Alkylation and inactivation of human glutathione transferase zeta (hGSTZ1-1) by maleylacetone and fumarylacetone

Article abstract of DOI:10.1021/tx025503s

Glutathione transferase zeta (GSTZ1-1) catalyzes the cis-trans isomerization of maleylacetoacetate or maleylacetone (MA) to fumarylacetoacetate or fumarylacetone (FA), respectively. GSTZ1-1 also catalyzes the glutathione-dependent biotransformation of a range of α-haloacids, including dichloroacetic acid. The objective of this study was to investigate the mechanism of inactivation of hGSTZ1-1 by MA and FA and to determine the covalent modification of hGSTZ1-1 by MA and FA in the presence and absence of glutathione. MA and FA (0.01-1 mM) inactivated all hGSTZ1-1 polymorphic variants in a concentration- and time-dependent manner, and this inactivation was blocked by glutathione. The C16A mutant of hGSTZ1c-1c was partially inactivated by MA and FA. Electrospray ionization-tandem mass spectrometry and SALSA (Scoring Algorithm for Spectral Analysis) analyses of tryptic digests of hGSTZ1 polymorphic variants revealed that the active site (SSC*SWR) and C-terminal (LLVLEAFQVSHPC*R) cysteine residues of hGSTZ1-1 were covalently modified by MA and FA. MA and FA adduction resulted in diagnostic 156-Da shifts in the masses of the modified peptide ions and in their MS-MS fragment ions. Alkylation of the active-site cysteine residues, but not of the C-terminal cysteine, was relatively less intense when hGSTZ1-1 polymorphic variants were incubated with MA or FA in the presence of S-methyl glutathione. These data indicate that MA and FA are substrate and product inactivators of hGSTZ1-1 and covalently modify hGSTZ1-1 at the active-site cysteine residue in the absence of glutathione. The observation that inactivation was blocked by glutathione indicates that binding of glutathione to the active site prevents reaction of MA or FA with the active-site cysteine residue. These data also indicate that MA and FA may covalently modify and inactivate other proteins that have accessible cysteine residues and may, thereby, contribute to dichloroacetic acid-induced or hypertyrosinemia type-I-associated toxicities.

Full text of DOI:10.1021/tx025503s

Chem. Res. Toxicol. 2002, 15, 707-716
707
Alk yla tion a n d In a ctiva tion of Hu m a n Glu ta th ion e
Tr a n sfer a se Zeta (h GSTZ1-1) by Ma leyla ceton e a n d
F u m a r yla ceton e
Hoffman B. M. Lantum,† 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, J ohn Curtin School of Medical
Research, Australian National University, Canberra ACT 2601, Australia
Received J anuary 2, 2002
Glutathione transferase zeta (GSTZ1-1) catalyzes the cis-trans isomerization of maleyl-
acetoacetate or maleylacetone (MA) to fumarylacetoacetate or fumarylacetone (FA), respec-
tively. GSTZ1-1 also catalyzes the glutathione-dependent biotransformation of a range of
R-haloacids, including dichloroacetic acid. The objective of this study was to investigate the
mechanism of inactivation of hGSTZ1-1 by MA and FA and to determine the covalent
modification of hGSTZ1-1 by MA and FA in the presence and absence of glutathione. MA and
FA (0.01-1 mM) inactivated all hGSTZ1-1 polymorphic variants in a concentration- and time-
dependent manner, and this inactivation was blocked by glutathione. The C16A mutant of
hGSTZ1c-1c was partially inactivated by MA and FA. Electrospray ionization-tandem mass
spectrometry and SALSA (Scoring Algorithm for Spectral Analysis) analyses of tryptic digests
of hGSTZ1 polymorphic variants revealed that the active site (SSC*SWR) and C-terminal
(LLVLEAFQVSHPC*R) cysteine residues of hGSTZ1-1 were covalently modified by MA and
FA. MA and FA adduction resulted in diagnostic 156-Da shifts in the masses of the modified
peptide ions and in their MS-MS fragment ions. Alkylation of the active-site cysteine residues,
but not of the C-terminal cysteine, was relatively less intense when hGSTZ1-1 polymorphic
variants were incubated with MA or FA in the presence of S-methyl glutathione. These data
indicate that MA and FA are substrate and product inactivators of hGSTZ1-1 and covalently
modify hGSTZ1-1 at the active-site cysteine residue in the absence of glutathione. The
observation that inactivation was blocked by glutathione indicates that binding of glutathione
to the active site prevents reaction of MA or FA with the active-site cysteine residue. These
data also indicate that MA and FA may covalently modify and inactivate other proteins that
have accessible cysteine residues and may, thereby, contribute to dichloroacetic acid-induced
or hypertyrosinemia type-I-associated toxicities.
tion, or bioactivation of xenobiotics (7-9). The conjuga-
tion of glutathione with chemotherapeutic agents may
be associated with increased drug resistance. Some GSTs
also show activities with endogenous substrates, such as
4-hydroxyalkenals (10), ∆5-3-ketosteroids in the prostag-
landin and leukotriene synthetic pathways (11), and
maleylacetoacetate (MAA) in the tyrosine degradation
pathway (12, 13).
In tr od u ction
Glutathione transferases (GSTs)1 are members of a
superfamily of Phase-II drug metabolizing enzymes that
catalyze the conjugation of glutathione with a range of
electrophilic substrates (1, 2). GSTs are expressed in
plants, microorganisms, and animals. Cytosolic GSTs
include alpha, mu, pi, theta, omega, sigma, and zeta
classes of GSTs (3-5). Microsomal and mitochondrial
GSTs have been also been identified (1, 6). The GST-
catalyzed conjugation of glutathione with electrophilic
substrates is the first step in the mercapturic acid
pathway that is associated with the detoxication, excre-
Glutathione transferase zeta (GSTZ1-1), which is
identical with maleylacetoacetate isomerase, catalyzes
the cis-trans isomerization of MAA to fumarylacetoac-
etate (FAA), the penultimate step in the tyrosine degra-
dation pathway (4, 13, 14). In many GST-catalyzed
reactions, glutathione is incorporated into the product
glutathione S-conjugates; in contrast, with GSTZ1-1 and
with DCA and MA as substrates, glutathione is required
for activity but is not consumed (15, 16, 17). MAA is labile
and undergoes decarboxylation to the more stable ma-
leylacetone (MA) (12, 18), which is a substrate for
GSTZ1-1 and is converted to fumarylacetone (FA) (15,
16, 19). GSTZ1-1 also catalyzes the glutathione-depend-
* To whom correspondence should be addressed. Phone: (585) 275-
1678. Fax: (585) 273-2652. E-mail: mw•anders@urmc.rochester.edu.
† University of Rochester Medical Center.
‡ University of Arizona.
§ Australian National University.
1
Abbreviations: MAA, maleylacetoacetate; FAA, fumarylacetoac-
etate; MA, maleylacetone; FA, fumarylacetone; SA, succinylacetone;
DCA, dichloroacetic acid; CFA, chlorofluoroacetic acid; NEM, N-
ethylmaleimide; GST, glutathione transferase; GSTZ1-1, glutathione
transferase zeta; SALSA, Scoring Algorithm for Spectral Analysis.
10.1021/tx025503s CCC: $22.00 © 2002 American Chemical Society
Published on Web 04/02/2002

708 Chem. Res. Toxicol., Vol. 15, No. 5, 2002
Lantum et al.
(22, 31, 32). 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.
ent biotransformation of dichloroacetic acid (DCA), chlo-
rofluoroacetic acid (CFA), and other dihaloacetic acids to
glyoxylate (17, 20). DCA is a mechanism-based inacti-
vator of GSTZ1-1 (21, 22).
Activities of h GSTZ1-1 w ith DCA a n d CF A a s Su b-
str a tes. The formation of glyoxylate from DCA and CFA was
measured spectrophotometrically, as previously described (20).
Reaction mixtures contained 1-2 µg of purified hGSTZ1-1
variants, 1 mM glutathione, and 0.1 M potassium phosphate
buffer (pH 7.4) in a final volume of 1 mL and were incubated at
37 °C. The reactions were initiated by addition of CFA (1 or 2
mM) or 1 mM DCA and were quenched by addition of 50 µL of
neat trifluoroacetic acid. Specific activities (nmol/min/mg pro-
tein) are reported as the mean ( SEM for three experiments.
Activities w ith MA a s Su bstr a te. The activities of hG-
STZ1-1 were determined by measuring the rate of formation of
FA from MA by HPLC analysis, as described previously (31).
Reaction mixtures contained enzyme (0.1-0.3 µg), 1 mM glu-
tathione, and 0.01 M potassium phosphate buffer (pH 7.4) in a
final volume of 0.5 mL. The reaction mixtures were incubated
for 5 min at 25 °C; the reaction was initiated by addition of 1
mM MA and was quenched after 30 s by addition of 50 µL of
concentrated HCl; 50 µL of a salicylic acid solution (1.37 mg in
1 mL methanol) was also added to each sample as an internal
standard. Samples (50 µL) were analyzed on a Hewlett-Packard
DCA-induced inactivation of GSTZ1-1 results in the
loss of activity and degradation of GSTZ1-1 (21, 22) with
the resultant perturbation of the tyrosine degradation
pathway such that the excretion of MAA-derived com-
pounds, MA, FA, and their saturated analogue, succiny-
lacetone (SA), is elevated (23). Plasma and urine con-
centrations of FA and SA are also elevated in patients
suffering from hypertyrosinemia type-I (24, 25). Hyper-
tyrosinemia type-I is a metabolic disorder that is associ-
ated with a loss-of-function mutation in fumarylacetoac-
etate hydrolase, which catalyzes the terminal step in the
tyrosine degradation pathway (26). The metabolic stabil-
ity and toxic effects of MA and FA, which are R,â-
unsaturated ketones, have not been investigated (27).
MA and FA are mixed inhibitors of hGSTZ1-1 with
CFA as substrate.2 These kinetic experiments also showed
that the specific activities of hGSTZ1-1 variants with MA
as substrate declined at MA concentrations greater than
1 mM (unpublished observations). These data indicated
that MA and FA were substrate and product inhibitors
of GSTZ1-1, respectively. Wong and Seltzer observed that
MA covalently modifies maleylacetone isomerase purified
from Escherichia coli by a non-Schiff-base mechanism
(28). Hence, we hypothesized that MA and FA covalently
modify and inactivate GSTZ1-1 by a Michael-addition
reaction with the active-site cysteine residue (4, 29, 30).
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 to 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 in the absence of glutathione. The retention
times of MA, FA, and salicylic acid were 9.6, 22.3, and 23.5 min,
respectively. The rate of nonenzymatic conversion of MA to FA
(0.021 nmol/min/mg of protein) was determined by analysis of
a solution containing MA (1 mM), glutathione (1 mM), and heat-
inactivated enzyme in 0.01 M potassium phosphate buffer (pH
7.4) and was subtracted from each sample. After acidification
with HCl, the nonenzymatic conversion of MA to FA and the
nonenzymatic reaction of glutathione with FA in 10 mM
potassium phosphate buffer containing 1 mM glutathione and
heat inactivated enzyme were undetectable permitting repro-
ducibility that was >95%. Specific activities (nmol/min/mg of
protein) are reported as the mean ( SEM for three experiments.
The objective of this study was to determine the
mechanism of inactivation of hGSTZ1-1 polymorphic
variants by MA and FA in the presence and absence of
glutathione. Substitution mutation analysis and LC-MS-
MS analysis were undertaken to determine the location
and type of MA- and FA-induced modifications. The data
show that MA and FA covalently modify cysteine residues
in hGSTZ1-1. These findings have implications with
respect to possible alkylating effects of MA and FA, which
are elevated in hypertyrosinemia-type-I patients or after
DCA-induced perturbations in tyrosine metabolism.
In a ctiva tion of h GSTZ1-1 by MA or F A. To investigate
the inactivation of hGSTZ1-1, 0-250 µM MA or FA, 0-100 µM
succinylacetone, or 0-100 µM N-ethylmaleimide were incubated
with 50-100 µg of hGSTZ1-1 in 0.1 M potassium phosphate
buffer (pH 7.4) in a final volume of 1 mL at 37 °C for 0-30 min
in the absence of glutathione. The reaction mixtures were
diluted 10-fold with ice-cold 0.1 M potassium phosphate buffer
(pH 7.4) containing 1.5 mM DTT, 0.5 mM EDTA, and 10%
glycerol, and the protein was recovered by three concentration-
dilution cycles with Centricon-10 concentrators (YM-10, 10 kDa
molecular mass cutoff; Millipore, Bedford, MA). Because of
significant losses of protein by this procedure, the reaction
mixtures were sometimes diluted 100-1000-fold in phosphate
buffer after inactivation before assaying for activity; this
permitted a quantitative recovery of protein for determination
of specific activities.
Exp er im en ta l P r oced u r es
Ma t er ia ls. Maleylacetone and fumarylacetone were a gift
from Dr. Peter Dedon, MIT, and were synthesized by the method
of Fowler and Seltzer (18); their properties were confirmed by
1H NMR and UV spectroscopic analyses. Dichloroacetic acid
(>99% pure) was obtained from Aldrich Chemical Co. (Milwau-
kee, WI). Chlorofluoroacetic acid (99% pure) was prepared by
hydrolysis of ethyl chlorofluoroacetate (Lancaster Synthesis,
Inc., Windham, NH), as described previously (20). Glutathione,
succinylacetone (4,6-dioxoheptanoic acid), 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 a n d th e h GSTZ1c-1c C16A Mu ta n t.
Recombinant N-terminal His-tagged hGSTZ1-1 polymorphic
variants and the C16A mutant of hGSTZ1c-1c were expressed
in E. coli M15[pREP4] cells (Qiagen Inc., Valencia, CA) and
purified with nickel affinity columns, as described previously
To determine whether glutathione and CFA block the MA-
and FA-induced inactivation of hGSTZ1-1, 0-1 mM glutathione
or CFA was added to the reaction mixtures, which were
incubated for 0-30 min; the activities were determined after
protein recovery. The kinetic data were analyzed by nonlinear
regression analysis (GraphPad Software, Inc., San Diego, CA).
P r ep a r a tion of P r otein Digests of Un m od ified a n d
Mod ified h GSTZ1-1. hGSTZ1-1 (100 µg) was incubated with
0, 0.1, or 1 mM MA or FA in the presence or absence of 1 mM
glutathione or S-methyl glutathione (Sigma, St. Louis, MO) in
2
Lantum, H. B. M., Board, P. G., and Anders, M. W. (2002) Kinetics
of the biotransformation of maleylacetone and chlorofluoroacetic acid
by polymorphic variants of human glutathione transferase zeta (hG-
STZ1-1) Chem. Res. Toxicol. (submitted for publication).

Alkylation and Inactivation of hGSTZ1-1
Chem. Res. Toxicol., Vol. 15, No. 5, 2002 709
0.1 M potassium phosphate buffer (pH 7.4) in a final volume of
1 mL for 1 h at 37 °C. The protein was recovered by three
concentration-dilution cycles as described above and suspended
in 100 µL of a denaturing solution containing 8 M urea, 0.4 M
ammonium bicarbonate, and 4 mM tris(2-carboxyethyl)phos-
phine (Pierce, Rockford, IL). The mixture was heated at 50 °C
for 45 min and diluted 4-fold with double-distilled water. The
proteins were then digested with 2 µg (∼1:50, w/w, enzyme:
protein ratio) of Sequencing Grade Modified Porcine Trypsin
(stock made by dissolving 20 µg in 200 µL of trypsin buffer
according to the manufacturer’s instructions; Promega, Madison,
WI) and incubated overnight at 37 °C. The digest was then
lyophilized and suspended in 200 µL of 50% acetonitrile/water
(v/v) solution.
Electr osp r a y Ion iza tion Ta n d em Ma ss Sp ectr om etr ic
An a lysis of h GSTZ1-1-Der ived P ep tid es. Samples of the
peptide digests were analyzed by reversed-phase LC-MS-MS,
as described previously (33,34) on a ThermoFinnigan LCQ mass
spectrometer in positive electrospray ionization mode. Briefly,
samples of the peptide digest (10-20 µL) were analyzed on a
ThermoSeparation Products liquid chromatograph equipped
with a Vydac 218TP51 C18, column (250 × 1.0 mm; 5 µm
particle size; Vydac, Hesperia, CA). The column was eluted with
a 0-95% gradient at a flow rate of 0.1 mL/min over 150 min;
solvent A contained 0.01% trifluoroacetic acid (Pierce) and 0.1
M formic acid (J . T. Baker, Phillipsburg, NJ ) in water (Burdick
& J ackson, Muskegon, MI), and solvent B contained 0.0085%
trifluoroacetic acid and 0.1 M formic acid in acetonitrile (Burdick
& J ackson). LC-MS data were acquired by data-dependent
scanning with automatic MS-MS analyses of precursor ions with
intensities above 35000 absolute ion counts. The LC-MS and
LC-MS-MS data were analyzed with ThermoFinnigan Xcalibur
software.
F igu r e 1. Kinetics of inactivation of hGSTZ1c-1c by MA.
hGSTZ1c-1c (50-100 µg/mL) was incubated with 25 (9), 40 (0),
60 (b), 100 (O), and 250 ([) µM MA in 0.1 M potassium
phosphate buffer (pH 7.4) at 37 °C for 0-30 min in the absence
of glutathione. hGSTZ1c-1c incubated in the absence of MA was
subjected to the same conditions and served as a control. The
reaction mixtures were placed on ice and diluted 100-1000-
fold, as described in Experimental Procedures. Activities with
CFA as substrate were determined with 2-5 µg of protein;
residual activities of the treated (A) were compared with those
of the untreated preparations (A0). The data were analyzed by
nonlinear regressions fitted to the second-order polynomial
equation. The data shown are the means of three experiments.
reaction that was independent of the concentration of
MA.
To determine whether this second-order reaction was
associated with the nonenzymatic conversion of MA to
FA that occurs in the absence of glutathione, the inac-
tivating effects of FA were also determined. FA also
inactivated GSTZ1-1 in a concentration- and time-de-
pendent manner; the time-dependent inactivation kinet-
ics for different concentrations of FA was also nonlinear
(data not shown). These data indicated that MA and FA
were nonmechanism based substrate and product inac-
tivators of hGSTZ1-1, that the rates of inactivation were
not linear with time, and that the data fit a second-order
rate equation.
Dep en d en ce on th e Olefin ic Bon d for In a ctiva -
tion of h GSTZ1-1. MA and FA are R,â-unsaturated
compounds that may react nonenymatically with thiols
(16). Their diketo moieties may mediate the formation
of Schiff-base adducts with proteins (28). Hence, the
inactivating effects of MA and FA were compared with
those of N-ethylmaleimide (NEM) and succinylacetone
SA. NEM is an R,â-unsaturated imide, and SA is the
saturated diketo analogue of MA and FA.
Sequest analysis of the LC-MS-MS data was done to deter-
mine the peptide coverage of the protein, to compare collision-
induced dissociation spectra of peptides with predicted spectra
from sequences in the database, and to screen for modifications.
The analysis of the data was done with TurboSequest, version
27, running under the Sequest Browser (ThermoFinnigan, San
J ose, CA); the Sequest searches used the NCBI nonredundant
FASTA human database; defining analysis parameters included
the endoprotease (trypsin) and possible modifications ((156 Da
for Cys or Lys residues).
To facilitate identification and characterization of unmodified
and modified hGSTZ1-1-derived peptides, peptide-sequence
motif searches of the LC-MS-MS spectral data were analyzed
with the Scoring Algorithm for Spectral Analysis (SALSA) (35,-
36). MS-MS scans with high scores were analyzed by matching
the identified y-ions with theoretical y-ions generated with
GPMAW software version 5.0 (Lighthouse Data, Odense, Den-
mark).
Resu lts
MA, FA, and NEM inactivated hGSTZ1c-1c in a
concentration-dependent manner after 30 min incuba-
tion; in contrast, the activity of hGSTZ1c-1c was not
reduced by SA (Figure 2). These data indicated that the
R,â-unsaturated carbonyl group, and less likely the oxo-
moiety, was required for MA- and FA-induced inactiva-
tion of hGSTZ1-1. Thus, the mechanism of inactivation
is likely to be associated with the reactivity of MA and
FA with cysteine residues of hGSTZ1-1.
P r otection of MA- a n d F A-In d u ced In a ctiva tion
of h GSTZ1-1 by Glu ta th ion e. The glutathione binding
site in the active site of hGSTZ1-1 has a highly conserved
sequence with a cysteine residue (14SSCSWRVIAL23) (4,
29, 31). We hypothesized that MA and FA would react
with the Cys-16 residue and, thereby, inactivate hG-
STZ1-1 and that glutathione, which is a substrate for
MA- a n d F A-In d u ced In a ctiva tion of h GSTZ1-1.
MA and FA were incubated with hGSTZ1-1 polymorphic
variants in the absence of glutathione, and the specific
activity of the protein recovered at different times was
determined with CFA as substrate. MA inactivated
hGSTZ1c-1c and all other polymorphic variants (data not
shown) in a time- and concentration-dependent manner
(Figure 1); maximal inactivation was observed within 20
for all variants and activities were undetectable after the
hGSTZ1-1 had been incubated with MA or FA at con-
centrations greater than 250 µM for 20 min. The time-
dependent effects showed second-order kinetics with
respect to residual activities (r2 > 0.99) for all concentra-
tions. These data indicated MA was a nonmechanism-
based inactivator of hGSTZ1-1 variants and that the
inactivating effects were determined by a second-order

710 Chem. Res. Toxicol., Vol. 15, No. 5, 2002
Lantum et al.
F igu r e 4. Protection of polymorphic variants of hGSTZ1-1 from
MA- and FA-induced inactivation. hGSTZ1-1 variants (100 µg/
mL) were incubated with 0 or 100 µM MA or FA in 0.1 M
potassium phosphate buffer (pH 7.4) at 37 °C for 30 min in the
absence or presence of 1 mM glutathione. The residual activities
with CFA as substrate were determined for each variant after
recovering protein by three dilution-concentration cycles, as
described in Experimental Procedures. The effects of NEM on
each variant are also shown. Data are shown as means ( SEM,
n ) 3; (*) p < 0.05, nonparametric repeated measures ANOVA
with Dunnett’s posttest to compare activities at each concentra-
tion of inhibitor with control.
F igu r e 2. Concentration-dependent effects of MA, FA, NEM,
and SA on hGSTZ1c-1c activities. hGSTZ1c-1c (100 µg/mL) was
incubated with the indicated concentrations of MA, FA, NEM,
and SA in 0.1 M potassium phosphate buffer (pH 7.4) at 37 °C
for 30 min in the absence of glutathione. The reaction mixture
was diluted 100-fold, and protein was recovered by three
concentration-dilution cycles; the residual activities were then
determined with CFA as substrate, as described in Experimen-
tal Procedures. (*) p < 0.05, nonparametric repeated measures
ANOVA with Dunnett’s posttest to compare activities of inhibi-
tor with control, n ) 3.
inactivation reaction (data not shown). These data indi-
cated that glutathione prevented MA and FA from
reacting with the active-site cysteine residue but do not
exclude the reaction of these compounds with the other
residues, particularly the four non-active-site cysteine
residues of hGSTZ1-1. Cys-16 is likely to be the residue
that was modified in the absence of glutathione.
P a r tia l In a ctiva tion of th e h GSTZ1c-1c C16A
Mu ta n t by MA a n d F A. To obtain additional informa-
tion about the possible reaction of MA and FA with the
active-site cysteine residue, the MA- and FA-induced
inactivation of the hGSTZ1c-1c C16A mutant was inves-
tigated. The activity of the N-His tagged C16A mutant
with DCA and CFA as substrate was higher than that
of the wild-type enzyme, whereas its activity with MA
as substrate was less than that of the wild-type enzyme
(Figure 5). The reason for these differences in activities
between the wild-type and C16A mutant hGSTZ1c-1c is
not known.
The inactivation of the wild-type and the C16A mutant
of hGSTZ1c-1c by MA and FA was compared (Figure 6).
With CFA as substrate, the wild-type enzyme had 3% or
0.5% residual activity after 30 min incubation with 100
µM MA or FA, respectively, whereas the C16A mutant
had 73% and 35% residual activity after 30 min incuba-
tion with 100 µM MA or FA, respectively (Figure 6A).
With MA as substrate, the wild-type enzyme had 17%
and 26% residual activity after 30 min incubation with
100 µM MA or FA, respectively; the C16A mutant had
86% and 60% residual activity after 30 min incubation
with 100 µM MA or FA, respectively (Figure 6B). Al-
though 1 mM glutathione blocked the MA- and FA-
induced inactivation of the wild-type enzyme by 80% as
shown above, 1 mM glutathione did not protect the C16A
mutant from inactivation by MA or FA (data not shown).
These data indicated that the C16A mutant enzyme was
partially resistant to inactivation by MA and FA and that
a residue other than the active-site Cys-16 residue was
a target for MA and FA.
F igu r e 3. Concentration-dependent protective effects of glu-
tathione on MA-induced inactivation of hGSTZ1c-1c. hGSTZ1c-
1c (50 µg/mL) was incubated with 0-1 mM glutathione in 0.1
M phosphate buffer (pH 7.4, 37 °C) for 5 min prior to adding
100 µM MA. After 30 min, the reaction mixture was diluted,
and the activities of the recovered protein were determined with
CFA as substrate, as described in Experimental Procedures. The
broken line indicates activities of hGSTZ1c-1c incubated with
1 mM glutathione for 35 min in the absence of MA. Data are
shown as means ( SEM, n ) 3; (*) p < 0.05, unpaired two-
tailed t-test to compare activities after inactivation for each
concentration of glutathione with control.
GSTZ1-1, would block these MA- and FA-induced inac-
tivation. Hence, the effects of glutathione on MA- and
FA-induced inactivation of hGSTZ1-1 were determined.
Incubation of hGSTZ1c-1c with 0.1-1 mM glutathione
before adding MA or FA protected hGSTZ1c-1c from
inactivation (Figure 3). The partial protective effects of
glutathione from inactivation by MA and FA were also
determined for the four polymorphic variants of hGSTZ1-
1; some variants were partially inactivated by MA and
FA in the presence of glutathione, and NEM inactivated
the polymorphic variants to different extents (Figure 4).
The inactivating effects of MA and FA were not blocked
by incubating hGSTZ1-1 with 1 mM CFA during the
Alk yla tion of h GSTZ1-1 P olym or p h ic Va r ia n ts by
MA a n d F A. Covalent modification of cysteine residues

Alkylation and Inactivation of hGSTZ1-1
Chem. Res. Toxicol., Vol. 15, No. 5, 2002 711
F igu r e 5. Activities of recombinant N-terminal His-tagged
wild-type and C16A mutant hGSTZ1c-1c with DCA, CFA, and
MA as substrates. Wild-type or mutant hGSTZ1c-1c was
incubated with 1 mM glutathione in 0.1 M potassium phosphate
buffer (pH 7.4) at 37 °C, and activities were determined with 1
mM DCA, CFA, or MA as substrates, as described in Experi-
mental Procedures. Data are shown as means ( SEM, n ) 3;
(*) p < 0.05, unpaired two-tailed t-test to compare activities of
the C16A mutant hGSTZ1c-1c with wild-type.
of hGSTZ1-1 polymorphic variants by MA or FA was
determined by LC-MS-MS analyses of tryptic peptides.
Polymorphic variants of hGSTZ1-1 were incubated with
100 µM MA or FA in 0.1 M potassium phosphate buffer
(pH 7.4) at 37 °C for 30 min, after which time the reaction
mixtures were diluted 20-fold and the proteins were
recovered as described in Experimental Procedures to
prevent aberrant modifications during the protein dena-
turing and digestion steps. The assumption was made
that MA or FA would modify only solvent-accessible
residues under these conditions.
F igu r e 6. Inactivation of wild-type and mutant C16A hG-
STZ1-1 by MA and FA. Wild-type and mutant C16A hGSTZ1c-
1c (50 µg/mL) were incubated with 100 µM MA and FA in the
absence of glutathione, and the residual activities were deter-
mined with (A) 1 mM CFA or (B) 1 mM MA as substrate. Data
are shown as means ( SEM, n ) 3; (*) p < 0.05, unpaired two-
tailed t-test to compare activities after inactivation with con-
trols.
The sequences of the expected cysteine-containing
tryptic peptides of hGSTZ1-1, their precursor ion masses,
and types of MA- and FA-induced modifications are
shown in Table 1. The three cysteine-containing tryptic
peptide fragments of hGSTZ1-1 are hereafter referred to
as peptides 1, 2, and 3 for the N-terminal-active site
peptide, the hydrophobic region peptide, and the C-
terminal peptide, respectively.
MS-MS scans for the unmodified and modified peptide 1
for all hGSTZ1-1 variants incubated in the absence or
presence of MA or FA, respectively (Table 1). The
identified y-ions were diagnostic of MA- or FA-induced
modification of the active-site cysteine residue and were
observed in all polymorphic variants. The Xcalibur
spectra and the Sequest and SALSA data for FA-modified
hGSTZ1-1 peptides were similar to those observed with
MA (data not shown).
Peptide 1 ionized relatively poorly (precursor ion count
) ∼1.03 × 105 ions), and the peak intensities after
collision-induced dissociation were of low abundance,
which limited interpretation of the b- and y-ion series of
the spectra (Figures 7 and 8). The base peak of the
unmodified peptide was at m/z 691.3, which corresponds
to the neutral loss of hydrogen sulfide (Figure 7) from
the precursor ion (M + H)+ ) 725 amu). LC-MS spectra
of the MA-modified peptide 1 showed a 156-amu shift in
the precursor ion mass (M + H)+ ) 881 amu, and the
LC-MS-MS base peak was at m/z 725, which indicates
the retro-Michael loss of 156 amu from the precursor ion;
the m/ z 691.3 amu peak was also intense and could
correspond to the neutral loss of hydrogen sulfide (34
amu) from 725 amu or the loss of 2-mercaptosuccinylac-
etone (2-mercapto-4,6-dioxo-heptanoic acid; 190 amu)
from m/ z 881 (Figure 8). The expected diagnostic shift
(Cys-residue mass + 156 amu) in the b3 (i.e., m/ z 278 to
434) and y4 (i.e., m/ z 551.2 to 707.3) ions was not
identified by visual inspection of the spectra of the
modified peptides. Sequest analysis failed to identify
peptide 1, and, hence, these data on peptide 1 are
inconclusive. SALSA analyses were, therefore, performed
to facilitate the identification of MS-MS scans corre-
sponding to modified peptides. SALSA analysis identified
Peptide 3 was readily identified from the LC-MS
Xcalibur raw data. Peptide 3 was doubly charged, and
the absolute ion count for the m/ z 806.5 and 886
precursor ions for the unmodified and modified peptides,
respectively, was greater than 1.2 × 106. The relative
abundances of the b- and y-ion series after collision-
induced dissociation were above 10%, thereby permitting
manual sequencing by visual inspection of the spectra.
The diagnostic 156-amu shifts of the b13- and y2-ions were
visible in the LC-MS-MS spectra; all the y-ions above y2
and only the b13 ion of the b-series shifted by the mass of
the adduct compared with the unmodified peptide, indi-
cating a Michael-addition adduct at the Cys-205 residue
(Figure 9). Sequest analyses showed MA- and FA-
associated modifications at Cys-205, which were seen in
all hGSTZ1-1 variants (data not shown). Sequest searches
also showed inconsistent modifications of Lys-191. SAL-
SA analyses also confirmed the 156-amu modification at
Cys-205 by providing diagnostic y-ions for the unmodified
and modified peptides; this modification was observed in
all variants. SALSA analysis failed to identify an MS-
MS scan corresponding to modification of Lys-191, and
its occurrence was, therefore, equivocal (Table 1).

712 Chem. Res. Toxicol., Vol. 15, No. 5, 2002
Lantum et al.
Ta ble 1. Sequ en ce a n d Dia gn ostic MA- a n d F A-In d u ced Mod ifica tion s of Th r ee Cystein e-Con ta in in g Tr yp tic P ep tid es of
h GSTZ1-1a
mass of
tryptic
peptides,
SALSA-identified peptidesc
(precursor ion, m/z; diagnostic
y-ions)
[M + H]+
Sequest-identified
modified peptidesb
peptide
sequencea
14SSCSWR19
([M + 2H]2+
)
peptide 1
725.8 (363.4)
NDf
([M + H]+ ) 881.5 m/z; 725,
794.4, 707.3, 448.2,
361.1, 638.2)d
([M + H]+ ) 725 m/z; 361.2,
448.2, 551.3, 638.33)e
ND
peptide 2
peptide 3
121QVGEEMQLTWAQNAITCG
FNALEQILQSTAGIYCVGD
EVTMADLCLVPQVANAER175
193LLVLEAFQVSHPCR206
5946 (2973)
1613 (807)
ND
LLVLEAFQVSHPC*Rg
([M + 2H]2+ ) 885.6 m/z;
531.2, 668.4, 755.3,
854.2, 982.3, 1129.5,
1200.5, 1329.6, 1442.6,
1541.6)d,e
other
178FKVDLTPYPTISSINK191
FKVDLTPYPTISSINK*g
ND
a
hGSTZ1-1 protein sequences were obtained by simulated digestion and fragmentation with GPMAW software; the precursor masses
of the tryptic peptides and the b- and y-ions for the MS-MS spectra of the unmodified and modified peptides were determined and compared
b
with the experimental spectra from Xcalibur files or after Sequest and SALSA analyses, as described in Experimental Procedures. LC-
MS-MS data in Xcalibur files were analyzed under the Sequest browser, as described in Experimental Procedures; more than 50% of the
hGSTZ1-1 sequence was covered; only the sequence of MA/FA-associated modified peptides are shown. c LC-MS-MS data in Xcalibur files
were analyzed and scored by SALSA with “SCSW” or “LVLEAFQVSHPC” sequences for the motif search. Precursor ions with high scores
were identified and their corresponding y-ions series were compared with theoretical data to determine whether the peptide was modified
d
by MA or FA. Precursor ion and corresponding diagnostic ions of tryptic peptides of MA- or FA-inactivated hGSTZ1-1 in the absence of
1 mM S-methylglutathione. e Precursor ion and corresponding diagnostic ions of tryptic peptides of MA- or FA-inactivated hGSTZ1-1 in
g
the presence of 1 mM S-methylglutathione. f ND, not detected. (*) 156 amu modified residue.
The effects of S-methylglutathione on MA- and FA-
induced modification of Cys-16 and Cys-205 were also
determined. S-Methylglutathione (1 mM) partially pro-
tected Cys-16, but not Cys-205, from MA- or FA-induced
modifications based on the intensities of the precursor
ion peaks of the LC-MS spectra (data not shown).
may be associated with FA that is formed nonenzymati-
cally during the incubations.
Discu ssion
This study describes the inactivation and alkylation
of hGSTZ1-1 by its substrate and product analogues, MA
and FA. The data indicated that the mechanism of
alkylation and, hence, inactivation was attributable to a
Michael-addition reaction of MA and FA with at least
two cysteine residues (Cys-16 and Cys-205) of hGSTZ1-1
variants. A two-site binding mechanism of inactivation
was initially indicated by the nonlinearity of the inacti-
vation kinetics and confirmed by substitution mutation
analysis and LC-MS analysis. These data explain why
MA and FA are mixed inhibitors of hGSTZ1-1 activities
with CFA as substrate and why the hGSTZ1-1 activities
are lower at concentrations of MA greater than 1 mM
(unpublished observations).
The finding that MA and FA formed Michael-addition
adducts with cysteine residues does not exclude other
mechanisms of covalent modification. Sequest analysis
of the LC-MS-MS data showed inconsistent modification
of a lysine residue (Table 1). The inhibition data showed
that SA did not inhibit hGSTZ1c-1c (Figure 2). Therefore,
although the formation of Schiff-base adducts may be
another mechanism of alkylation and inactivation, it does
not appear to be the primary mechanism. Previous
studies have shown that SA nonenzymatically forms
stable adducts with proteins and free amino acids,
especially lysine, and that the stability of the adducts is
enhanced by treatment with sodium borohydride, indica-
tive of Schiff-base formation (37). Wong and Seltzer
showed, however, that sodium borohydride does not
enhance labeling of GSTZ1-1 by [14C]MA (28). These
findings and observations reported herein indicate that
Schiff-base formation of GSTZ1-1 by MA and FA is a less
important mechanism of inactivation than a Michael-
addition mechanism.
These data indicated that MA and FA formed Michael-
addition adducts with the cysteine residues in peptides
1 and 3. Cys-16, but not Cys-205, was protected from MA-
and FA-induced alkylation by S-methylglutathione. The
Sequest analyses provided equivocal evidence that MA
and FA may form Schiff-base adducts with Lys-191.
These data do not exclude the formation of adducts
with peptide 2, which was not identified. Polymorphic
variants of hGSTZ1-1 were, therefore, digested with
endoproteinase Glu-C (Sigma, St. Louis, MO) to obtain
smaller fragments of peptide 2 to facilitate identification
of modifications of cysteine residues. Sequest analyses
showed that the peptide coverage of GLU-C digests of
hGSTZ1-1 variants (n ) 4) was less than 5%, and SALSA
motif searches failed to identify any fragments of peptide
2 (data not shown). Hence, the Glu-C digests were not
investigated in more detail.
Rea ctivity of MA a n d F A w ith Glu ta th ion e. FA
was apparently a more potent inactivator of hGSTZ1-1
variants than MA (Figures 2, 4, and 6). This may be
associated with relative differences in the reactivities of
MA and FA with thiols. To investigate this point, the
reactivities of MA and FA with 10 mM glutathione in
0.1 M potassium phosphate buffer (pH 8.5) were studied
by continuous UV-spectral analysis over 10 min at room
temperature. The absorbance of 400 µM FA at 312 nm
decreased rapidly after addition of glutathione, whereas
the decrease in the absorbance of 400 µM MA at 312 nm
was much slower (Figure 10). These data indicate dif-
ferences in the reactivities of MA compared with FA with
thiols. Furthermore, some of the observed effects of MA

Alkylation and Inactivation of hGSTZ1-1
Chem. Res. Toxicol., Vol. 15, No. 5, 2002 713
F igu r e 7. LC-MS-MS spectra of the unmodified active-site peptide (peptide 1) of hGSTZ1c-1c. hGSTZ1c-1c (50 µg/mL) was incubated
in 0.1 M potassium phosphate buffer (pH 7.4) at 37 °C for 30 min in the absence of MA, FA, and glutathione. The protein was
recovered by three dilution-concentration cycles, digested with trypsin, and analyzed by LC-MS-MS, as described in Experimental
Procedures. Inserted text shows the mass of the precursor ions ([M + H]+) and the sequence of peptide 1 with its corresponding b-
and y-ions for the unmodified peptide.
F igu r e 8. LC-MS-MS spectra of the MA-modified active-site peptide (peptide 1) of hGSTZ1c-1c. hGSTZ1c-1c (50 µg/mL) was incubated
for 30 min with 100 µM MA in 0.1 M potassium phosphate buffer (pH 7.4) at 37 °C for 30 min in the absence of glutathione. The
protein was recovered by three dilution-concentration cycles, digested with trypsin, and analyzed by LC-MS-MS, as described in
Experimental Procedures. Inserted text shows the mass of the precursor ions ([M + H]+) and the sequence of peptide 1 with
corresponding b- and y-ions for the MA-modified peptide; (*) MA-adducted cysteine residues.
Although complete inactivation of hGSTZ1-1 by MA
and FA was observed in the absence of glutathione, the
inactivation of wild-type hGSTZ1-1 variants by MA and
FA was blocked by glutathione in a concentration-
dependent manner. Furthermore, the C16A mutant of
hGSTZ1c-1c underwent less inactivation than the wild-
type enzyme, indicating that MA and FA modified Cys-
16. These data indicate that Cys-16 is a target for
alkylation and that alkylation of Cys-16 by MA and FA
may interfere with binding of glutathione at the active
site of the enzyme. Alternatively, glutathione may be
bound to the Cys-16 modified enzyme but not activated
to the reactive thiolate that is required for catalytic
activity.
The partial inactivation of C16A hGSTZ1c-1c and some
variants of wild-type hGSTZ1-1 by MA and FA in the
presence of glutathione indicated that another cysteine
residue was modified. LC-MS analysis showed that Cys-
205 was also modified. The 3D structure of hGSTZ1-1,
which was solved in a glutathione- and DTT-containing
buffer, shows that Cys-205 lies in a mobile loop (H2
) in
the C-terminal domain. Cys-205, but none of the other
cysteine residues, formed a mixed disulfide with DTT,
indicating that Cys-205 is solvent accessible; the Cys-

714 Chem. Res. Toxicol., Vol. 15, No. 5, 2002
Lantum et al.
F igu r e 9. LC-MS-MS spectra of the MA-modified C-terminal peptide (peptide 3) of hGSTZ1c-1c. hGSTZ1c-1c (50 µg/mL) was incubated
with 100 µM MA or FA in 0.1 M potassium phosphate buffer (pH 7.4) at 37 °C for 30 min in the absence of glutathione. The protein
was recovered by three dilution-concentration cycles, digested with trypsin, and analyzed by LC-MS-MS as described in Experimental
Procedures. Inserted text shows the mass of the precursor ions ([M + 2H]2+) and the sequence of peptide 3 with corresponding b-
and y-ions for the MA-modified peptide; (*) MA-adducted cysteine residue.
data indicate that alkylation of Cys-205 is associated with
inactivation of hGSTZ1-1, perhaps by steric effects that
impede substrate access to the active site.
The mechanism by which glutathione protected hG-
STZ1-1 from inactivation is not known. Structural analy-
sis of hGSTZ1-1 shows that the thiol of Cys-16 points
toward the active site and is shielded by glutathione with
respect to the substrate-binding site (29). Glutathione
may, therefore, prevent inactivation by the substrate or
product during the catalytic cycle. This proposal is
supported by the data from LC-MS experiments, which
showed that MA or FA did not modify the Cys-16 when
incubated with S-methylglutathione, whereas Cys-205
was modified in the presence and absence of S-methyl
glutathione. The data do not, however, exclude the
possibility that the mechanism of protection by glu-
tathione may be associated with the nonenzymatic reac-
tion of glutathione with MA and FA (12, 16). This is
highly unlikely to be the only mechanism of protection
from inactivation, particularly for Cys-205, because MA
and FA inactivated the C16A mutant in the absence and
presence of glutathione.
Given that MA and FA completely inactivated hG-
STZ1-1 in the absence of glutathione, the data support
the view that alkylation of the active-site Cys-16 is the
major mechanism of inactivation in the absence of
glutathione. Certain diseases, such as hereditary ty-
rosinemia type-I and glutathione synthetase deficiency,
are associated with low hepatic glutathione concentra-
tions (38). Many xenobiotics form glutathione-conjugates
F igu r e 10. Reactivity of MA and FA with glutathione. MA
(panel A) and FA (panel B) were diluted in 1 mL 0.1 M
and, thereby, reduce cellular glutathione concentrations
(39). Thus, alkylation of the active-site peptide may occur
in these disease states. Physiologically, however, glu-
tathione should be constantly bound to hGSTZ1-1 making
adduction of MA and FA to Cys-205 the most likely
mechanism that will occur in the cell.
potassium phosphate buffer (pH 8.5) to give a final concentration
of 400 µM, and glutathione was added to give a final concentra-
tion of 10 mM. The changes in absorbance were measured by
UV-spectral analysis. The beginning spectra are marked t0, and
the spectra recorded after 10 min are marked t10
.
205-containing loop is located near the active site of
hGSTZ1c-1c (29). These structural data and the present
Inactivation of hGSTZ1-1 by FA indicates a mechanism
of feedback inhibition of GSTZ1-1 activities in patients

Alkylation and Inactivation of hGSTZ1-1
Chem. Res. Toxicol., Vol. 15, No. 5, 2002 715
with fumarylacetoacetate hydrolase (FAH) deficiency.
FAH catalyzes the hydrolysis of fumarylacetoacetate to
fumarate and acetoacetate, the terminal step in the
tyrosine degradation pathway. Hypertyrosinemia type-I
patients (40, 41) and mice with FAH-deficiencies (42, 43)
excrete elevated amounts of FAA-derivatives, notably
succinylacetoacetate and SA (44), and the mercapturic
acid of fumarylacetone (S-2-fumarylacetone N-acetylcys-
teine) (45) indicating a build-up of FAA (24). FAA, like
FA, may also covalently modify and inactivate GSTZ1-
1. A mechanism of competitive feedback inhibition of
GSTZ1-1 by an FAA-glutathione conjugate has also been
proposed (45).
Inactivation of GSTZ1-1 by FAA and the ensuing
accumulation of MAA-derivatives may be associated with
the disorders characteristic of hypertyrosinemia type-I,
such as hepatocellular neoplasia. Indirect support for this
contention is based on the observation that DCA, a
mechanism-based inactivator of GSTZ1-1 (22), causes
hepatic neoplasia in rats and mice (46, 47) and systemic
toxicities (48-51) in rodents similar to those described
in patients with hypertyrosinemia type-I (26, 40). Previ-
ous studies have shown that DCA-treated rats have
reduced GSTZ1-1 activities and protein concentrations
(21), and these effects result in the elevated excretion of
MA and SA (23). The alkylating properties of MA and
FA demonstrated in this study indicate that MA- and FA-
induced alkylation of macromolecules may contribute to
the multiorgan disorders associated with hypertyrosine-
mia type-I and prolonged exposure to DCA (26, 48, 50-
52).
of GSTZ1-1 may inhibit tyrosine degradation under
pathological conditions, but the observed prevention of
inactivation by glutathione indicates that FA and MA
likely produce little inactivation under physiological
conditions. The alkylating effects of MA and FA may
contribute to disorders associated with DCA-induced
perturbations of tyrosine metabolism and disorders of
hypertyrosinemia type-I.
Ack n ow led gm en t. The authors thank Dr. George
Tsaprailis, Director of the Southwest Environmental
Health Sciences Center Proteomics Core Facility, for his
assistance with the LC-MS analyses, and Laura Tis-
careno for her assistance with the SALSA and Sequest
analyses. The purified recombinant hGSTZ1-1 variants
were prepared by Wayne B. Anderson. This work was
funded in part by the University of Rochester J ames P.
Wilmot Cancer Research Fellowship (H.B.M.L.) and by
the National Institute of Environmental Health Sciences
Grants ES03127 (M.W.A.), ES10056, and ES06694
(D.C.L.).
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The active-site peptide of GSTZ1-1 is highly conserved
among species and all contain the Cys-16 residue. In
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(14). The LC-MS data confirmed that all polymorphic
variants of hGSTZ1-1 contain Cys-205. Humans may,
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than other species. The polymorphic variants of hG-
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differing extents, and some variants were not completely
protected from inactivation by glutathione (Figure 4).
These data also indicate that humans may have varying
susceptibilities to the GSTZ1-1 inactivating effects of MA
and FA.
In conclusion, MA and FA are substrate and product
inactivators of hGSTZ1-1. These effects are associated
with the formation of Michael-addition adducts with at
least Cys-16 and Cys-205. The FA-induced inactivation

716 Chem. Res. Toxicol., Vol. 15, No. 5, 2002
Lantum et al.
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