Paul M. Weiss

Effect of the presence of a reversible inhibitor on the time course of slow-binding inhibition☆

The half-time for the initial burst seen when a slow-binding inhibitor is present in an enzyme assay decreases from 0.693/k4 to 0.693/(k3 + k4) as the concentration of the slow-binding inhibitor is increased from zero to infinity (k3 and k4 are forward and reverse rate constants for the isomerization causing the slow-binding behavior). If the inhibitor solution contains a classical reversible inhibitor in addition to the slow-binding one, the half-time decreases from the same limit at zero inhibitor to a level which is higher at infinite inhibitor concentration (k3 is divided by (1 + xKi/Kj), where x is the ratio of classical and slow-binding inhibitor concentrations, and Ki and Kj are their initial inhibition constants before the slow-binding phase). Thus if one is using a racemic inhibitor, both enantiomers of which inhibit initially but only one of which shows slow-binding behavior, one will not obtain the correct parameters for the pure slow-binding inhibitor. A similar situation would apply if one were using a mixture of inhibitors such as antibiotics, several of which inhibit initially, but only one of which is a slow-binding inhibitor. This theory is illustrated by determining the half-times for the slow-binding inhibition of yeast hexokinase by various levels of TmATP in the presence and absence of HoATP, which shows little slow-binding behavior.