John Wiley & Sons, Inc.Voet Biochemistry 3e© 2004 John Wiley & Sons, Inc.Voet Biochemistry 3e© 2004 John Wiley & Sons, Inc.Voet Biochemistry 3e© 2004 John Wiley & Sons, Inc.Voet Biochemistry 3e© 2004 John Wiley & Sons, Inc.Lineweaver-Burk EquationVoet Biochemistry 3e© 2004 John Wiley & Sons, Inc.Voet Biochemistry 3e© 2004 John Wiley & Sons, Inc.Figure 14-9 A double reciprocal (Lineweaver–Burk) plot.Page 480Voet Biochemistry 3e© 2004 John Wiley & Sons, Inc.Table 14-1 Values of KM, kcat, and kcat/KMfor Some Enzymes and Substrates.Page 480Control of Enzyme ActivityAmount of enzymesynthesis(eg inducible enzymes)degradationdegradationInhibitionAllosteric RegulationVoet Biochemistry 3e© 2004 John Wiley & Sons, Inc.Voet Biochemistry 3e© 2004 John Wiley & Sons, Inc.Voet Biochemistry 3e© 2004 John Wiley & Sons, Inc.Figure 14-11 Competitive inhibition.Page 484Voet Biochemistry 3e© 2004 John Wiley & Sons, Inc.Figure 14-12 Lineweaver–Burk plot of the competitively inhibited Michaelis–Menten enzyme described by Fig. 14-11.Page 484Voet Biochemistry 3e© 2004 John Wiley & Sons, Inc.ESIUncompetitive Inhibition –inhibitor binds only to ESVoet Biochemistry 3e© 2004 John Wiley & Sons, Inc.Figure 14-13 Lineweaver–Burk plot of a simple Michaelis–Menten enzyme in the presence of uncompetitive inhibitor.Page 485Mixed (= uncompetitive) InhibitionVoet Biochemistry 3e© 2004 John Wiley & Sons, Inc.Voet Biochemistry 3e© 2004 John Wiley & Sons, Inc.Figure 14-14 Lineweaver–Burk plot of a simple Michaelis-Menten enzyme in the presence of a mixed inhibitor.Page 486Voet Biochemistry 3e© 2004 John Wiley & Sons, Inc.Catalytic EfficiencyKcat = Vmax/[E]TThis is the same as the “TURNOVER NUMBER”Or the number of substrate molecules converted