Abstract
The structural and functional role of Arg111 in GSTU4-4 from Glycine max (GmGSTU4-4) was studied by chemical modification followed by site-directed mutagenesis. The arginine-specific reagent 2,3-butanedione (BTD) inactivates the enzyme in borate buffer at pH 8.0, with pseudo-first-order saturation kinetics. The rate of inactivation exhibited a non-linear dependence on the concentration of BTD which can be described by reversible binding of reagent to the enzyme (K-D 81.2 +/- 9.2 mM) prior to the irreversible reaction, with maximum rate constants of 0.18 +/- 0.01 min(-1). Protection from inactivation was afforded by substrate analogues demonstrating the specificity of the reaction. Structural analysis suggested that the modified residue is Arg111, which was confirmed by protein chemistry experiments. Site-directed mutagenesis was used in dissecting the role of Arg111 in substrate binding, specificity and catalytic mechanism. The mutant Arg111Ala enzyme exhibited unchanged K-m value for GSH but showed reduced affinity for the xenobiotic substrates, higher k(cat) and specific activities towards aromatic substrates and lower specific activities towards aliphatic substrates. The biological significance of the specific modification of Arg111 by dicarbonyl compounds and the role of Arg111 as a target for engineering xenobiotic substrate specificity were discussed. (C) 2016 Elsevier B.V. All rights reserved.