ANTICHYMOTRYPSIN INTERACTION WITH CHYMOTRYPSIN - PARTITIONING OF THE COMPLEX

The interactions of bovine pancreatic chymotrypsin (Chtr) and recombin ant alpha1-antichymotrypsin (rACT) and rACT variants were studied by k inetic and gel electrophoretic analyses, leading to the formulation of a general kinetic scheme that accounts for all known results concerni ng this serpin-protease pair, as well as for results obtained with oth er such pairs. Incubation of rACT and Chtr leads rapidly to the format ion-of an inhibited complex, Chtr.rACT, that is stable toward sodium dodecyl sulfate denaturation and boiling. The extent of release of act ive Chtr from this complex increases markedly as ionic strength, mu, i s raised. The kinetic scheme quantitatively accounts for this effect o n the basis of a partitioning of Chtr.rACT between dissociation of th e complex to yield active enzyme and cleaved rACT, and Chtr.catalyzed conversion of the complex to a form that is much more resistant to rel ease of active enzyme. Sodium dodecyl sulfate-polyacrylamide gel elect rophoresis analyses of reaction mixtures of rACT and Chtr are consiste nt with the scheme. Also consistent are the results of experiments mea suring the effects of 1) Chtr.rACT concentration, 2) uncomplexed Chtr , and 3) added alpha2-Macroglobulin on active Chtr release from Chtr.r ACT. Proteolysis of Chtr.rACT* to give a resistant complex is also ca talyzed by human neutrophil elastase, a process with potential physiol ogical relevance. Comparison of the rates of Chtr dissociation from th e complexes formed with rACT and with rACT variants mutated at the P1 site suggests that such rates are more sensitive to P1 substitution at low mu than at high mu. Several equivalents of the L358R-rACT variant are required for full inhibition of Chtr. This observation is also qu antitatively accounted for by the proposed kinetic scheme, on the basi s of another partitioning step between L358R-rACT acting as a substrat e or as an inhibitor toward Chtr.