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

Triose Phosphate Isomerase Reaction Progression

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

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 V 0 = V max [S]/([S] + K M ) E + S ↔ ES → E + P Assumptions Single subunit & substrate Product low (V 0 ) ES constant (steady state) Definitions V max = k 2 [E] T ([E] T = [E] + [ES]) K M = (k -1 + k 2 )/k 1 (Michaelis constant) k 1 k 2 k -1 When does V 0 = ½V max ? What is K m ? What is V 0 when S is much smaller than K m ? What is V 0 when S is much larger than K m ?

Lineweaver-Burk Double-Reciprocal Plot 1/ V 0 = K M / V max 1/S + 1/ V max To calculate K m and V max Y = m X + b

Michaelis Constant Value: K m A higher K m value means what?

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

Substrate Preference with Chymotrypsin K cat /K M 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 V max and K M ?

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 → V max unchanged since V max = k 2 [E] T Inhibitor binds in the active site → K M increases since K M = (k -1 + k 2 )/k 1 How is Lineweaver-Burk plot altered?

Competitive Inhibition: Lineweaver- Burk Plot V max Unaltered K M Increased

Noncompetitive Enzyme Inhibition Is V max affected? How is K M influenced? Reaction Pathway

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

Noncompetitive Inhibition – Lineweaver-Burk Plot V max Lowered K M Unchanged

Uncompetitive Enzyme Inhibition Is V max affected? How is K M influenced? Reaction Pathway

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

Uncompetitive Inhibition – Lineweaver-Burk Plot V max Lowered K M Lowered

Virus Inhibition via Reverse Transcriptase Activity HIV that causes AIDS

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

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 hydrophobicpockets 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?

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

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

Feedback Inhibition of Phosphofructokinase

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

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

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

Lineweaver-Burk Plot Which reaction has the highest K M and V max value?

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