Kinetic studies on rat liver microsomal glutathione transferase: consequences of activation

Abstract

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-dinitrobenzene and glutathione. The formation constants were similar for the unactivated and activated enzyme ((15 ± 1) · 10 3 and (14 ± 1)· 10 3 M −, 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 k cat, 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 K f-values for Meisenheimer complex formation exhibited pK a values close to 6 for both the activated and unactivated microsomal glutathione transferase. The pH profile of k cat (with saturating 1-chloro-2,4-dinitrobenzene and variable GSH concentrations) showed apparent pK a 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.