Benzoic acid derivatives induce recovery of catalytic activity in the partially inactive Met208Lys mutant of human glutathione transferase A1-1.

Human glutathione transferase A1-1 (GST A1-1) is a detoxifying enzyme catalyzing the conjugation of glutathione with a variety of hydrophobic, electrophilic substrates. When the role of the hydrophobic substrate-binding site residue Met208 was investigated by random mutagenesis, introduction of charged amino acid residues had the greatest deleterious effect on enzyme activity. However, in the lysine mutant some of the lost activity could be regained by the addition of a benzoic acid derivative to the reaction mixture. The activating molecule has now been optimized such that all activity is recovered. The most potent activator, 4-propylbenzoic acid, has been used in studies of the mechanism behind the activation. A heterodimeric species of GST A1-1, containing only one activatable subunit, has been constructed. The heterodimer shows a strictly additive activation curve when compared to its parental forms, indicating that the activation is not due to co-operativity between the subunits. Furthermore, a novel electrophilic substrate, 4-chloro-3,5-dinitrobenzoic acid, with a carboxylate group expected to interact with residue 208 gives a higher kcat value with the lysine mutant than with wild-type GST A1-1. All results obtained in the here support the view that the positive charge introduced into the lysine mutant adversely affects the structure of the C-terminal helix of this enzyme, preventing it from adopting the conformation needed for full activity. The negatively charged carboxylate group of the activator probably neutralizes the positive charge of the side-chain amino group and thereby restores the substrate-binding site to a form that is favorable for the catalytic function. Copyright 1999 Academic Press.