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J Biol Chem, Vol. 273, Issue 28, 17459-17462, July 10, 1998

Antichymotrypsin Interaction with Chymotrypsin
REACTIONS FOLLOWING ENCOUNTER COMPLEX FORMATION

Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6323

Serpins, serine proteinase inhibitors, form enzymatically inactive, 1:1 complexes (denoted E*I*) with their target proteinases, that only slowly release I*, in which the P1-P1' linkage is cleaved. Recently we presented evidence that the serpin antichymotrypsin (ACT, I) reacts with the serine proteinase chymotrypsin (Chtr, E) to form an E*I* complex via a three-step mechanism, E + I E ·I  EI'  E*I* in which EI', which retains the P1-P1' linkage, is formed in a partly or largely rate-determining step, depending on temperature (O'Malley, K. H, Nair, S. A., Rubin, H., and Cooperman, 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' residue) to 398 (the C terminus), coupled with rapid quench flow kinetic analysis. We show that the E·I encounter complex of wild type-rACT and Chtr forms both E*I* and postcomplex fragment with the same rate constant, so that both species arise from EI' conversion to E*I*. These results support our earlier conclusion that the P1-P1' linkage is preserved in EI' and imply that E*I* corresponds to a covalent adduct of E and I, either acyl enzyme or the tetrahedral intermediate formed by water attack on acyl enzyme. Furthermore, we show that the A347R (P12) variant of rACT, which is a substrate rather than an inhibitor of Chtr, 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 proposed scheme for ACT interaction with Chtr that is considered in the light of relevant results from studies of other serpin-serine proteinase pairs.

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