Contribution of Linear Free Energy Relationships to Isozyme- and pH-Dependent Substrate Selectivity of Glutathione S-Transferases:  Comparison of Model Studies and Enzymatic Reactions

Abstract

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. Brønsted coefficients (βnuc) for these reactions in 10% DMF:90% H2O were determined for each electrophile; βnuc ranged from 0.16 to 0.93. In 30% DMF:70% H2O, the βnuc values increased relative to 10% DMF and ranged from 0.29 to 1.04. Numerical simulations demonstrate that these ranges of βnuc 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 Brønsted 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. βnuc 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 Brønsted analysis. Thus, even for a reaction characterized by a low βnuc value, variation of the pK a of enzyme-bound GSH leads to changes in the intrinsic reactivity of the nucleophilic GS-, according to the Brønsted free energy relationship. In principle, variation of the pK a of GSH may contribute to isozyme-dependent substrate selectivity.

Rapid biocompatible macrocyclization of peptides with decafluoro-diphenylsulfone

“…2C ) exist in other S N Ar reactions between peptides and perfluoroarenes 18 , 19 because S N Ar reactions with thiols exhibit large Brønsted coefficients. 20 Substrate-dependence was the most pronounced when solvation was poor ( e.g. , high CH 3 CN content); increased solvation in solutions with high aqueous content masked the effect ( Fig.…”

“…). While glutathione transferase can be used to catalyze S N Ar-reactions in water, 20 the enzyme acts only on peptides that contain unnatural N-terminal-α-Glu. 20 , 21 Hence, improved fAr cross-linking agents provide the ability to access diverse chemical modalities through native polypeptide sequences.…”

Correlation analyses for bimolecular nucleophilic substitution reactions of chloroacetanilide herbicides and their structural analogs with environmentally relevant nucleophiles

“…e E a from the potential for the half-reaction PhSSPh ϩ 2e Ϫ ϭ 2PhS Ϫ [11]. f pK a ϭ 6.9 [29]. g Estimate of E n from the potential for the half-reaction 5S(s) ϩ 2e Ϫ ϭ S [27].…”

Mechanisms of promiscuity among drug metabolizing enzymes and drug transporters

“…An interesting extension of the reactive cofactor role in substrate promiscuity is the possibility that multiple isoforms of an enzyme with slightly different chemical reactivities of their cofactor could collectively achieve greater promiscuity and afford better protection from the widest range of toxins. This theoretical possibility has been considered with GSTs . Specifically, the Bronsted relationship predicts that there is a trade‐off between lowering the pK a of GSH to generate more GS ‐ and decreasing the nucleophilicity of the resulting thiolate .…”

“…Specifically, the Bronsted relationship predicts that there is a trade‐off between lowering the pK a of GSH to generate more GS ‐ and decreasing the nucleophilicity of the resulting thiolate . In fact, different GST isoforms with different substrate selectivities exhibit different pK a s for their bound GSH and this could contribute to the collective substrate promiscuity of GSTs . Regardless of this speculative possibility, it is noteworthy that evolution has poised differentially the pK a s of these GSTs near the maximum predicted by Bronsted behavior for a range of electrophilic substrates.…”