Modulation of serpin reaction through stabilization of transient intermediate by ligands bound to α-helix F

Abstract

Mechanism-based inhibition of proteinases by serpins involves enzyme acylation and fast insertion of the reactive center loop (RCL) into the central β-sheet of the serpin, resulting in mechanical inactivation of the proteinase. We examined the effects of ligands specific to α-helix F (αHF) of plasminogen activator inhibitor-1 (PAI-1) on the stoichiometry of inhibition (SI) and limiting rate constant (klim) of RCL insertion for reactions with β-trypsin, tissue-type plasminogen activator (tPA), and urokinase. The somatomedin B domain of vitronectin (SMBD) did not affect SI for any proteinase or klim for tPA but decreased the klim for β-trypsin. In contrast to SMBD, monoclonal antibodies MA-55F4C12 and MA-33H1F7, the epitopes of which are located at the opposite side of αHF, decreased klim and increased SI for every enzyme. These effects were enhanced in the presence of SMBD. RCL insertion for β-trypsin and tPA is limited by different subsequent steps of PAI-1 mechanism as follows: enzyme acylation and formation of a loop-displaced acyl complex (LDA), respectively. Stabilization of LDA through the disruption of the exosite interactions between PAI-1 and tPA induced an increase in the klim but did not affect the SI. Thus it is unlikely that LDA contributes significantly to the outcome of the serpin reaction. These results demonstrate that the rate of RCL insertion is not necessarily correlated with SI and indicate that an intermediate, different from LDA, which forms during the late steps of PAI-1 mechanism, and could be stabilized by ligands specific to αHF, controls bifurcation between the inhibitory and the substrate pathways. © 2007 by The American Society for Biochemistry and Molecular Biology, Inc.