Studies on the mechanism of binding of serpins and serine proteases.

The serpin family of inhibitors have an important role in the control of coagulation and fibrinolysis. For a full understanding of how these pathways operate in vivo and correct measurement of enzyme and inhibitor activity, in vitro knowledge of the mechanism of action of serpins is essential. Using alpha 2-antiplasmin as a model inhibitor we find, in contrast to most previous reports, a reversible mechanism: E + I in equilibrium with EI in equilibrium with EI', where complex formation is two stepped, but both steps are reversible. Our work with plasmin in the presence of 50 mM aminohexanoic acid shows that binding of alpha 2-antiplasmin is very tight (but reversible) with an overall Ki (Ki final) = 4.0 pM. With chymotrypsin (a model serine protease) Ki final = 100 pM, so as expected binding of alpha 2-antiplasmin is weaker with chymotrypsin. However, analysis of the individual rate constants shows that the difference in strength of binding is accounted for by the dissociation rate constant for the second step (k-2) = 1.9 x 10(-6) s-1 for plasmin and 1.1 x 10(-4) s-1 for chymotrypsin. Thus k-2, the rate constant previously ignored, explains the different affinities of alpha 2-antiplasmin for these two enzymes. Furthermore, this model of two (or more) step, reversible binding is accepted for protease inhibitors of other families. With one of these, aprotinin (a Kunitz inhibitor) with plasmin we also obtain a two-stage reversible mechanism with a Ki final = 200 pM and the strength of inhibition is also largely determined by k-2 = 3.5 x 10(-5) s-1.(ABSTRACT TRUNCATED AT 250 WORDS)