Functions of his107 in the catalytic mechanism of human glutathione S-transferase hGSTM1a-1a

Domain interchange analyses and site-directed mutagenesis indicate that the His107 residue of the human subunit hGSTM1 has a pronounced influence on catalysis of nucleophilic aromatic substitution reactions, and a H107S substitution accounts for the marked differences in the properties of the homologous hGSTM1-1 (His107) and hGSTM4-4 (Ser107) glutathione S-transferases. Reciprocal replacement of His107 and Ser107 in chimeric enzymes results in reciprocal conversion of catalytic properties. With 1-chloro-2,4-dinitrobenzene as a substrate, the His107 residue primarily influences the pH dependence of catalysis by lowering the apparent pK(a) of k(cat)/K-m from 7.8 for the Ser-107-containing enzymes to 6.3 for the His-107 containing enzymes. There is a parallel shift in the pK(a) for thiolate anion formation of enzyme-bound GSH. Y6F mutations have no effect on the pK(a) for these enzymes. Crystal structures of hCSTM1a-1a indicate that the imidazole ring of His107 is oriented toward the substrate binding cleft approximately 6 Angstrom from the GSH thiol group. Thus, His107 has the potential to act as a general base in proton transfer mediated through an active site water molecule or directly following a modest conformational change, to promote thiolate anion formation. All wild-type enzymes and H107S chimera have nearly identical equilibrium constants for formation of enzyme-GSH complexes (K-d values of 1-2 x 10(-6) M); however, K-m(GSH) and K-i values for S-methylglutathione inhibition determined by steady-state kinetics are nearly 100-fold higher. The functions of His107 of hGSTM1a-1a are unexpected in view of a substantial body of previous evidence that excluded participation of histidine residues in the catalytic mechanisms of other glutathione S-transferases. Consequences of His 107 involvement in catalysis are also substrate-dependent; in contrast to 1-chloro-2,4-dinitrobenzene, for the nucleophilic addition reaction of GSH to ethacrynic acid, the H107S substitution has no effect on catalysis presumably because product release is rate-limiting.