Ascenzi P, Menegatti E, Guarneri M, Antonini E
Mol Cell Biochem. 1983;56(1):33-8. doi: 10.1007/BF00228766.
Pre-steady-state and steady-state kinetics for the p.p. elastase-catalysed hydrolysis of ZAlaONp, one of the most favourable substrates for this serine protease, have been studied between pH 4.0 and 8.0. The results are consistent with the minimum three-step mechanism: (formula; see text) Under pre-steady-state conditions, where [E0] much greater than [S0], the values of the dissociation constant of the E X S complex (Ks = k-1/k+1) and of the individual rate constants for the catalytic steps (k+2 and k+3) have been determined over the whole pH range explored. Under steady-state conditions, where [S0] much greater than [E0], the values of kcat and Km have been obtained over the same pH range. The pH profiles of k+2, k+3, k+2/Ks, kcat, kcat/Km reflect the ionization of a group, probably His57, with a pKa value of 6.85 +/- 0.10. The values of Ks and Km are pH independent. The steady-state parameters for the p.p. elastase-catalysed hydrolysis of a number of p-nitrophenyl esters of N-alpha-carbobenzoxy-L-amino acids have been also determined between pH 4.0 and 8.0 and compared with those of b.beta-trypsin and b.alpha-chymotrypsin. For all the substrates examined the acylation step (k+2) is rate limiting in the p.p. elastase catalysis, between pH 4.0 and 8.0. The different catalytic behaviours of p.p. elastase, b.beta-trypsin and b.alpha-chymotrypsin are consistent with the known three-dimensional structures of these serine proteases.
对木瓜蛋白酶催化水解 Z - 丙氨酸对硝基苯酯（该丝氨酸蛋白酶最适宜的底物之一）的预稳态和稳态动力学，在 pH 4.0 至 8.0 之间进行了研究。结果与最少三步机制相符：（公式；见原文）在预稳态条件下，即[E₀]远大于[S₀]时，已在整个探索的 pH 范围内测定了 E·S 复合物的解离常数（Ks = k⁻¹/k⁺¹）以及催化步骤的各个速率常数（k⁺²和 k⁺³）。在稳态条件下，即[S₀]远大于[E₀]时，已在相同的 pH 范围内获得了 kcat 和 Km 的值。k⁺²、k⁺³、k⁺²/Ks、kcat、kcat/Km 的 pH 图谱反映了一个基团（可能是 His57）的电离，其 pKa 值为 6.85 ± 0.10。Ks 和 Km 的值与 pH 无关。还在 pH 4.0 至 8.0 之间测定了木瓜蛋白酶催化水解多种 N - α - 苄氧羰基 - L - 氨基酸对硝基苯酯的稳态参数，并与β - 胰蛋白酶和α - 胰凝乳蛋白酶的参数进行了比较。对于所研究的所有底物，在 pH 4.0 至 8.0 之间，木瓜蛋白酶催化中的酰化步骤（k⁺²）是限速步骤。木瓜蛋白酶、β - 胰蛋白酶和α - 胰凝乳蛋白酶不同的催化行为与这些丝氨酸蛋白酶已知的三维结构一致。
