3.4.1 Principle of Enzyme Inhibition Kinetics

Enzyme inhibition assays measure the rate of an enzymatic reaction — product formation or substrate consumption — as a function of inhibitor...

Enzyme inhibition assays measure the rate of an enzymatic reaction — product formation or substrate consumption — as a function of inhibitor concentration. The concentration of inhibitor that reduces enzyme activity by 50% relative to an uninhibited control is termed the IC50, determined from the sigmoidal dose-response curve generated by testing a range of inhibitor concentrations. The true inhibition constant, Ki, is a thermodynamic measure of binding affinity independent of substrate concentration, and is derived from IC50 using the Cheng-Prusoff equation once the mode of inhibition is known.

Mechanistically, inhibition may be competitive (the inhibitor competes with substrate at the active site, increasing the apparent Michaelis constant, Km, while maximal velocity, Vmax, is unchanged), non-competitive (the inhibitor binds a site distinct from the active site and reduces Vmax while Km is unchanged), uncompetitive (the inhibitor binds only the enzyme-substrate complex), or mixed. Distinguishing between these modes, typically via Lineweaver-Burk or Michaelis-Menten analysis at multiple substrate concentrations, informs both the mechanism of action and the likely in-vivo behaviour of the inhibitor.