Biochemical Reaction Rate Enzyme Kinetics Lipitor inhibits HMGCo

Biochemical Reaction Rate: Enzyme Kinetics Lipitor inhibits HMG-Co. A 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

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

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

Michaelis-Menten Enzyme Kinetics k 1 k 2 E + S ↔ ES → E + P k-1 Assumptions • Single subunit & substrate • Product low (V 0) • ES constant (steady state) V 0 = Vmax [S]/([S] + KM) Definitions Vmax = k 2[E]T ([E]T = [E] + [ES]) KM = (k-1 + k 2)/k 1 (Michaelis constant) When does V 0 = ½Vmax? What is Km? What is V 0 when S is much smaller than Km? What is V 0 when S is much larger than Km?

Lineweaver-Burk Double-Reciprocal Plot To calculate Km and Vmax 1/ V 0 = KM/ Vmax 1/S + 1/ Vmax Y=m. X+b

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

Enzyme Turn-Over Number: Kcat is the substrate turnover to product when the enzyme is fully saturated Vmax = k 2[E]T Kcat = Vmax/[E]T k 1 k 2 E + S ↔ ES → E + P k-1

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

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?

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

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

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

Reversible Inhibitors: Competitive Inhibition Reaction Pathway

Competitive Enzyme Inhibition Reaction Pathway Substrate can out compete inhibitor → Vmax unchanged since Vmax= k 2[E]T Inhibitor binds in the active site → KM increases since KM= (k-1 + k 2)/k 1 How is Lineweaver-Burk plot altered?

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

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

Noncompetitive Enzyme Inhibition Taking enzyme out of circulation → Vmax lowered since Vmax= k 2[E]T Inhibitor binds both E and ES → KM unchanged since KM= (k-1 + k 2)/k 1 How is Lineweaver-Burk plot altered? Reaction Pathway

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

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

Uncompetitive Enzyme Inhibition Taking enzyme out of circulation → Vmax lowered since Vmax= k 2[E]T Inhibitor binds to E after the substrate binds → KM decreased since KM= (k-1 + k 2)/k 1 How is Lineweaver-Burk plot altered? Reaction Pathway

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

Virus Inhibition via Reverse Transcriptase Activity HIV that causes AIDS

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

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

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

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?