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, 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 thiol 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.
Chang
M
M
Department of Medicinal Chemistry and Pharmacognosy (M/C 781), College of Pharmacy, University of Illinois at Chicago, 833 South Wood Street, Chicago, Illinois 60612-7231, USA.
Shin
Y G
YG
van Breemen
R B
RB
Blond
S Y
SY
Bolton
J L
JL
D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D003545:Cysteine / Q000235:genetics; D004220:Disulfides / Q000378:metabolism; D004228:Dithionitrobenzoic Acid / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000494:pharmacology*; D004855:Equilenin / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology*; D002393:Estrogens, Catechol / Q000378:metabolism*; D016166:Free Radical Scavengers / Q000494:pharmacology; D051549:Glutathione S-Transferase pi; D005982:Glutathione Transferase / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D006639:Histidine / Q000235:genetics; D006736:Horses; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D019163:Reducing Agents / Q000378:metabolism; D013329:Structure-Activity Relationship; D013438:Sulfhydryl Compounds / Q000378:metabolism; D013439:Sulfhydryl Reagents / Q000378:metabolism; D017186:Titrimetry
D004220:Disulfides; D004791:Enzyme Inhibitors; D002393:Estrogens, Catechol; D016166:Free Radical Scavengers; D007527:Isoenzymes; D011994:Recombinant Proteins; D019163:Reducing Agents; D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents; D006639:Histidine; C065250:4-hydroxy-equilenin; D004228:Dithionitrobenzoic Acid; C496556:GSTP1 protein, human; D051549:Glutathione S-Transferase pi; D005982:Glutathione Transferase; D003545:Cysteine; D004855:Equilenin