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 (K(D)(GSH) approximately 10 mM), followed by slower formation of thiolate (k(2) approximately 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-fold in the activated enzyme, contributing to an increased k(cat) (3.6 s(-1)). Interestingly, in the activated enzyme, thiolate formation and proton release from the enzyme are not strictly coupled, because proton release (as well as k(cat)) was found to be approximately 4 times slower than GSH thiolate formation in an unbuffered system. Solvent kinetic isotope effect measurements demonstrated a 2-fold decrease in the rate constant (k(2)) for thiolate formation and k(cat) (in the reaction with 1-chloro-2,4-dinitrobenzene) for both unactivated and activated MGST1. This indicates that thiolate formation contributes to k(cat) for the activated enzyme, as suggested previously for unactivated MGST1. The stoichiometry of thiolate formation, proton release, and burst kinetics suggested utilization of one GSH molecule per enzyme trimer.