Sluis-Cremer N, Dirr H
Department of Biochemistry, University of the Witwatersrand, Johannesburg, South Africa.
FEBS Lett. 1995 Sep 4;371(2):94-8. doi: 10.1016/0014-5793(95)00826-u.
A highly reactive cysteine residue in class pi glutathione S-transferases enhances their susceptibility to chemical alkylation and oxidative stress. Alkylation of the reactive Cys45 in the porcine class pi enzyme (pGSTP1-1) with either N-iodoacetyl-N'-(5-sulpho-1-naphthyl)ethylenediamine or iodoacetamide results in a loss of enzyme activity and glutathione-binding function. Similarly, oxidation of pGSTP1-1 with hydrogen peroxide (H2O2) also results in a loss of catalytic and glutathione-binding function, but these effects are reversed by the addition of 5 mM glutathione or dithiothreitol. Analysis by SDS-PAGE of the H2O2-oxidised enzyme indicates oxidation-induced formation of disulphide bonds involving Cys45. Equilibrium-unfolding studies with guanidinium chloride indicate that the unfolding of Cys45-alkylated and H2O2-oxidised pGSTP1-1 can be described by a two-state model in which the predominant thermodynamically stable species are the folded dimer and unfolded monomer. Unfolding transition curves suggest that the introduction of a large and bulky AEDANS at Cys45 does not affect the unfolding pathway for pGSTP1-1. H2O2-oxidised pGSTP1-1, on the other hand, appears to follow a different unfolding pathway. This appears not to be a result of the introduction of disulphide bonds since the reduction of these bonds in the oxidised protein with dithiothreitol does not affect the unfolding transition. Furthermore, the conformational stability of the oxidised protein is significantly diminished (delta G(H2O) = 11.6 kcal/mol) when compared with unmodified and AEDANS-alkylated enzyme (delta G(H2O) = 22.5 kcal/mol).
π类谷胱甘肽S-转移酶中一个高反应性半胱氨酸残基增强了它们对化学烷基化和氧化应激的敏感性。用N-碘乙酰基-N'-(5-磺基-1-萘基)乙二胺或碘乙酰胺对猪π类酶（pGSTP1-1）中反应性半胱氨酸45进行烷基化，会导致酶活性和谷胱甘肽结合功能丧失。同样，用过氧化氢（H2O2）氧化pGSTP1-1也会导致催化和谷胱甘肽结合功能丧失，但添加5 mM谷胱甘肽或二硫苏糖醇可逆转这些影响。对H2O2氧化的酶进行SDS-PAGE分析表明，氧化诱导形成了涉及半胱氨酸45的二硫键。用氯化胍进行的平衡去折叠研究表明，半胱氨酸45烷基化和H2O2氧化的pGSTP1-1的去折叠可以用两态模型来描述，其中主要的热力学稳定物种是折叠的二聚体和未折叠的单体。去折叠转变曲线表明，在半胱氨酸45处引入大而庞大的5-二甲氨基萘-1-磺酰基（AEDANS）不影响pGSTP1-1的去折叠途径。另一方面，H2O2氧化的pGSTP1-1似乎遵循不同的去折叠途径。这似乎不是引入二硫键的结果，因为用二硫苏糖醇还原氧化蛋白中的这些键不会影响去折叠转变。此外，与未修饰和AEDANS烷基化的酶（ΔG(H2O)=22.5 kcal/mol）相比，氧化蛋白的构象稳定性显著降低（ΔG(H2O)=11.6 kcal/mol）。
