Stopped-flow studies on the association of achymotrypsin with pancreatic trypsin inhibitor were performed with the proflavine displacement method in a broad range of inhibitor concentration. When all corrections arising from the coupling of indicator binding were applied, good correspondence with other methods was observed. Results at neutral pH are in agreement with a mechanism in which a fast preequilibrium (diffusion-controlled association rate and dissociation equilibrium constant 5 X 10-4 m) is followed by

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Modified trypsin kallikrein inhibitor (I*), with the reactive‐site peptide bond Lys‐15–Ala‐16 split, reacts with α‐chymotrypsin (E) via an intermediate X to the stable tetrahedral complex C: E + I*⇌ X → C. Formation of X constitutes a fast pre‐equilibrium (equilibrium constant Kx= 7 × 10−5 M. association rate constant kx= 4 × 103 M−1 s−1) to the slow reaction X → C (rate constant kc= 2 × 10−3 s−1), all values at pH 7.5. No intermediate X is observed when α‐chymotrypsin reacts with I*‐OMe in which the carboxyl group of Lys‐15 is esterified by methanol. This observation as well as the different pH dependence of the overall association rate constants in the case of I* and I*‐OMe indicate that formation of X precedes formation of the acyl enzyme in the catalytic pathway. The data are compared to the similar results obtained with β‐trypsin and I* or I*‐OMe.

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Equilibrium measurements of the binding of reactive-site-cleaved (modified) bovine trypsinkallikrein inhibitor (Kunitz) to a-chymotrypsin and p-trypsin show a stoichiometric 1 : 1 association with high binding constants. At least in the case of chymotrypsin much evidence is presented that the reaction with modified inhibitor leads to the same complex as the reaction with virgin inhibitor does. The association rate constant of modified inhibitor with chymotrypsin at pH 7, 22.5"C is 15.8 M-' s-'. This is about 2 x 104 times slower than the binding of virgin inhibitor to that enzyme. In the analogous reaction of modified inhibitor with P-trypsin, however, the association rate constant (1.2 x 104 M-' s-l at pH 6.9, 22.5"C) is of about the same order of magnitude as it is in the reaction of virgin inhibitor and trypsin. These and analogous phenomena observed in the reactions of virgin and modified soybean trypsin inhibitor (Kunitz) with a-chymotrypsin and /I-trypsin suggest that the specificity of both inhibitors to trypsin is strongly reflected in the association rate constants of the modified forms.The dissociation rate constants of the complexes of trypsin-kallikrein inhibitor with chymotrypsin or with trypsin towards the modified inhibitor are estimated to be unmeasurably slow (half-life times ,of 45 or 1.5 x 104 years, respectively).A recent kinetic study of the interaction of bovine trypsin-kallikrein inhibitor (Kunitz) with a-chymotrypsin [l] exhibited a close similarity of its mechanism with that of the system soybean trypsin inhibitor/ trypsin [2]. In the latter system and for many other inhibitors of this type a substrate hydrolysis by serine-histidine proteinases. Many unsuccessful attempts to detect the modified species for the trypsin-kallikrein inhibitor (Kunitz) lead to the conclusion that the equilibrium lies completely on the side of the unmodified inhibitor with the reactive-site peptide bond Lys-15 -Ala-16 intact [5]. Only an upper limit of &yd < lo-* was estimated by mass spectral investigations [6]. Based on earlier findings [7] that the reactive peptide bond can be cleaved when the neighbouring S-S bridge in position 14 is split, the modified inhibitor was prepared by selective reduction, tryptic hydrolysis and reformation of the S-S bridge [5,8,9].It is now possible to compare the kinetics of the reaction of modified inhibitor with serine-histidine proteinases with that of virgin inhibitor. In the latter case the formation of a stable inhibitor . enzyme Eur. J. Biochem. 52 (1975)