Widersten M, Björnestedt R, Mannervik B
Department of Biochemistry, Uppsala University, Sweden.
Biochemistry. 1996 Jun 18;35(24):7731-42. doi: 10.1021/bi9601619.
The present study proposes the participation of both carboxylate groups of the glutathione molecule as functional entities in the catalytic apparatus of human glutathione transferase (GST) A1-1. Functional studies in combination with structural data provide evidence for the alpha-carboxylate of the Glu residue of glutathione acting as a proton acceptor in the catalytic mechanism. The Glu carboxylate is hydrogen-bonded to a protein hydroxyl group and a main-chain NH, as well as to a water molecule of low mobility in the active site region. The Glu alpha-carboxylate of glutathione is bound in a similar manner to the active sites of mammalian glutathione transferases of classes Alpha, Mu, and Pi, for which three-dimensional structures are known. Mutation of the hydroxyl group that is hydrogen-bonded to the alpha-carboxylate of the Glu residue of glutathione (Thr68->Val) caused a shift of the pH dependence of the enzyme-catalyzed reaction, suggesting that the acidic limb of the pH-activity profile reflects the ionization of the carboxylate of the Glu residue of glutathione. The second carboxylate group of glutathione, which is part of its Gly residue, interacts with two Arg side chains in GST A1-1. One of these residues (Arg45) may influence an ionic interaction (Arg221/Asp42), which appears to contribute to binding of the second substrate by fixing the C-terminal alpha-helix as a lid over the active site. Removal of the Gly residue from the glutathione molecule caused a 13-fold increase in the KM value for the electrophilic substrate. Thus, the Gly carboxylate of glutathione, by way of influencing the topology of the active site, contributes to the binding of the second substrate of the enzyme. Consequently, the glutathione molecule has several functions in the glutathione transferase catalyzed reactions, not only as a substrate providing the thiol group for different types of chemical reactions but also as a substrate contributing a carboxylate that acts as a proton acceptor in the catalytic mechanism and a carboxylate that modulates binding of the second substrate to the enzyme.
本研究提出，谷胱甘肽分子的两个羧基作为功能实体参与了人谷胱甘肽转移酶（GST）A1-1的催化机制。功能研究与结构数据相结合，为谷胱甘肽中Glu残基的α-羧基在催化机制中作为质子受体提供了证据。Glu羧基与一个蛋白质羟基、一个主链NH以及活性位点区域中低迁移率的水分子形成氢键。谷胱甘肽的Gluα-羧基以类似方式与已知三维结构的α、μ和π类哺乳动物谷胱甘肽转移酶的活性位点结合。与谷胱甘肽中Glu残基的α-羧基形成氢键的羟基发生突变（Thr68→Val），导致酶催化反应的pH依赖性发生偏移，这表明pH-活性曲线的酸性部分反映了谷胱甘肽中Glu残基羧基的电离。谷胱甘肽的第二个羧基是其Gly残基的一部分，在GST A1-1中与两个Arg侧链相互作用。其中一个残基（Arg45）可能影响一种离子相互作用（Arg221/Asp42），这种相互作用似乎通过将C末端α-螺旋固定为活性位点上方的盖子来促进第二种底物的结合。从谷胱甘肽分子中去除Gly残基会使亲电子底物的KM值增加13倍。因此，谷胱甘肽的Gly羧基通过影响活性位点的拓扑结构，有助于酶的第二种底物的结合。因此，谷胱甘肽分子在谷胱甘肽转移酶催化的反应中有多种功能，不仅作为为不同类型化学反应提供硫醇基团的底物，还作为在催化机制中作为质子受体的羧基以及调节第二种底物与酶结合的羧基的底物。
