Reaction of human chymase with reactive site variants of alpha 1-antichymotrypsin. Modulation of inhibitor versus substrate properties.

Inhibition of human chymase by alpha 1-antichymotrypsin produces 3.5 mol of degraded inhibitor for every mol of chymase inhibited, resulting in a stoichiometry of inhibition (SI) of 4.5. In the present study, the substrate versus inhibitor properties of this reaction were examined further using wild type and mutant recombinant antichymotrypsins (rACT). Titration of chymase hydrolytic activity with rACT-L358 (wild type) and reactive site (P1) variants of ACT, L358W, L358M, and L358F revealed that the SI was sensitive to P1 residue replacements. SI values increased in the order of Trp < Met < Leu < Phe where SI values were 1.5, 2, 4, and 7, respectively. Chymase inhibitor complex and cleaved inhibitor were demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis for all variants; the relative intensities of each band were consistent with SI values established by titration. NH2-terminal sequence analyses of the products formed in the reaction of chymase with rACT-L358F indicated that the P1-P1' bond was the primary site of cleavage resulting in the hydrolysis and inactivation of this variant. The apparent second-order rate constant for chymase inhibition (k'/[I]) by rACT also was affected by P1 substitution. k'/[I] values increased in an order opposite that obtained for SI values (Phe < Leu < Met < Trp). The reactive loop mutant (rACT-P3P3') produced by replacing the reactive site region of ACT (Thr356-Val361) with that of alpha 1-proteinase inhibitor (Ile356-Pro361) revealed a different reaction pattern. Although its SI was near 1, the value for k'/[I] was the lowest among variants. rACT-L358R, another P1 variant, did not inhibit chymase. These results are evaluated with respect to the substrate preferences of human chymase and with respect to partitioning schemes proposed to explain SI values greater than 1.