Enzyme kinetics and inhibition

HIV Protease

Dimeric “Aspartyl Protease”, two identical subunits w/ active site ASP residues, flaps close and structure rigifies, major drug target of anti virals

HIV protease inhibitors

Indinavir that mimics transition state: prefers transition state (maximum binding interaction

Enzyme Kinetics

Michaelis-Menten graph

Michaelis Constant (Km)

Ratio of rate constants for elementary reaction steps, strength of ES complex formation

for rapid equilibrium K_m = K_d

Michaelis-Menten Assumptions

1. [enzyme] does not change

2. k2 >> k-2 (no back reaction, easy at early time points)

3. [E]total << [S] (excess substrate!)

4. [S] does not change much with time

5. Steady-state of [ES]

Steady-State of the Michaelis complex [ES]

production and consumption of the ES complex proceeds at the same rate, the concentration of ES is constant

Monitoring Kinetics under Steady-State Conditions

enzyme is binding to substrate → converts to product → unbinds → binds to new substrate

Michaelis Menten Equation

double the enzyme concentration → Vmax doubles, but Km does not change

V0 is half-maximal at a concentration of S that is equal to Km

Saturation effect

observed in enzyme catalysis when plotting the initial velocity (Vo) against the substrate concentration([S])

Initial velocity (Vo)

measured at the beginning of the enzyme-catalyzed reaction, when substrate concentration can be considered constant, will decrease as the reaction progresses.

low concentrations of substrate

Vo increases almost linearly with an increase in [S]

Lineweaver-Burk (Double Reciprocal) Plot

Km of some enzymes

lower the Km, the less Substrate the enzyme requires, which is associated with more efficient catalysis

most Km values range between 10-7 to 10-1 M

catalytic rate constant/turnover number (k₂/k_cat)

best, most efficient, enzyme has a maximal value of kcat

Catalytic Efficiency

V0 = kcat/Km [E]_T [S]

Defines the efficiency of the enzyme Higher values, more efficient

catalytic perfection

kcat/Km approaches diffusion limitted maximum (in water) ~ 10⁸-10⁹ M-1 sec-1

Bioavailablity

how much enzyme is around to ctalayse reactions; controlled by biochemical processes involved in protein synthesis and localization

Catalytic Efficiency (regulation)

determined by protein modifications; Binding of regulatory molecules, Covalent modification, Proteolytic processing

Allosteric inhibition

“feedback inhibition”

Phosphorylation

Proteolytic cleavage

Pepsinogen is a protease “zymogen” that is activated by autocleavage

competitive inhibitor

binds at the active site (usually) and thus prevents the substrate from binding

affects Km but not Vmax; high levels of substrate relieve inhibition

uncompetitive inhibitor

binds only to the enzyme–substrate complex

decreases Km and Vmax; cannot be overcome at high concentrations

Noncompetitive/Mixed inhibitors

bind to both the enzyme and enzyme-substrate complex

Km unchanged; Vmax unattainable; reduces total enzyme concentration