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Mass Spectral Characterization of Dichloroacetic Acid-Modified Human Glutathione Transferase Zeta Wayne B. Anderson,† Daniel C. Liebler,‡ Philip G. Board,§ and M. W. Anders*,† Department of Pharmacology and Physiology, University of Rochester Medical Center, 601 Elmwood Avenue, Box 711, Rochester, New York 14642, Southwest Environmental Health Sciences Center, College of Pharmacy, University of Arizona, Tucson, Arizona 85721, and Molecular Genetics Group, Division of Molecular Medicine, John Curtin School of Medical Research, Australian National University, Canberra ACT 2601, Australia Received May 6, 2002
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 R-haloalkanoic acids. GSTZ1-1-catalyzed biotransformation of fluorine-lacking R,R-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) × 102, and the kcat 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 ∼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 [1-14C]DCA binding to C16A hGSTZ1c1c. 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.
Introduction Glutathione transferases catalyze the conjugation of glutathione with a range of electrophilic xenobiotics (1, 2). The formation of glutathione S-conjugates may be associated with either detoxication or bioactivation to reactive intermediates (3). Several cytosolic and microsomal classes of glutathione transferases are known (1). Glutathione transferase zeta (GSTZ1-1) is an evolutionarily conserved class of cytosolic glutathione transferases that was first identified in 1997 (4). Four allelic variants of hGSTZ1-1 (1a-1a, 1b-1b, 1c-1c, and 1d-1d)1 have been identified (5, 6), and hGSTZ1c-1c is the most frequent. A fifth allelic variant, hGSTZ1e-1e, may also exist (6). GSTZ1-1 is identical with maleylacetoacetate isomerase (7) and catalyzes the cis-trans isomerization of maley* Address correspondence to this author at the Department of Pharmacology and Physiology, University of Rochester Medical Center, 601 Elmwood Ave., Box 711, Rochester, NY 14642. Telephone: 585275-1678; Fax: 585-273-2652; Email:
[email protected]. † University of Rochester Medical Center. ‡ University of Arizona. § Australian National University. 1 Abbreviations: DCA, dichloroacetic acid; CFA, chlorofluoroacetic acid; hGSTZ1c-1c, human glutathione transferase zeta variant c; TCEP, tris(2-carboxyethyl)phosphine; MALDI-TOF MS, matrix-assisted laser-desorption-ionization time-of-flight mass spectrometry; MS3, MS/MS/MS.
lacetoacetate to fumarylacetoacetate, the penultimate step in the tyrosine degradation pathway (8). GSTZ1-1 has little activity with a range of model GST substrates (4), but catalyzes the biotransformation of many R-haloalkanoic acids (9). GSTZ1-1 converts R-monohaloalkanoic acids to stable glutathione conjugates and dihaloacetic acids to glyoxylate (9). Glyoxylate may be oxidized to oxalate, reduced to glycolate, transaminated to glycine, or metabolized to carbon dioxide (10). Several R-haloalkanoic acids, including bromochloroacetic acid and dichloroacetic acid (DCA), are drinking water disinfection byproducts (11). DCA may be present in drinking water at concentrations up to 160 µg/L (12), which corresponds to an approximate DCA exposure of 4 µg/kg/day (13). DCA is a metabolite of tetrachloroethene, trichloroethene, trichloroacetic acid, and chloral hydrate (14-16). DCA is hepatocarcinogenic in rats (17) and mice (18). DCA is toxic to the testes and the kidney and causes a reversible peripheral neuropathy [for a review, see (13)]. DCA activates pyruvate dehydrogenase secondary to its inhibition of pyruvate dehydrogenase kinase (19) and is used for the clinical management of congenital and acquired forms of lactic acidosis (20). Fluorine-lacking R,R-dihaloalkanoic acids are mechanism-based inactivators of GSTZ1-1 (21, 22). The rate of inactivation (kinact) of the polymorphic forms of hGSTZ1-1
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follows the order: 1a-1a < 1b-1b = 1c-1c = 1d-1d (22). Mechanism-based inactivation of GSTZ1-1 by DCA is associated with covalent modification and degradation of the enzyme in vivo (21). Covalent modification of hGSTZ1-1 is characterized both by the glutathionedependent binding of DCA and by the DCA-dependent binding of glutathione to the protein (22). C16A hGSTZ1c1c catalyzes the biotransformation of DCA, but is resistant to inactivation by DCA (23). 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. DCA-inactivated hGSTZ1c-1c was analyzed by a combination of MALDI-TOF MS, LC/MS/MS, substitution mutation, and [1-14C]DCA binding analyses. The results indicated a single site of DCA modification at the active site cysteine-16 residue. The structure and location of the modification account for the observed inactivation of hGSTZ1-1 by DCA.
Experimental Procedures Materials. DCA (>99% pure), sodium glyoxylate (>99% pure), sinapinic acid, R-cyano-4-hydroxycinnamic acid, glutathione, bovine serum albumin, phenylhydrazine, potassium ferricyanide, mono- and dibasic potassium phosphate, and iodoacetamide were purchased from Sigma-Aldrich-Fluka (Milwaukee, WI). TCEP hydrochloride was purchased from Pierce (Rockford, IL). [1-14C]DCA (57 mCi/mmol) was obtained from American Radiolabeled Chemicals (St. Louis, MO) and was diluted 10-fold with nonradiolabeled DCA to give 5.7 mCi/mmol specific activity. [14C]Methylated molecular-weight markers were obtained from Amersham Biosciences (Piscataway, NJ). CFA was prepared by acid hydrolysis of chlorofluoroacetic acid ethyl ester (PCR Inc., Gainesville, FL), as previously described (21). Dithiothreitol was purchased from Eastman Kodak (Rochester, NY). Sequencing-grade trypsin was obtained from Roche Molecular Biochemicals (Indianapolis, IN) and from Promega (Madison, WI). Dye-binding reagent for Bradford protein analysis was purchased from Bio-Rad (Hercules, CA). HPLC-grade water was purchased from Burdick and Jackson (Muskegon, MI). HPLC-grade acetonitrile and TFA were obtained from J. T. Baker (Philipsburg, NJ). Other reagents were purchased from commercial suppliers. Expression and Purification of Recombinant hGSTZ1c1c and C16A hGSTZ1c-1c. Recombinant 6X N-terminal Histagged hGSTZ1c-1c and 6X N-terminal His-tagged C16A hGSTZ1c-1c were expressed in Escherichia coli and purified by nickel affinity chromatography, as previously described (5, 22). A mammalian protease inhibitor cocktail (Sigma) was added immediately prior to cell lysis to reduce proteolytic degradation of the enzymes. [The bacterial protease inhibitor cocktail (Sigma) was not used because it contains EDTA that interferes with nickel affinity chromatography.] The enzymes were stored at -20 °C in 20 mM potassium phosphate buffer (pH 7.4) containing 50 mM sodium chloride, 0.5 mM EDTA, 1.5 mM dithiothreitol, and 10% glycerol. Enzyme purity, as assessed by SDS-PAGE with Coomassie R-250 staining, was >95% (see Figure 8A). A protein with a molecular mass of ∼50 kDa was identified as the hGSTZ1c-1c dimer by Western blot analysis with rabbit anti-rGSTZ1-1 antibody (data not shown). Determination of Enzyme Concentration. hGSTZ1c-1c and C16A hGSTZ1c-1c concentrations were determined by the method of Bradford (24) with Bio-Rad dye-binding reagent and bovine serum albumin as the standard. The concentration of the hGSTZ1c-1c stock solution was also determined from the absorbance at 280 nm. A portion of the enzyme stock solution was dialyzed against 0.1 M potassium phosphate (pH 7.4) and diluted 10-fold with 0.1 M potassium phosphate (pH 7.4). The
Anderson et al. concentration of the diluted stock solution was determined by the Bradford method and by measuring the absorbance at 280 nm. A molar extinction coefficient of 19 940 M-1 cm-1 was predicted from the sequence of the recombinant enzyme (all cysteine residues in reduced form) based on the method of Pace et al. (25) and employed in the A280 calculations. The exact mass (25 351 daltons) of the recombinant 6X N-terminal His-tagged hGSTZ1c-1c was used for conversion to molar concentration. Inactivation of hGSTZ1c-1c and C16A hGSTZ1c-1c by DCA. hGSTZ1c-1c and C16A hGSTZ1c-1c were incubated with glutathione (1 mM) and DCA in 0.1 M potassium phosphate (pH 7.4) for 2 h at 37 °C. Enzyme and DCA concentrations are indicated in the figure legends. Control reaction mixtures lacked DCA. For some experiments, [1-14C]DCA (5.7 mCi/mmol) was used. To assess inhibition by glyoxylate, sodium glyoxylate (0100 DCA molar excess, 0-94.7 mM) was included in reaction mixtures that contained glutathione (1 mM), DCA (0.95 mM), and hGSTZ1c-1c (20 µg/mL). DCA stock solutions were brought to pH 6.5-7.5 with sodium hydroxide. The pH of the reaction mixture was measured at the highest DCA and sodium glyoxylate concentrations and did not differ from the buffer pH. For the DCA-concentration dependency (Figure 1) and glyoxylate inhibition (Figure 3) experiments, each replicate reaction mixture series was prepared with a different DCA or sodium glyoxylate stock solution. Reaction mixtures were prepared and kept on ice, initiated by addition of DCA, and immediately transferred to a 37 °C water bath. The incubation time [2 h or ∼12 inactivation half-lives (22)] was chosen to ensure either complete inactivation of the enzyme or complete consumption of the substrate. Unbound substrate and product were removed immediately after incubation by ultrafiltration at 4 °C with YM10 ultrafiltration devices (Microcon, Centricon, or Centriplus depending on the reaction mixture volume; Millipore, Bedford, MA) with three dilution cycles. Dilutions were carried out with a volume of 0.1 M potassium phosphate (pH 7.4) equal to or greater than the initial reaction volume. For any given experiment, ultrafiltration retentate volumes were determined gravimetrically and adjusted to equal volume by addition of 0.1 M potassium phosphate (pH 7.4) prior to quantification of protein concentrations. Protein recovery from the ultrafiltration devices was 65-95% and varied depending on the initial protein concentration, reaction volume, and the type of ultrafiltration device employed. For the stoichiometry of the [1-14C]DCA binding experiment (Figure 4A), ultrafiltration retentates were dialyzed overnight with Slide-A-Lyzer Mini-Dialysis devices (3000 molecular-weight cut-off; Pierce, Rockford, IL) against 1 L of 0.1 M potassium phosphate (pH 7.4) with 2 buffer changes to ensure complete removal of unbound radioactivity. Activity with CFA as Substrate. Residual hGSTZ1c-1c activity was determined by a discontinuous assay (26) with CFA as substrate; 2 or 2.5 µg of enzyme was used per assay. Reactions were quenched 20 min after substrate addition. The concentration of glyoxylate was measured by the spectrometric method of Vogels and Van Der Drift (27). Tryptic Digestion of hGSTZ1c-1c Preparations. Reaction mixtures that contained hGSTZ1c-1c and had been incubated with glutathione or with both glutathione and DCA were ultrafiltered as described to remove unbound substrate and product. The enzymes (0.1-0.5 µg/µL) were incubated with trypsin (0.005-0.02 µg/µL) in 0.1 M ammonium bicarbonate for 14-18 h at 37 °C. The hGSTZ1c-1c/trypsin ratio (w/w) was typically 25 but was maintained between 10 and 50 for all incubations. Lyophilized trypsin was reconstituted in 1 mM HCl or the manufacturer’s resuspension buffer immediately prior to use. Immediately after incubation, the digestion mixtures were lyophilized or dried with a SpeedVac Concentrator (set to low temperature setting; Thermo Savant, Holbrook, NY) and stored at -20 °C until analyzed by HPLC. HPLC Analysis of hGSTZ1c-1c Tryptic Digests. Tryptic digests were reconstituted in a small volume of HPLC starting solvent so that the concentration of hGSTZ1c-1c-derived tryptic peptides was ∼2.5 pmol each/µL. One hundred microliter
Characterization of DCA-Modified hGSTZ1-1 samples (∼250 pmol) were analyzed on a HPLC system (Gilson, Middletown, WI) equipped with a Vydac 218TP52 C18 column (250 × 2.1 mm; 5-µm particle size; 300-Å pore; Grace Vydac, Hesperia, CA) and 200-µL loop. The HPLC system was controlled by Gilson 712 HPLC Controller software (v. 1.30). The column was eluted with a gradient at a flow rate of 0.5 mL/ min. From 0 to 3 min, the column was eluted with 100% Solvent A (2% acetonitrile, 98% water, 0.1% TFA), and at 3.01 min the mobile phase was changed to 5% Solvent B (98% acetonitrile, 2% water, 0.1% TFA). A linear gradient to 45% Solvent B at min 45 was run, and the gradient was then increased to 100% Solvent B at min 60. The absorbance of the eluate was measured at 215 nm (5-nm bandwidth) with a LC-235 diode-array detector (Perkin-Elmer, Shelton, CT). Fractions were collected every minute from 0 to 11 min and from 45 to 60 min and every 30 s from 11 to 45 min. Fractions to be analyzed by MALDI-TOF MS were dried in a SpeedVac Concentrator (set to low temperature setting) and stored at -20 °C until analyzed. Liquid Scintillation Analysis. For the stoichiometry of [1-14C]DCA binding experiment (Figure 4A), 7.5 µg of hGSTZ1c1c from each replicate incubation was analyzed in duplicate. Dialysate corresponding to 7.5 µg of hGSTZ1c-1c was transferred to 6-mL scintillation vials and diluted to 0.5 mL with distilled water. Five milliliters of Ecoscint A scintillation cocktail (National Diagnostics, Atlanta, GA) was added, and the mixture was mixed thoroughly. The samples were analyzed for 2 min each with a Wallac model 1409 liquid scintillation analyzer (Perkin-Elmer Life Sciences, Boston, MA) set to count in DPM mode. An external standard was used to correct for counting efficiency and quench. Background DPM was taken as the DPM determined for the samples incubated in the absence of [1-14C]DCA. Background-corrected DPM values were converted to moles of [1-14C]DCA bound per mole of hGSTZ1c-1c. HPLC fractions (0.25 or 0.5 mL) were mixed with 4 mL of the scintillation cocktail, mixed, and assayed as described above. MALDI-TOF MS Analysis of Intact hGSTZ1c-1c. Intact (undigested) hGSTZ1c-1c samples (3 µg, prepared as described in Figure 1) were incubated in the presence or absence of 50 mM TCEP (in a final volume of 16.8 µL) for 30 min in the dark. After incubation, 5% TFA was added so that the final concentration was 0.5% TFA. The samples were applied to C18 ZipTips (Millipore, Bedford, MA) that had been conditioned according to the manufacturers’ instructions. The bound enzyme samples were desalted by filling and emptying the ZipTips twice with 20 µL of 0.1% TFA, and the bound enzyme was eluted with acetonitrile/water (50:50) containing 10 mg/mL sinapinic acid and 0.1% TFA so that the final enzyme concentration was ∼5 pmol/µL. Two microliters of the eluate was spotted on the MALDI sample plate and allowed to air-dry. MALDI-TOF MS analyses were carried out with a Voyager-DE STR mass spectrometer (Perseptive Biosystems, Foster City, CA) run in positive ion, delayed extraction, and linear modes. Ion extraction delay, grid voltage, and laser intensity were adjusted to achieve optimal resolution, which was typically 300-350 full-width halfmaximum for the peak corresponding to unmodified hGSTZ1c1c. The instrument default calibration setting was used, and
