title: Glutathione transferase zeta-catalyzed bioactivation of dichloroacetic acid: reaction of glyoxylate with amino acid nucleophiles.
abstract: Dichloroacetic acid (DCA) is a drinking water contaminant, a therapeutic agent, and a rodent carcinogen. Glutathione transferase zeta (GSTZ1-1) catalyzes the biotransformation of a range of alpha-haloalkanoates and the cis-trans isomerization of maleylacetoacetate. GSTZ1-1 catalyzes the bioactivation of fluorine-lacking dihaloacetates to S-(alpha-halocarboxymethyl)glutathione, a reactive intermediate that covalently modifies and inactivates the enzyme or is hydrolyzed to glyoxylate. The purpose of this study was to examine the GSTZ1-1-catalyzed bioactivation of DCA, including the reaction of DCA-derived glyoxylate with amino acid nucleophiles and the characterization of the structures and kinetics of adduct formation by LC/MS. The binding of [1-(14)C]DCA-derived label to bovine serum albumin required both GSTZ1-1 and GSH, whereas the binding to dialyzed rat liver cytosolic protein was increased in the presence of GSH. Studies with model peptides (antiflammin-2 and IL-8 inhibitor) indicated that glyoxylate, rather than S-(alpha-chlorocarboxymethyl)glutathione, was the reactive species that modified amino acid nucleophiles. Both addition (+74 Da) and addition-elimination (+56 Da) adducts of glyoxylic acid were observed. Addition adducts (+74 Da) could not be characterized completely by mass spectrometry, whereas addition-elimination adducts (+56 Da) were characterized as 2-carboxy-4-imidazolidinones. 2-Carboxy-4-imidazolidinones were formed by the rapid equilibrium reaction of glyoxylate with the N-terminal amino group of antiflammin-2 to give an intermediate carbinolamine (K(eq) = 0.63 mM(-1)), which slowly eliminated water to give an intermediate imine (k(2) = 0.067 hour(-1)), which rapidly cyclized to give the 2-carboxy-4-imidazolidinone. Glucose 6-phosphate dehydrogenase was inactivated partially by glyoxylate when reactants were reduced with sodium borodeuteride, which may indicate that glyoxylate reacts with selective lysine epsilon-amino groups. The results of the present study demonstrate that GSTZ1-1 catalyzes the bioactivation of DCA to the reactive metabolite glyoxylate. The reaction of glyoxylate with cellular macromolecules may be associated with the multiorgan toxicity of DCA.
authors.affiliation: Department of Pharmacology and Physiology, University of Rochester Medical Center, 601 Elmwood Avenue, Box 711, Rochester, New York 14642, USA.
authors.forename: Wayne B
authors.initials: WB
authors.lastname: Anderson
authors.forename: Philip G
authors.initials: PG
authors.lastname: Board
authors.forename: M W
authors.initials: MW
authors.lastname: Anders
mesh_terms: D000090:Acetoacetates / Q000378:metabolism; D000588:Amines / Q000378:metabolism; D000596:Amino Acids / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D001711:Biotransformation; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D003999:Dichloroacetic Acid / Q000378:metabolism*; D005982:Glutathione Transferase / Q000378:metabolism*; D006016:Glycolates / Q000737:chemistry / Q000378:metabolism*; D006868:Hydrolysis; D007536:Isomerism; D007700:Kinetics; D008099:Liver / Q000378:metabolism; D008239:Lysine / Q000737:chemistry; D013058:Mass Spectrometry; D009842:Oligopeptides / Q000494:pharmacology; D010446:Peptide Fragments / Q000494:pharmacology; D051381:Rats
chemical_list: D000090:Acetoacetates; D000588:Amines; D000596:Amino Acids; D006016:Glycolates; D009842:Oligopeptides; D010446:Peptide Fragments; C064647:antiflammin P2; D003999:Dichloroacetic Acid; C498692:Gstz1 protein, rat; D005982:Glutathione Transferase; D008239:Lysine