Kinetics of trypsin inhibition by its specific inhibitors.

The kinetics of inhibition of trypsin by its specific inhibitors, pancreatic trypsin inhibitor, ovomucoid trypsin inhibitor, and soybean trypsin inhibitor, has been studied by following the hydrolysis of benzoylarginine ethyl ester in the presence of the inhibitor, and the results have been analyzed with the method described previously [Tian & Tsou (1982) Biochemistry 21, 1028]. The results obtained are consistent with the following: (a) The enzyme binds with the pancreatic inhibitor irreversibly to form an inactive complex. (b) The binding with the ovomucoid inhibitor to form the inactive complex is reversible. (c) An intermediate is formed before the relatively stable inactive complex with the soybean inhibitor, and both steps are reversible. The respective microscopic rate constants are determined by suitable plots of the apparent rate constants under different substrate and inhibitor concentrations. The second-order rate constants for the initial binding step thus obtained are in accord with the apparent inactivation rate constants determined by measuring the activity remaining with a stopped-flow apparatus equipped with a multimixing system after the enzyme-inhibitor mixture has been incubated for different time intervals.