C-terminal region amino acid substitutions contribute to catalytic differences between murine class alpha glutathione transferases mCSTA1-1 and mCSTA2-2 toward anti-diol epoxide isomers of benzo [c]phenanthrene

The molecular basis for catalytic differences between structurally closely related murine class alpha glutathione (GSH) transferases mGSTA1-1 and mGST A2-2 in the GSH conjugation of anti-diol epoxide isomers of benzo[c]phenant hrene (anti-B[c]PDE) was investigated. GSH conjugation of both (-)- and (+) -enantiomers of anti-B[c]PDE was observed in the presence of mGSTA1-1 (60 a nd 40% GSH conjugation, respectively), whereas mGSTA2-2 exhibited a prefere nce for the (-)-anti-isomer (>97%). In addition, the specific activity of m GSTA2-2 toward the (-)-anti-B[c]PDE isomer was relatively higher than that of mGSTA1-1. The amino acid sequences of mGSTA1-1 and mGSTA2-2 differ at 10 positions that are distributed in three sections. Section I contains amino acid residues in positions 65 and 95; section II contains residues in posi tions 157, 162, and 169, and section III contains residues in positions 207 , 213, 218, 221, and 222. Enzyme activity measurements with chimeras of mGS TA1-1 and mGSTA2-2 revealed that amino acid substitutions in section III ac count for their differential enantioselectivity and catalytic activity towa rd anti-B[c]PDE, Site-directed mutagenesis of amino acid residues in sectio n III of mGSTA2-2 with corresponding residues of mGSTA1-1 followed by activ ity measurements of the wild type and mutated enzymes indicates that leucin e 207 and phenylalanine 221 may be critical for the high catalytic activity of mGSTA2-2 toward (-)-anti-B[c]PDE. Molecular modeling studies demonstrat ed that the active site of mGSTA1-1 accommodates both enantiomers of anti-B [c]PDE, whereas the (-)-anti-isomer interacts more favorably with active si te residues in mGSTA2-2, The results of this study clearly indicate that am ino acid substitutions in the C-terminal region contribute to catalytic dif ferences between mGSTA1-1 and mGSTA2-2 with respect to anti-B[c]PDE.