Glutathione transferase zeta (GSTZ1-1) is widely expressed in eukaryotic species, and four human allelic variants of hGSTZ1-1 have been described. GSTZ1-1 catalyzes the cis-trans isomerization of maleylacetoacetate to fumarylacetoacetate and the biotransformation of a range of alpha-haloalkanoic acids. GSTZ1-1-catalyzed biotransformation of fluorine-lacking alpha,alpha-dihaloalkanoic acids, including dichloroacetic acid (DCA), results in the mechanism-based inactivation and covalent modification of the enzyme. The objective of this study was to investigate further the DCA-induced inactivation of hGSTZ1c-1c and to explore the mechanism of inactivation by characterization of the sites and types of DCA-induced covalent modifications. The partition ratio for the DCA-induced, mechanism-based inactivation of hGSTZ1c-1c was (5.7 +/- 0.5) x 10(2), and the k(cat) for the biotransformation of DCA was 39 min(-)(1). Inactivation of hGSTZ1c-1c in vitro was limited at high enzyme concentrations and was inhibited by glyoxylate. The stoichiometry of DCA binding to hGSTZ1c-1c was approximately 0.5 mol of DCA/mol of enzyme monomer. A single DCA-derived adduct was observed and was assigned to cysteine-16 by a combination of matrix-assisted laser-desorption-ionization time-of-flight and electrospray-ionization quadrupole ion-trap mass spectrometry and by analysis of [1-(14)C]DCA binding to C16A hGSTZ1c-1c. The DCA-derived adduct contained both glutathione and the carbon skeleton of DCA, presumably in a dithioacetal linkage. Also, cysteine-16 formed a mixed disulfide bond with glutathione. These data support a mechanism of inactivation whereby glutathione displaces a chlorine atom from DCA, and cysteine-16 in the enzyme active site displaces the second chlorine atom to result in a covalently modified and inactivated enzyme. These findings explain the DCA-induced inactivation of GSTZ1-1 observed in humans and rats.