Sluis-Cremer N, Naidoo N, Dirr H
Department of Biochemistry, University of the Witwatersrand, Johannesburg, South Africa.
Eur J Biochem. 1996 Dec 1;242(2):301-7. doi: 10.1111/j.1432-1033.1996.0301r.x.
The oxidation of protein sulphydryls to disulphides and their reduction back to sulphydryls is an early cellular response to oxidative stress. Hydrogen-peroxide-mediated oxidation of class-pi glutathione S-transferase results in the formation of disulphide bonds, which inhibits its catalytic function yet allows it to retain its non-substrate-ligand-binding properties. The overall hydrodynamic volume of the oxidised class-pi glutathione S-transferase type-1 homodimer (GSTP1-1) is decreased, and its tertiary-structural and secondary-structural elements are changed with respect to the native protein. Structural differences appear to be prominent in domain 1 of oxidised GSTP1-1, in that the exposure of both tryptophan residues is increased, while the electric potential about one of them is altered. Treatment of the oxidised protein with dithiothreitol or glutathione restores its enzymatic capabilities, albeit with lower specific activities for 1-chloro-2,4-dinitrobenzene and ethacrynic acid. The hydrophobic binding site (H-site) for electrophilic substrates is negatively affected in that the K(m) and catalytic-efficiency values are diminished significantly with respect to those values obtained for the native protein. The dithiothreitol-treated oxidised GSTP1-1 is able to regain its overall hydrodynamic volume; however, both its secondary-structural and tertiary-structural elements remain modified with respect to the native protein, as do both tryptophanyl environments. Furthermore, the oxidised glutathione S-transferase and dithiothreitol-treated oxidised glutathione S-transferase are less thermostable (tM = 55.5 degrees C and 56.3 degrees C, respectively) than the native enzyme (tM = 59 degrees C). These results indicate that the class-pi glutathione S-transferase is unable to regain its native conformation after oxidative inactivation.
蛋白质巯基氧化为二硫键以及它们还原回巯基是细胞对氧化应激的早期反应。过氧化氢介导的π类谷胱甘肽S-转移酶氧化导致二硫键形成，这抑制了其催化功能，但使其保留非底物配体结合特性。氧化的π类谷胱甘肽S-转移酶1型同二聚体（GSTP1-1）的整体流体动力学体积减小，其三级结构和二级结构元件相对于天然蛋白质发生了变化。氧化的GSTP1-1的结构差异在结构域1中似乎很突出，因为两个色氨酸残基的暴露增加，而其中一个的电势发生了改变。用二硫苏糖醇或谷胱甘肽处理氧化的蛋白质可恢复其酶活性，尽管对1-氯-2,4-二硝基苯和依他尼酸的比活性较低。亲电子底物的疏水结合位点（H-位点）受到负面影响，因为相对于天然蛋白质获得的值，Km和催化效率值显著降低。二硫苏糖醇处理的氧化GSTP1-1能够恢复其整体流体动力学体积；然而，其二级结构和三级结构元件相对于天然蛋白质仍然被修饰，两个色氨酸环境也是如此。此外，氧化的谷胱甘肽S-转移酶和二硫苏糖醇处理的氧化谷胱甘肽S-转移酶的热稳定性低于天然酶（分别为tM = 55.5℃和56.3℃，天然酶tM = 59℃）。这些结果表明，π类谷胱甘肽S-转移酶在氧化失活后无法恢复其天然构象。
