chapter 6.3: enzyme kinetics

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Chapter 6.3: Enzyme Kinetics. CHEM 7784 Biochemistry Professor Bensley. CHAPTER 6.3 Enzyme Kinetics. description of enzyme kinetics by examining the Michaelis-Menten theory. Today’s Objectives : (To learn and understand the). What is (are?) Enzyme Kinetics?. - PowerPoint PPT Presentation

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  • Chapter 6.3: Enzyme KineticsCHEM 7784BiochemistryProfessor Bensley

  • CHAPTER 6.3Enzyme Kineticsdescription of enzyme kinetics by examining the Michaelis-Menten theoryTodays Objectives: (To learn and understand the)

  • What is (are?) Enzyme Kinetics?Kinetics is the study of the rate at which compounds reactRate of enzymatic reaction is affected byEnzymeSubstrateEffectorsTemperature

  • How to Take Kinetic Measurements

  • Effect of Substrate Concentration Ideal Rate:

    Deviations due to:Limitation of measurementsSubstrate inhibitionSubstrate prep contains inhibitorsEnzyme prep contains inhibitors

  • Plot V0 vs. [S]Michaelis-Menten EquationDescribes rectangular hyperbolic plot

    Vo = Vmax [S] Km + [S]

  • Km = [S] @ Vmax (units moles/L=M)(1/2 of enzyme bound to S)Vmax = velocity where all of the enzyme is bound to substrate (enzyme is saturated with S)

  • Measurements made to measure initial velocity (vo). At vo very little product formed. Therefore, the rate at which E + P react to form ES is negligible and k-2 is 0. Therefore

    Initial Velocity AssumptionE + S ESE + Pk1k-1k2ES+E+P

  • Steady State AssumptionSteady state Assumption = [ES] is constant. The rate of ES formation equals the rate of ES breakdownES+E+P

  • Rate of ES formationRate = k1 [E] [S]

  • Rate of ES breakdownRate = (k2 [ES]) + (k-1[ES])Rate = [ES](k2 + k-1)

  • Thereforeif the rate of ES formation equals the rate of ES breakdown

    1) k1[E][S] = [ES](k-1+ k2)2) (k-1+ k2) / k1 = [E][S] / [ES]3) (k-1+ k2) / k1 = Km (Michaelis constant)

  • What does Km mean?Km = [S] at VmaxKm is a combination of rate constants describing the formation and breakdown of the ES complexKm is usually a little higher than the physiological [S]

  • What does Km mean?Km represents the amount of substrate required to bind of the available enzyme (binding constant of the enzyme for substrate)Km can be used to evaluate the specificity of an enzyme for a substrate (if obeys M-M)Small Km means tight binding; high Km means weak bindingGlucoseKm = 8 X 10-6AlloseKm = 8 X 10-3MannoseKm = 5 X 10-6HexokinaseGlucose + ATP Glucose-6-P + ADP

  • What does kcat mean?

  • What does kcat/Km mean?

  • Arent Enzymes Kinetics Fun?!The final form of M-M equation in the case of a single substrate is

    kcat (turnover number): how many substrate molecules can one enzyme molecule convert per secondKm (Michaelis constant): an approximate measure of substrates affinity for enzyme

  • Limitations of M-MSome enzyme catalyzed rxns show more complex behaviorE + SESEZEP E + PWith M-M can look only at rate limiting stepOften more than one substrate E+S1ES1+S2ES1S2EP1P2 EP2+P1 E+P2 Must optimize one substrate then calculate kinetic parameters for the otherAssumes k-2 = 0 Assume steady state conditions

  • V maxKmKm ~ 1.3 mM

    Vmax ~ 0.25

  • -1/Km = -0.8Km = 1.23 mM1/Vmax = 4.0Vmax = 0.25 1/ 1/

    ****FIGURE 6-10 Initial velocities of enzyme-catalyzed reactions. A theoretical enzyme catalyzes the reaction S P, and is present at a concentration sufficient to catalyze the reaction at a maximum velocity, Vmax, of 1 M/min. The Michaelis constant, Km (explained in the text), is 0.5 M. Progress curves are shown for substrate concentrations below, at, and above the Km. The rate of an enzyme-catalyzed reaction declines as substrate is converted to product. A tangent to each curve taken at time = 0 defines the initial velocity, V0, of each reaction.**FIGURE 6-11 Effect of substrate concentration on the initial velocity of an enzyme-catalyzed reaction. The maximum velocity, Vmax, is extrapolated from the plot, because V0 approaches but never quite reaches Vmax. The substrate concentration at which V0 is half maximal is Km, the Michaelis constant. The concentration of enzyme in an experiment such as this is generally so low that [S] >> [E] even when [S] is described as low or relatively low. The units shown are typical for enzyme-catalyzed reactions and are given only to help illustrate the meaning of V0 and [S]. (Note that the curve describes part of a rectangular hyperbola, with one asymptote at Vmax. If the curve were continued below [S] = 0, it would approach a vertical asymptote at [S] = Km.)***FIGURE 6-12 Dependence of initial velocity on substrate concentration. This graph shows the kinetic parameters that define the limits of the curve at high and low [S]. At low [S], Km >> [S] and the [S] term in the denominator of the Michaelis-Menten equation (Eqn 6-9) becomes insignificant. The equation simplifies to V0 = Vmax[S]/Km and V0 exhibits a linear dependence on [S], as observed here. At high [S], where [S] >> Km, the Km term in the denominator of the Michaelis-Menten equation becomes insignificant and the equation simplifies to V0 = Vmax; this is consistent with the plateau observed at high [S]. The Michaelis-Menten equation is therefore consistent with the observed dependence of V0 on [S], and the shape of the curve is defined by the terms Vmax/Km at low [S] and Vmax at high [S].*BOX 6-1 FIGURE 1 A double-reciprocal or Lineweaver-Burk plot.**