Inhibitory mechanism of serpins. Identification of steps involving the active-site serine residue of the protease.

The role of the protease active-site serine residue in the formation of protease-serpin complexes has been investigated by using a mutant of thrombin in which Ser195 was mutated to alanine (S195A). The structural integrity of S195A was established by examining the kinetics of its interaction with the inhibitor hirudin, which does not have substantial interactions with Ser195. The affinity of S195A for hirudin was only tenfold less than that of thrombin and the kinetic constants for the formation of the S195A-hirudin complex were very similar to those observed with thrombin. In contrast to hirudin, the dissociation constants (Ki) for S195A with serpins (antithrombin, protease nexin-1 and alpha1-antitrypsin with a P1 arginine) were 2 x 10(3) to 2 x 10(5)-fold higher than those observed with thrombin. These results indicate a critical role for interactions with Ser195 in stabilizing the thrombin-serpin complexes. The cofactor heparin compensated partially for the loss of interactions with Ser195; it increased the affinity of S195A for protease nexin-1 and antithrombin by 140-fold and 1000-fold, respectively. In the case of heparin/antithrombin, the increase in affinity could be attributed mainly to interactions outside the active site of S195A. Kinetic studies with antithrombin and protease nexin-1 in the presence of heparin indicated that Ser195 was not involved in any rate-limiting process in the formation of protease-serpin complexes. Interactions with Ser195 increased the stability of the complex by markedly reducing its rate of breakdown rather than by increasing its rate of formation. Overall, the results of the kinetic studies were consistent with a mechanism in which the binding of the protease induces a rate-limiting conformational change in the serpin and interactions with the protease's active-site serine residue, occurring in a subsequent faster step, greatly stabilize the complex.