Heterogeneity in Serpin−Protease Complexes As Demonstrated by Differences in the Mechanism of Complex Breakdown

Abstract

Serpins trap their target proteases in the form of an acyl-enzyme complex. The trap is kinetic, however, and thus serpin-protease complexes ultimately break down, releasing a cleaved inactive serpin and an active protease. The rates of this deacylation process vary greatly depending on the serpin-protease pair with half-lives ranging from minutes to months. The reasons for the diversity in breakdown rates are not clearly understood. In the current study, pH and solvent isotope effects were utilized to probe the mechanism of breakdown for an extremely stable complex and several unstable complexes. Two different patterns for the pH dependence of k(bkdn), the first-order rate constant of breakdown, were found. The stable complex, which breaks down at neutral pH with a half-life of approximately 2 weeks, exhibited a pH-k(bkdn) profile consistent with solvent-hydroxide ion mediated ester hydrolysis. There was no evidence for the participation of the catalytic machinery in the breakdown of this complex, suggesting extensive distortion of the active site. The unstable complexes, which break down with half-lives ranging from minutes to hours, exhibited a bell-shaped pH profile for k(bkdn), typical of the pH-rate profiles of free serine proteases. In the low to neutral pH range k(bkdn) increased with increasing pH in a manner characteristic of His57-mediated catalysis. In the alkaline pH range a decrease in k(bkdn) was observed, consistent with the titration of the Ile16-Asp194 salt bridge (chymotrypsinogen numbering). The alkaline pH dependence was not exhibited in pH-rate profiles of free or substrate-bound HNE, indicating that the salt bridge was significantly destabilized in the complexed protease. These results indicate that breakdown is catalytically mediated in the unstable complexes although, most likely, the protease is not in its native conformation and the catalytic machinery functions inefficiently. However, a mechanism in which breakdown is determined by the equilibrium between distorted and undistorted forms of the complexed protease cannot be completely dismissed. Overall, the results of this study suggest that the protease structure in unstable complexes is distorted to a lesser extent than in stable complexes.

Kinetic Characterization of the Protein Z-dependent Protease Inhibitor Reaction with Blood Coagulation Factor Xa

“…Analysis of the pH dependence of this dissociation provided evidence that dissociation was mediated by His-57 and Ile-16 catalytic residues of the protease, consistent with factor Xa retaining residual function in the conformationally trapped acyl-intermediate complex to catalyze its deacylation. This contrasts with the pH dependence of dissociation of the antithrombin-factor Xa complex and other serpin-protease acyl-intermediate complexes that show no evidence for catalytic residue involvement in the dissociation (14,15). Dissociation rate constants for such complexes are thus Ͼ100-fold lower than that found for the ZPI-factor Xa complex and increase ϳ10-fold per unit increase in pH without bound, characteristic of an uncatalyzed hydroxide-mediated hydrolysis of the acyl bond (14).…”

“…Our findings suggest that ZPI does in fact inhibit factor Xa by the established serpin inhibitory mechanism in which the serpin conformationally traps the protease at the acyl-intermediate stage of cleavage of an exposed serpin-reactive bond. However, our studies demonstrate that this conformational trapping fails to fully inactivate the catalytic machinery of the protease as it does in other serpin-protease complexes (14,15), thereby accounting for the apparent anomalous behavior of the ZPI-factor Xa reaction. Our findings further reveal a catalytic role for protein Z in promoting the membrane-associated ZPI-factor Xa reaction, similar to the role of heparin in promoting antithrombin-protease reactions (16).…”

“…Such a dependence is diagnostic of the participation of two key residues that regulate catalysis by chymotrypsin family serine proteases, His-57 and Ile-16 (chymotrypsin numbering) (14,15). His-57 is part of the catalytic triad whose function depends on the residue being in the unprotonated basic form, whereas Ile-16 is the N-terminal residue formed upon activation of the zymogen whose positively charged amine group in the protonated state forms an activating salt bridge with Asp-194 at the active site (27).…”

Comparative Trajectories of Active and S195A Inactive Trypsin upon Binding to Serpins

“…We may therefore suggest that our two conformers are also in equilibrium. This hypothesis could provide an alternative explanation to the observations of Plotnick et al (38), who showed that different serpin-proteinase pairs have different breakdown mechanisms. If one assumes on the one hand that there is an equilibrium between two forms of the final complex, one in which some catalytic activity of the proteinase is left and one in which the catalytic activity is fully abolished and on the other hand that the proportion of these two forms varies with each serpin-proteinase pair, we may easily explain the data of Plotnick et al (38).…”

“…This hypothesis could provide an alternative explanation to the observations of Plotnick et al (38), who showed that different serpin-proteinase pairs have different breakdown mechanisms. If one assumes on the one hand that there is an equilibrium between two forms of the final complex, one in which some catalytic activity of the proteinase is left and one in which the catalytic activity is fully abolished and on the other hand that the proportion of these two forms varies with each serpin-proteinase pair, we may easily explain the data of Plotnick et al (38). Our data may also provide an explanation to the apparently contradictory results obtained by another team in two consecutive papers relating cross-linking experiments on the same serpin-proteinase pair (40,41).…”