KINETIC-STUDIES ON RAT-LIVER MICROSOMAL GLUTATHIONE TRANSFERASE - CONSEQUENCES OF ACTIVATION

Rat liver microsomal glutathione transferase is activated by sulfhydry l reagents and proteolysis. This property varies, however, depending o n the combination, concentration and reactivity of the substrates. Thu s, a multi-dimensional diagram can be envisioned in which the paramete rs affecting enzyme activity and activation are visualized. In princip le activation could stem from an alteration in enzyme mechanism, trans ition-state complementarity, product release rate or pH-rate behaviour . These studies appear to rule out these possibilities and an alternat e hypothesis is suggested based on the following experiments: (i) alte rnate substrate diagnosis of the kinetic mechanism of microsomal gluta thione transferase indicates a random sequential mechanism. Non-activa ted 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 c onstants were similar for the unactivated and activated enzyme ((15 +/ - 1). 10(3) and (14 +/- 1). 10(3) M(-1), respectively, at pH 8). Inasm uch as the Meisenheimer complex resembles the transition state there i s no evidence for an increased stabilization upon activation. (iii) Th e catalytic rate constant k(cat) does not vary with the viscosity in t he assay medium. Thus, product release is not rate limiting for the un activated and activated microsomal glutathione transferase (with satur ating 1-chloro-2,4-dinitrobenzene and varying GSH). (iv) The pH depend ence of the K-f-values for Meisenheimer complex formation exhibited pK (a) values close to 6 for both the activated and unactivated microsoma l glutathione transferase. The pH profile of k(cat) (with saturating 1 -chloro-2,4-dinitrobenzene and variable GSH concentrations) showed app arent 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 requi rement 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 enta ils a more efficient utilization of GSH. It is suggested that a higher rate of formation of the glutathione thiolate anion occurs in the act ivated enzyme.