Stenberg G, Board P G, Carlberg I, Mannervik B
Department of Biochemistry, University of Uppsala, Sweden.
Biochem J. 1991 Mar 1;274 ( Pt 2)(Pt 2):549-55. doi: 10.1042/bj2740549.
Glutathione transferase (GST) epsilon (also known as GST2 or GST B1B1), the major Class Alpha GST in human liver has been subjected to oligonucleotide-directed site-specific mutagenesis. Four arginine residues, R13, R20, R69 and R187, of which all but R69 are strictly conserved through GST Classes Alpha, Mu and Pi have been replaced by Ala. The mutant enzymes have been expressed in Escherichia coli, purified by affinity chromatography and characterised. Compared with the wild-type enzyme, all mutant GSTs had altered catalytic properties. All mutants had decreased specific activity with 1-chloro-2,4-dinitrobenzene (CDNB). Mutants R13A, R69A and R187A also showed decreased activities with other substrates such as cumene hydroperoxide (CuOOH) and androstenedione. In contrast, mutant R20A had an increased peroxidase activity and an isomerase activity essentially the same as that of the wild-type GST. With the substrates used, kcat./Km values were decreased for all mutant GSTs. Increases in the [S0.5] values were most significant for glutathione (GSH), while values for CDNB and CuOOH were less markedly affected. Thus, various kinetic data indicate that the GSH affinity has been reduced by the mutations and that this loss of affinity is linked to the decreased specific activities. Inhibition studies showed an increased sensitivity towards S-hexyl-GSH; this was particularly marked for mutant R69A. Mutant R20A had a lowered [I50] value but, in contrast, also the highest [I80] value as compared with the wild-type enzyme. Towards bromosulphophthalein, mutants R20A and R69A had a markedly increased sensitivity, about 35-fold in comparison with the wild-type. The inhibition properties of mutant R187A were similar to those of the wild-type enzyme and the properties of mutant R13A were in between. The increased sensitivity to S-hexyl-GSH, in contrast with the decreased affinity for GSH, was suggested to be due to an altered distribution between conformational states of the enzyme induced by the mutations. The arginine residues in positions 13, 20 and 69 all seem to be important for the catalytic properties of GST. Further, the inhibition studies indicate a role of arginine residues in the stabilisation of conformational states of the enzyme.
谷胱甘肽转移酶（GST）ε（也称为GST2或GST B1B1）是人类肝脏中主要的α类GST，已进行了寡核苷酸定向的位点特异性诱变。四个精氨酸残基，即R13、R20、R69和R187，除R69外，其余在α类、μ类和π类GST中都严格保守，现已被丙氨酸取代。突变酶已在大肠杆菌中表达，通过亲和层析纯化并进行了表征。与野生型酶相比，所有突变型GST的催化特性均发生了改变。所有突变体对1-氯-2,4-二硝基苯（CDNB）的比活性均降低。突变体R13A、R69A和R187A对其他底物如氢过氧化异丙苯（CuOOH）和雄烯二酮的活性也降低。相比之下，突变体R20A的过氧化物酶活性增加，异构酶活性与野生型GST基本相同。对于所使用的底物，所有突变型GST的kcat./Km值均降低。谷胱甘肽（GSH）的[S0.5]值增加最为显著，而CDNB和CuOOH的值受影响较小。因此，各种动力学数据表明，突变降低了GSH亲和力，且这种亲和力的丧失与比活性降低有关。抑制研究表明对S-己基-GSH的敏感性增加；这在突变体R69A中尤为明显。突变体R20A的[I50]值降低，但相比之下，与野生型酶相比，其[I80]值也最高。对于溴磺酞，突变体R20A和R69A的敏感性显著增加，与野生型相比约为35倍。突变体R187A的抑制特性与野生型酶相似，突变体R13A的特性介于两者之间。与对GSH亲和力降低相反，对S-己基-GSH敏感性增加被认为是由于突变诱导的酶构象状态分布改变所致。13、20和69位的精氨酸残基似乎对GST的催化特性都很重要。此外，抑制研究表明精氨酸残基在稳定酶的构象状态中起作用。
