Slow-binding inhibition of chymotrypsin and cathepsin G by the peptide aldehyde chymostatin

Abstract

The microbial, peptide-derived aldehyde chymostatin is a potent, competitive inhibitor of chymotrypsin and cathepsin G: Ki = 4 .times. 10-10 and 1.5 .times. 10-7 M, respectively. It is a "slow-binding inhibitor" of both proteases and, as such, allows determination of rate constants for its association with and dissociation from these proteases. Inhibition kinetics indicate second-order rate constants for the association of chymostatin with chymotrypsin and cathepsin G of 360,000 and 2000 M-1 s-1, respectively, and a first-order rate constant for the dissociation of both protease-chymostatin complexes of approximately 0.0002 s-1. Thus, the extreme difference in potency of chymostatin as an inhibitor of chymotrypsin and cathepsin G originates entirely in kon. Solvent deuterium isotope effects (SIE) were determined to probe the reaction step that rate limits kon. For the reaction of chymotrypsin with chymostatin, the SIE for kon is 1.6 .+-. 0.1, while for the reaction of chymotrypsin with the peptide substrates Ala-Ala-Phe-pNA and Suc-Ala-Ala-Pro-Phe-pNA, the SIE''s for kc/Km are 2.8 .+-. 0.2 and 1.9 .+-. 0.1, respectively. These results suggest that kon for the association of chymotrypsin with chymostatin is at least partially rate limited by a reaction step involving proton transfer. Combined with results for the inhibition of chymotrypsin by Bz-Phe-H [Kennedy, W. P., and Schultz, R. M. (1979) Biochemistry 18, 349-56], these data suggest a mechanism for inhibition by chymostatin involving the general-base-catalyzed formation of an enzyme-bound hemiacetal, followed by a conformational change of this intermediate that produces the final, stable complex of enzyme and inhibitor. For the inhibition of chymotrypsin, the transition states of these two reaction steps are of similar energy, and each partially rate limits the overall process governed by kon.