EXPERIMENTAL-METHOD FOR THE KINETIC-STUDY OF UNSTABLE AND SITE-DIRECTED IRREVERSIBLE INHIBITORS AND ITS APPLICATION TO THE INACTIVATION OF CHYMOTRYPSIN BY PHENYLMETHYLSULFONYL FLUORIDE

The inactivation of enzymes by unstable irreversible inhibitors has only been experimentally characterized by means of discontinuous methods involving preincubation of the enzyme with the inhibitor and the removal of aliquots for further measurements of residual activity. A recent theoretical work proposed a continuous method for the kinetic study of these inhibitors in the presence of an auxiliary substrate. This method was based on approximate expressions for the evolution of the product concentration, which contained series expansions with five or more exponential terms, which severely complicates their use in practice. In this paper, a new experimental method has been developed for the kinetic study of unstable and site-directed irreversible inhibitors. This new method considers the operation of the enzymatic inactivation system in two different ranges of inhibitor concentration. Thus, at low inhibitor concentrations exact analytical equations describe the kinetic behaviour of the system from the rates of the corresponding initial and final steady states. At high inhibitor concentrations, however, the product accumulation follows an exact uniexponential equation. The simplicity and efficiency of the method are illustrated by the study of the inactivation of chymotrypsin by phenylmethylsulfonyl fluoride, whose instability has been seriously under estimated in the literature.