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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• 作者：Minsun Chang ; Young Geun Shin ; Richard B. van Breemen ; Sylvie Y. Blond ; and Judy L. Bolton
• 刊名：Biochemistry
• 出版年：2001
• 出版时间：April 17, 2001
• 年：2001
• 卷：40
• 期：15
• 页码：4811 - 4820
• 全文大小：138K
• 年卷期：v.40,no.15(April 17, 2001)
• ISSN：1520-4995

文摘

The inactivation mechanism(s) of human glutathione S-transferase P1-1 (hGST P1-1) by thecatechol metabolite of Premarin estrogens, 4-hydroxyequilenin (4-OHEN), was (were) studied by meansof site-directed mutagenesis, electrospray ionization mass spectrometric analysis, titration of free thiolgroups, kinetic studies of irreversible inhibition, and analysis of band patterns on nonreducing sodiumdodecyl 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 attackand/or oxidative damage leading to loss of enzymatic activity. To investigate the role of cysteine residuesin the 4-OHEN-mediated inactivation of this enzyme, one or a combination of cysteine residues wasreplaced by alanine residues (C47A, C101A, C47A/C101A, C14A/C47A/C101A, and C47A/C101A/C169Amutants). Mutation of Cys 47 decreased the affinity for the substrate GSH but not for the cosubstrate1-chloro-2,4-dinitrobenzene (CDNB). However, the Cys 47 mutation did not significantly affect the rateof 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-OHENshowed that a single molecule of 4-OHEN-o-quinone attached to the proteins, with the exception of theC14A/C47A/C101A mutant where no covalent adduct was detected. 4-OHEN also caused oxidative damageas demonstrated by the appearance of disulfide-bonded species on nonreducing SDS-PAGE and protectionof 4-OHEN-mediated enzyme inhibition by free radical scavengers. The studies of thiol group titrationand irreversible kinetic experiments indicated that the different cysteines have distinct reactivity for4-OHEN; Cys 47 was the most reactive thiol group whereas Cys 169 was resistant to modification. Theseresults 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 bydisulfide bond formation.