Báo cáo khóa học: S-(2,3-Dichlorotriazinyl)glutathione A new affinity label for probing the structure and function of glutathione transferases potx

Labrou Laboratory of Enzyme Technology, Department of Agricultural Biotechnology, Agricultural University of Athens, Greece S-2,3-Dichlorotriazinylglutathione SDTG was synthes-ized and s

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 an 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 of 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 dependence on SDTG concentration,

consistent with the formation of a reversible complex with

the enzyme (Kd 107.9 ± 2.1 lM for the fast phase, and

224.5 ± 4.2 lM 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 analogues, demonstrating

the specificity of the reaction When the enzyme was

inacti-vated (42% residual activity),
1 mol SDTG per mol

dimeric enzyme was incorporated Amino-acid analysis,

molecular modelling, and site-directed mutagenesis studies suggested that the modifying residue is Met121, which is located at the end of a-helix H¢¢¢3 and forms part of the xenobiotic-binding site The results reveal an unexpected structural communication between subunits, which consists

of mutually exclusive modification of Met residues across enzyme subunits Thus, modification of Met121 on one subunit prevents modification of Met121 on the other sub-unit This communication is governed by Phe51, which is located at the dimer interface and forms part of the hydro-phobic lock-and-key intersubunit motif The ability of SDTG to inactivate other glutathione-binding enzymes and GST isoenzymes was also investigated, and it was concluded that this new reagent may have general applicability as an affinity reagent for other enzymes with glutathione-binding sites

Keywords: affinity labelling; chlorotriazine; herbicides; xenobiotics

Glutathione transferases (GSTs; EC 2.5.1.18) comprise a

large family of glutathione (GSH)-binding enzymes which

catalyse the conjugation of GSH with a variety of

hydro-phobic electrophiles through the formation of a thioether

bond [1,2] These enzymes offer protection against toxic

xenobiotics and byproducts of oxidative metabolism In

addition to their catalytic activities, plant GSTs are also

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 helices

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 Omega [3,4,9] Whereas Zeta, Theta and Omega classes

of GSTs are found in plants and animals, the large Phi and Tau classes are unique to plants [9] In maize (Zea mays L),

42 GST isoenzymes have been identified so far [12] Some of them and their subunits have been characterized in detail [12–15] The isoenzyme GST I (or ZmGSTF1, according to 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 protein of 214 amino acids [12]

Affinity labelling is a useful tool for the identification and probing of specific catalytic and regulatory sites in purified enzymes and proteins [17–20] Affinity labelling experiments 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 analogues, such as S-(4-succinimidyl)benzophenone [21,22], S-(2-nitro-4-azidophenyl)glutathione [23], S-(4-bro-mo-2,3-dioxobutyl)glutathione [24], S-azidophenacylgluta-thione [25]

Detailed studies of GSTs are justified by their consid-erable agronomic and therapeutic potential For example, they are candidates for the development of transgenic plants with increased resistance to biotic and abiotic stress [26,27] In addition, they are promising candidates for

Correspondence to N E Labrou, Laboratory of Enzyme Technology,

Department of Agricultural Biotechnology, Agricultural University of

Athens, Iera Odos 75, GR-11855-Athens, Greece.

Tel./Fax: +30 2105294308, E-mail: lambrou@aua.gr

Abbreviations: CDNB, 1-chloro-2,4-dinitrobenzene; GSH,

glutathi-one; GST, glutathione S-transferase; G-site, glutathione-binding site;

H-site, xenobiotic-binding site; SDTG,

S-(2,3-dichlorotri-azinyl)glutathione.

Enzyme: Glutathione S-transferase (GST; EC 2.5.1.18).

(Received 20 April 2004, revised 8 July 2004, accepted 12 July 2004)

developing anticancer gene therapy drugs for protecting

normal cells from chemotherapeutics [28] Recently,

GST I has been successfully applied as an analytical

enzyme for the determination of herbicides in solution

[29] Therefore, detailed characterization of these enzymes

is of great importance In this study, a new alkylating

affinity label was designed and synthesized, and its

reaction with GST I investigated An unexpected

mech-anism of structural communication between the enzyme’s

subunits is revealed, which was obscure despite the

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 obtained from Boehringer,

Mannheim, Germany Molecular biology reagents, kits,

and Pfu DNA polymerase were from Promega Cyanuric

chloride (1,3,5-sym-trichlorotriazine), GSH (99%),

1-chloro-2,4-dinitrobenzene (CDNB; 99%), glutathione

reductase from Saccharomyces cerevisiae [300 unitsÆ(mg

protein))1] and L-lactate dehydrogenase from bovine

heart [1000 unitsÆ(mg protein))1] were from

Sigma-Aldrich Co

Synthesis, purification and analysis of SDTG

SDTG (Fig 1A) was synthesized by substituting the

chlorine atom of cyanuric chloride with GSH as reported

by Katusz et al [24] to produce

S-(4-bromo-2,3-dioxobu-tyl)glutathione, with the following modifications: cyanuric

chloride (1.6 mmol) was added to 30 mL cold (2C) water/

acetone (1 : 1, v/v) The pH was adjusted to 4.0 To the

above mixture was slowly added aqueous GSH (1.6 mmol;

5 mL) The pH was maintained throughout the reaction at

4.0 After the reaction was complete (1–2 h; 5C), the

mixture was extracted five times with chloroform

(5· 50 mL) The aqueous phase was collected and

concen-trated on a rotary evaporator until a solid powder appeared

The solid powder was stored desiccated at )20 C The

course of the reaction was followed and the products were

analysed by ascending analytical TLC on silica gel 60 plates

with a fluorescent indicator, using the solvent system

propan-2-ol/acetic acid/water (4 : 1 : 1, v/v/v) The product

contained primary amines (ninhydrin and

2,4,6-trinitroben-zenesulfonic acid tests) and no free thiol groups

(5,5-dithio-bis-(2-nitrobenzoic) acid test)

SDTG was purified by HPLC on a C18reverse-phase S5

ODS2 Spherisorb silica column (250 mm· 4.6 mm

inter-nal diameter) using a water/acetonitrile linear gradient

containing trifluoroacetic acid (0.1%, v/v) The starting

solvent system was 10% (v/v) acetonitrile and 90% (v/v)

water containing trifluoroacetic acid (0.1%, v/v) The purity

of the product was assessed by ascending analytical TLC on

silica gel 60 plates with a fluorescent indicator, using

propan-2-ol/acetic acid/water (4 : 1 : 1, v/v/v) as the solvent

system, and by HPLC on a C18 reverse-phase column It

was found to be at least 98.4% pure

SDTG was also analysed by positive ionization nano-electrospray MS using the Q-Tof (Micromass UK Ltd, Manchester, UK) mass spectrometer A capillary voltage of

1000 V and a sampling cone voltage of 40 V were used Data were acquired over 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 mM

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 decomposition of SDTG in a buffer identical with that used in the inactivation studies (100 mM potas-sium phosphate buffer, pH 6.5) was determined by meas-urement of 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) Enzyme (2 units) was incubated in the absence (m) or presence of 14.5 l 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 SDTG (j) At the times indicated, aliquots were withdrawn and assayed for activity.

Expression and purification of maize GST I and other

enzymes

Maize GST I was cloned into a pQE70 expression vector to

yield the pQEGST expression plasmid as described by

Labrou et al [14] Expression and purification of wild-type

GST I were performed as described [14] Expression of

mutants was also performed as described by Labrou et al

[14], but purification was achieved by using the affinity

adsorbent Cibacron blue 3GA–Sepharose, adsorbed at

0.1Mpotassium phosphate buffer, pH 7.0, and eluted with

0.1M potassium phosphate buffer, pH 7.0, containing

5 mM GSH Recombinant rat GST A1-1 [32] and human

GST A1-1 [33] were expressed in Escherichia coli and

purified on a hexyl-GSH column as described previously

[34] The expression vectors for rat GST A1-1 and human

GST A1-1 were much appreciated gifts from W M Atkins

(Department of Medicinal Chemistry, University of

Wash-ington, Seattle, WA, USA) Glutathione synthase from

S cerevisiae was purified to homogeneity as described

previously [35]

Site-directed mutagenesis

Site-directed mutagenesis was performed as described

by Weiner et al [36] The pairs of oligonucleotide primers

used in the PCRs were as follows: Phe51Ala mutation,

5¢-CGGAACCCCGCAGGTCGAGTTTCC-3¢ and 5¢-GA

CGAGGTGCTCGGGGCTCTT-3¢; Met121Ala

muta-tion, 5¢-ATCAGTCCGGCACTTGGGGGAACC-3¢ and

5¢-GAGGACGTCGAAGAGGATGGGTTACAG-3¢

The mutation (codon underlined above) was confirmed

by DNA sequencing on Applied Biosystems Sequencer

373A with the DyeDeoxy Terminator Cycle sequencing kit

Assay of enzyme activity and protein

Enzyme assays were performed by monitoring the

forma-tion of the conjugate of CDNB (1 mM) and GSH (2.5 mM)

at 340 nm (e¼ 9.6 mM )1 cm)1) at 30C according to a

published method [13,14] Observed reaction velocities were

corrected for spontaneous reaction rates when necessary

All initial velocities were determined in triplicate in buffers

equilibrated at constant temperature Protein concentration

was determined by the method of Bradford [37] using BSA

(fraction V) as standard

Enzyme inactivation studies

GST I was inactivated at 25C in 1 mL incubation mixture

containing potassium phosphate buffer, pH 6.5 (100 lmol),

SDTG (0–218.2 nmol) and enzyme (2 units, GST assay at

30C) The rate of inactivation was followed by periodically

removing samples (20 lL) for assay of enzymatic activity

[17,20]

Rate constants for the reaction exhibiting biphasic

kinetics were calculated from log(% remaining activity)

vs time (min), using the equation [19,20]:

Remaining activity¼ ð1  FÞek fast tþ Fek slow t

where F represents the fractional residual activity of the

partial active enzyme intermediate, and k and k are

the rate constants for the slow and fast phase of the reaction Analysis was performed using the GRAFIT (Erithacus Software Ltd, Horley, Surrey, UK) computer program

Kdwas determined as described previously [19,20] Studies of inactivation of GST I by SDTG in the presence of S-methyl-GSH and S-nitrobenzyl-GSH were performed at 25C 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 at

30C)

Inactivation of other enzymes (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 mM

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 dehydrogenase), SDTG (98.2 nmol) and enzyme (typically 2 units)

Kinetic analysis Steady-state kinetic measurements of native and SDTG-modified GST I were performed at 30C in 0.1M

potassium phosphate buffer, pH 6.5 Initial velocities were determined in the presence of 2.5 mM GSH; CDNB was used in the concentration range 0.06–1.2 mM Alternat-ively, CDNB was used at a fixed concentration (1 mM), and the GSH concentration varied in the range 0.15– 2.2 mM Solutions of GSH and analogues were freshly prepared each day and stored on ice under N2 All initial velocities were determined in triplicate in buffers equili-brated at constant temperature The apparent kinetic parameters kcat and Km were determined by fitting the collected steady-state data to the Michaelis–Menten equation by nonlinear regression analysis using theGRAFIT

computer program

Stoichiometry of SDTG binding to GST I GST I (100 lg) in 100 mM potassium phosphate buffer,

pH 6.5, was inactivated with 51.2 nmol SDTG at 25C The SDTG-modified enzyme was separated from the 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 mM potassium phosphate buffer, pH 6.5 The solution with SDTG–GST I covalent complex was then lyophilized and stored at )20 C The lyophilized SDTG-modified enzyme was dissolved in 8Murea and incubated with N-ethylmaleimide

to block free -SH groups, and then with Woodward’s reagent K (5 mM) or 2,4,6-trinitrobenzenesulfonic acid (5 mM) 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 determined 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]

UV spectroscopic studies

Far-UV spectra were measured with a Perkin–Elmer

Lamda 16 recording spectrophotometer at 25C The

enzyme (typically 0.02–0.05 mgÆmL)1) was dialysed against

0.01M potassium phosphate buffer, pH 7.0, and its UV

spectra were recorded between 250 and 190 nm

Molecular modelling

Structure manipulations and analysis were performed using

thePYMOLsoftware [41]

Results and Discussion

Synthesis of SDTG

SDTG was synthesized by nucleophile displacement of a

chlorine atom of the 1,3,5-sym-trichlorotriazine ring by the

-SH group of GSH The 1,3,5-triazine scaffold is of special

interest because of its synthetic accessibility, i.e one can take

advantage of the temperature-dependent successive

dis-placement of the chloride atoms by different nucleophiles

[18,42,43] Other advantages of triazine-based affinity labels

are their high stability in neutral buffer solutions and the

presence of electron-rich nitrogen sites on the triazine ring,

which increase the capability of forming additional

hydro-gen bonds with amino-acid residues within protein binding

sites [42,43] We have previously described the use of such

triazine-based probes to stoichiometrically label the active

site of oxaloacetate decarboxylase [18] and Clostridium

histolyticumclostripain [43]

The overall yield in the synthesis of SDTG from GSH

was
55% SDTG was purified by HPLC on a C18

reverse-phase S5 ODS2 Spherisorb silica column The product was

eluted at 8.8 min SDTG purity was assessed as described in

Experimental procedures The product showed a single spot

with Rf¼ 0.55, a UV absorption spectrum with a peak at

230 nm, and was negative in the

5,5-dithio-bis-(2-nitro-benzoic) acid test for free -SH groups and positive in the

ninhydrin and 2,4,6-trinitrobenzenesulfonic acid test for

primary amine The chloride content was found to be 2 mol

per mol SDTG Purified SDTG was also analysed by 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 25C, 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

(pH 6.5) was the same as that necessary for high GST

activity This probably affords an enzyme conformation

similar to that adopted during catalysis, thus creating more

favourable conditions for ligand binding The kinetics of

inactivation were biphasic (Fig 1B), with the rapid reaction

occurring immediately on exposure of the enzyme to SDTG

and the slow inactivation continuing to yield enzyme with a final residual activity of 42 ± 3% At all concentrations of SDTG used, biphasic kinetics were observed The rate of inactivation for the fast and slow phases was dependent on SDTG concentration, as illustrated in Fig 2 For both phases, a plot of 1/kobsvs 1/[SDTG] yields a straight line This indicates that the reaction obeys pseudo-first-order saturation kinetics and is consistent with reversible binding

of reagent before covalent modification according to the following equation [17–20]:

Eþ SDTG Ðkd E:SDTG!k3

E-SDTG where E represents the free enzyme, E:SDTG is the reversible complex, and E-SDTG is the covalent product The steady-state rate equation for the interaction is [23–25]:

1=kobs¼ 1=k3þ kd=ðk3½SDTGÞ where kobs is the rate of enzyme inactivation for a given concentration of SDTG, k3is the maximal rate of inacti-vation (min)1), and Kdis the apparent dissociation constant

of the E:SDTG complex From the data shown in Fig 2, Kd values of 107.9 ± 2.1 lMand 224.5 ± 4.2 lM, for the fast and slow reactions, respectively, were estimated Apparent maximal rate constants were determined to be 0.049 ± 0.002 min)1for the fast reaction, and 0.0153 ± 0.001 min)1 for the slower reaction

The stability of SDTG against hydrolysis was demon-strated by measuring the rate of chloride released from the molecule in conditions identical with those used in the inac-tivation experiments The results showed that the first-order rate constant for SDTG hydrolysis was 1.2· 10)5min)1 This corresponds to 0.07% and 0.03% of the rate observed for the slow and fast phase, respectively This suggests that

Fig 2 Dependence of the pseudo-first-order rate constant for the fast (j) and slow phase (r) of inactivation on the concentration of SDTG, expressed as a double-reciprocal plot GST I (2 units) was incubated at

pH 6.5 and 25 C with various concentrations of SDTG (14.5– 219.3 l M ), and the rate constants were calculated as described in the text The slope and intercept of the double-reciprocal plot were cal-culated by unweighted linear regression analysis.

the slow phase of inactivation observed is not due to the

decomposition of SDTG but is the direct result of SDTG–

enzyme interaction [31]

To determine the stoichiometry of incorporation of

SDTG, modified and unmodified GST I were treated with

Woodward’s reagent K and 2,4,6-trinitrobenzenesulfonic

acid, and the amount of covalently bound SDTG was

determined by subtraction of the total number of carboxy

and amino groups in the modified and unmodified enzyme

The results of this experiment are shown in Table 1 As

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 analogues

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 The 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 enzyme exhibits kinetic properties

that are different from that of the unmodified enzyme The

results are shown in Table 2 The modified enzyme exhibits

about threefold reduced affinity for GSH and about

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 site-directed mutagenesis Direct amino-acid sequence determination of the modified peptide was not possible because of its instability during Edman degradation reac-tions The results from a typical amino-acid analysis indicated that the modified enzyme exhibits loss of 1 mol Met per mol enzyme From analysis of the crystal structure

of the enzyme in complex with S-atrazine–GSH conjugate [11], it is evident that Met121 is within or close to the binding site and accessible for covalent modification (Fig 3) It is located at the end of a-helix H¢¢¢3and forms part of the xenobiotic-binding site [11] Although the thioether bond of methionine is usually considered to be

of low reactivity, a number of pieces of experimental evidence from affinity labelling experiments, suggest that in

Table 1 Stoichiometry of SDTG binding to GST I Total carboxy and

primary amino groups for the modified and unmodified enzyme were

determined by the Woodward’s Reagent K and

2,4,6-trinitro-benzenesulfonic acid assays.

Unmodified

GST I

SDTG-modified GST I

SDTG-modified Phe51Ala mutant Carboxy groups 26.8 ± 0.3 29.2 ± 0.2 31.2 ± 0.3

Primary amino

groups

14.9 ± 0.1 16.2 ± 0.2 17.3 ± 0.3

Table 2 Steady-state kinetic parameters of unmodified and

SDTG-modified GST I for the CDNB conjugation reaction at pH 6.5 and

30
C.

GST I

K m (m M )

k cat · 10)2 (s)1)

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 drawn 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 spacefill representation and Phe51 is shown in blue.

several enzymes may act as a reactive 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-dinitrophenyl)hydroxylamine, respectively

To provide further experimental evidence and establish

the involvement of Met121 in the reaction with SDTG,

site-directed mutagenesis experiments were carried out Met121

was mutated to Ala, and the resulting mutant was subjected

to inactivation studies The Met121Ala mutant was resistant

to inactivation by SDTG (90 lM) at pH 6.5 and 25C,

compared with the wild-type enzyme Comparison of the

far-UV difference spectra of native and mutated enzyme

indicated the absence of any structural perturbation caused

by the mutation (Fig 4) This rules out the possibility that

the resistance of the Met121Ala mutant to inactivation is

due to conformational changes in its structure These

observations imply that SDTG binds at one site at all stages

of the reaction The best explanation for these results may

be that the reaction of SDTG at the binding site of one

subunit changes the conformation of the other subunit,

thereby completely abolishing reaction of SDTG with the

second subunit

Analysis of the crystal structure of the enzyme in complex

with the S-atrazine–GSH conjugate [11] provides a

struc-tural explanation for the intrasubunit communication

observed on reaction of Met121 with SDTG Although

the H-sites of neighbouring subunits are distant (Fig 3B), a

plausible mode of communication between them exists

Structural examination reveals that the key residue bridging

the dimer interface, Phe51, may have an important role in

intrasubunit communication This residue forms the

lock-and-key motif responsible for a highly conserved

hydro-phobic interaction in the subunit interface 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 alternate subunit are largely found in a single kinked a-helix H¢¢3(Fig 3B), the signal may be transmitted via the helix to H-site residues such as Met121, Ile118, Leu122 and Phe114, which are located at the end of this helix Conformational changes in these residues would then change the affinity for CDNB binding, which is supported by the finding that the Kmof 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¢¢¢3of the adjacent subunit which contains Met121

To confirm the key role of Phe51 in this hypothesis, site-directed mutagenesis was used The mutant Phe51Ala was expressed, purified, and subjected to inactivation studies (Fig 5) Upon reaction with SDTG at pH 6.5 and

25C, the mutant was progressively inactivated to a final residual activity of about 1.9% (Fig 5) Comparison of the far-UV difference spectra of native and mutated enzyme (data not shown) indicates the absence of any structural perturbation caused by the mutation Amino-acid analysis of the SDTG-modified Phe51Ala mutant and determination of its total amino and carboxy content suggests that the modified residue is also methionine, and

2 mol SDTG per mol enzyme was incorporated (Table 1) This provides strong evidence that the inability

of SDTG to attack the other subunit in native GST I is the indirect result of the interaction between the two enzyme subunits, and that this subunit interaction is absent in the Phe51Ala mutant

The hydrophobic lock-and-key intersubunit motif involving Phe51 is the major structural feature conserved

Fig 4 Far-UV difference spectroscopy of the wild-type GST I

(a, 0.05 mgÆmL-1) and mutant Met121Ala (b, 0.0375 mgÆmL-1) Spectra

were measured at 25 C in 0.01 M potassium phosphate buffer at

pH 7.0.

Fig 5 Time course of inactivation of wild-type GST I and mutant Phe51Ala by SDTG Wild-type (r) and mutant Phe51Ala (j) were incubated in the presence of 72.7 and 92 l M SDTG, respectively, at

pH 6.5 and 25 C At the times indicated, aliquots were withdrawn and assayed for enzymatic activity.

at the dimer interface of GST I Similar lock-and-key motifs

have also been observed for the classes Alpha, Mu and Pi

GSTs [48–50] The conserved hydrophobic interaction

formed by the side chain of the Phe51 residue, which

protrudes from the loop in domain I of one monomer into

the hydrophobic pocket of domain II of the other

mono-mer, physically anchors the two subunits together at either

end of the interface

Explanation of the biphasic kinetics

An average incorporation of 0.5 mol reagent per mol

enzyme subunit indicates that reaction of SDTG with one

Met121 prevents the reaction of the Met121 of the second

subunit The biphasic kinetics observed may be explained

by assuming that the two subunits, or at least the

conformation of the Met121 side chains in each subunit,

are not equivalent regarding the reaction with SDTG, and

exhibit different reactivity The existence of such a

nonsymmetrical arrangement of GST 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 between 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

identified The plot shows significant differences in several

regions between chains A and B, including a-helix H¢¢¢3

(residues 188–122) A large difference is centred on

Met121 In particular, the mean B-factors of Met121 at

the A and B chains are 26.67 A˚2 and 49.26 A˚2, and the

B-factors of S atoms are 38.14 A˚2 and 79.30 A˚2,

respect-ively It is therefore reasonable to propose that

conform-ational changes and changes in dynamics may also

contribute to the observed biphasic kinetics

Results from steady-state kinetics using CDNB and

1,2-dichloro-4-nitrobenzene as electrophilic substrates for GSTs

from several classes are consistent with the idea of two noncooperative binding sites However, the large bulky aflatoxin–GSH conjugate [51] and the product analogue glutathionyl S-[4-(succinimidyl)benzophenone] [22] have been shown to bind to mouse Alpha class 2-2 and rat liver GST enzymes with a stoichiometry of 1 mol per mol enzyme dimer, and binding 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 35C, whereas negative cooperativity occurs below 25C [52] These results suggest that the two binding sites may not be independent and further support the

cooperative self-preservation mechanism proposed by Ricci et al [54] for the human P1-1 enzyme According to this mechanism, a cooperativity is utilized by the enzyme to provide self-preservation against inhibitors or physical factors that threaten its catalytic efficiency This 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 that the modification of one enzyme subunit of the GST I homodimer prevents modification of the other subunit, which suggests that the two enzyme active sites are co-ordinated

Reaction of SDTG with other GSH-binding enzymes

To demonstrate the wide applicability of SDTG as an affinity label for other GSH-binding enzymes such as

S cerevisiaeglutathione reductase, glutathione synthase, rat GST A1-1 and human GST A1-1, inactivation studies were carried out The pseudo-first-order rates of inactivation observed at a SDTG concentration of 98.2 lMand in the presence and absence of 1 mMS-methyl-GSH are summar-ized in Table 3 All enzymes were susceptible to inactivation

by SDTG The protective effect of S-methyl-GSH suggests that the reaction is specific It is interesting to note that the human and rat GST A1-1 isoenzymes obeyed biphasic kinetics, with residual activity after labelling of 48% and 33%, respectively, which confirms the conclusions on maize

Fig 6 Structural flexibility of GST I A plot of the crystallographic

B-factors along the polypeptide chains A and B obtained from the

crystal structure of GST I in complex with S-atrazine–GSH conjugate

(PDB code 1bye [11]) The plot was produced by the WHAT IF software

package [53] The height at each residue position indicates the average

B-factor of all atoms in the residue.

Table 3 Observed rates of inactivation (k obs ) of GSH-binding enzymes and bovine heart L -lactate dehydrogenase by SDTG in the presence and absence of 1 m M S-methyl-GSH NI, No inactivation.

Enzyme

k obs · 10)3 (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 ± 0.1a 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

a Fast phase inactivation rate.

GST I In addition, the ability of SDTG to inactivate a

non-GSH-dependent enzyme such as bovine heart L-lactate

dehydrogenase was investigated SDTG did not inactivate

L-lactate dehydrogenase and did not show any inhibitory

effect on its catalytic reaction This finding strengthens the

view that the SDTG acts as a true affinity label for the

GSH-binding site and indicates that this new reagent may have

wider applicability as an affinity label for other enzymes

with GSH-binding sites

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