Inhibitors Enzyme I
Biology 3515 / Chemistry 3515 Biological Chemistry Lab 2/26/2009 Jean M. Bower, PhD
Some material from slides David P. Goldenberg, 2003
Velocity of enzymatic reactions generally follows hyperbolic kinetics
Km in the Michaelis-Menten mechanism
For enzymes that utilize the true Michaelis-Menten mechanism, kcat is rate-limiting. In this case, ... kcat<<k2 Km k2 / k1 = [E][S] / [ES] = ES dissociation constant Km is an apparent dissociation constant of ES
What is an "apparent" value?
Basically a value that is acknowledged to be altered from the "true" value by some factor(s) Examples:
Km (but not =) dissociation constant of ES if kcat is ratelimiting The presence of an inhibitor can affect the values you determine for Km and/or Vmax of a reaction, but they don't affect the true values inherent to the enzyme (obtained in the absence of inhibitor) Apparent Km = K'm Apparent Vmax = V'max
Reversible Inhibitors
Can bind to free enzyme and/or enzyme-substrate complex (usually non-covalently)
Classification of Reversible Inhibitors
Competitive bind only to free enzyme Uncompetitive bind only to enzyme-substrate complex Mixed bind to both free enzyme and enzymesubstrate complex; affinities may differ Noncompetitive bind equally well to free enzyme and enzyme-substrate complex
Inhibitor classes exert differential effects on apparent Km and Vmax
Competitive Noncompetitive
Uncompetitive
Treatment of methanol poisoning with ethanol
Competitive inhibition
Why does a competitive inhibitor have these effects?
Km = [S] when V = (1/2) Vmax In the presence of a competitive inhibitor, more substrate is required to reach the uninhibited velocity But, given high enough [S], the same Vmax can still be reached
Why does a competitive inhibitor have these effects?
"When stress is applied to a system at chemical equilibrium, the equilibrium will shift in the direction tends that to relieve or counteract that stress."
Le Chtelier's Principle
A competitive inhibitor reduces [E]
Equilibrium shifts toward [E]
[ E ][ S ] ' K m increases [ ES ]
Noncompetitive inhibition
Why does a noncompetitive inhibitor have these effects?
E and ES are bound equally by the noncompetitive inhibitor Amount of uninhibited ES is decreased, so V'max is decreased E vs. ES equilibrium is not altered, so K'm is unchanged Amount of substrate required to reach Vmax is unchanged, so K'm is unchanged
Uncompetitive inhibition
Why does an uncompetitive inhibitor have these effects?
Only ES complex is bound by the uncompetitive inhibitor Decrease in ES means less activity, so apparent maximal velocity (V'max) decreases E vs. ES equilibrium shifts toward ES
[ E ][ S ] ' K m decreases [ ES ]
One very successful and lucrative class of competitive inhibitors: statins
Lipitor, Crestor, Zocor, etc. Structural analogs of HMG-CoA Compete for HMG-CoA reductase sites, reducing production of cholesterol
(HMG-CoA)
Statins: From fungus to pharma. P.A. Rea, 2008. American Scientist 96(5):48
Experiment 4, Day 1
What type of r...