Kinetics of the biotransformation of maleylacetone and chlorofluoroacetic acid by polymorphic variants of human glutathione transferase zeta (hGSTZ1-1)

Article abstract of DOI:10.1021/tx010095y

Glutathione transferase zeta (GSTZ1-1) catalyzes the cis-trans isomerization of maleylacetoacetate and the biotransformation of a range of α-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 k_cat 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 k_cat 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 (K_ic^MA) for all variants was about 100 μM and the mean uncompetitiv- inhibition constant (K_iu^MA) was about 201 μM. Hence, MA and α-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 (<5%). This is the first kinetic analysis of polymorphic variants of hGSTZ1-1, and the similarity of the kinetic constants for hGSTZ1-1 variants with either MA or CFA as substrates indicates that few differences in DCA-induced perturbations of tyrosine metabolism would likely be observed in humans.

Full text of DOI:10.1021/tx010095y

Chem. Res. Toxicol. 2002, 15, 957-963
957
Kin etics of th e Biotr a n sfor m a tion of Ma leyla ceton e a n d
Ch lor oflu or oa cetic Acid by P olym or p h ic Va r ia n ts of
Hu m a n Glu ta th ion e Tr a n sfer a se Zeta (h GSTZ1-1)
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, J ohn 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 maleylaceto-
acetate 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 (<5%). This is the first kinetic analysis of polymorphic variants of
hGSTZ1-1, and the similarity of the kinetic constants for hGSTZ1-1 variants with either MA
or CFA as substrates indicates that few differences in DCA-induced perturbations of tyrosine
metabolism would likely be observed in humans.
necessitated the use of substrate analogues to determine
enzyme activities. Maleylacetone (MA) (8), maleylpyru-
vate (9), and cis-â-acetylacrylic acid (10) are substrates
for GSTZ1-1 purified from beef and rat liver and from
Vibrio 01.
In tr od u ction
Glutathione transferase zeta (GSTZ1-1)1 (1), which is
identical with maleylacetoacetate isomerase (2), is a novel
cytosolic glutathione transferase that catalyzes the glu-
tathione-dependent isomerization of maleylacetoacetate
(MAA) to fumarylacetoacetate (FAA), the penultimate
step in the phenylalanine and tyrosine degradation
pathway (3), and the biotransformation of a range of
R-haloacids (4, 5). GSTZ1-1 is highly phylogenetically
conserved: mRNA and proteins have been detected in
plants, microorganisms, and mammals. Four polymorphic
variants of hGSTZ1-1 (1a-1a, 1b-1b, 1c-1c, and 1d-1d)
have been identified by expressed sequence tag database
analysis (6). The occurrence of the different variants in
the sampled Caucasian population is 1c-1c . 1b-1b >
1d-1d > 1a-1a (6).
Dichloroacetic acid (DCA) is a mechanism-based inac-
tivator 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 pos-
sibility 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 loss-
of-function mutations in fumarylacetoacetate hydrolase
(15, 16). Accumulation of the diketoacids fumarylacetoac-
etate, succinylacetoacetate, and succinylacetone (SA) in
the body is associated with these perturbations in ty-
rosine 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).
MAA has been prepared enzymatically (7), but appar-
ently 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: 585-
275-1678; Fax: 585-273-2652; E-mail: mw_anders@urmc.rochester.edu.
† University of Rochester Medical Center.
‡ Australian National University.
1
Abbreviations: MAA, maleylacetoacetate; FAA, fumarylaceto-
acetate; MA, maleylacetone; FA, fumarylacetone; DCA, dichloroacetic
acid; CFA, chlorofluoroacetic acid; GSTZ, glutathione transferase zeta;
hGSTZ1-1, human glutathione transferase zeta.
10.1021/tx010095y CCC: $22.00 © 2002 American Chemical Society
Published on Web 06/12/2002

958 Chem. Res. Toxicol., Vol. 15, No. 7, 2002
Lantum et al.
permitted simultaneous determination of substrate loss, which
was <10% under the assay conditions, and product formation.
The rate of nonenzymatic conversion of MA to FA was deter-
mined by analysis of a solution containing MA (1 mM), GSH (1
mM), and heat-denatured protein in 0.01 M potassium phos-
phate buffer (pH 7.4) and was insignificant after a 30 s reaction
time.
Activities w ith CF A a s Su bstr a te. The formation of
glyoxylate from CFA was measured spectrophotometrically, as
previously described (5). Reaction mixtures contained 1-2 µg
of purified hGSTZ1-1 variants and 1 mM GSH in 1 mL final
volume of 0.1 M potassium phosphate buffer (pH 7.4) and were
incubated at 37 °C for 20 min. The reactions were initiated by
addition of CFA (0-2 mM) and were quenched by addition of
50 µL of trifluoroacetic acid. Under these conditions, less than
10% of substrate was consumed in 20 min.
The pH dependencies of the activities of the variants were
determined in 0.05 M potassium phosphate or 0.05 M borate-
KCl buffer over the range of 6-10 pH units. The assay mixtures
contained 2 mM CFA and 1 mM GSH and were incubated as
described above.
To investigate the effects of MA and FA on the activities of
each polymorphic variant with CFA as substrate, the reactions
were initiated by addition of CFA simultaneously with 0-400
µM MA or FA, and the amount of glyoxylate formed over 15-
20 min was determined as described above.
The kinetic data were analyzed by nonlinear regression
analysis (GraphPad Software, Inc., San Diego, CA) or fitted to
the Michaelis-Menten equation with EnzFitter software (Bio-
soft; Ferguson, MO). The type of inhibition of MA and FA for
the different polymorphic variants with CFA as substrate was
determined from Lineweaver-Burk plots; competitive inhibition
constants (Kic) and uncompetitive inhibition constants (Kiu) of
MA and FA for the different polymorphic variants were obtained
from Dixon plots (1/v vs [I]) and Cornish-Bowden plots ([S]/v vs
[I]), respectively (25).
In a ctiva tion of Recom bin a n t h GSTZ1-1 by DCA. Each
variant was inactivated by incubating the enzyme (50 µg/mL)
with GSH (1 mM) and DCA (1 mM) in 0.1 M potassium
phosphate buffer (pH 7.4) at 37 °C in the presence or absence
of bovine serum albumin (100 µg/mL) for 2-4 h. The residual
activity of the enzyme incubated with DCA was determined
either after recovery by three concentration-dilution cycles with
Centricon-10 concentrators (YM-10, 10 kDa molecular mass
cutoff; Millipore, Bedford, MA) or after 100-fold dilution before
assaying for isomerase activities. Protein concentrations of the
recovered enzyme solutions were determined with the Bio-Rad
protein assay (Bio-Rad Laboratories, Hercules, CA) with bovine
serum albumin as the standard.
The effects of MAA or MA on the biotransformation of
R-haloacids or the effects of R-haloacids on the isomer-
ization 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 biotrans-
formation of CFA by each variant and the effects of DCA-
induced inactivation of hGSTZ1-1 on the isomerase
activities of each variant were also determined.
Exp er im en ta l P r oced u r es
Ma ter ia ls. 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 con-
firmed by 1H NMR and UV spectroscopic analyses. Dichloro-
acetic 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 previ-
ously (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.
Exp r ession a n d P u r ifica tion of Recom bin a n t h GSTZ1-1
P olym or p h ic Va r ia n ts. 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).
Act ivit ies w it h MA a s Su bst r a t e. 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
Sta tistica l An a lysis. Pearson’s correlation coefficients of the
kcat, Km, and kcat/Km for MA and CFA as substrates of each
polymorphic variant were determined with GraphPad Prism
(GraphPad Software, Inc., San Diego, CA). Differences among
the means of the kinetic constants of each variant were tested
for significance by one-way ANOVA and Bonferroni’s post-test
between pairs of values.
(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
Resu lts
quantified with
a calibration curve prepared with known
Kin etics of h GSTZ1-1 Va r ia n ts w ith MA a s Su b-
str a te. The activities of hGSTZ1-1 variants with MA as
substrate were determined by measuring the rate of
formation of FA. The Vmax of the four variants varied
appreciably: rates of formation of FA were 1b-1b ≈ 1c-
1c > 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 differ-
ences 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
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 determi-
nation of the activities at temperatures above 25 °C and at assay
times longer than 30 s. MA and FA could be clearly distin-
MA
FA
guished by their retention times (tR
min) and UV spectra (λmaxMA ) 275 nm; λmaxFA ) 312 nm); this
) 9.6 min; tR ) 22.3

Kinetics of Biotransformation of MA and CFA by hGSTZ1-1
Chem. Res. Toxicol., Vol. 15, No. 7, 2002 959
Ta ble 1. Kin etic Con sta n ts of Recom bin a n t h GSTZ1-1
P olym or p h ic Va r ia n ts w ith MA a s Su bstr a tea
Vmax [µmol min-1
Km
(µM)
kcat
k
cat/Km
variant
(mg of protein)-1
]
(s-1
)
(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 (0.1-
0.3 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
b
shown as means ( SEM, n ) 3. Means of these variants differ
significantly from the mean of hGSTZ1c-1c. c Means for each
variant do not differ significantly from one another.
Ta ble 2. Kin etic Con sta n ts of Recom bin a n t h GSTZ1-1
P olym or p h ic Va r ia n ts w ith CF A a s Su bstr a tea
Vmax [µmol min-1
variant (mg of protein)-1
k
cat/Km
]
Km (µM)
kcat (s-1
)
(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
6.1b
1.0
0.8
0.8
4.3 × 103
4.7 × 103
5.0 × 103
4.1 × 103
F igu r e 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.
193 ( 23
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.
Ta ble 3. Com p etitive (Kic) a n d Un com p etitive (Kiu
)
In h ibition Con sta n ts of MA a n d F A for h GSTZ1-1
Va r ia n tsa
MA
FA
numbers (kcat) ranged from 130 to 780 s-1 for the four
variants. The catalytic efficiencies (kcat/Km) for the four
1a-1a 1b-1b 1c-1c 1d-1d 1a-1a 1b-1b 1c-1c 1d-1d
variants ranged from 8 × 105 to 21 × 105 M-1 s-1
,
K
K
ic (µM)
90
110
180
90
90
230
70
260
80
90
100
120
80
105
iu (µM) 155
240
indicating a high catalytic efficiency of the hGSTZ1-1
variants with MA as substrate. The nonenzymatic con-
version of MA to FA under assay conditions was insig-
nificant, and no correction was necessary.
a
The competitive inhibition constants (Kic) were determined
from Dixon plots (1/v vs [I]) with inhibitor concentrations of 0-400
µM and substrate concentrations of 10-2000 µM. The uncompeti-
tive inhibition constants (Kiu) were determined from Cornish-
Bowden 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.
Kin etics of h GSTZ1-1 Va r ia n ts w ith CF A a s Su b-
str a te. The activities for each variant with CFA as
substrate were determined by measuring the rate of
glyoxylate formation. The Vmax for hGSTZ1a-1a was sig-
nificantly 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 hGSTZ1b-
1b, -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.
Cor r ela t ion An a lysis of MA a n d CF A for
h GSTZ1-1 Va r ia n ts. 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. Nonethe-
less, the analysis showed that hGSTZ1a-1a had different
kinetic properties compared with the other variants.
Effect of p H on h GSTZ1-1 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
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).
Mech a n ism s of In h ibition of CF A Tu r n over by
MA a n d F A. 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

960 Chem. Res. Toxicol., Vol. 15, No. 7, 2002
Lantum et al.
F igu r e 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.
F igu r e 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.
FA
mean Kiu values for the hGSTZ1-1 variants were Kic
)
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-In d u ced In a ctiva tion of h GSTZ1-1
Va r ia n ts on MA Tu r n over . hGSTZ1c-1c inactivation
FA
83 ( 6 mM and Kiu ) 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 consis-
tent with the observed mixed inhibition of hGSTZ1-1 by

Kinetics of Biotransformation of MA and CFA by hGSTZ1-1
Chem. Res. Toxicol., Vol. 15, No. 7, 2002 961
F igu r e 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 hGSTZ1a-
1a, -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.
F igu r e 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 poly-
morphic 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.
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 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; 1d-
1d: Glu-32, Gly-42, Met-82) (6, 33). The crystal structure
of hGSTSZ1b-1b shows that Lys-32 lies in the â2-sheet
sandwiched between two R-helices defining a hydrophobic
pocket, Arg-42 is in the large mobile loop that links the
â2-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 substitu-
tion mutations on the crystal structure of hGSTZ1-1 are
not known.
The effects of DCA-induced inactivation on the isom-
erase activities of hGSTZ1-1 polymorphic variants were
also characterized (Figure 4). After a 4 h incubation,
isomerase activities were determined after protein re-
covery by centrifugal filtration. Residual isomerase ac-
tivities were reduced by more than 95% for hGSTZ1b-
1b, -1c-1c, and -1d-1d and by 88% for hGSTZ1a-1a.
Discu ssion
p H-Dep en d en ce of h GSTZ1-1 Activities. hGSTZ1a-
1a, -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 deter-
mined 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.
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 vari-
ants 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-His-
tagged 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.
hGSTZ1-1 has five cysteine residues. One (Cys-16) is
located in the GSH binding site, and the cysteine thiolate

962 Chem. Res. Toxicol., Vol. 15, No. 7, 2002
Lantum et al.
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 observa-
tions). Mixed disulfide formation between GSH and the
active-site cysteine residue may be a confounding factor
for assessing pH-dependence at high pH values.
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 DCA-
induced inactivation than the other variants (12). There-
fore, subjects expressing hGSTZ1a-1a may be less sus-
ceptible to the effects of DCA-induced perturbations in
tyrosine metabolism than those expressing other vari-
ants.
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 biotransforma-
tion 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 sub-
strates, few differences in catalytic efficiencies of
hGSTZ1-1 polymorphic variants were observed, indicat-
ing that humans would likely have similar responses to
the inactivating effects of DCA on hGSTZ1-1.
Differ en ces betw een th e Kin etic Con sta n ts for
MA a n d CF A. 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
MA
CFA
reflected in the kcat/Km
and kcat/Km
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.
Va r ia n t-Dep en d en t Differ en ces in Kin etic P a -
r a m eter s. 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 -1c-
1c with MA as the substrate, and the Vmax of hGSTZ1a-
1a was higher than that of the other variants with CFA
as the substrate. With MA as substrate, hGSTZ1c-1c had
Ack n ow led gm en t. We thank Wayne B. Anderson
for preparation of the purified recombinant hGSTZ1-1
variants and Larry J olivette for his helpful suggestions.
This research was supported in part by the University
of Rochester J ames P. Wilmot Cancer Research Fellow-
ship (H.B.M.L.) and by the National Institute of Envi-
ronmental Health Sciences Grant ES03127 (M.W.A.).
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