A novel application of linear free energy relationships is described in which the; substrate selectivities and pH dependencies of glutathione S-transferases (GSTs) are correlated to the pK(a) of glutathione (GSH) at the active site. To determine whether the variation in the thiol pK(a) of GSH at the active sites of GST isozymes can contribute to their differential selectivity for electrophilic substrates, model studies were performed with 4-substituted thiobenzenes, with pK(a) values ranging from 4.5 to 7.5. Second-order rate constants were determined for the specific base-catalyzed reaction of each thiol with a diverse range of GST electrophilic substrates. Bronsted coefficients (beta(nuc)) for these reactions in 10% DMF:90% H2O were determined for each electrophile; beta(nuc) ranged from 0.16 to 0.93. In 30% DMF:70% H2O, the beta(nuc) values increased relative to 10% DMF and ranged from 0.29 to 1.04. Numerical simulations demonstrate that these ranges of beta(nuc) values along with the isozyme-dependent variation in GSH pK(a) could account for a 7.5-fold difference;in relative turnover rates for GST catalysis of some electrophilic substrates. To challenge the predictions of this Bronsted analysis, electrophiles for which chemical steps are rate limiting in enzyme turnover were used as a substrate in reactions with a series of GSTA1-1 mutants with variable GSH pK(a). beta(nuc) values were determined to be 0.16 +/- 0.05 for cumene hydroperoxide (CHP) and 0.25 +/- 0.06 for 1-chloro-2,4-dinitrobenzene,in excellent agreement with the model studies. Furthermore, the dependence of the relative rates of CHP turnover on GSH pK(a) was well correlated, at pH 6.5, 7.4, and 8.0 with the relative rates predicted by the Bronsted analysis. Thus, even for a reaction characterized by a low beta(nuc) value, variation of the pK(a) of enzyme-bound GSH leads to changes in the intrinsic reactivity of the nucleophilic GS(-), according to the Bronsted free energy relationship. In principle, variation of the pK(a) of GSH may contribute to isozyme-dependent substrate selectivity.