Kinetic analysis of the slow ionization of glutathione by microsomal glutathione transferase MGST1.

An important aspect of the catalytic mechanism of microsomal glutathione transferase (MGST1) is the activation of the thiol of bound glutathione (GSH). GSH binding to MGST1 as measured by thiolate anion formation, proton release, and Meisenheimer complex formation is a slow process that can be described by a rapid binding step (K(GSH)d = 47 +/- 7 mM) of the peptide followed by slow deprotonation (k2 = 0.42 +/- 0.03 s(-1). Release of the GSH thiolate anion is very slow (apparent first-order rate k(-2) = 0.0006 +/- 0.00002 s(-)(1)) and thus explains the overall tight binding of GSH. It has been known for some time that the turnover (kcat) of MGST1 does not correlate well with the chemical reactivity of the electrophilic substrate. The steady-state kinetic parameters determined for GSH and 1-chloro-2,4-dinitrobenzene (CDNB) are consistent with thiolate anion formation (k2) being largely rate-determining in enzyme turnover (kcat = 0.26 +/- 0.07 s(-1). Thus, the chemical step of thiolate addition is not rate-limiting and can be studied as a burst of product formation on reaction of halo-nitroarene electrophiles with the E.GS- complex. The saturation behavior of the concentration dependence of the product burst with CDNB indicates that the reaction occurs in a two-step process that is characterized by rapid equilibrium binding ( = 0.53 +/- 0.08 mM) to the E.GS- complex and a relatively fast chemical reaction with the thiolate (k3 = 500 +/- 40 s(-1). In a series of substrate analogues, it is observed that log k3 is linearly related (rho value 3.5 +/- 0.3) to second substrate reactivity as described by Hammett sigma- values demonstrating a strong dependence on chemical reactivity that is similar to the nonenzymatic reaction (rho = 3.4). Microsomal glutathione transferase 1 displays the unusual property of being activated by sulfhydryl reagents. When the enzyme is activated by N-ethylmaleimide, the rate of thiolate anion formation is greatly enhanced, demonstrating for the first time the specific step that is activated. This result explains earlier observations that the enzyme is activated only with more reactive substrates. Taken together, the observations show that the kinetic mechanism of MGST1 can be described by slow GSH binding/thiolate formation followed by a chemical step that depends on the reactivity of the electrophilic substrate. As the chemical reactivity of the electrophile becomes lower the rate-determining step shifts from thiolate formation to the chemical reaction.