EXPERIMENTAL APPROACH TO THE KINETIC-STUDY OF UNSTABLE SITE-DIRECTED IRREVERSIBLE INHIBITORS - KINETIC ORIGIN OF THE APPARENT POSITIVE COOPERATIVITY ARISING FROM INACTIVATION OF TRYPSIN BY P-AMIDINOPHENYLMETHANESULPHONYL FLUORIDE

Experimental characterization of enzyme inactivation by unstable irrev ersible inhibitors has only previously been carried out by using disco ntinuous methods involving preincubation, removal of samples and furth er residual activity assays. A continuous method for the kinetic study of these inhibitors in the presence of an auxiliary substrate was rec ently proposed in a theoretical study. This method was based on approx imate expressions for the evolution of the product concentration, whic h contained series expansions with five or more exponential terms, ser iously complicating their use in practice. In the present paper, a new experimental method has been developed for the kinetic study of unsta ble and site-directed irreversible inhibitors, considering two differe nt ranges of inhibitor concentration. Thus at low inhibitor concentrat ions, the system evolves from an initial to a final steady state, the rates of which are described by exact analytical equations. At high in hibitor concentrations, however, the product accumulation can be descr ibed by an exact uniexponential equation. This simple and efficient me thod has been applied to the kinetic study of trypsin inactivation by p-amidinophenylmethanesulphonyl fluoride, near the optimum pH of the e nzyme. The dependence of the final steady-state rate on the substrate concentration shows apparent positive cooperativity which has not prev iously been reported. The kinetic origin of this type of co-operativit y is predicted by one of the exact analytical equations derived here. The instability of new protein and non-protein irreversible inhibitors has to be carefully characterized to prevent true unstable irreversib le inhibitors being wrongly described as allosteric reversible inhibit ors.