First-sphere and second-sphere electrostatic effects in the active site of a class Mu glutathione transferase

The activation of the thiol of glutathione (GSH) bound in the active site of the class mu glutathione transferase M1-1 from rat involves a hydrogen-bonding network that includes a direct (first-sphere) interaction between the hydroxyl group of Y6 and the sulfur of GSH and second-sphere interactions involving a hydrogen bond between the main-chain amide N-H of L12 and the hydroxyl group of Y6 and an on-face hydrogen bond between the hydroxyl group of T13 and the pi-electron cloud of Y6 (i.e., T13-OH---pi-Y6-OH---SG). The functions of these hydrogen bonds have been examined with a combination of site-specific mutagenesis and X-ray crystallography. The hydroxyl group of Y6 has a normal pK(a) of about 10 even though it is shielded from solvent and is in a largely hydrophobic environment. The apparent pK(a) of GSH in the binary Y6F . GSH complex is increased by 1.6 log units, land the reactivity of the enzyme-bound nucleophile is reduced, The catalytic properties of the Y6L mutant are identical to those of Y6F, suggesting that the weakly polar on-edge interaction between the aromatic ring and sulfur has no influence on catalysis. The refined three-dimensional structure of the Y6F mutant in complex with GSH shows no major structural perturbation of the protein other than a change in the coordination environment of the sulfur. Removal of the second-sphere influence of the on-face hydrogen bond between the hydroxyl group of T13 as in the T13V and T13A mutants elevates the pK(a) of enzyme-bound GSH by about 0.7 pK(a) units. Crystal structures of these mutants show that structural changes in the active site are minor and suggest that the changes in pK(a) of E . GSH are due to the presence or absence of the on-face hydrogen bond. The T13S mutant has a completely different side-chain hydrogen-bonding geometry than T13 in the native enzyme and catalytic properties similar to the T13A and T13V mutants consistent with the absence of an on-face hydrogen bond. The gamma-methyl group of T13 is essential in enforcing the on-face hydrogen bond geometry and preventing the hydroxyl group from forming more favorable conventional hydrogen bonds.