The role of residue M232 in the C-terminal helix of human glutathione transferase T1-1
Glutathione transferases (GSTs) catalyze the glutathione (GSH) conjugation of various xenobiotics and endogenous compounds. GSTs play a pivotal role in cellular defence, as they are the main contributors to the inactivation of genotoxic compounds. A primary determinant for divergent substrate acceptance among GSTs appears to be the electrophilic binding site or “H-site”. Shokeer et al. (2010) reported that replacement of residue methionine 232 by alanine in mouse GST T1-1 greatly altered the enzyme’s catalytic activities. The corresponding residue in human enzyme is also methionine, but, in other mammalian GST T1-1 orthologues, arginine (horse; Equus caballus) or leucine (pig; Sus scrofa) residues are found at this position. The goal of the present study aims to investigate the role of residue 232 (located in the C-terminal α-helix) in the structure and function of human GST T1-1 protein. Three variants (M232R, M232L and M232W) were constructed and expressed in Escherichia coli. The effects of these mutations on protein expression, catalytic activity and structure were tested. Variants M232R and M232L results were generally unremarkable, showing only minor differences from the wild-type protein. However, variant M232W showed significant decreases in protein expression, had reduced specific activities and showed substantial changes in the CD spectrum. The spectral shift was consistent with the interpretation that the C-terminal α-helix unwinds into a random-coil conformation. Unlike horse and pig, no mammalian species is known to have a GST T1-1 enzyme with a tryptophan residue at position 232. Results from this study shows that residue 232 does not critically affect the structure and function of human GST T1-1.