Shviadas V Iu, Galaev I Iu, Berezin I V
Biokhimiia. 1980 Apr;45(4):629-35.
The hydrolysis of L-tryptophane ethyl ester catalyzed by alpha-chymotrypsin and the effect of ethyl ster of D-tryptophane on the course of the reaction were studied. A kinetic pattern of a three-step enzymatic reaction based on the assumption that the enzyme complex with the protonated form of the substrate is the only reactive one, was proposed. It was shown that the limiting step of the enzymatic reaction consists in a formation of intermediate acyl enzyme. The pH-dependence of the bimolecular rate constant (kcat/Km) for the enzymatic hydrolysis of L-tryptophane ethyl ester is bell-shaped and is described by a pattern including ionization of two groups with pKa values of 7,0 +/- 0,1; and 7,5 +/- 0,1; the value of pKa equal to 7,5 +/- 0,1 corresponds to substrate ionization (pKas for the amino group of L-tryptophane ethyl ester is 7,6). The constants for the binding of protonated and non-protonated substrate forms by the enzyme were calculated from the step-wise dependence of the Km values of pH. An analysis of the bell-shaped dependence of the catalytic constant of enzymatic hydrolysis included determination of pK values of the ionogenic groups of the enzyme--substrate complex (pK'a = 6,8 +/- 0,1 and pK''a = 7,3 +/- 0,1).
研究了α-胰凝乳蛋白酶催化L-色氨酸乙酯的水解作用以及D-色氨酸乙酯对反应进程的影响。基于底物质子化形式的酶复合物是唯一反应性复合物这一假设，提出了一个三步酶促反应的动力学模式。结果表明，酶促反应的限速步骤在于形成中间酰基酶。L-色氨酸乙酯酶促水解的双分子速率常数（kcat/Km）的pH依赖性呈钟形，由一个包含两个pKa值分别为7.0±0.1和7.5±0.1的基团电离的模式描述；pKa值为（7.5±0.1）对应底物电离（L-色氨酸乙酯氨基的pKas为7.6）。根据Km值对pH的逐步依赖性计算了酶与质子化和非质子化底物形式的结合常数。对酶促水解催化常数的钟形依赖性分析包括测定酶-底物复合物离子基团的pK值（pK'a = 6.8±0.1和pK''a = 7.3±0.1）。
