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 inEscherichia coliand 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,KmGSH, 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 inkcat/KmGSHwhich increases with an apparent pKavalue 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 theKmGSHvalue (0.72 mM at pH 7.5), as well as the pKa(8.1) derived from the pH dependence ofkcat/KmGSH, are lower with this substrate. In contrast,kcatandkcat/Kmelectrophiledisplay either a maximum or a plateau at pH 7.0–7.5, and an apparent pKavalue of 5.7 was determined for the pH dependence ofkcatwith both 4-nitrophenethyl bromide and 1,2-epoxy-3-(4-nitrophenoxy)propane as electrophilic substrates. This pKavalue reflects an ionization of enzyme-bound GSH, most probably involving the sulfhydryl group, whose pKavalue 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).