Detailed Mechanism of Interaction of Bovine β-Trypsin with Soybean Trypsin Inhibitor (Kunitz)

Abstract The rate of formation of the stable trypsin-inhibitor complex from bovine β-trypsin and either virgin or modified soybean trypsin inhibitor (Kunitz) was monitored by following the change in absorbance at 260 nm after mixing of equimolar reactants in a stopped flow apparatus. At low reactant concentrations both reactions are second order both with respect to concentration and to time. As the concentration of reactants is raised both the time and concentration dependence gradually switch to first order. These results coupled with other data suggest that the simplest possible mechanism of interaction is see PDF for equation On the assumption that the equilibration to form the loose noncovalent complexes is rapid, values of Kl* = κ-1/κ1 and Kl* = κ4/κ-4 and of the rate constants κ2 and κ-3 were computed from the data. Within the limits of rather large experimental error pKl = pKl* = 4.65 independent of pH in the 4.5 to 8 range. The rate constants κ2 and κ-3 increase with increasing pH in this range and reach plateau values of 140 and 10 per s, respectively, near pH 8. Both rate constants show similar pH dependence with another plateau in the pH 5 to 6 range. The magnitude and pH dependence of these parameters is roughly consistent with Ks values and catalytic rate constants for hydrolysis of excellent amide and peptide substrates. Measurements of the dissociation of the stable complex between pH 1.7 and 3.5 were also made. The dissociation rate constant is very fast near pH 2 (approximately 100 per s) and declines sharply as the pH is raised. Preliminary measurements indicate significant differences in the rate of association of soybean trypsin inhibitor with α- and with β-trypsin as well as in the rate of dissociation of α-trypsin and of β-trypsin complexes.