Hubbard C D, Shoupe T S
J Biol Chem. 1977 Mar 10;252(5):1633-8.
The kinetics of the acylation of alpha-chymotrypsin by a series of substituted phenyl p-nitrobenzoates have been studied by stopped flow and conventional spectrophotometry. Electron withdrawal in the leaving group accelerates the rate of acylation, and the p value obtained for eight esters is +1.96. The pH- and pD-independent acylation rate constants are, respectively, 1.40 X 10(4) M-1S-1 and 1.23 X 10(4) M-1S-1 for p-nitrophenyl p-nitrobenzoate, and, respectively, 2.19 X 10(3) M-1S-1 and 1968 X 10(3) M-1S-1 for p-nitrophenyl benzoate at 25 degrees. An analysis of structure-reactivity results and kinetic solvent isotope effects indicates a mechanism for acylation by phenylbenzoates in which initial reaction is a nucleophilic attack by an imidazole of the enzyme (His 57). Subsequently, there is rapid transfer of the acylating group to the serine 195 from the acylimidazole species. The kinetic solvent isotope effects for acylation by p-nitrophenyl phenyl acetate and p-nitrophenyl phenyl acetate and p-nitrophenyl hydrocinnamate, in 5%, v/v, acetonitrile, are 1.3 and 2.0, respectively. The latter ester is inhibited more than is p-nitrophenyl benzoate when 5%, v/v, dioxane is substituted for 5%, v/v, acetonitrile as co-solvent. In the presence of 5%, v/v, dioxane a change in the kinetic solvent isotope effect to 1.7 is found for p-nitrophenyl benzoate and p-nitrophenyl phenylacetate while that for the analogous hysdrocinnamate ester is unaffected. The results for the latter substrate are in accord with a general base-catalysed mechanism. Electron-withdrawal groups in the phenyl ring of phenyl acetates accelerate the enzyme acylation yielding a leaving group p of 2.05. The kinetic solvent isotope effects for acylation by p-nitrophenyl thiolacetate and by p-nitrophenyl acetate are close to 2.0. The mechanism of acylation of chymotrypsin by phenyl acetates is not unambiguously defined using these data.
通过停流分光光度法和传统分光光度法研究了一系列取代苯基对硝基苯甲酸酯对α-糜蛋白酶的酰化动力学。离去基团中的吸电子作用加速了酰化速率，八种酯的哈米特方程ρ值为 +1.96。在25℃下，对硝基苯基对硝基苯甲酸酯的pH和pD无关的酰化速率常数分别为1.40×10⁴ M⁻¹s⁻¹和1.23×10⁴ M⁻¹s⁻¹，对硝基苯基苯甲酸酯的分别为2.19×10³ M⁻¹s⁻¹和1968×10³ M⁻¹s⁻¹。对结构 - 反应性结果和动力学溶剂同位素效应的分析表明，苯基苯甲酸酯的酰化机制是酶的咪唑（His 57）进行亲核攻击引发初始反应，随后酰基从酰基咪唑物种快速转移到丝氨酸195上。在5%（v/v）乙腈中，对硝基苯基苯乙酸酯和对硝基苯基氢化肉桂酸酯酰化的动力学溶剂同位素效应分别为1.3和2.0。当用5%（v/v）二氧六环代替5%（v/v）乙腈作为共溶剂时，后一种酯比对硝基苯基苯甲酸酯受到的抑制作用更强。在5%（v/v）二氧六环存在下，对硝基苯基苯甲酸酯和对硝基苯基苯乙酸酯的动力学溶剂同位素效应变为1.7，而类似的氢化肉桂酸酯的则不受影响。后一种底物的结果符合一般碱催化机制。苯乙酸酯苯环上的吸电子基团加速了酶的酰化，得到的离去基团ρ值为2.05。对硝基苯基硫代乙酸酯和对硝基苯基乙酸酯酰化的动力学溶剂同位素效应接近2.0。利用这些数据不能明确界定苯乙酸酯对糜蛋白酶的酰化机制。
