Vargo M A, Colman R F
Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716, USA.
J Biol Chem. 2001 Jan 19;276(3):2031-6. doi: 10.1074/jbc.M008212200. Epub 2000 Oct 12.
Rat liver glutathione S-transferase, isozyme 1-1, catalyzes the glutathione-dependent isomerization of Delta(5)-androstene-3,17-dione and also binds steroid sulfates at a nonsubstrate inhibitory steroid site. 17beta-Iodoacetoxy-estradiol-3-sulfate, a reactive steroid analogue, produces a time-dependent inactivation of this glutathione S-transferase to a limit of 60% residual activity. The rate constant for inactivation (k(obs)) exhibits a nonlinear dependence on reagent concentration with K(I) = 71 microm and k(max) = 0.0133 min(-1). Complete protection against inactivation is provided by 17beta-estradiol-3,17-disulfate, whereas Delta5-androstene-3,17-dione and S-methylglutathione have little effect on k(obs). These results indicate that 17beta-iodoacetoxy-estradiol-3-sulfate reacts as an affinity label of the nonsubstrate steroid site rather than of the substrate sites occupied by Delta5-androstene-3,17-dione or glutathione. Loss of activity occurs concomitant with incorporation of about 1 mol 14C-labeled reagent/mol enzyme dimer when the enzyme is maximally inactivated. Isolation of the labeled peptide from the chymotryptic digest shows that Cys(17) is the only enzymic amino acid modified. Covalent modification of Cys(17) by 17beta-iodoacetoxy-estradiol-3-sulfate on subunit A prevents reaction of the steroid analogue with subunit B. These results and examination of the crystal structure of the enzyme suggest that the interaction between the two subunits of glutathione S-transferase 1-1, and the electrostatic attraction between the 3-sulfate of the reagent and Arg(14) of subunit B, are important in binding steroid sulfates at the nonsubstrate steroid binding site and in determining the specificity of this affinity label.
大鼠肝脏谷胱甘肽 S-转移酶同工酶 1-1 催化 Δ⁵-雄烯-3,17-二酮的谷胱甘肽依赖性异构化反应，并且还在非底物抑制性类固醇位点结合类固醇硫酸盐。17β-碘乙酰氧基雌二醇-3-硫酸盐是一种反应性类固醇类似物，它会使这种谷胱甘肽 S-转移酶发生时间依赖性失活，残余活性限制在 60%。失活速率常数（k(obs)）对试剂浓度呈非线性依赖，K(I) = 71 μM，k(max) = 0.0133 min⁻¹。17β-雌二醇-3,17-二硫酸盐可完全保护酶不被失活，而 Δ⁵-雄烯-3,17-二酮和 S-甲基谷胱甘肽对 k(obs) 影响很小。这些结果表明，17β-碘乙酰氧基雌二醇-3-硫酸盐作为非底物类固醇位点的亲和标记物起反应，而不是与由 Δ⁵-雄烯-3,17-二酮或谷胱甘肽占据底物位点发生反应。当酶被最大程度失活时，活性丧失与每摩尔酶二聚体掺入约 1 摩尔¹⁴C 标记试剂同时发生。从胰凝乳蛋白酶消化产物中分离出标记肽表明，半胱氨酸（Cys）(17) 是唯一被修饰的酶促氨基酸。17β-碘乙酰氧基雌二醇-3-硫酸盐对亚基 A 上的 Cys(17) 进行共价修饰可阻止类固醇类似物与亚基 B 反应。这些结果以及对该酶晶体结构的研究表明，谷胱甘肽 S-转移酶 1-1 两个亚基之间的相互作用，以及试剂的 3-硫酸盐与亚基 B 的精氨酸（Arg）(14) 之间的静电吸引，对于在非底物类固醇结合位点结合类固醇硫酸盐以及确定这种亲和标记物的特异性很重要。
