Studies on the mechanism of binding of serpins and serine proteases

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Abstract
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 a2-antiplasmin as a model inhibitor we find, in contrast to most previous reports, a reversible mechanism: E +1 ⇋ EI ⇋ 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 a:-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 a3-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 × 10−6 s−’ for plasmin and 1.1 × 10−4 s−' for chymotrypsin. Thus k_2i the rate constant previously ignored, explains the different affinities of α2-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 K( final = 200 pM and the strength of inhibition is also largely determined by k_2 = 3.5 × 10−5 s−1. These results indicate a common mechanism of action for these diverse families of small protein protease inhibitors. These findings have important implications for serpin action in vivo and measurement of activity in vitro, and these are discussed.

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