The activation of the thiol of glutathione (GSH) bound in theactive site of the class
muglutathione transferase M1-1 from rat involves a hydrogen-bondingnetwork that includes a direct (first-sphere) interaction between the hydroxyl group of Y6 and the sulfur ofGSH and second-sphere interactionsinvolving a hydrogen bond between the main-chain amide N-H of L12 andthe hydroxyl group of Y6 andan on-face hydrogen bond between the hydroxyl group of T13 and the
-electron cloud of Y6 (i.e., T13-OH- - -
-Y6-OH- - -
-SG). The functions ofthese hydrogen bonds have been examined with a combinationof site-specific
mutagenesis and X-ray crystallography. Thehydroxyl group of Y6 has a normal p
Kaofabout 10 even though it is shielded from solvent and is in a largelyhydrophobic environment. The apparentp
Ka of GSH in the binary Y6F·GSH complexis increased by 1.6 log units, and the reactivity of theenzyme-bound nucleophile is reduced. The catalytic properties ofthe Y6L
mutant are identical to thoseof Y6F, suggesting that the weakly polar on-edge interaction betweenthe aromatic ring and sulfur has noinfluence on catalysis. The refined three-dimensional structure ofthe Y6F
mutant in complex with GSHshows no major structural perturbation of the protein other than achange in the coordination environmentof the sulfur. Removal of the second-sphere influence of theon-face hydrogen bond between the hydroxylgroup of T13 as in the T13V and T13A
mutants elevates thep
Ka of enzyme-bound GSH by about 0.7p
Kaunits. Crystal structures of these
mutants show that structuralchanges in the active site are minor andsuggest that the changes in p
Ka of E·GSHare due to the presence or absence of the on-face hydrogenbond. The T13S
mutant has a completely different side-chainhydrogen-bonding geometry than T13 inthe native enzyme and catalytic properties similar to the T13A and T13V
mutants consistent with theabsence of an on-face hydrogen bond. The
-methyl group of T13is essential in enforcing the on-facehydrogen bond geometry and preventing the hydroxyl group from formingmore favorable conventionalhydrogen bonds.