Jemth P, Mannervik B
Department of Biochemistry, Uppsala University, Sweden.
Arch Biochem Biophys. 1997 Dec 15;348(2):247-54. doi: 10.1006/abbi.1997.0357.
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 kcat/K(m)GSH which increases with an apparent pKa 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 pKa (8.1) derived from the pH dependence of kcat/K(m)GSH, are lower with this substrate. In contrast, kcat and kcat/K(m)electrophile display either a maximum or a plateau at pH 7.0-7.5, and an apparent pKa value of 5.7 was determined for the pH dependence of kcat with both 4-nitrophenethyl bromide and 1,2-epoxy-3-(4-nitrophenoxy)propane as electrophilic substrates. This pKa value reflects an ionization of enzyme-bound GSH, most probably involving the sulfhydryl group, whose pKa value thus is lowered 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).
重组人θ类谷胱甘肽转移酶T1-1已在大肠杆菌中进行了异源表达，并描述了一种简单的纯化方法，该方法包括固定化铁离子亲和色谱法和橙黄A染料色谱法。就底物选择性和动力学参数而言，该酶的催化特性与其他谷胱甘肽转移酶（即使在θ类中）也有显著差异。除了先前用于监测该酶的底物1,2-环氧-3-(4-硝基苯氧基)丙烷外，人谷胱甘肽转移酶T1-1对天然存在的苯乙基异硫氰酸酯也有活性，并且对氢过氧化异丙苯还具有谷胱甘肽过氧化物酶活性。此外，该酶对4-硝基苄基氯和4-硝基苯乙基溴有活性，但对化学反应性更强的1-氯-2,4-二硝基苯没有可检测到的活性。以1,2-环氧-3-(4-硝基苯氧基)丙烷作为第二底物时，谷胱甘肽的米氏常数K(m)GSH在低pH值时较高，但在较高pH值时降低。这反映在kcat/K(m)GSH上，其随着表观pKa值9.0而增加，反映了溶液中谷胱甘肽硫醇基团的电离。以4-硝基苯乙基溴作为亲电底物时也得到相同的结果，尽管该底物的K(m)GSH值（pH 7.5时为0.72 mM）以及从kcat/K(m)GSH的pH依赖性推导的pKa（8.1）较低。相比之下，kcat和kcat/K(m)亲电试剂在pH 7.0 - 7.5时显示最大值或平台期，并且以4-硝基苯乙基溴和1,2-环氧-3-(4-硝基苯氧基)丙烷作为亲电底物时，确定kcat的pH依赖性表观pKa值为5.7。这个pKa值反映了酶结合的谷胱甘肽的电离，很可能涉及巯基，其pKa值因此被酶降低。与先前发表的序列相比，在cDNA中发现了三个差异。其中一个差异导致推导的氨基酸序列发生变化，涉及编码氨基酸126的核苷酸三联体，确定为GAG（Glu），而不是发表的GGG（Gly）。
