Role of the glutamyl α-carboxylate of the substrate glutathione in the catalytic mechanism of human glutathione transferase A1-1
Role of the glutamyl α-carboxylate of the substrate glutathione in the catalytic mechanism of human glutathione transferase A1-1
复制标题
DOI：
10.1021/bi010429i
复制
发表时间：
2001-12-25
期刊：
影响因子：
2.9
通讯作者：
Mannervik, B
中科院分区：
文献类型：
--
作者：
Gustafsson, A;Pettersson, PL;Mannervik, B
谷胱甘肽的谷胱甘肽羧酸盐有助于谷胱甘肽转移酶的催化功能。人谷胱甘肽转移酶A1-1 （GST A1-1）与1-氯-2,4-二硝基苯偶联反应的催化效率降低了15000倍，如果使用谷胱甘肽的脱羧类似物dGSH （GABA-Cys-Gly）作为替代硫醇底物。减少的部分原因是由于酶不能促进dGSH的电离。天然底物谷胱甘肽与GST A1-1结合后，巯基的pK(a)值从9.2降低到6.7。然而，dGSH中缺乏Glu α -羧酸盐使酶促反应中硫醇的pKa值提高到非酶促反应的pKa值。K-M（dGSH）比K-M（GSH）高100倍。活性位点残基Thr68与谷胱甘肽的Glu α -羧酸形成氢键。通过T68E突变将羧酸盐引入GST A1-1，使dGSH的催化效率提高了10倍，并使活性位点结合的dGSH的pK(a)值降低了约1个pH单位。改变的pK(a)值与羧酸盐促进谷胱甘肽巯基电离的催化机制一致。以δ(5)-雄烯-3,17-二酮为底物时，酶的效率降低了24000倍，而以4-硝基氨基醛（NCA）为底物时，酶的效率降低不到150倍。在后一种反应中，NCA接受一个质子，并且与所研究的其他反应不同，它可能不依赖于Glu α -羧酸盐来使巯基去质子化。谷氨酸-羧酸盐的另一个功能可能是在活性位点内产生谷胱甘肽。
The Glu alpha-carboxylate of glutathione contributes to the catalytic function of the glutathione transferases. The catalytic efficiency of human glutathione transferase A1-1 (GST A1-1) in the conjugation reaction with 1-chloro-2,4-dinitrobenzene is reduced 15000-fold if the decarboxylated analogue of glutathione, dGSH (GABA-Cys-Gly), is used as an alternative thiol substrate. The decrease is partially due to an inability of the enzyme to promote ionization of dGSH. The pK(a) value of the thiol group of the natural substrate glutathione decreases from 9.2 to 6.7 upon binding to GST A1-1. However, the lack of the Glu alpha-carboxylate in dGSH raised the pKa value of the thiol in the enzymatic reaction to that of the nonenzymatic reaction. Furthermore, K-M(dGSH) Was 100-fold higher than K-M(GSH). The active-site residue Thr68 forms a hydrogen bond to the Glu alpha-carboxylate of glutathione. Introduction of a carboxylate into GST A1-1 by a T68E mutation increased the catalytic efficiency with dGSH 10-fold and reduced the pK(a) value of the active site bound dGSH by approximately 1 pH unit. The altered pK(a) value is consistent with a catalytic mechanism where the carboxylate contributes to ionization of the glutathione thiol group. With Delta(5)-androstene-3,17-dione as substrate the efficiency of the enzyme is, decreased 24000-fold while with 4-nitrocinnamaldehyde (NCA) the decrease is less than 150-fold. In the latter reaction NCA accepts a proton and, unlike the other reactions studied, may not be dependent on the Glu alpha-carboxylate for deprotonation of the thiol group. An additional function of the Glu alpha-carboxylate may be productive orientation of glutathione within the active site.