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

An important aspect of the catalytic mechanism of microsomal glutathione tr ansferase (MGST1) is the activation of the thiol of bound glutathione (GSH) . GSH binding to MGST1 as measured by thiolate anion formation, proton rele ase, and Meisenheimer complex formation is a slow process that can be descr ibed by a rapid binding step (K-d(GSH) = 47 +/- 7 mM) of the peptide follow ed by slow deprotonation (k(2) = 0.42 +/- 0.03 s(-1)). Release of the GSH t hiolate 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 bee n known for some time that the turnover (k(cat)) of MGST1 does not correlat e well with the chemical reactivity of the electrophilic substrate. The ste ady-state kinetic parameters determined for GSH and 1-chloro-2,4-dinitroben zene (CDNB) are consistent with thiolate anion formation (k(2)) being large ly rate-determining in enzyme turnover (k(cat) = 0.26 +/- 0.07 s(-1)). Thus , the chemical step of thiolate addition is not rate-limiting and can be st udied as a burst of product formation on reaction of halo-nitroarene electr ophiles with the E.GS(-) complex. The saturation behavior of the concentrat ion dependence of the product burst with CDNB indicates that the reaction o ccurs in a two-step process that is characterized by rapid equilibrium bind ing (K-d(CDNB) = 0.53 +/- 0.08 mM) to the E.GS(-) complex and a relatively fast chemical reaction with the thiolate (k(3) = 500 +/- 40 s(-1)). In a se ries 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 Ham mett sigma (-) values demonstrating a strong dependence on chemical reactiv ity that is similar to the nonenzymatic reaction (rho = 3.4). Microsomal gl utathione 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 th e first time the specific step that is activated. This result explains earl ier observations that the enzyme is activated only with more reactive subst rates. 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 substrat e. As the chemical reactivity of the electrophile becomes lower the rate-de termining step shifts from thiolate formation to the chemical reaction.