Andersson C, Piemonte F, Mosialou E, Weinander R, Sun T H, Lundqvist G, Adang A E, Morgenstern R
Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden.
Biochim Biophys Acta. 1995 Mar 15;1247(2):277-83. doi: 10.1016/0167-4838(94)00239-d.
Rat liver microsomal glutathione transferase is activated by sulfhydryl reagents and proteolysis. This property varies, however, depending on the combination, concentration and reactivity of the substrates. Thus, a multi-dimensional diagram can be envisioned in which the parameters affecting enzyme activity and activation are visualized. In principle activation could stem from an alteration in enzyme mechanism, transition-state complementarity, product release rate or pH-rate behaviour. These studies appear to rule out these possibilities and an alternate hypothesis is suggested based on the following experiments: (i) alternate substrate diagnosis of the kinetic mechanism of microsomal glutathione transferase indicates a random sequential mechanism. Non-activated and activated enzyme follow the same mechanism by these criteria. (ii) The microsomal glutathione transferase stabilizes a Meisenheimer complex between 1,3,5-trinitrobenzene and glutathione. The formation constants were similar for the unactivated and activated enzyme ((15 +/- 1).10(3) and (14 +/- 1).10(3) M-1, respectively, at pH 8). Inasmuch as the Meisenheimer complex resembles the transition state there is no evidence for an increased stabilization upon activation. (iii) The catalytic rate constant kcat does not vary with the viscosity in the assay medium. Thus, product release is not rate limiting for the unactivated and activated microsomal glutathione transferase (with saturating 1-chloro-2,4-dinitrobenzene and varying GSH). (iv) The pH dependence of the Kf-values for Meisenheimer complex formation exhibited pKa values close to 6 for both the activated and unactivated microsomal glutathione transferase. The pH profile of kcat (with saturating 1-chloro-2,4-dinitrobenzene and variable GSH concentrations) showed apparent pKa values of 5.7 +/- 0.5 and 6.3 +/- 0.4 for the unactivated and activated enzyme, respectively, indicative of a very similar requirement for deprotonation of the enzyme-GSH-1-chloro-2,4-dinitrobenzene complex. (v) Examination of the kinetic parameters (obtained with GSH as the variable substrate against increasingly reactive electrophilic substrates) in Hammett plots shows that the activation mechanism entails a more efficient utilization of GSH. It is suggested that a higher rate of formation of the glutathione thiolate anion occurs in the activated enzyme.
大鼠肝脏微粒体谷胱甘肽转移酶可被巯基试剂和蛋白水解作用激活。然而，这种特性会因底物的组合、浓度和反应活性而有所不同。因此，可以设想一个多维图，其中影响酶活性和激活的参数得以可视化。原则上，激活可能源于酶机制、过渡态互补性、产物释放速率或pH-速率行为的改变。这些研究似乎排除了这些可能性，并基于以下实验提出了另一种假设：(i) 对微粒体谷胱甘肽转移酶动力学机制的替代底物诊断表明是随机顺序机制。根据这些标准，未激活和激活的酶遵循相同的机制。(ii) 微粒体谷胱甘肽转移酶稳定了1,3,5-三硝基苯与谷胱甘肽之间的迈森海默络合物。未激活和激活的酶的形成常数相似（在pH 8时，分别为(15±1)·10³和(14±1)·10³ M⁻¹）。由于迈森海默络合物类似于过渡态，因此没有证据表明激活后稳定性增加。(iii) 催化速率常数kcat不随测定介质中的粘度而变化。因此，对于未激活和激活的微粒体谷胱甘肽转移酶（使用饱和的1-氯-2,4-二硝基苯和不同的谷胱甘肽），产物释放不是限速步骤。(iv) 迈森海默络合物形成的Kf值的pH依赖性对于激活和未激活的微粒体谷胱甘肽转移酶均显示出接近6的pKa值。kcat的pH曲线（使用饱和的1-氯-
