Allen K G, Stewart J A, Johnson P E, Wettlaufer D G
Eur J Biochem. 1978 Jul 3;87(3):575-82. doi: 10.1111/j.1432-1033.1978.tb12409.x.
The pH dependence of papain catalysis was analyzed by a scheme which evaluates the kinetic contribution of both protonated and unprotonated species of functional groups involved in catalysis. Kinetic measurements were made at constant pH, without buffers, by automatic titration. The rate-determining step for papain-catalyzed hydrolysis of alpha-N-benzoyl-L-arginine ethyl ester, determined by nucleophile competition, changed from acylation below pH 6.5 to mixed acylation-deacylation above pH 6.5. Kinetic analysis indicated that three prototropic groups governed the pH-specificity of alpha-N-benzoyl-L-arginine ethyl ester hydrolysis. These prototropic groups had pKa values of 4.8, 6.5 to 6.7, and 8.7. Theoretical treatment of the kinetics provided an excellent fit with the experimentally found profile when the contribution of all three prototropic groups was considered. Analysis showed that, in acid, the pathways of papain catalysis were functional with either two or three active-site protons. In base, a single functional ionic pathway is associated with an active site with only one proton. Pathways involving an unprotonated active site are catalytically inoperative in both acid and base. These results indicate that papain exhibits several catalytically functional ionic pathways. The results are discussed in terms of pKa assignments, and the mechanism of papain catalysis.
通过一种评估参与催化的官能团的质子化和非质子化物种动力学贡献的方案，分析了木瓜蛋白酶催化作用对pH的依赖性。在无缓冲液的恒定pH条件下，通过自动滴定进行动力学测量。通过亲核试剂竞争确定，木瓜蛋白酶催化α-N-苯甲酰-L-精氨酸乙酯水解的速率决定步骤在pH 6.5以下从酰化转变为pH 6.5以上的混合酰化-脱酰化。动力学分析表明，三个质子转移基团决定了α-N-苯甲酰-L-精氨酸乙酯水解的pH特异性。这些质子转移基团的pKa值分别为4.8、6.5至6.7和8.7。当考虑所有三个质子转移基团的贡献时，动力学的理论处理与实验发现的曲线非常吻合。分析表明，在酸性条件下，木瓜蛋白酶催化途径在有两个或三个活性部位质子时起作用。在碱性条件下，单一的功能性离子途径与只有一个质子的活性部位相关。涉及非质子化活性部位的途径在酸性和碱性条件下均无催化活性。这些结果表明，木瓜蛋白酶表现出几种具有催化功能的离子途径。根据pKa归属和木瓜蛋白酶催化机制对结果进行了讨论。
