Plotnick Michael I, Samakur Madhurika, Wang Zhi Mei, Liu Xhuzuo, Rubin Harvey, Schechter Norman M, Selwood Trevor
Pulmonary, Allergy, and Critical Care Division, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
Biochemistry. 2002 Jan 8;41(1):334-42. doi: 10.1021/bi015650+.
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.
丝氨酸蛋白酶抑制剂（Serpins）以酰基 - 酶复合物的形式捕获其靶蛋白酶。然而，这种捕获是动力学性质的，因此丝氨酸蛋白酶抑制剂 - 蛋白酶复合物最终会分解，释放出一个裂解的无活性丝氨酸蛋白酶抑制剂和一个活性蛋白酶。这种去酰化过程的速率因丝氨酸蛋白酶抑制剂 - 蛋白酶对的不同而有很大差异，半衰期从几分钟到几个月不等。分解速率多样性的原因尚不清楚。在当前的研究中，利用pH值和溶剂同位素效应来探究一种极其稳定的复合物以及几种不稳定复合物的分解机制。发现了分解的一级速率常数k(bkdn)对pH值依赖性的两种不同模式。这种稳定的复合物在中性pH下分解，半衰期约为2周，其pH - k(bkdn)曲线与溶剂 - 氢氧根离子介导的酯水解一致。没有证据表明催化机制参与了这种复合物的分解，这表明活性位点发生了广泛的扭曲。那些半衰期从几分钟到几小时不等的不稳定复合物，其k(bkdn)呈现出钟形的pH曲线，这是游离丝氨酸蛋白酶典型的pH - 速率曲线。在低至中性pH范围内，k(bkdn)随着pH值的升高而增加，呈现出由His57介导催化的特征。在碱性pH范围内，观察到k(bkdn)下降，这与Ile16 - Asp194盐桥（按照胰凝乳蛋白酶原编号）的滴定一致。游离的或与底物结合的人嗜酸性粒细胞弹性蛋白酶（HNE）的pH - 速率曲线中未表现出碱性pH依赖性，这表明在复合蛋白酶中盐桥显著不稳定。这些结果表明，在不稳定复合物中分解是由催化介导的，尽管很可能蛋白酶并非处于其天然构象且催化机制功能效率低下。然而，一种认为分解由复合蛋白酶扭曲和未扭曲形式之间的平衡所决定的机制也不能被完全排除。总体而言，这项研究的结果表明，不稳定复合物中的蛋白酶结构比稳定复合物中的扭曲程度要小。
