Role of Active-Site Residues 107 and 108 of GlutathioneS-Transferase mGSTA4-4 in Determining the Catalytic Properties of the Enzyme for 4-Hydroxynonenal

The murine α-class glutathioneS-transferase mGSTA4-4 displays a high catalytic activity with 4-hydroxynonenal (4-HNE), a cytotoxic product of lipid peroxidation. The X-ray crystal structure of mGSTA4-4 was used to design mutations targeting the 4-HNE binding site, with the goal of defining the structural elements of the mGSTA4-4 protein necessary for the high conjugative activity with 4-HNE. Two candidate positions, 107 and 108, were investigated. Of these, residue 108 appears to be significant in codetermining the catalytic properties of mGSTA4-4 toward 4-HNE. Systematic mutagenesis of amino acid 108 indicated that high activity toward 4-HNE is contingent on the presence of an aliphatic, hydrophobic side chain in this position. In particular, replacement of the wild-type V108 with leucine led to a more than fivefold increase in both absolute activity of the enzyme for 4-HNE and its selectivity for 4-HNE over the model substrate 1-chloro-2,4-dinitrobenzene, due to a selective increase of the turnover number for 4-HNE with no change in the affinity of the protein for this substrate and no changes in the kinetic parameters for 1-chloro-2,4-dinitrobenzene. In contrast, the A107L mutation decreased activity of the enzyme for both 4-HNE and CDNB and partially reversed the positive effect of the V108L mutation in a double mutant.