The pH dependence of serpin-proteinase complex dissociation reveals a mechanism of complex stabilization involving inactive and active conformational states of the proteinase which are perturbable by calcium

Serpin family protein proteinase inhibitors trap proteinases at the acyl-intermediate stage of cleavage of the serpin as a proteinase substrate by undergoing a dramatic conformational change, which is thought to distort the proteinase active site and slow deacylation. To investigate the extent to which proteinase catalytic function I. Ls defective in the serpin-proteinase complex, we compared the pH dependence of dissociation of several serpin-proteinase acyl-complexes with that of normal guanidinobenzoyl-proteinase acyl-intermediate complexes. Whereas the apparent rate constant for dissociation of guanidinobenzoyl-proteinase complexes (k(diss, app)) showed a pH dependence characteristic of His-57 catalysis of complex deacylation, the pH dependence of k(diss, app) for the serpin-proteinase complexes showed no evidence for His-57 involvement in complex deacylation and was instead characteristic of a hydroxide-mediated deacylation similar to that observed for the hydrolysis of tosylarginine methyl ester. Hydroxylamine enhanced the rate of serpin-proteinase complex dissociation but with a rate constant for nucleophilic attack on the acyl bond several orders of magnitude slower than that of hydroxide, implying limited accessibility of the acyl bond in the complex. The addition of 10-100 mm Ca2+ ions stimulated up to 80-fold the dissociation rate constant of several serpin-trypsin complexes in a saturable manner at neutral pH and altered the pH dependence to a pattern characteristic of His-57-catalyzed complex deacylation. These results support a mechanism of kinetic stabilization of serpin-proteinase complexes wherein the complex is trapped as an acyl-intermediate by a serpin conformational change-induced inactivation of the proteinase catalytic function, but suggest that the inactive proteinase conformation in the complex is in equilibrium with an active proteinase conformation that can be stabilized by the preferential binding of an allosteric ligand such as Ca2+.