Impact of enzyme concentration and residence time on apparent activity recovery in jump dilution analysis

Jump dilution analysis is commonly used to evaluate the reversibility of inhibition and to quantify the residence time of the inhibitor–enzyme complex. During hit and lead characterization, one sometimes observes apparently linear progress curves after jump dilution that display activity recoveries that are intermediate between those expected for fully reversible and irreversible inhibition. Computer simulations of progress curves after jump dilution indicate that seemingly linear progress curves can result when dealing with tight-binding inhibitors if substoichiometric concentrations of inhibitor are preincubated with enzyme. In this situation, the activity recovered is comparable to that expected for instantaneously reversible inhibitors. In addition, simulations demonstrate that intermediate values of activity recovery may be observed for compounds with modestly slow dissociation rates (i.e., residence times >0 min but ⩽20 min) when the attending curvature of the data is not accounted for. The observation of intermediate values of recovery can, thus, serve as an indication of either modest residence time or a contaminating inactivator within an inhibitor sample, in either case prompting greater scrutiny of the test compound.