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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?. Product Formation Versus Time.

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

  3. 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

  4. Triose Phosphate Isomerase Reaction Progression

  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. Complex Interactions Require Regulation

  14. Enzyme Regulation Strategies • Catalytic step with large negative ∆G • Early in the metabolic pathway • Common point of branch pathways

  15. Allosteric Enzymes: Catalysts and Information Sensors • Features • Do not conform to Michaelis-Menten kinetics • Sigmoidal not hyperbolic curve • Quaternary structure change • T (tense) and R (relaxed) state • T – less active; R – more active

  16. Regulators of Catalysis for Aspartate Trans-Carbamoylase What nucleotide stabilizes the T & R configurations?

  17. Cleland Representation of Bi-substrate Reactions • Sequential Reactions – all substrates enzyme bind before product release (in, in – out, out) • Double-Displacement (aka ping pong) – one product released before all substrates enzyme bind (in-out, in-out)

  18. What type of reaction is this?

  19. Concerted Model for Allosteric Enzymes • Features • Multiple active sites on different polypeptides • S binds more readily to R than T • Allosteric constant ([T] >>>[R]) • No subunit hybrids (symmetry rule)

  20. Sequential Model for Allosteric Enzymes • Features • Multiple active sites on different polypeptides • S binds more readily to R than T • Subunit hybrids exist (induced changes in neighbors)

  21. Questions: 1, 3, 5, 6, 8, 9, and 10

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