Kinetic evidence for a two-state, hybrid model for the trypsin activation by modifiers.

Kinetic investigation of the activation of alpha- and beta-trypsins by tosyl-L-arginine methyl ester, in the presence of benzamidine at concentrations higher than 1 mM, indicates that each could exist as a two-state hybrid allosteric system, based on at least two enzyme forms, E and E* (or E and F). Form E* shows much higher affinity for ligands, thus giving rise to ternary complexes of the types SE*S, ME*S, ME*M, SE*M, where the right position identifies binding at the active site, whereas the left position represents binding to the allosteric site. Using a large number of experimental points, measured at different combinations of substrate and modifier concentrations, it was possible to evaluate quantitatively the parameters needed for the description of the model with considerable precision. At high concentrations, benzamidine competes with tosyl-L-arginine methyl ester for an allosteric site, blocking substrate activation of beta-trypsin, but allowing activation of alpha-trypsin. These results imply the participation of an induced fit step during ternary complex formation that gives rise to complexes of the form E*S2 or E*SI, where E* stands for the conformationally changed E* in the complex. The overall picture is that of a two-state model combined with induced-fit. Negative cooperativity (nH = 0.80) was found for the binding of benzamidine (over 1 mM) to trypsin in the presence of substrate. The proposed model is allowing the design of experiments that should lead to an understanding of the mechanism of trypsin activation by modifiers.