Kinetic characterization of thiolate anion formation and chemical catalysis of activated microsomal glutathione transferase 1.

Microsomal glutathione transferase 1 (MGST1) displays the unique ability to be activated, up to 30-fold, by the reaction with sulfhydryl reagents, e.g., N-ethylmaleimide. Analysis of glutathione (GSH) thiolate formation, which occurs upon mixing activated MGST1 with GSH, reveals biphasic kinetics, where the rapid phase dominated at higher GSH concentrations. The kinetic behavior suggests a two-step mechanism consisting of a rapid GSH-binding step (KDGSH ≈ 10 mM), followed by slower formation of thiolate (k2 ≈ 10 s-1). The release rate (or protonation of the enzyme GSH thiolate complex) of GS- was slow (k-2 = 0.016 s-1), consistent with overall tight binding of GSH. Electrophilic second substrates react rapidly with the E•GS- complex, and again, a two-step mechanism is suggested. In comparison to the unactivated enzyme [Morgenstern et al. (2001) Biochemistry 40, 3378−3384], the mechanisms of GSH thiolate formation and electrophile interaction are similar; however, thiolate anion formation is enhanced 30-fo...