Structural and functional consequences of inactivation of human glutathione S-transferase P1-1 mediated by the catechol metabolite of equine estrogens, 4-hydroxyequilenin
Structural and functional consequences of inactivation of human glutathione S-transferase P1-1 mediated by the catechol metabolite of equine estrogens, 4-hydroxyequilenin
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DOI：
10.1021/bi002513o
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发表时间：
2001-04-17
期刊：
影响因子：
2.9
通讯作者：
Bolton, JL
中科院分区：
文献类型：
--
作者：
Chang, MS;Shin, YG;Bolton, JL
人谷胱甘肽S-转移酶P1-1的失活机制采用定点突变、电喷雾质谱分析、巯基滴定、不可逆抑制动力学研究非还原性十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）带型分析。四个半胱氨酸为了研究半胱氨酸残基在4-OHEN介导的该酶失活中的作用，将半胱氨酸残基中的一个或组合替换为丙氨酸残基（C47 A、C101 A、C47 A/C101 A、C14 A/C47 A/C101 A和C47 A/C101 A/C169 A突变体）。突变的Cys 47降低了对底物GSH的亲和力，但不降低对共底物1-氯-2，4-二硝基苯（CDNB）的亲和力。然而，Cys 47突变没有显著影响催化速率，因为突变体的V-max值与野生型的V-max值相似或更高。用4-OHEN处理的野生型和突变型酶的电喷雾电离质谱分析显示，除了C14 A/C47 A/C101 A突变体没有检测到共价加合物之外，单个分子的4-OHEN-邻醌附着于蛋白质。4-OHEN还引起氧化损伤，如在非还原性SDS-PAGE上二硫键结合的物质的出现和自由基清除剂对4-OHEN介导的酶抑制的保护所证明的。盗基滴定和不可逆动力学实验的研究表明，不同的半胱氨酸具有不同的反应性4-OHEN; Cys 47是最具反应性的巯基，而Cys 169是耐修饰。这些结果表明，hGST P1-1通过两种可能的机制被4-OHEN灭活：（1）半胱氨酸残基的共价修饰和（2）氧化损伤，导致蛋白质通过二硫键形成而灭活。
The inactivation mechanism(s) of human glutathione S-transferase P1-1 (hGST P1-1) by the catechol metabolite of Premarin estrogens, 4-hydroxyequilenin (4-OHEN), was (were) studied by means of site-directed mutagenesis, electrospray ionization mass spectrometric analysis, titration of free thiol groups, kinetic studies of irreversible inhibition, and analysis of band patterns on nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The four cysteines (Cys 14, Cys 47, Cys 101, and Cys 169 in the primary sequence) in hGST P1-1 are susceptible to electrophilic attack and/or oxidative damage leading to loss of enzymatic activity, To investigate the role of cysteine residues in the 4-OHEN-mediated inactivation of this enzyme, one or a combination of cysteine residues was replaced by alanine residues (C47A, C101A, C47A/C101A, C14A/C47A/C101A, and C47A/C101A/C169A mutants). Mutation of Cys 47 decreased the affinity for the substrate GSH but not for the cosubstrate 1-chloro-2,4-dinitrobenzene (CDNB). However, the Cys 47 mutation did not significantly affect the rate of catalysis since V-max values of the mutants were similar or higher compared to that of wild type. Electrospray ionization mass spectrometric analyses of wild-type and mutant enzymes treated with 4-OHEN showed that a single molecule of 4-OHEN-o-quinone attached to the proteins, with the exception of the C14A/C47A/C101A mutant where no covalent adduct was detected. 4-OHEN also caused oxidative damage as demonstrated by the appearance of disulfide-bonded species on nonreducing SDS-PAGE and protection of 4-OHEN-mediated enzyme inhibition by free radical scavengers. The studies of thief group titration and irreversible kinetic experiments indicated that the different cysteines have distinct reactivity for 4-OHEN; Cys 47 was the most reactive thiol group whereas Cys 169 was resistant to modification. These results demonstrate that hGST P1-1 is inactivated by 4-OHEN through two possible mechanisms: (1) covalent modification of cysteine residues and (2) oxidative damage leading to proteins inactivated by disulfide bond formation.