ANTICHYMOTRYPSIN INTERACTION WITH CHYMOTRYPSIN - REACTIONS FOLLOWING ENCOUNTER COMPLEX-FORMATION

Serpins, serine proteinase inhibitors, form enzymatically inactive, 1: 1 complexes (denoted EI*) with their target proteinases, that only sl owly release I, in which the P1-P1' linkage is cleaved. Recently we p resented evidence that the serpin antichymotrypsin (ACT, I) reacts wit h the serine proteinase chymotrypsin (Chtr, E) to form an EI* complex via a three-step mechanism, E + I reversible arrow E .I reversible ar row EI' reversible arrow EI* in which EI', which retains the P1-P1' l inkage, is formed in a partly or largely rate-determining step, depend ing on temperature (O'Malley, K. H, Nair, S, A., Rubin, H., and Cooper man, B, S, (1997) J. Biol. Chem. 272, 5354-5359). Here we extend these studies through the introduction of a new assay for the formation of the postcomplex fragment, corresponding to ACT residues 359 (the P1' r esidue) to 398 (the C ter minus), coupled with rapid quench flow kinet ic analysis. We show that the E I encounter complex of wild type-rACT and Chtr forms both EI* and postcomplex fragment with the same rate c onstant, so that both spe cies arise from EI' conversion to EI*. Thes e results sup port our earlier conclusion that the P1-P1' linkage is p reserved in EI' and imply that EI* corresponds to a covalent adduct o f E and I, either acyl enzyme or the tetrahedral intermediate formed b y water attack on acyl enzyme. Furthermore, we show that the A347R (P1 2) variant of rACT, which is a substrate rather than an inhibitor of C htr, has a rate constant for postcomplex fragment formation from the E I complex very similar to that observed for WT-rACT, implying that EI ' is the common intermediate from which partitioning to inhibitor and substrate pathways occurs. These results are used to elaborate a propo sed scheme for ACT interaction with Chtr that is considered in the lig ht of relevant results from studies of other serpin-serine proteinase pairs.