S
-(2,3-Dichlorotriazinyl)glutathione
A new affinity label for probing the structure and function of glutathione transferases
Georgia A. Kotzia and Nikolaos E. Labrou
Laboratory of Enzyme Technology, Department of Agricultural Biotechnology, Agricultural University of Athens, Greece
S-(2,3-Dichlorotriazinyl)glutathione (SDTG) was synthes-
ized and shown to be a n effective alkylating affinity label
for recombinant maize glutathione S-transferase I (GST I).
Inactivation of GST I by SDTG at pH 6 .5 followed biphasic
pseudo-first-order saturation kinetics. The biphasic kinetics
can be described in terms o f a fast initial phase of inactiva-
tion followed by a slower phase, leading to 42 ± 3%
residual activity. The rate of inactivation for both phases
exhibits nonlinear d ependence o n SDTG c oncentration,
consistent with the formation of a r eversible complex with
the enzyme (K
d
107.9 ± 2.1 l
M
for the fast phase, and
224.5 ± 4.2 l
M
for the slow phase) before irreversible
modification with maximum rate constants of
0.049 ± 0.002 min
)1
and 0.0153 ± 0.001 min
)1
for the
fast and slow phases, respectively. Protection from inacti-
vation was afforded by substrate a nalogues, demonstrating

involved in the response to different biotic and abiotic
stresses, and can be specifically induced in response to a
variety of stimuli, such as pathogens and chemicals [3–7].
The cytosolic GSTs are homodimers or heterodimers. Each
monomer has two domains, an a/b domain which includes
a1–a3, and a large a-helical domain comprising h elices
a4–a9. The former contains a GSH-binding site (G-site) on
top of the a domain. A hydrophobic pocket (H-site) lies
between the domains, in which a generally hydrophobic
substrate binds and reacts with GSH [8–16].
In plants, GSTs are grouped into five classes based on
their amino-acid sequences, namely Theta, Zeta, Phi, Tau
and O mega [3,4,9]. Whereas Zeta, Theta and Omega classes
of GSTs are found in plants and animals, the large Phi and
Tau classes a re unique to plants [9]. In maize ( Zea m ays L),
42 GST isoenzymes h ave been identified so f ar [12]. Some of
them and their subunits have been characterized in detail
[12–15]. The isoenzyme GST I (or ZmGSTF1, a ccording t o
the nomenclature of Edwards et al. [3]) has been the major
focus of interest as a model for herbicide detoxification.
Known to be the most abundant maize GST, it shows
constitutive expression in maize seedlings and is a homo-
dimer p rotein of 214 a mino acids [12].
Affinity labelling is a useful tool for t he ide ntification and
probing of specific catalytic and regulatory sites in purified
enzymes and proteins [17–20]. Affinity labelling e xperiments
complement the results from crystallography and provide
structural information on proteins in free solution. This
approach has been widely used to characterize GST
isoenzymes using electrophilic or photoactivated GSH

available kinetic [13] and crystallographic data [10,11].
The results may also be useful in the design of specially
engineered forms of GST I with potential application in
medicine and agrobiotechnology.
Experimental procedures
Materials
Crystalline BSA (fraction V ) was obtai ned from B oehringer,
Mannheim, Germany. Molecular biology reagents, kits,
and Pfu DNA polymerase were from Promega. C yanuric
chloride (1,3,5-sym-trichlorotriazine), GSH (99%),
1-chloro-2,4-dinitrobenzene (CDNB; 99%), glutathione
reductase from Saccharomyce s cerevisiae [300 unitsÆ(mg
protein)
)1
]and
L
-lactate dehydrogenase f rom bovine
heart [1000 unitsÆ(mg protein)
)1
]werefromSigma-
Aldrich C o.
Synthesis, purification and analysis of SDTG
SDTG (Fig . 1A) was s ynthesized by substituting the
chlorine ato m of cyanuric chloride with GSH as reported
by Katusz et al. [24] to produce S-(4-bromo-2,3-dioxobu-
tyl)glutathione, with t he follo wing modifications: cyanuric
chloride (1.6 mmol) was a dded to 30 mL c old (2 °C) water/
acetone (1 : 1, v/v). The pH was adjusted to 4.0. To the
above mixture was s lowly added aqueous GSH (1.6 mmol;
5 mL). The pH was maintained throughout the reaction at

1000 V and a sampling cone voltage of 40 V were used.
Data were acquired o ver the m/z range 100–3000.
Chloride content was measured using the assay devel-
oped by Zall et al. [30], as modified by Hu and Colman
[31]. The absorption coefficient was measured in 50 m
M
potassium phosphate buffer, pH 7.0, on the basis of the
SDTG concentrations determined from the primary amine
content.
Determination of the stability of SDTG
The rate of d ecomposition of SDTG in a buffer identical
with that used in the inactivation studies (100 m
M
potas-
sium phosphate b uffer, p H 6.5) was determined by meas-
urement o f the time dependence of chloride release from the
molecule using the method of Zall et al. [30], as modified by
Hu & Colman [31].
Fig. 1. Structure of SDTG (A) and time course of inactivation of
recombinant GST I by SDTG at pH 6.5 and 25 °C(B).En zyme
(2 units) was i ncubat ed in the absence (m)orpresenceof14.5l
M
SDTG (d), 36.36 l
M
SDTG (h), 72.7 l
M
SDTG (s), 145.5 l
M
SDTG (n) or 219.3 l
M

Site-directed mutagenesis was pe rformed a s described
by Weiner et al. [36]. The pairs of oligonucleotide primers
used in the PCRs were as follows: Phe51Ala mutation,
5¢-CGGAACCCC
GCAGGTCGAGTTTCC-3¢ and 5 ¢-GA
CGAGGTGCTCGGGGCTCTT-3¢; Met121Ala muta-
tion, 5 ¢-ATCAGTCCG
GCACTTGGGGGAACC-3¢ and
5¢-GAGGACGTCGAAGAGGATGGGTTACAG-3¢.
The mutatio n (codon underlined above) was confirmed
by DNA sequencing on Applied Biosystems Sequencer
373A with th e D yeDeoxy T erminator Cycle s equencing k it.
Assay of enzyme activity and protein
Enzyme assays were performed by monitoring the forma-
tion of the c onjugate of CDNB ( 1 m
M
)andGSH(2.5m
M
)
at 340 nm (e ¼ 9.6 m
M
)1
.cm
)1
)at30°C according to a
published method [13,14]. Observed reaction v elocities w ere
corrected for spontaneous reaction rates when necessary.
All initial velocities were determined in triplicate in buffers
equilibrated at c onstant t emperature. P rotein concentration
was determined by t he method of Bradford [37] using BSA

K
d
was d etermined as described previously [19,20].
Studies of i nactivation of GST I b y SDTG in t he
presence of S-methyl-GSH and S-nitrobenzyl-GSH were
performed at 25 °C in 1 mL incubation mixture contain-
ing potassium phosphate buffer, pH 6.5 (100 lmol),
SDTG (218.2 nmol), S-methyl-GSH or S-nitrobenzyl-
GSH (0.5 lmol) and enzyme (2 units, GST assay a t
30 °C).
Inactivation of other enzy mes (S. cerevisiae glutathione
reductase, S . cerevisiae glutathione synthase, rat GST A1-1,
human GST A1-1 and bovine heart
L
-lactate dehydro-
genase) was performed (in the absence or presence of 1 m
M
S-methyl-GSH) in 1 mL incubation mixture containing
100 lmol potassium phosphate buffer, pH 6.5 (for rat GST
A1-1 and human GST A1-1) or 100 lmol potassium
phosphate buffer, pH 7.5 ( for glutathione reductase, gluta-
thione synthase and
L
-lactate deh ydrogenase), S DTG
(98.2 nmol) and enzyme (typically 2 units).
Kinetic analysis
Steady-state kinetic measurements of native and SDTG-
modified GST I were performed at 30 °Cin0.1
M
potassium phosphate buffer, pH 6.5. Initial velocities w ere

potassium phosphate buffer,
pH 6.5, was inactivated with 51.2 nmol SDTG at 25 °C.
The SDTG-modified e nzyme was separated from t he free
SDTG by ultrafiltration (in an Amicon stirred cell 8050
carrying a Diaflo YM10 ultrafiltration membrane; cut-off
10 kDa) after extensive washing with 100 m
M
potassium
phosphate buffer, pH 6.5. The s olution w ith S DTG–GST I
covalent complex was then lyophilized and stored at
)20 °C. The lyop hilized SDTG-modified e nzyme was
dissolved in 8
M
urea and i ncubated w ith N-ethylmaleimide
to block free -SH groups, and then with Woodward’s
reagent K (5 m
M
) or 2 ,4,6-trinitrobenzenesulfonic acid
(5 m
M
) for the determination of total carboxyl and primary
amino groups, respectively. The same treatment was also
applied to the unmodified GST I, as a control. Total
carboxy and primary amino groups in the modified and
unmodified enzyme were d etermined at 340 nm as described
previously [38,39].
Amino-acid analysis of native and SDTG-modified
GST I was performed by the method of Davey & Ersser [40].
Ó FEBS 2004 Affinity labelling of maize GST I (Eur. J. Biochem. 271) 3505
UV spectroscopic studies

histolyticum clostripain [43].
The overall yield in the synthesis of SDTG f rom GSH
was % 55%. SDTG was p urified by HPLC on a C
18
reverse-
phase S5 ODS2 Spherisorb silica column. The product was
eluted at 8.8 min. SDTG purity w as assessed as described in
Experimental procedures. The product showed a single spot
with R
f
¼ 0.55, a UV absorption spectrum w ith a peak at
230 nm, and w as negative in the 5,5-dithio-bis-(2-nitro-
benzoic) acid test for f ree -SH grou ps and positive in the
ninhydrin and 2,4,6-trinitrobenzenesulfonic acid test for
primary a mine. The chloride content w as found to be 2 mol
per mol SDTG. P urified SDTG was a lso analysed b y positive
ionization nano-electrospray MS. Evidence for one major
ion at m/z 457.4 was found, indicating a molecular mass of
456.3 Da, which corresponds well to the mass of SDTG
(455.28 Da).
Kinetics of reaction of SDTG with GST I
When maize GST I was incubated with SDTG at pH 6.5
and 25 °C, it was progressively inactivated (Fig. 1B),
whereas, in the absence of SDTG, virtually no change in
activity was observed. This inactivation was irreversible,
and activity was not restored by extensive dialysis or gel
filtration on Sephadex G-25. The pH used for inactivation
(pH6.5)wasthesameasthatnecessaryforhighGST
activity. This probably affords an enzyme conformation
similar to that adopted during catalysis, t hus creating more

d
=ðk
3
½SDTGÞ
where k
obs
is the rate of enzyme inactivation for a given
concentration of S DTG, k
3
isthemaximalrateofinacti-
vation (min
)1
), and K
d
is the apparent dissociation constant
of the E :SDTG complex. F rom the data shown in F ig. 2, K
d
values of 107.9 ± 2.1 l
M
and 224.5 ± 4.2 l
M
,forthefast
and slow reactions, respectively, were estimated. Apparent
maximal rate constants were determined to be 0.049 ±
0.002 min
)1
for the fast reaction, and 0.0153 ± 0.001 min
)1
for t he slower reaction.
The stability of SDTG against hydrolysis was demon-

indicated by the data, 1 mol SDTG is bound per mol wild-
type enzyme at 42% inactivation.
The specificity of a protein chemical modification
reaction can be indicated by the ability of natural
ligands or active-site-directed reagent to protect against
inactivation [17–24]. The effect of GSH a nalogues S-
methyl-GSH and S-nitrobenzyl-GSH on the reaction of
SDTG with GST I was investigated. S-Methyl GSH
and S-nitrobenzyl GSH protect GST I against inacti-
vation by SDTG. T he protective effect afforded by S-
nitrobenzyl GSH was more significant than that
afforded by S-methyl GSH, at comparable concentra-
tions, which is in agreement with their relative affinity
constants.
Kinetic analysis of the modified enzyme (42% remaining
activity) showed that the e nzyme exhibits kinetic properties
that are different from that of the unmodified enzyme. The
results are shown in T able 2. The modified e nzyme exhibits
about threefold reduced affinity for GSH and a bout
twofold increased affinity for CDNB. A final activity of
less than 50% (e.g. 42%) accords with the incorporation of
SDTG into one subunit, producing a change in the
unmodified subunit which alters its activity to a small
degree (% 7%).
Identification of GST I residue modified by SDTG
To identify which residue in GST I became modified by
SDTG, we used amino-acid analysis, molecular modelling
and s ite-directed mutagenesis. Direct amino-acid sequence
determination o f the modified peptide was not possible
because of its instability during E dman degradation reac-

GST I
K
m
(m
M
)
k
cat
· 10
)2
(s
)1
)
GSH CDNB
Unmodified 1.1 ± 0.20 1.60 ± 0.10 29.3
SDTG-modified 2.9 ± 0.15 0.78 ± 0.02 11.2
Fig. 3. Structural representation depicting important residues of maize
GST I. (A) Bound S-atrazine–GSH conjugate is shown in red. Met121
is dr awn in a spacefill representation. (B) Possible mode of commu-
nication between subunits. Bound S-atrazine–GSH conjugate is shown
in red. Met121 is drawn in a sp acefill representation and P he51 is
shown in b lue .
Ó FEBS 2004 Affinity labelling of maize GST I (Eur. J. Biochem. 271) 3507
several enzymes may act as a r eactive nucleophile. For
example, a methionine residue is modified in isocitrate
dehydrogenase [44], in human uterine progesterone receptor
[45], and
D
-amino acid oxidase [46,47] by reaction with
iodoacetate, 16a-(bromoacetoxy)progesterone and O-(2 ,4-

and-key motif responsible for a highly conserved hydro-
phobic interaction in the subunit i nterface. This residue
makes contact with a hydrophobic patch on the alternate
subunit, comprising, in part, Trp97, Val96, Val100 and
Gln104. As the interface contacts on the a lternate subunit
are largely found in a single kinked a-helix H¢¢
3
(Fig. 3B),
the s ignal may be transmitted via the helix to H-site residues
such as Met121, Ile118, Leu122 and Phe114, which are
located at the end of t his helix. Conformational changes in
these residues would then change the affinity for CDNB
binding, which is supported by the finding that the K
m
of the
modified enzyme for CDNB is lower (see Table 2), and
abolish reaction of SDTG with Met121 at the second
subunit. Thus, the observed intrasubunit communication is
probably directed via Phe51 of the monomer–monomer
contact region, to a-helix H¢¢¢
3
of the a djacent subunit which
contains Met121.
To confirm the key role of Phe51 in this hypothesis,
site-directed mutagenesis was used. T he mutant Phe51Ala
was expressed, p urified, and subjected t o i nactivation
studies (Fig. 5). Upon reaction with SDTG at pH 6.5 and
25 °C, the mutant was progressively inactivated to a final
residual activity of about 1.9% (Fig. 5). Comparison of
the far-UV difference spectra of n ative and mutated

3508 G. A. Kotzia and N. E. Labrou (Eur. J. Biochem. 271) Ó FEBS 2004
at the dimer interface of GST I. Similar l ock-and-key motifs
have also been observed for the classes Alpha, Mu and Pi
GSTs [48–50]. T he conserved h ydrophobic i nteraction
formed by the side chain of the Phe51 residue, which
protrudes from the loop in domain I of one monomer into
the h ydrophobic pocket of domain I I o f t he other mono-
mer, physically anchors the two subunits together at either
end o f the interface.
Explanation of the biphasic kinetics
An average incorporation of 0.5 mol reagent per mol
enzyme subunit indicates that reaction o f SDTG with one
Met121 prevents the reaction of the Met121 of the s econd
subunit. The biphasic kinetics observed may be explained
by assuming that the two subunits, o r at least the
conformation of the Met121 side chains in each subunit,
are not equivalen t regarding the reaction with SDTG, and
exhibit differen t reactivity. The existence of such a
nonsymmetrical arrangement of G ST I subunits has been
observed in the crystal structures [10,11]. The two subunits
of GST I complexed with various product analogues
show some structural differences betw een them, suggesting
that the two substrate-binding sites in the enzyme dimer
may not act independently [10,11]. Furthermore, other
important factors must be considered with regard to the
dynamics of this enzyme. A plot of the crystallographic B-
factors along the polypeptide chain can give an indication
of the relative flexibility of the different portions of the
protein (Fig. 6). GST I displays a well-defined flexibility
pattern. Several regions with high mobility can be

enzyme dimer, and b inding of this ligand completely
abolished the catalytic activity of both enzyme subunits.
In addition, binding studies of GSH to the human P1-1
enzyme have shown that binding displays positive cooper-
ativity above 35 °C, whereas n egative cooperativity occurs
below 25 °C [52]. These results suggest t hat t he two binding
sites may not be independent and further support the
Ôcooperative self-preservationÕ mechanism proposed by
Ricci et al. [54] for the human P1-1 enzyme. According to
this mechanism, a c ooperativity is utilized by the e nzyme to
provide s elf-preservation against inhibitors or physical
factors t hat threaten i ts catalytic e fficiency. T his mechanism
is based on a structural intersubunit communication by
which one subunit, as a consequence of an inactivating
modification, triggers a defence arrangement in the other
subunit to prevent modification [54]. In the present study,
we observe th at the modification of one enzyme subunit of
the GST I homodimer prevents modification of the other
subunit, which suggests t hat the two e nzyme a ctive sites are
co-ordinated.
Reaction of SDTG with other GSH-binding enzymes
To demonstrate the wide applicab ility o f S DTG as an
affinity label for other GSH-binding enzymes such as
S. cerevisiae glutathione reductase, glutathione synthase, rat
GST A1-1 a nd hum an GST A 1-1, inactivation studies were
carried out. T he pseu do-first-order rates of inactivation
observed at a SDTG concentration of 98.2 l
M
and in the
presence and a bsence of 1 m

(min
)1
)
(in the absence of
S-methyl-GSH)
% Protection from
inactivation
(in the presence of
S-methyl-GSH)
Rat GST A1-1 1.12 ± 0.1
a
85.5
Human GST A1-1 2.83 ± 0.1
a
84.3
S. cerevisiae glutathione
reductase
3.5 ± 0.2 86.5
S. cerevisiae glutathione
synthase
1.9 ± 0.1 85.4
Bovine heart
L
-lactate
dehydrogenase
NI –
a
Fast phase inactivation rate.
Ó FEBS 2004 Affinity labelling of maize GST I (Eur. J. Biochem. 271) 3509
GST I. In a ddition, the ability o f SDTG t o inactivate a non-

glutathione S-transferase that interacts with flav onoids. Plant J .
36, 433–442.
7.Dixon,D.P.,Cummins,I.,Cole,D.J.&Edwards,R.(1998)
Glutathione-mediated detoxification systems in plants. Curr. Opin.
Plant Biol. 1, 258–266.
8. Armstrong, R.N. (1997) Structure, catalytic mechanism, and
evolution of the glu tathione transferases. Chem. Res. Toxicol. 10,
2–18.
9. Dixon, D., Laptthorn, A. & Edwards, R. (2002) Pla nt glutathione
transferases. Genome Biol. 3, 1–10.
10. Neuefeind, T., Huber, R., Dasenbrock, H., Prade, L. & Bieseler,
B. (1997) Crystal structure of herbicide-detoxifying maize glu-
tathione S-transferase-I in complex with lactoylglutathione: evi-
dence f or an induced-fit mech anism. J. Mol. Biol. 274, 446–453.
11. Prade, L., Huber, R. & Bieseler, B. (1998) Structures of herbicides
in complex with t heir detoxifying enzyme glutathione S-transfer-
ase: explanations for the selectivity of the enzyme in plants.
Structure 6, 1445–1452.
12. McGonigle,B.,Keeler,S.J.,Lau,S.M.,Koeppe,M.K.&O’Keefe,
D.P. (2000) A genomics approach to the com prehensive analysis
of the glutathione S-transferase gene family in soybean and maize.
Plant Physiol. 124, 1 105–1120.
13. Labrou, N.E., Mello, L.V. & Clonis, Y .D. (2001) Functional and
structural roles of the glutathione -binding residues in maiz e ( Zea
mays) glutathione S-t ransferase I. Bioc hem. J. 358 , 101 –110.
14. Labrou, N.E., Mello, L.V. & Clonis, Y.D. (2001) The conserved
Asn49 of maize glutathione S-transferase I m odulates substrate
binding, catalysis and intersubunit communication . Eur. J. Bio-
chem. 26 8, 3950–3957.
15. Labrou, N.E., Rigden, D.J. & Clonis, Y.D. (2004)

the p hotoaffinity label glu tathionyl S-[4-(succinimid yl)-benzophe-
none]. J. Biol. Chem. 27 5 , 5493–5503.
23. Cooke, R.J., Bjornestedt, R., Douglas, K.T., M cKie, J .H., King,
M.D., Coles, B., Ketterer, B. & Mannervik, B. (1994) Photo-
affinity labelling of the active site of the rat glutathione transferases
3–3 and 1–1 and human glutathione transferase A1–1. Biochem. J.
302, 383–390.
24. Katusz,R.M.,Bono,B.&Colman,R.F.(1991)S-(4-Bromo-2,3-
dioxobutyl)glutathione: a new a ffinity label f or the 4–4 isoenzyme
of rat liver glutathion e S-transferase. Biochemistry 30, 11230–
11238.
25. Hoesch, R.M. & Boyer, T.D. (1 989) Localization of a portion of
the a ctive site of two rat liver glutathione S-transferases using a
photoaffinity l ab el. J. Biol. Chem . 264 , 1 7712–17717.
26. Milligan, A.S., Daly, A., Parry, M.A.J., Lazzeri, P.A. & Jepson, I.
(2001) The expression of a maize glutathione S-transferase in
transgenic w heat confers herb icide tolerance, both in planta and
in vivo. Mol. Breed. 7, 3 01–305.
27. Roxas, V.P., Smith, R.K., Allen, E.R. & Allen, R.D. (1997)
Overexpression of glutathione S-transferase/glutathione peroxi-
dase enhances the growth of transgenic tobacco seedlings during
stress. Nat. Biotechnol. 15, 988 –991.
28. Yue, H., Corley, N. & Shah, P. (2003) Human glutathione-S-
transferase. United States Patent No. 6 506 571.
29. Andreou, V. & Clonis, Y.D. (2002) Novel fiber-optic biosensor
based on immobilized glutathione S-transferase and sol–gel en-
trapped bromcresol g reen for t he determination o f atrazine. Anal.
Chim. Acta 460 , 1 51–161.
30. Zall, D.M., Fisher, D. & Garner, M.Q . (1956) P hotometric
determin at ion of chlo rid es in wat er. Anal. Chem. 28, 1665–1668.

siological flu ids by h igh-performance liquid chromatography with
phenylisothiocyanate derivatization and comparison with ion-
exchange chromatography. J. Chromatogr. 528, 9–2 3.
41. DeLano, W.L. (2004) The PyMOL molecular graphics s ystem on
word wide web. http: //www.pymol.org.
42. Labrou,N.E.&Clonis,Y.D.(2002)Immobilisedsyntheticdyesin
affinity chromatography. In Biochromatography: Theory and
Practice (Vijayalakshmi, M.A., ed.), pp. 235–251. Taylor &
Francis Publishers, London.
43.Labrou,N.E.&Rigden,D.J.(2004)Thestructure–function
relationship in the c lostripain family of peptidases. Eur. J. Bio-
chem. 27 1, 983–992.
44. Colman, R. (1968) Effect of modification of a methionyl residue
on the kinetic and molecular properties of isocitrate dehydro-
genase. J. Bio l. C hem. 24 3 , 2454–2464.
45. Holmes, S.D. & Smith, R.G. (1983) Identification of histidine and
methionine residues in the active site of the human u terine pro-
gesterone receptor with the affinity labels 11a- and 16a-(bromo-
acetoxy) progesterone. Bi ochemistry 22 , 1729–1734.
46. D’Silva, C., Williams, C.H. & Massey, V. (1986) Electrophilic
amination o f a single methionine residu e located at the a ctive site
of
D
-amino acid oxidase b y O-(2,4-dinitrophenyl) hydroxylamine.
Biochemistry 25, 5602–5608.
47. D’Silva, C., W illiams, C.H. & Massey, V. (1987) Identification of
methionine-110 as the residue covalently modified in the electro-
philic inactivation of
D
-amino acid oxidase by O-(2,4-dinitro-