Comstock K E, Widersten M, Hao X Y, Henner W D, Mannervik B
Department of Biochemistry and Molecular Biology, Oregon Health Sciences University, Portland 97201.
Arch Biochem Biophys. 1994 Jun;311(2):487-95. doi: 10.1006/abbi.1994.1266.
The multigene family of cytosolic glutathione S-transferases (GSTs) consists of four classes (Alpha, Mu, Pi, and Theta), all involved in the detoxication of reactive electrophiles. The human Mu class GSTs consist of at least four expressed isozyme subunits, GST M1, GST M2, GST M3, and GST M4, which have 70-90% amino acid sequence identity. The gene and cDNA sequences for GST M4 have been determined recently (K. E. Comstock, K. J. Johnson, D. Rifenbery, and W. D. Henner, J. Biol. Chem. (1993) 268, 16958-16965). Cloning of GST M4 cDNA into an Escherichia coli expression system permitted the production of the corresponding protein. The enzyme was purified and shown to have a relatively low specific activity with the standard GST substrate 1-chloro-2,4-dinitrobenzene (1.4 +/- 0.2 mumol min-1 mg-1 protein), but an activity equivalent to other Mu class enzymes with other tested substrates. The protein forms functional dimers composed of subunits with a M(r) of approximately 26,400. A detailed comparison of the activity with various substrates and inhibitors was performed between GST M4-4 and other human Mu class GSTs, GST M1a-1a, GST M2-2, and GST M3-3, produced in bacterial expression systems. Despite the high level of amino acid sequence identity, the enzymatic properties of these enzymes were quite different. Comparisons with the crystallographic structure of a homologous rat GST, GST 3-3, indicate that a number of the nonconserved amino acid residues can be assigned to the putative active site of GST M4-4. This suggests that diversification in the evolution of these genes has occurred primarily in the substrate binding regions to cope with an increasing variety of foreign compounds.
胞质谷胱甘肽S-转移酶（GSTs）的多基因家族由四类（α、μ、π和θ）组成，它们都参与活性亲电试剂的解毒作用。人类μ类GSTs至少由四个表达的同工酶亚基组成，即GST M1、GST M2、GST M3和GST M4，它们具有70-90%的氨基酸序列同一性。GST M4的基因和cDNA序列最近已被确定（K.E.康斯托克、K.J.约翰逊、D.里芬贝里和W.D.亨纳，《生物化学杂志》（1993年）268，16958-16965）。将GST M4 cDNA克隆到大肠杆菌表达系统中可产生相应的蛋白质。该酶经过纯化，结果表明其对标准GST底物1-氯-2,4-二硝基苯的比活性相对较低（1.4±0.2 μmol min-1 mg-1蛋白质），但与其他μ类酶对其他测试底物的活性相当。该蛋白质形成由亚基组成的功能性二聚体，亚基的相对分子质量约为26,400。对在细菌表达系统中产生的GST M4-4与其他人类μ类GSTs（GST M1a-1a、GST M2-2和GST M3-3）的活性与各种底物和抑制剂进行了详细比较。尽管氨基酸序列同一性水平很高，但这些酶的酶学性质却有很大差异。与同源大鼠GST（GST 3-3）的晶体结构比较表明，一些非保守氨基酸残基可归属于GST M4-4的假定活性位点。这表明这些基因在进化过程中的多样化主要发生在底物结合区域，以应对越来越多的外来化合物。
