Björnestedt R, Stenberg G, Widersten M, Board P G, Sinning I, Jones T A, Mannervik B
Department of Biochemistry, Uppsala University, Biomedical Center, Sweden.
J Mol Biol. 1995 Apr 7;247(4):765-73. doi: 10.1016/s0022-2836(05)80154-8.
Arg15 is a conserved active-site residue in class Alpha glutathione transferases. X-ray diffraction studies of human glutathione transferase A1-1 have shown that N epsilon of this amino acid residue is adjacent to the sulfur atom of a glutathione derivative bound to the active site, suggesting the presence of a hydrogen bond. The phenolic hydroxyl group of Tyr9 also forms a hydrogen bond to the sulfur atom of glutathione, and removal of this hydroxyl group causes partial inactivation of the enzyme. The present study demonstrates by use of site-directed mutagenesis the functional significance of Arg15 for catalysis. Mutation of Arg15 into Leu reduced the catalytic activity by 25-fold, whereas substitution by Lys caused only a threefold decrease, indicating the significance of a positively charged residue at position 15. Mutation of Arg15 into Ala or His caused a substantial reduction of the specific activity (200 or 400-fold, respectively), one order of magnitude more pronounced than the effect of the Tyr9-->Phe mutation. Double mutations involving residues 9 and 15 demonstrated that the effects of mutations at the two positions were additive except for the substitution of His for Arg15, which appeared to cause secondary structural effects. The pKa value of the phenolic hydroxyl of Tyr9 was determined by UV absorption difference spectroscopy and was found to be 8.1 in the wild-type enzyme. The corresponding pKa values of mutants R15K, R15H and R15L were 8.5, 8.7 and 8.8, respectively, demonstrating the contribution of the guanidinium group of Arg15 to the electrostatic field in the active site. Addition of glutathione caused an increased pKa value of Tyr9; this effect was not obtained with S-methylglutathione. These results show that Tyr9 is protonated when glutathione is bound to the enzyme at physiological pH values. The involvement of an Arg residue in the binding and activation of glutathione is a feature that distinguishes class Alpha glutathione transferases from members in other glutathione transferase classes.
精氨酸15是α类谷胱甘肽转移酶中一个保守的活性位点残基。对人谷胱甘肽转移酶A1-1的X射线衍射研究表明，该氨基酸残基的ε-氨基与结合在活性位点的谷胱甘肽衍生物的硫原子相邻，提示存在氢键。酪氨酸9的酚羟基也与谷胱甘肽的硫原子形成氢键，去除该羟基会导致酶部分失活。本研究通过定点诱变证明了精氨酸15对催化作用的功能重要性。将精氨酸15突变为亮氨酸使催化活性降低了25倍，而用赖氨酸替代仅导致三倍的降低，表明15位带正电荷残基的重要性。将精氨酸15突变为丙氨酸或组氨酸导致比活性大幅降低（分别为200倍或400倍），比酪氨酸9突变为苯丙氨酸的效应明显高一个数量级。涉及残基9和15的双突变表明，除了用组氨酸替代精氨酸15似乎会引起二级结构效应外，两个位置的突变效应是相加的。通过紫外吸收差光谱法测定了酪氨酸9酚羟基的pKa值，发现野生型酶中该值为8.1。突变体R15K、R15H和R15L的相应pKa值分别为8.5、8.7和8.8，证明精氨酸15的胍基对活性位点静电场的贡献。添加谷胱甘肽会使酪氨酸9的pKa值升高；用S-甲基谷胱甘肽则未观察到这种效应。这些结果表明，在生理pH值下，当谷胱甘肽与酶结合时，酪氨酸9处于质子化状态。精氨酸残基参与谷胱甘肽的结合和激活是α类谷胱甘肽转移酶与其他谷胱甘肽转移酶类成员相区别的一个特征。
