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Biochemical Reaction Rate: Enzyme Kinetics

Biochemical Reaction Rate: Enzyme Kinetics. Lipitor inhibits HMG-CoA reductase, a critical step in cholesterol biosynthesis. What affect do enzymes and enzyme inhibitors have on enzyme catalysis on a quantitative level?. Triose Phosphate Isomerase Reaction Progression.

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Biochemical Reaction Rate: Enzyme Kinetics

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  1. Biochemical Reaction Rate: Enzyme Kinetics Lipitor inhibits HMG-CoA reductase, a critical step in cholesterol biosynthesis What affect do enzymes and enzyme inhibitors have on enzyme catalysis on a quantitative level?

  2. Triose Phosphate Isomerase Reaction Progression

  3. Product Formation Versus Time Determining the reaction velocity dependents on what?

  4. Reaction Velocity versus Substrate A → P V = -d[A]/dt = d[P]/dt V = k[A] A + B → P V = k[A][B] 2 A → P V = k[A]2

  5. Michaelis-Menten Enzyme Kinetics k1k2 k-1 E + S ↔ ES → E + P Assumptions • Single subunit & substrate • Product low (V0) • ES constant (steady state) Definitions Vmax = k2[E]T([E]T = [E] + [ES]) KM = (k-1 + k2)/k1(Michaelis constant) V0 = Vmax [S]/([S] + KM) When does V0 = ½Vmax? What is Km? What is V0 when S is much smaller than Km? What is V0 when S is much larger than Km?

  6. Lineweaver-Burk Double-Reciprocal Plot To calculate Km and Vmax 1/ V0 = KM/ Vmax 1/S + 1/ Vmax Y = m X + b

  7. Michaelis Constant Value: Km A higher Km value means what?

  8. Enzyme Turn-Over Number: Kcat Kcat is the substrate turnover to product when the enzyme is fully saturated Vmax = k2[E]TKcat = Vmax/[E]T k1k2 k-1 E + S ↔ ES → E + P

  9. Substrate Preference with Chymotrypsin • Kcat/KM is a measure of Catalytic Efficiency

  10. Varying the Enzyme For a one-substrate, enzyme-catalyzed reaction, which of the family of curves would you expect to be obtained? Hint: What are the equations for Vmax and KM?

  11. Not a Michaelis-Menten Enzyme-Kinetics Reaction Why not?

  12. Not a Michaelis-Menten Enzyme-Kinetics Reaction Why not?

  13. Not a Michaelis-Menten Enzyme-Kinetics Reaction An irreversible inhibitor is present

  14. Reversible Inhibitors: Competitive Inhibition Reaction Pathway

  15. Competitive Enzyme Inhibition Reaction Pathway Substrate can out compete inhibitor → Vmax unchanged since Vmax= k2[E]T Inhibitor binds in the active site → KM increases since KM= (k-1 + k2)/k1 How is Lineweaver-Burk plot altered?

  16. Competitive Inhibition: Lineweaver-Burk Plot • Vmax Unaltered • KM Increased

  17. Noncompetitive Enzyme Inhibition Reaction Pathway Is Vmax affected? How is KM influenced?

  18. Noncompetitive Enzyme Inhibition Reaction Pathway Taking enzyme out of circulation → Vmax lowered since Vmax= k2[E]T Inhibitor binds both E and ES → KM unchanged since KM= (k-1 + k2)/k1 How is Lineweaver-Burk plot altered?

  19. Noncompetitive Inhibition – Lineweaver-Burk Plot • Vmax Lowered • KM Unchanged

  20. Uncompetitive Enzyme Inhibition Reaction Pathway Is Vmax affected? How is KM influenced?

  21. Uncompetitive Enzyme Inhibition Reaction Pathway Taking enzyme out of circulation → Vmax lowered since Vmax= k2[E]T Inhibitor binds to E after the substrate binds → KM decreased since KM= (k-1 + k2)/k1 How is Lineweaver-Burk plot altered?

  22. Uncompetitive Inhibition – Lineweaver-Burk Plot • Vmax Lowered • KM Lowered

  23. Virus Inhibition via Reverse Transcriptase Activity HIV that causes AIDS

  24. Early Developed HIV Drugs Propose how these molecules function at inhibitors of HIV.

  25. Reverse Transcriptase Inhibition by Non-Nucleoside Analog Noncompetitive inhibitor binds to a hydrophobic patch on surface of reverse transcriptase

  26. HIV Protease Cartoon Structure Substrate with side chains binding in hydrophobic pockets Catalytic Asp residues

  27. HIV Protease Inhibitors Rational drug design based on a detailed knowledge of the enzyme structure Why is it preferable to select a target inhibitor that is unique to the virus and not the host?

  28. Transition State Analogue Inhibitor Why is the transition state analogue an inhibitor?

  29. Transition State Analogue Inhibitor Why is the transition state analogue an inhibitor?

  30. Feedback Inhibition of Phosphofructokinase

  31. Allosteric Enzyme Regulation of Phosphofructokinase What is the difference on these two curves?

  32. Mechanism of Allosteric Inhibition for Phosphofructokinase Green structure w/o PEP; Red configuration w/ PEP

  33. Kinetic Relationships Draw the curve for the appropriate connection between variables.

  34. Lineweaver-Burk Plot Which reaction has the highest KM and Vmax value?

  35. Chapter 7 Problems: 5, 7, 18, 19, 21, 25, 27, 29 and 31

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