Structural and functional consequences of inactivation of human glutathione S-transferase P1-1 mediated by the catechol metabolite of equine estrogens, 4-hydroxyequilenin
- Authors
- 장민선; Shin, YG; van Breemen, RB; Blond, SY; Bolton, JL
- Issue Date
- Apr-2001
- Publisher
- American Chemical Society
- Keywords
- SITE-DIRECTED MUTAGENESIS; CLASS-PI; CYSTEINE RESIDUES; 3-DIMENSIONAL STRUCTURE; CATALYTIC MECHANISM; HYDROGEN-PEROXIDE; SULFHYDRYL-GROUP; OXYGEN RADICALS; PROTEIN DAMAGE; DISULFIDE BOND
- Citation
- Biochemistry, v.40, no.15, pp 4811 - 4820
- Pages
- 10
- Journal Title
- Biochemistry
- Volume
- 40
- Number
- 15
- Start Page
- 4811
- End Page
- 4820
- URI
- https://scholarworks.sookmyung.ac.kr/handle/2020.sw.sookmyung/149622
- DOI
- 10.1021/bi002513o
- ISSN
- 0006-2960
- Abstract
- 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.
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