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 the specificity of the reaction. When the enzyme was inacti- vated (42% residual activity), % 1 m ol 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. T hus, modification of Met121 on one subunit prevents modification of M et121 on the other sub- unit. This communication is governed by Phe51, which is located at t he dimer i nterface and forms part of the h ydro- phobic lock-and-key intersubunit motif. The ability of SDTG to inactivate other glutathione-binding enzymes a nd GST isoenzymes was also investig ated, 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; h erbicides; 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 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 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. F or 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. Labro u, Laboratory of E n zyme Technology, Department of Agricultural Biotechnology, Agricultural University of Athens, I era Odos 75, GR-11855-Athens, Greece. Tel./Fax: +30 2105294308, E-mail: lambro u @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, r evised 8 July 2004, accepted 12 July 2004) Eur. J. Biochem. 271, 3503–3511 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04285.x developing anticancer gene therapy drugs for protecting normal cells from chemotherapeutics [28]. Recently, GST I has b een successfully applied as a n a nalytical enzyme for the determination of herbicides in solution [29]. Therefore, detailed characterization of these en zymes is of great importance. In this study, a new a lkylating affinity label was designed and synthesized, and its reaction with GST I investigated. A n unexpected mech- anism of structural communication between the enzyme’s subunits is revealed, w hich 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 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 4.0. After the reaction was complete (1–2 h; 5 °C), the mixture was extracted five times with chloroform (5 · 50 mL). The a queous phase was collected and concen- trated on a r otary evaporator until a solid powder a ppeared. The solid powder was stored desiccated at )20 °C. The course of the reaction was followed and the p roducts were analysed by ascending analytical TLC on silica g el 60 plates with a fluorescent indicator, using t he solvent s ystem propan-2-ol/acetic acid/water (4 : 1 : 1 , v /v/v). The product contained primary amines (ninhydr in 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 C 18 reverse-phase S5 ODS2 Spherisorb silica c olumn (250 mm · 4.6 mm i nter- nal diameter) using a water/acetonitrile linear gradient containing trifluoroacetic acid (0.1%, v/v). T he starting solvent system was 10% (v/v) acetonitrile and 90% (v/v) water c ontaining trifluoroacetic acid (0.1%, v/v). The purity of the product w as 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) a s the solvent system, and by HPLC on a C 18 reverse-phase column. It was found to be at least 98.4% pure. SDTG was also analysed by positive ionization nano- electrospray MS u sing 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 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 SDTG (j). At the times indicated, aliqu ots were withdrawn and assa yed f or ac tivity. 3504 G. A. Kotzia and N. E. Labrou (Eur. J. Biochem. 271) Ó FEBS 2004 Expression and purification of maize GST I and other enzymes Maize G ST I w as 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]. E xpression of mutants was also performed as described by Labrou et al. [14], but purification was achieved by using the a ffinity adsorbent Cibacron blue 3GA–Sepharose, adsorbed at 0.1 M potassium phosphate buffer, pH 7.0, and eluted with 0.1 M potassium p hosphate buffer, pH 7.0, containing 5m M GSH. Recombinan t rat GST A 1-1 [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 a ppreciated gifts from W. M. Atkins (Department of Medicinal Chemistry, University of Wash- ington, Seattle, WA, U SA). Glutathione synthase from S. cerevisiae was purified to homogeneity as described previously [35]. Site-directed mutagenesis 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 (fraction V ) as standard. Enzyme inactivation studies GST I was inactivated at 25 °C i n 1 mL incubation mixture containing potass ium phosphate buffer, pH 6.5 ( 100 lmol), SDTG (0–218.2 nmol) and enzyme (2 units, GST assay at 30 °C). T he rate of inactivation was followed by p eriodically 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. t ime (min), using the equation [19,20]: Remaining a ctivity ¼ð1 À FÞe Àk fast t þ Fe Àk slow t where F represents the fractional residual activity of the partial active enzyme intermediate, and k fast and k slow are the rate constants for the slow a nd fast phase of the reaction. Analysis was performed using the GRAFIT (Erithacus Software L td, Horley, Surrey, UK) computer program. 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 determined in the presence of 2.5 m M GSH; CDNB was used in the concentration range 0.06–1.2 m M .Alternat- ively, CDNB was used at a fixed concentration (1 m M ), and the GSH concentration varied in the range 0.15– 2.2 m M . Solutions of GSH and an alogues were f reshly prepared each day and stored on ice under N 2 . All initial velocities were determined in triplicate in buffers equili- brated at constant temperature. The apparent kinetic parameters k cat and K m were determined by fitting the collected steady-state data to the Michaelis–Menten equation by nonlinear regression analysis using t he GRAFIT computer program. Stoichiometry of SDTG binding to GST I GST I (100 lg) in 100 m M 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 Far-UV spectra were measured with a Perkin–Elmer Lamda 16 recording spectrophotometer at 25 °C. The enzyme (typically 0.02–0.05 mgÆmL )1 ) was dialysed against 0.01 M 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 the PYMOL software [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 i ts synthetic accessibility, i.e . one can t ake advantage of the temperature-dependent successive dis- placement of the chloride atoms b y different nucleophiles [18,42,43]. O ther advantages o f t riazine-based a ffinity 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 bin ding sites [42,43]. We have previously described the use of s uch triazine-based probes to stoichiometrically label t he active site of oxaloacetate decarboxylase [18] and Clostridium 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 favourable conditions for ligand binding. The kinetics of inactivation were biphasic (Fig. 1B), wit h t he r apid reaction occurring immediately on e xposure of the enzyme to SDTG and the slow inactivation continuing to yield enzyme with a final residual activity of 42 ± 3%. A t all concentrations of SDTG used, biphasic kinetics were observed. The rate of inactivation for the fast and slow phases was dependent on SDTG c oncentration, as illustrated in F ig. 2. F or both phases, a plot of 1/k obs vs. 1/[SDTG] yields a straight line. This indicates that t he 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]: E þ SDTG Ð k d E:SDTG À! k 3 E-SDTG whereErepresentsthefreeenzyme,E:SDTGisthe reversible complex, and E-SDTG is the covalent product. The steady-state rate equation f or the interaction is [2 3–25]: 1=k obs ¼ 1=k 3 þ k 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- strated b y m easuring the rate o f c hloride released f rom the molecule in conditions identical with those used i n t he inac- tivation e xperiments. The results showed t hat t he fir st-order rate constant for SDTG hydrolysis was 1.2 · 10 )5 min )1 . This corresponds to 0.07% and 0.03% of the rate ob served 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 a s a doub le-reciprocal plot. GS T I (2 units) was i ncubated 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 d escribed in the text. The slope and intercept of the double -reciprocal plot were cal- culated by u nweight ed linear r egression analysis . 3506 G. A. Kotzia and N. E. Labrou (Eur. J. Biochem. 271) Ó FEBS 2004 the slow phase of inactivation observed is not due to the decomposition o f SDTG but is the direct result of S DTG– enzyme inte raction [31]. To determine the stoichiometry of i ncorporation of SDTG, modified and unmodified G ST 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 o f the total number of c arboxy and amino groups in the modified and un modified 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 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- 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 t he 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¢¢¢ 3 and forms part of the xenobiotic-binding site [11]. A lthough 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 bin ding to GST I. Total carboxy and primary am ino groups for the mo dified and un modified enzyme were determined by the Woodward’s Reagent K and 2,4,6-trinitro- benzenesulfonic a cid 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 ) 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- dinitrophenyl)hydroxylamine, respectively. To provide further experimental evidence and establish the i nvolvement of Met121 in the reaction with SDTG, site- directed mutagenesis experiments were carried out. Met121 was m utated to Ala, and the resulting mutant was subjected to inactivation studies. T he Met121Ala mutant w as resistant to inactivation by SDTG (90 l M )atpH6.5and25°C, compared with the wild-type enzyme. Comparison of the far-UV difference spe ctra 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 o ther subunit, thereby completely abolishing reaction of SDTG with the second subunit. Analysis of the crystal structu re of t he enzyme in co mplex with the S-atrazine–GSH conjugate [11] provides a struc- tural explanation for the intrasubunit c ommunication observed on reaction of Met121 with SDTG. Although the H -sites of neighbouring subunits are distant (Fig. 3B), a plausible mode o f communication between them exists. Structural examin ation r eveals 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 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 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 c ontent suggests that the modified residue is also methionine, and % 2 mol SDTG per mol enzyme was incorporated (Table 1). This provides strong evidenc e 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 °Cin0.01 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)andmutantPhe51Ala(j)were incubated i n the presence of 72.7 and 92 l M SDTG, respectively, at pH 6.5 and 25 °C. At th e times indicated, aliquo ts were with drawn and assayed f or e nzymatic a ctivity. 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 identified. The plot shows significant d ifferences in several regions between chains A and B, including a-he lix H¢¢¢ 3 (residues 188–122). A large difference is cent red 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 ,andthe 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 f rom steady-state kinetics using CDNB and 1,2- dichloro-4-nitrobenzene as e lectro philic 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 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 M S-methyl-GSH a re summar- ized in Table 3. All enzymes were susceptible to inactivation by SDTG. The protective effect of S-methyl-GSH suggests that the r eaction is s pecific. It i s interesting to note that the human and r at GST A1-1 isoenzymes obeyed b iphasic kinetics, with residual activity after labelling of 48% and 33%, respectively, which confirms the conclusions o n maize Fig. 6. Structural flexibility of GST I. A plot of the crystallographic B-factors along the p olypeptide chains A and B obtained from the crystal structure of GST I i n complex with S-atrazine–GSH conjugate (PDB code 1bye [11]). The plot was produced by the WHAT IF software package [53]. The height at e ac h residue position indicates the average B-factor of al l atoms in the residue. Table 3. Observed rates of inactivation (k obs ) of GSH-binding enzymes and bovine heart L -lactate dehydrogenase b y SDTG in t he 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 ± 0.1 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- 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 a s a true affinity label for the GSH- binding site and i ndicates t hat t his new reagent may have wider applicability as an affinity label for other enzymes with GSH-binding sites. References 1. 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