Structural and functional consequences of inactivation of human glutathione S-transferase P1-1 mediated by the catechol metabolite of equine estrogens, 4-hydroxyequilenin

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, electr ospray ionization mass spectrometric analysis, titration of free thiol grou ps, kinetic studies of irreversible inhibition, and analysis of band patter ns on nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The four cysteines (Cys 14, Cys 47, Cys 101, and Cys 169 in th e primary sequence) in hGST P1-1 are susceptible to electrophilic attack an d/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 r esidues (C47A, C101A, C47A/C101A, C14A/C47A/C101A, and C47A/C101A/C169A mut ants). Mutation of Cys 47 decreased the affinity for the substrate GSH but not for the cosubstrate 1-chloro-2,4-dinitrobenzene (CDNB). However, the Cy s 47 mutation did not significantly affect the rate of catalysis since V-ma x values of the mutants were similar or higher compared to that of wild typ e. Electrospray ionization mass spectrometric analyses of wild-type and mut ant enzymes treated with 4-OHEN showed that a single molecule of 4-OHEN-o-q uinone 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 non reducing SDS-PAGE and protection of 4-OHEN-mediated enzyme inhibition by fr ee radical scavengers. The studies of thief group titration and irreversibl e 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 mo dification of cysteine residues and (2) oxidative damage leading to protein s inactivated by disulfide bond formation.