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

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• 作者：Michael I. Plotnick ; Madhurika Samakur ; Zhi Mei Wang ; Xhuzuo Liu ; Harvey Rubin ; Norman M. Schechter ; and Trevor Selwood
• 刊名：Biochemistry
• 出版年：2002
• 出版时间：January 8, 2002
• 年：2002
• 卷：41
• 期：1
• 页码：334 - 342
• 全文大小：99K
• 年卷期：v.41,no.1(January 8, 2002)
• ISSN：1520-4995

文摘

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 serpinand 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 breakdownrates are not clearly understood. In the current study, pH and solvent isotope effects were utilized toprobe 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, werefound. 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 wasno evidence for the participation of the catalytic machinery in the breakdown of this complex, suggestingextensive distortion of the active site. The unstable complexes, which break down with half-lives rangingfrom minutes to hours, exhibited a bell-shaped pH profile for k_bkdn, typical of the pH-rate profiles of freeserine proteases. In the low to neutral pH range k_bkdn increased with increasing pH in a manner characteristicof His57-mediated catalysis. In the alkaline pH range a decrease in k_bkdn was observed, consistent withthe titration of the Ile16-Asp194 salt bridge (chymotrypsinogen numbering). The alkaline pH dependencewas not exhibited in pH-rate profiles of free or substrate-bound HNE, indicating that the salt bridge wassignificantly destabilized in the complexed protease. These results indicate that breakdown is catalyticallymediated in the unstable complexes although, most likely, the protease is not in its native conformationand the catalytic machinery functions inefficiently. However, a mechanism in which breakdown isdetermined by the equilibrium between distorted and undistorted forms of the complexed protease cannotbe completely dismissed. Overall, the results of this study suggest that the protease structure in unstablecomplexes is distorted to a lesser extent than in stable complexes.