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Kinetics of the Biotransformation of Maleylacetone and Chlorofluoroacetic Acid by Polymorphic Variants of Human Glutathione Transferase Zeta (hGSTZ1-1) Hoffman B. M. Lantum,† 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, and Molecular Genetics Group, Division of Molecular Medicine, John Curtin School of Medical Research, Australian National University, Canberra ACT 2601, Australia Received May 21, 2001
Glutathione transferase zeta (GSTZ1-1) catalyzes the cis-trans isomerization of maleylacetoacetate and the biotransformation of a range of R-haloacids. The objective of this study was to determine the kinetics of the biotransformation of maleylacetone (MA), an analogue of the natural substrate maleylacetoacetate, and chlorofluoroacetic acid (CFA) by polymorphic variants of recombinant hGSTZ1-1. The kcat of the four variants of hGSTZ1-1 with MA as the substrate followed the order: 1c-1c > 1b-1b > 1d-1d > 1a-1a whereas the kcat for the biotransformation of CFA followed the order: 1a-1a > 1b-1b ∼ 1c-1c ∼ 1d-1d. The turnover rates of MA were much higher than those of CFA for each variant and ranged from 22-fold (1a-1a) to 980-fold differences (1c-1c). The catalytic efficiencies of hGSTZ1-1 variants with MA as the substrate were much greater than those with CFA as the substrate, but little difference among the polymorphic variants was observed. MA was a mixed inhibitor of all variants with CFA as substrate: the mean competitive inhibition constant (KicMA) for all variants was about 100 µM, and the mean uncompetitive inhibition constant (KiuMA) was about 201 µM. Hence, MA and R-haloacids apparently compete for the same active site on the enzyme. DCA-induced inactivation of the four variants showed that the inactivated enzymes show markedly reduced isomerase activities. The residual activities were different for each variant: 1a-1a (12%) > 1b-1b ∼ 1c-1c ∼ 1d-1d ( 1d-1d > 1a-1a (6). MAA has been prepared enzymatically (7), but apparently not by chemical synthesis, and its high lability has * 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; E-mail:
[email protected]. † University of Rochester Medical Center. ‡ Australian National University. 1 Abbreviations: MAA, maleylacetoacetate; FAA, fumarylacetoacetate; MA, maleylacetone; FA, fumarylacetone; DCA, dichloroacetic acid; CFA, chlorofluoroacetic acid; GSTZ, glutathione transferase zeta; hGSTZ1-1, human glutathione transferase zeta.
necessitated the use of substrate analogues to determine enzyme activities. Maleylacetone (MA) (8), maleylpyruvate (9), and cis-β-acetylacrylic acid (10) are substrates for GSTZ1-1 purified from beef and rat liver and from Vibrio 01. Dichloroacetic acid (DCA) is a mechanism-based inactivator of rat and human GSTZ1-1 (11, 12). The kinetics of the DCA-induced inactivation of hGSTZ differ among the four polymorphic variants (12), indicating the possibility of differences in the kinetics among the variants with DCA and other substrates. Perturbations of tyrosine metabolism that occur in hypertyrosinemia type 1 (13, 14) are associated with lossof-function mutations in fumarylacetoacetate hydrolase (15, 16). Accumulation of the diketoacids fumarylacetoacetate, succinylacetoacetate, and succinylacetone (SA) in the body is associated with these perturbations in tyrosine metabolism (17, 18). DCA-induced inactivation of GSTZ1-1 also perturbs tyrosine metabolism in rats and leads to the formation and excretion of MA and SA (19), which are the decarboxylated and both decarboxylated and reduced analogues of MAA, respectively. Humans are exposed to DCA from a variety of sources: it is a common drinking water contaminant (20), it is used for the treatment of congenital lactic acidosis (21), and it is a metabolite of trichloroacetic acid and chloral hydrate (22).
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The effects of MAA or MA on the biotransformation of R-haloacids or the effects of R-haloacids on the isomerization of MAA or MA have not been investigated. Understanding the variant-dependent effects on the turnover of the different substrates should increase our knowledge about DCA-induced perturbations of tyrosine metabolism that may be associated with polymorphisms of GSTZ1-1 in the human population. The objective of this study was to examine the kinetics of the biotransformation of MA and chlorofluoroacetic acid (CFA) by polymorphic variants of recombinant hGSTZ1-1. In addition, the effects of MA on the biotransformation of CFA by each variant and the effects of DCAinduced inactivation of hGSTZ1-1 on the isomerase activities of each variant were also determined.
Experimental Procedures Materials. Maleylacetone and fumarylacetone (FA) were a gift from Dr. Peter Dedon, MIT, and were synthesized by the method of Fowler and Seltzer (8); their properties were confirmed by 1H NMR and UV spectroscopic analyses. Dichloroacetic acid (>99% pure) was obtained from Aldrich Chemical Co. (Milwaukee, WI). Chlorofluoroacetic acid (99% pure) was prepared by hydrolysis of chlorofluoroacetic acid ethyl ester (Lancaster Synthesis, Inc., Windham, NH), as described previously (5). Glutathione, phenylhydrazine, potassium ferricyanide, and mono- and dibasic potassium phosphate were purchased from Sigma Chemical Co. (St. Louis, MO). Other reagents were obtained from commercial suppliers. Expression and Purification of Recombinant hGSTZ1-1 Polymorphic Variants. Recombinant N-terminal His-tagged hGSTZ1-1 polymorphic variants were expressed in Escherichia coli M15[pREP4] cells (Qiagen Inc., Valencia, CA) and purified with nickel affinity columns, as described previously (12). The purified 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 DTT, and 10% glycerol (v/v). Activities with MA as Substrate. The activities of hGSTZ1-1 polymorphic variants were determined by measuring the rate of formation of FA from MA. Reaction mixtures contained enzyme (0.1-0.3 µg) and GSH (1 mM) in a final volume of 0.5 mL of 0.01 M potassium phosphate buffer (pH 7.4). The reaction mixtures were incubated for 5 min at 25 °C; the reaction was initiated by adding 0-1 mM MA and was quenched after 30 s by adding 50 µL of concentrated HCl; 50 µL of a salicylic acid solution (1.37 mg in 1 mL of methanol) was also added to each sample as an internal standard. Samples (50 µL) were analyzed on a Hewlett-Packard 1090 liquid chromatograph equipped with a µBondapak C18-column (3.9 mm × 300 mm, 10 µm particle size; Waters, Milford, MA). The column was eluted with a 0-30% methanol gradient at a flow rate of 0.75 mL/min over 30 min; solvent A contained 0.075% acetic acid in water, and solvent B contained 0.075% acetic acid and 60% methanol in water. The absorbances of MA and FA in the eluate were measured with a diode-array detector at 312 nm. Concentrations of FA in the reaction mixtures were quantified with a calibration curve prepared with known concentrations of FA. The analytical method described above afforded greater specificity and sensitivity than methods that measure the decrease in the absorbance of MAA or MA (3, 23, 24) in the sense that FA is more stable than MA in acid aqueous solvents and has a higher molar absorptivity at 312 nm than MA (8). The limit of detection of FA was 10 µM, and GSH conjugates of FA or other metabolites of FA (e.g., SA) did not interfere in the assay. The rapid turnover of MA precluded accurate determination of the activities at temperatures above 25 °C and at assay times longer than 30 s. MA and FA could be clearly distinguished by their retention times (tRMA ) 9.6 min; tRFA ) 22.3 min) and UV spectra (λmaxMA ) 275 nm; λmaxFA ) 312 nm); this
Lantum et al. permitted simultaneous determination of substrate loss, which was 1a-1a ≈1d-1d (Table 1). No other intermediates, such as SA or GSH conjugates of FA that have detectable absorbance at 275 nm, were observed in the eluate under the reaction conditions. This indicated that the differences among the activities of the variants were unlikely to be associated with the formation of other products. The Km for MA ranged from 95 to 540 µM, and the turnover
Kinetics of Biotransformation of MA and CFA by hGSTZ1-1
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Table 1. Kinetic Constants of Recombinant hGSTZ1-1 Polymorphic Variants with MA as Substratea variant
Vmax [µmol min-1 (mg of protein)-1]
Km (µM)
kcat (s-1)
kcat/Km (M-1 s-1)
1a-1a 1b-1b 1c-1c 1d-1d
318 ( 91b 1010 ( 220 1860 ( 720 464 ( 215b
177 ( 73c 213 ( 35 540 ( 243 95 ( 29
134 427 784 196
7.6 × 105 20 × 105 14.5 × 105 20.6 × 105
a MA (0-1 mM) was incubated with hGSTZ1-1 variants (0.10.3 µg/mL) and GSH (1 mM) in 0.01 M potassium phosphate buffer (pH 7.4) at 25 °C for 30 s, and the amount of FA formed was determined by HPLC analysis, as described under Experimental Procedures. Data were fitted to the Michaelis-Menten equation to determine the kinetic constants for each enzyme. Data are shown as means ( SEM, n ) 3. b Means of these variants differ significantly from the mean of hGSTZ1c-1c. c Means for each variant do not differ significantly from one another.
Table 2. Kinetic Constants of Recombinant hGSTZ1-1 Polymorphic Variants with CFA as Substratea variant
Vmax [µmol min-1 (mg of protein)-1]
Km (µM)
kcat (s-1)
kcat/Km (M-1 s-1)
1a-1a 1b-1b 1c-1c 1d-1d
14.5 ( 0.4b 2.3 ( 0.2 1.9 ( 0.3 1.9 ( 0.3c
1440 ( 80b 204 ( 19 164 ( 6 193 ( 23
6.1b 1.0 0.8 0.8
4.3 × 103 4.7 × 103 5.0 × 103 4.1 × 103
a CFA (0-2 mM) was incubated with hGSTZ1-1 variants (1-2 µg/mL) and glutathione (1 mM) in 0.1 M potassium phosphate buffer (pH 7.4) at 37 °C for 15-20 min, and the amount of glyoxylate formed was determined spectrophotometrically as described under Experimental Procedures. Data were fitted to the Michaelis-Menten equation to determine the kinetic constants for each enzyme. Data are shown as means ( SEM, n ) 3. b hGSTZ1a-1a differs significantly from the other variants. c Means for each variant do not differ significantly from one another.
numbers (kcat) ranged from 130 to 780 s-1 for the four variants. The catalytic efficiencies (kcat/Km) for the four variants ranged from 8 × 105 to 21 × 105 M-1 s-1, indicating a high catalytic efficiency of the hGSTZ1-1 variants with MA as substrate. The nonenzymatic conversion of MA to FA under assay conditions was insignificant, and no correction was necessary. Kinetics of hGSTZ1-1 Variants with CFA as Substrate. The activities for each variant with CFA as substrate were determined by measuring the rate of glyoxylate formation. The Vmax for hGSTZ1a-1a was significantly larger than those of hGSTZ1b-1b, -1c-1c, and -1d-1d, which were not significantly different from each other (Table 2). The Km values for CFA with hGSTZ1b1b, -1c-1c, and -1d-1d were similar, whereas the Km for hGSTZ1a-1a was significantly larger. The kcat/Km values differed little among the variants and were of the order of 103 M-1 s-1. The kcat for hGSTZ1a-1a was larger than that of the other variants. Correlation Analysis of MA and CFA for hGSTZ1-1 Variants. Pearson’s correlation coefficients for the kcat, Km, and kcat/Km of each polymorphic variant with MA and CFA as substrates were determined. No significant correlations were observed for the kcat, Km, and kcat/Km when all four variants were analyzed. Nonetheless, the analysis showed that hGSTZ1a-1a had different kinetic properties compared with the other variants. Effect of pH on hGSTZ1-1 Activities. Potassium phosphate and borate-KCl (50 mM) buffers with pH values ranging from 6.0 to 9.6 were used to determine the pH dependence of each variant in the presence of 1 mM GSH and 2 mM CFA. Under these saturating conditions, hGSTZ1a-1a, -1b-1b, and -1c-1c had optimal
Figure 1. Effect of pH on the specific activities of hGSTZ1-1 variants with CFA as substrate. hGSTZ1a-1a (A), -1b-1b (B), -1c-1c (C), and -1d-1d (D) variants (1-2 µg/mL) were incubated at 37 °C for 15-20 min with glutathione (1 mM) and CFA (2 mM) in 0.05 M potassium phosphate (b) or 0.05 M borate-KCl (O) buffers with pH values ranging from 6.0 to 10, and the rate of glyoxylate formation was determined, as described under Experimental Procedures. Table 3. Competitive (Kic) and Uncompetitive (Kiu) Inhibition Constants of MA and FA for hGSTZ1-1 Variantsa MA
FA
1a-1a 1b-1b 1c-1c 1d-1d 1a-1a 1b-1b 1c-1c 1d-1d Kic (µM) Kiu (µM)
90 155
110 180
90 240
90 230
70 260
80 90
100 120
80 105
a The competitive inhibition constants (K ) were determined ic from Dixon plots (1/v vs [I]) with inhibitor concentrations of 0-400 µM and substrate concentrations of 10-2000 µM. The uncompetitive inhibition constants (Kiu) were determined from CornishBowden plots ([S]/v vs [I]) with inhibitor concentrations of 0-400 µM and substrate concentrations of 10-2000 µM. The mean velocities (n ) 3) at each substrate and inhibitor concentration were used for the plots to generate a single value for each variant.
activities at pH 7.8-8.2, whereas hGSTZ1d-1d had the highest activity at pH 7.4 (Figure 1). Buffers containing 0.05 M glycine, tricine, or glycylglycine inhibited the activities of hGSTZ1-1 variants. Preliminary experiments indicated that hGSTZ1c-1c was stable at pH 6 and 10 (data not shown). Mechanisms of Inhibition of CFA Turnover by MA and FA. The effects of MA on the turnover of CFA were examined for each variant. CFA (0-2 mM) was incubated with 1-2 µg of hGSTZ1-1/mL and 1 mM GSH in the presence of 0-400 µM MA, and the amount of glyoxylate formed was determined after 15-20 min, as indicated under Experimental Procedures. MA was a mixed inhibitor of all hGSTZ1-1 variants (Figure 2); the mean Kic of all variants (95 ( 5 µM) was smaller than the mean Kiu (201 ( 20 µM) (Table 3). To evaluate the effects of FA formed from MA during the 15-20 min incubation time, assays were also done in the presence of 0-400 µM FA under similar reaction conditions. FA was also a mixed inhibitor of all four hGSTZ1-1 variants with CFA as the substrate (Figure 3); the mean Kic and
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Figure 2. Effects of MA on the biotransformation of CFA by hGSTZ1-1 variants. CFA (0-2 mM) and 0 (0), 50 (9), 100 (]), 200 ([), and 400 (O) µM MA were added simultaneously to 0.1 M potassium phosphate buffer (pH 7.4) containing glutathione (1 mM) and 1-2 µg/mL (A) hGSTZ1a-1a, (B) hGSTZ1b-1b, (C) hGSTZ1c-1c, and (D) hGSTZ1d-1d and were incubated for 15-20 min at 37 °C. Lineweaver-Burk plots were used to determine the type of inhibition.
Figure 3. Effects of FA on the biotransformation of CFA by hGSTZ1-1 variants. CFA (0-2 mM) and 0 (0), 50 (9), 100 (]), 200 ([), and 400 (O) µM FA were added simultaneously to 0.1 M potassium phosphate buffer (pH 7.4) containing glutathione (1 mM) and 1-2 µg/mL (A) hGSTZ1a-1a, (B) hGSTZ1b-1b, (C) hGSTZ1c-1c, and (D) hGSTZ1d-1d and were incubated for 15-20 min at 37 °C. Lineweaver-Burk plots were used to determine the type of inhibition.
mean Kiu values for the hGSTZ1-1 variants were KicFA ) 83 ( 6 mM and KiuFA ) 144 ( 39. FA formed from MA may contribute to the inhibitory effects of MA. Recent experiments show that MA and FA alkylate both Cys-16 and Cys-205 of hGSTZ1-1 variants (26), which is consistent with the observed mixed inhibition of hGSTZ1-1 by
MA and FA. FA reacts nonenzymatically with GSH to form a GSH S-conjugate (8, 27); it is uncertain whether the formation of this GSH S-conjugate may also inhibit hGSTZ1-1 activity. Effects of DCA-Induced Inactivation of hGSTZ1-1 Variants on MA Turnover. hGSTZ1c-1c inactivation
Kinetics of Biotransformation of MA and CFA by hGSTZ1-1
Figure 4. Variant-dependent differences in the DCA-induced inactivation of hGSTZ1-1 on the isomerase activities. Each variant was incubated for 4 h at 37 °C with glutathione (1 mM) and DCA (1 mM) in 0.1 M potassium phosphate buffer (pH 7.4) containing 100 µg/mL albumin. The inactivated protein was recovered as described under Experimental Procedures, and activities were determined with MA as substrate. Control samples were processed similarly but were not incubated with DCA. The data represent the residual activities of each variant as a percent of control values. The control values for hGSTZ1a1a, -1b-1b, -1c-1c, and -1d-1d were 53 ( 0.4, 177 ( 5, 179 ( 11, and 174 ( 12 µmol min-1 (mg of protein)-1, respectively. Data are shown as means ( SEM, n ) 3.
was studied in incubation mixtures containing 1 mM DCA and 1 mM glutathione in the absence or presence of 100 µg/mL bovine serum albumin. (Albumin was added to increase the recovery of protein after centrifugal filtration.) Inactivation of hGSTZ1c-1c was greater in the presence of albumin than in its absence, but the specific activities of the recovered proteins from control samples were similar [∼175 µmol min-1 (mg of protein)-1] (data not shown). The activity of hGSTZ1c-1c was reduced to 58% and 11% of control in the absence and presence of albumin, respectively. The effects of DCA-induced inactivation on the isomerase activities of hGSTZ1-1 polymorphic variants were also characterized (Figure 4). After a 4 h incubation, isomerase activities were determined after protein recovery by centrifugal filtration. Residual isomerase activities were reduced by more than 95% for hGSTZ1b1b, -1c-1c, and -1d-1d and by 88% for hGSTZ1a-1a.
Discussion The isomerase activity of GSTZ1-1 with a variety of diketoacids as substrates has been determined with enzymes from different microorganisms (23, 24, 28-30) and rat liver (3, 31). Little is known, however, about the kinetics of hGSTZ1-1 with MA as substrate. The objective of the present study was to examine the kinetics of polymorphic variants of recombinant hGSTZ1-1 with MA and CFA as substrates. MA is a stable analogue of MAA, the endogenous substrate of GSTZ1-1 (8), and CFA is a noninactivating substrate of GSTZ1-1 (11, 12). Nickel affinity-purified N-His-tagged polymorphic variants of recombinant hGSTZ1-1 were used because GSTZ1-1 has a low affinity for GSH affinity columns (1). The effects of the N-terminal His-tag on hGSTZ1-1 activities have not been evaluated. Based on the crystal structure of hGSTZ1b-1b, the N-terminal histidines are not in close proximity to the active site, and the N-Histagged hGSTZ1b-1b adopts the canonical GST fold (32). It is, therefore, unlikely that the N-terminal His-tag may alter the kinetic properties of GSTZ1-1.
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Figure 5. 3D backbone model of hGSTZ1b-1b showing the location of the active-site serine residue (red) and the amino acid residues Lys-32, Gly-42, and Thr-82 (yellow) that are associated with the single nucleotide polymorphisms of hGSTZ1-1. The amino acid residues of other hGSTZ1-1 polymorphic variants are: 1a-1a: Lys-32, Arg-42, and Thr-82; 1c-1c: Glu-32, Gly-42, and Thr-82; and 1d-1d: Glu-32, Gly-42, and Met-82. The PDB file 1FW1 was accessed and modified with RasMol version 2.7.1.1 (Bernstein + Sons, Bellport, NY) to create the model.
The polymorphic variants of hGSTZ1-1 are associated with nonsynonymous substitution mutations at codons 32, 42, and 82 (1a-1a: Lys-32, Arg-42, Thr-82; 1b-1b: Lys-32, Gly-42, Thr-82; 1c-1c: Glu-32, Gly-42, Thr-82; 1d1d: Glu-32, Gly-42, Met-82) (6, 33). The crystal structure of hGSTSZ1b-1b shows that Lys-32 lies in the β2-sheet sandwiched between two R-helices defining a hydrophobic pocket, Arg-42 is in the large mobile loop that links the β2-strand to the R2-helix near the entrance of the active site, and Thr-82 is in the R3-helix close to the linker region of the N- and C-terminal domains at the base of the active site (Figure 5) (32). The effects of the substitution mutations on the crystal structure of hGSTZ1-1 are not known. pH-Dependence of hGSTZ1-1 Activities. hGSTZ1a1a, -1b-1b, and -1c-1c had optimal activities at about pH 8, whereas hGSTZ1d-1d had optimal activity at pH 7.4. The measured pH optima for hGSTZ1-1 variants are lower than those observed with most other GSTs, which have pH optima at about pH 8.5 (34, 35). There may, however, be some uncertainty in the measured pH optima of hGSTZ variants because the pH optima were determined with buffers of different ionic strengths and, with hGSTZ1a-1a, the experiments were conducted with less than saturating conditions of CFA. The effect of these differences on the activities of hGSTZ1-1 variants is uncertain. The reason for the lower pH optimum of hGSTZ1d-1d compared with the other variants is not known. The only structural factor that distinguishes hGSTZ1d-1d from other variants is the methionine at Thr-82 (6, 33). This residue is located at the linker region between the N- and C-terminal domains at the base of the active site (Figure 5) (32) and may indirectly affect the pKa of active-site residues. hGSTZ1-1 has five cysteine residues. One (Cys-16) is located in the GSH binding site, and the cysteine thiolate
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is 2.8 Å from the thiolate of glutathione (32). This orientation may allow the formation of a GSH-cysteine disulfide bond, which has been observed during mass spectral analysis of hGSTZ1-1 incubated with GSH (W. B. Anderson and M. W. Anders, unpublished observations). Mixed disulfide formation between GSH and the active-site cysteine residue may be a confounding factor for assessing pH-dependence at high pH values. Differences between the Kinetic Constants for MA and CFA. The kinetic constants with MA as substrate differed from those with CFA as the substrate for the four hGSTZ1-1 variants. The Vmax values for the biotransformation of MA were 22-fold (hGSTZ1a-1a) to 977-fold (hGSTZ1c-1c) higher than for CFA. The marked difference in the kcat of MA compared with CFA is also reflected in the kcat/KmMA and kcat/KmCFA values, which were much larger for MA than for CFA. The differences in the turnover numbers and catalytic efficiencies for MA relative to CFA may have been even greater given that the activities with MA as substrate were determined at 25 °C compared with 37 °C with CFA as the substrate. Hence, the catalytic efficiency of hGSTZ1-1 with MA as the substrate is much higher than with R-haloacids. Variant-Dependent Differences in Kinetic Parameters. The only parameter that differed significantly among the variants was Vmax: the Vmax of hGSTZ1a-1a and -1d-1d was lower than that of hGSTZ1b-1b and -1c1c with MA as the substrate, and the Vmax of hGSTZ1a1a was higher than that of the other variants with CFA as the substrate. With MA as substrate, hGSTZ1c-1c had the highest kcat, and hGSTZ1a-1a had the lowest kcat. With CFA as substrate, hGSTZ1a-1a had the highest kcat, and hGSTZ1b-1b, -1c-1c, and -1d-1d had the lowest kcat. Under physiological conditions, saturating substrate concentrations of MAA or MA, formed by decarboxylation of MAA, are not likely to be achieved. Hence, significant differences among individuals expressing different hGSTZ1-1 variants would likely not be observed. These observations show that the hGSTZ1a-1a variant is an outlier. hGSTZ1a-1a possesses an A to G mutation at nucleotide 124 that results in a glycine to arginine change at codon 42 (1, 6). This arginine residue is located at the entrance of the active site in the 3D-structure (32). This mutation seems to favor a higher turnover of CFA and a lower turnover of MA compared with the other variants that have glycine residues at codon 42. Despite these differences in rates of turnover, the kcat/Km values were similar among the four variants. Thus, mutations associated with the variants at codon 42, and less likely at codons 32 and 82, affect the chemical step involving the rate of formation and stabilization of the enzymesubstrate complex or the rate of release of the product from the enzyme and, thereby, alter the turnover rate. Competition between R-Haloacids and MA for the Active Site. The inhibition studies showed that MA and FA were mixed inhibitors of GSTZ1-1 activities with CFA as the substrate. The competitive inhibition constants were lower than the uncompetitive inhibition constants, indicating that MA and FA compete for a common active site of GSTZ1-1 with CFA as the substrate but that these compounds may bind to another site on the enzyme. Indeed, recent mass spectral characterization of hGSTZ1-1 variants that had been incubated with MA and FA shows that MA and FA alkylate both Cys-16 and Cys205 (26).
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It is also pertinent to note that the residual isomerase activity of hGSTZ1a-1a was higher than that of the other variants and that hGSTZ1a-1a is less susceptible to DCAinduced inactivation than the other variants (12). Therefore, subjects expressing hGSTZ1a-1a may be less susceptible to the effects of DCA-induced perturbations in tyrosine metabolism than those expressing other variants. In conclusion, this is the first kinetic analysis of polymorphic variants of hGSTZ1-1 with MA and CFA as substrates. The kinetic constants for the biotransformation of MA and CFA show that MA is a much better substrate, as reflected in the kcat/Km values, than CFA for all polymorphic variants. With MA or CFA as substrates, few differences in catalytic efficiencies of hGSTZ1-1 polymorphic variants were observed, indicating that humans would likely have similar responses to the inactivating effects of DCA on hGSTZ1-1.
Acknowledgment. We thank Wayne B. Anderson for preparation of the purified recombinant hGSTZ1-1 variants and Larry Jolivette for his helpful suggestions. This research was supported in part by the University of Rochester James P. Wilmot Cancer Research Fellowship (H.B.M.L.) and by the National Institute of Environmental Health Sciences Grant ES03127 (M.W.A.).
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