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Chapter 6.3: Enzyme Kinetics

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?.

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Chapter 6.3: Enzyme Kinetics

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  1. Chapter 6.3: Enzyme Kinetics CHEM 7784 Biochemistry Professor Bensley

  2. CHAPTER 6.3Enzyme Kinetics • description of enzyme kinetics by examining the Michaelis-Menten theory Today’s Objectives: (To learn and understand the)

  3. What is (are?) Enzyme Kinetics? • Kineticsis the study of the rate at which compounds react • Rate of enzymatic reaction is affected by • Enzyme • Substrate • Effectors • Temperature

  4. How to Take Kinetic Measurements

  5. Effect of Substrate Concentration • Ideal Rate: • Deviations due to: • Limitation of measurements • Substrate inhibition • Substrate prep contains inhibitors • Enzyme prep contains inhibitors

  6. Plot V0 vs. [S] • Michaelis-Menten Equation • Describes rectangular hyperbolic plot • Vo = Vmax [S] • Km + [S]

  7. Vmax = velocity where all of the enzyme is bound to substrate (enzyme is saturated with S) Km = [S] @ ½ Vmax (units moles/L=M) (1/2 of enzyme bound to S)

  8. E S k-2 Initial Velocity Assumption • Measurements made to measure initial velocity (vo). At vovery little product formed. Therefore, the rate at which E + P react to form ES is negligible and k-2 is 0. Therefore E + S E + P k1 k2 E + S ES E + P k-1

  9. E S k1 k2 E + S ESE + P k-1 Steady State Assumption Steady state Assumption = [ES] is constant. The rate of ES formation equals the rate of ES breakdown E + S E + P

  10. E S E + S k1 E + S ES Rate of ES formation Rate = k1 [E] [S]

  11. E E S S E + S E + P k-1 k2 ES E + S ES E + P • Rate of ES breakdown Rate = (k2 [ES]) + (k-1[ES]) Rate = [ES](k2 + k-1)

  12. Therefore………if 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)

  13. What does Km mean? • Km = [S] at ½ Vmax • Km is a combination of rate constants describing the formation and breakdown of the ES complex • Km is usually a little higher than the physiological [S]

  14. What does Km mean? • Km represents the amount of substrate required to bind ½ 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 binding Hexokinase Glucose + ATP <-> Glucose-6-P + ADP Glucose Km = 8 X 10-6 Allose Km = 8 X 10-3 Mannose Km = 5 X 10-6

  15. k1 kcat E + S ES E + P k-1 What does kcat mean?

  16. What does kcat/Km mean?

  17. Aren’t 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 second • Km (Michaelis constant): an approximate measure of substrate’s affinity for enzyme

  18. Limitations of M-M • Some enzyme catalyzed rxns show more complex behavior E + S<->ES<->EZ<->EP<-> E + P With M-M can look only at rate limiting step • Often more than one substrate E+S1<->ES1+S2<->ES1S2<->EP1P2<-> EP2+P1<-> E+P2 Must optimize one substrate then calculate kinetic parameters for the other • Assumes k-2 = 0 • Assume steady state conditions

  19. V max Km Km ~ 1.3 mM Vmax ~ 0.25

  20. 1/ 1/ -1/Km = -0.8 Km = 1.23 mM 1/Vmax = 4.0 Vmax = 0.25

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