REACTION OF HUMAN CHYMASE WITH REACTIVE-SITE VARIANTS OF ALPHA-1-ANTICHYMOTRYPSIN - MODULATION OF INHIBITOR VERSUS SUBSTRATE PROPERTIES

Inhibition of human chymase by alpha1-antichymotrypsin produces 3.5 mo l of degraded inhibitor for every mol of chymase inhibited, resulting in a stoichiometry of inhibition (SI) of 4.5. In the present study, th e substrate versus inhibitor properties of this reaction were examined further using wild type and mutant recombinant antichymotrypsins (rAC T). Titration of chymase hydrolytic activity with rACT-L358 (wild type ) and reactive site (P1) variants of ACT, L358W, L358M, and L358F reve aled that the SI was sensitive to P1 residue replacements. SI values i ncreased in the order of Trp < Met < Leu < Phe where SI values were 1. 5, 2, 4, and 7, respectively. Chymase inhibitor complex and cleaved in hibitor were demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis for all variants; the relative intensities of each ba nd were consistent with SI values established by titration. NH2-termin al sequence analyses of the products formed in the reaction of chymase with rACT-L358F indicated that the P1-P1' bond was the primary site o f cleavage resulting in the hydrolysis and inactivation of this varian t. The apparent second-order rate constant for chymase inhibition (k'/ [I]) by rACT also was affected by P1 substitution. k'/[I] values incre ased in an order opposite that obtained for SI values (Phe < Leu < Met < Trp). The reactive loop mutant (rACT-P3P3') produced by replacing t he reactive site region of ACT (Thr356 Val) with that of alpha1-protei nase 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. The se results are evaluated with respect to the substrate preferences of human chymase and with respect to partitioning schemes proposed to exp lain SI values greater than 1.