Kinetic characterization of recombinant human glutathione transferase T1-1, a polymorphic detoxication enzyme

Recombinant human theta class glutathione transferase T1-1 has been heterologously expressed in Escherichia coli and a simple purification method involving immobilized ferric ion affinity chromatography and Orange A dye chromatography is described. The catalytic properties of the enzyme differ significantly from those of other glutathione transferases, also within the theta class, with respect to both substrate selectivity and kinetic parameters, In addition to 1,2-epoxy-3-(4-nitrophenoxy)propane, the substrate used previously to monitor the enzyme, human glutathione transferase T1-1 has activity with the naturally occurring phenethylisothiocyanate and also displays glutathione peroxidase activity with cumene hydroperoxide. Further, the enzyme is active with 4-nitrobenzyl chloride and 4-nitrophenethyl bromide, but shows no detectable activity with the more chemically reactive 1-chloro-2,4-dinitrobenzene. The Michaelis constant for glutathione, K-m(GSH), with 1,2-epoxy-3-(4-nitrophenoxy)propane as second substrate, is high at low pH values but decreases at higher pH values. This is mirrored in k(cat)/K-m(GSH) which increases with an apparent pK(a) value of 9.0, reflecting the ionization of the thiol group of glutathione in solution, The same results are obtained with 4-nitrophenethyl bromide as electrophilic substrate, although the K-m(GSH) value (0.72 mM at pH 7.5), as well as the pK(a) (8.1) derived from the pH dependence of k(cat)/K-m(GSH), are lower with this substrate. In contrast, k(cat) and k(cat)/K-m(electrophile) display either a maximum or a plateau at pH 7.0-7.5, and an apparent pK(a) value of 5.7 was determined for the pH dependence of k(cat) with both 4-nitrophenethyl bromide and 1,2-epoxy-3-(4-nitrophenoxy)propane as electrophilic substrates. This pK(a) value reflects an ionization of enzyme-bound GSH, most probably involving the sulfhydryl group, whose pK(a) value thus is lowered by 3 pH units by the enzyme. Three differences in the cDNA as compared to the sequence previously published were found. One of these differences causes a change in the deduced amino acid sequence and involves the nucleotide triplet encoding amino acid 126, which was determined as GAG (Glu), instead of the published GGG (Gly). (C) 1997 Academic Press.