Zhou J M, Liu C, Tsou C L
Laboratory of Molecular Enzymology, Academia Sinica, Beijing, China.
Biochemistry. 1989 Feb 7;28(3):1070-6. doi: 10.1021/bi00429a022.
The kinetics of inhibition of trypsin by its specific inhibitors, pancreatic trypsin inhibitor, ovomucoid trypsin inhibitor, and soybean trypsin inhibitor, has been studied by following the hydrolysis of benzoylarginine ethyl ester in the presence of the inhibitor, and the results have been analyzed with the method described previously [Tian & Tsou (1982) Biochemistry 21, 1028]. The results obtained are consistent with the following: (a) The enzyme binds with the pancreatic inhibitor irreversibly to form an inactive complex. (b) The binding with the ovomucoid inhibitor to form the inactive complex is reversible. (c) An intermediate is formed before the relatively stable inactive complex with the soybean inhibitor, and both steps are reversible. The respective microscopic rate constants are determined by suitable plots of the apparent rate constants under different substrate and inhibitor concentrations. The second-order rate constants for the initial binding step thus obtained are in accord with the apparent inactivation rate constants determined by measuring the activity remaining with a stopped-flow apparatus equipped with a multimixing system after the enzyme-inhibitor mixture has been incubated for different time intervals.
通过在抑制剂存在的情况下跟踪苯甲酰精氨酸乙酯的水解，研究了胰蛋白酶的特异性抑制剂（胰腺胰蛋白酶抑制剂、卵类粘蛋白胰蛋白酶抑制剂和大豆胰蛋白酶抑制剂）对胰蛋白酶的抑制动力学，并采用先前描述的方法[田和邹（1982年）《生物化学》21卷，第1028页]对结果进行了分析。所得结果与以下情况一致：（a）酶与胰腺抑制剂不可逆地结合形成无活性复合物。（b）与卵类粘蛋白抑制剂结合形成无活性复合物是可逆的。（c）在与大豆抑制剂形成相对稳定的无活性复合物之前会形成一个中间体，且两个步骤都是可逆的。通过在不同底物和抑制剂浓度下对表观速率常数进行合适的作图来确定各自的微观速率常数。由此获得的初始结合步骤的二级速率常数与通过在酶 - 抑制剂混合物孵育不同时间间隔后，使用配备多混合系统的停流装置测量剩余活性所确定的表观失活速率常数一致。
