KINETIC CHARACTERIZATION OF RECOMBINANT HUMAN GLUTATHIONE TRANSFERASET1-1, A POLYMORPHIC DETOXICATION ENZYME

Recombinant human theta class glutathione transferase T1-1 has been he terologously expressed in Escherichia coli and a simple purification m ethod involving immobilized ferric ion affinity chromatography and Ora nge A dye chromatography is described. The catalytic properties of the enzyme differ significantly from those of other glutathione transfera ses, also within the theta class, with respect to both substrate selec tivity and kinetic parameters, In addition to 1,2-epoxy-3-(4-nitrophen oxy)propane, the substrate used previously to monitor the enzyme, huma n glutathione transferase T1-1 has activity with the naturally occurri ng phenethylisothiocyanate and also displays glutathione peroxidase ac tivity with cumene hydroperoxide. Further, the enzyme is active with 4 -nitrobenzyl chloride and 4-nitrophenethyl bromide, but shows no detec table activity with the more chemically reactive 1-chloro-2,4-dinitrob enzene. The Michaelis constant for glutathione, K-m(GSH), with 1,2-epo xy-3-(4-nitrophenoxy)propane as second substrate, is high at low pH va lues 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 s ubstrate, 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(electro phile) display either a maximum or a plateau at pH 7.0-7.5, and an app arent 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)p ropane as electrophilic substrates. This pK(a) value reflects an ioniz ation of enzyme-bound GSH, most probably involving the sulfhydryl grou p, whose pK(a) value thus is lowered by 3 pH units by the enzyme. Thre e differences in the cDNA as compared to the sequence previously publi shed were found. One of these differences causes a change in the deduc ed amino acid sequence and involves the nucleotide triplet encoding am ino acid 126, which was determined as GAG (Glu), instead of the publis hed GGG (Gly). (C) 1997 Academic Press.