Abstract
Study of the interaction of glutathione transferase F1-1 from Zea mays (ZmGSTF1-1) with Cu(II), in the presence of ascorbate showed that the enzyme was rapidly inactivated. The inactivation was time and Cu(II) concentration dependent. The rate of inactivation showed non-linear dependence on Cu(II) concentration, indicating that a reversible complex with the enzyme (KD 84.5 ± 6.5 μM) was formed. The inhibitors S-nitrobenzyl-glutathione or S-methyl-glutathione competes with Cu(II), suggesting the specificity of the chemical modification reaction. SDS-PAGE analysis of the inactivated enzyme showed that the enzyme is fragmented and two new bands of 13 and 11 kDa are formed. This shows that ZmGSTF1-1 was specifically cleaved at a single site, by the locally generated free radicals, through a Fenton-type reaction. Sequencing of the fragments allowed the identification of the Cu(II) binding site on ZmGSTF1-1. The three-dimensional structure of ZmGSTF1-1 reveals that the Cu(II) binding site is localized within the glutathione-binding site (G-site) and His40 and Gln53 are most likely the residues that provide the coordination sites for the Cu(II) binding. These findings were confirmed by site-directed mutagenesis. This copper-induced oxidative cleavage reaction of ZmGSTF1-1 may function as a detoxification route for Cu(II) for protecting plant cells from copper-induced deleterious effects.