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• W2050168058 abstract "In plants, glutathione accumulates in response to different stress stimuli as a protective mechanism, but only limited biochemical information is available on the plant enzymes that synthesize glutathione. Glutamatecysteine ligase (GCL) catalyzes the first step in glutathione biosynthesis and plays an important role in regulating the intracellular redox environment. Because the putative Arabidopsis thaliana GCL (AtGCL) displays no significant homology to the GCL from bacteria and other eukaryotes, the identity of this protein as a GCL has been debated. We have purified AtGCL from an Escherichia coli expression system and demonstrated that the recombinant enzyme catalyzes the ATP-dependent formation of γ-glutamylcysteine from glutamate (Km = 9.1 mm) and cysteine (Km = 2.7 mm). Glutathione feedback inhibits AtGCL (Ki ∼1.0 mm). As with other GCL, buthionine sulfoximine and cystamine inactivate the Arabidopsis enzyme but with inactivation rates much slower than those of the mammalian, bacterial, and nematode enzymes. The slower inactivation rates observed with AtGCL suggest that the active site differs structurally from that of other GCL. Global fitting analysis of initial velocity data indicates that a random terreactant mechanism with a preferred binding order best describes the kinetic mechanism of AtGCL. Unlike the mammalian GCL, which consists of a catalytic subunit and a regulatory subunit, AtGCL functions and is regulated as a monomeric protein. In response to redox environment, AtGCL undergoes a reversible conformational change that modulates the enzymatic activity of the monomer. These results explain the reported posttranslational change in AtGCL activity in response to oxidative stress. In plants, glutathione accumulates in response to different stress stimuli as a protective mechanism, but only limited biochemical information is available on the plant enzymes that synthesize glutathione. Glutamatecysteine ligase (GCL) catalyzes the first step in glutathione biosynthesis and plays an important role in regulating the intracellular redox environment. Because the putative Arabidopsis thaliana GCL (AtGCL) displays no significant homology to the GCL from bacteria and other eukaryotes, the identity of this protein as a GCL has been debated. We have purified AtGCL from an Escherichia coli expression system and demonstrated that the recombinant enzyme catalyzes the ATP-dependent formation of γ-glutamylcysteine from glutamate (Km = 9.1 mm) and cysteine (Km = 2.7 mm). Glutathione feedback inhibits AtGCL (Ki ∼1.0 mm). As with other GCL, buthionine sulfoximine and cystamine inactivate the Arabidopsis enzyme but with inactivation rates much slower than those of the mammalian, bacterial, and nematode enzymes. The slower inactivation rates observed with AtGCL suggest that the active site differs structurally from that of other GCL. Global fitting analysis of initial velocity data indicates that a random terreactant mechanism with a preferred binding order best describes the kinetic mechanism of AtGCL. Unlike the mammalian GCL, which consists of a catalytic subunit and a regulatory subunit, AtGCL functions and is regulated as a monomeric protein. In response to redox environment, AtGCL undergoes a reversible conformational change that modulates the enzymatic activity of the monomer. These results explain the reported posttranslational change in AtGCL activity in response to oxidative stress. Regulation of the intracellular redox environment is critical in cellular physiology for influencing signaling pathways and cell fate in response to stress (1Finkel T. Curr. Opin. Cell Biol. 2003; 15: 247-254Crossref PubMed Scopus (1229) Google Scholar). In plants, as in other organisms, glutathione plays multiple roles as protection against various environmental stresses (2Noctor G. Foyer C.H. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1998; 49: 249-279Crossref PubMed Scopus (4562) Google Scholar). As an antioxidant, glutathione quenches reactive oxygen species and is involved in the ascorbate-glutathione cycle that eliminates peroxide (2Noctor G. Foyer C.H. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1998; 49: 249-279Crossref PubMed Scopus (4562) Google Scholar). Plants use glutathione for the detoxification of xenobiotics (3Matringe M. Scalla R. Plant Physiol. 1988; 86: 619-622Crossref PubMed Google Scholar), herbicides (4May M.J. Vernoux T. Leaver C. Van Montagu M. Inze D. J. Exp. Bot. 1998; 49: 649-667Google Scholar), air pollutants such as sulfur dioxide and ozone (5Madamanchi N.R. Alscher R.G. Plant Physiol. 1991; 97: 88-93Crossref PubMed Scopus (106) Google Scholar, 6Sen-Gupta A. Alscher R.G. McCune D. Plant Physiol. 1991; 96: 650-655Crossref PubMed Scopus (113) Google Scholar), and heavy metals (7Grill E. Winnacker E.L. Zenk M.H. Science. 1985; 230: 674-676Crossref PubMed Scopus (1000) Google Scholar). Although glutathione accumulates in response to different stress stimuli in plants, the structural and kinetic properties of the plant enzymes responsible for its production remain biochemically uncharacterized. Glutathione synthesis occurs in two ATP-dependent steps. In the first reaction, glutamate-cysteine ligase (GCL) 1The abbreviations used are: GCL, glutamate-cysteine ligase (alternate name: γ-glutamylcysteine synthetase); AtGCL, Arabidopsis thaliana glutamate-cysteine ligase; DTT, D/L-dithiothreitol; ESI, electrospray ionization; MOPSO, 3-(N-morpholino)-2-hydroxypropanesulfonic acid; MS, mass spectrometry; Ni-NTA, nickel-nitrilotriacetic acid; TOF, time-of-flight; ter, ternary. (EC 6.3.2.2) catalyzes formation of the dipeptide γ-glutamylcysteine from cysteine and glutamate (Scheme I). Addition of glycine to the dipeptide occurs in a second reaction, catalyzed by glutathione synthetase. Of the two enzymes, GCL appears to be rate-limiting (8Richman P.G. Meister A. J. Biol. Chem. 1975; 250: 1422-1426Abstract Full Text PDF PubMed Google Scholar). Exposure to heavy metals increases the levels of GCL mRNA in Brassica juncea and activates transcription of both GCL and glutathione synthetase in Arabidopsis thaliana (9Schafer H.J. Greiner S. Rausch T. Haag-Kerwer A. FEBS Lett. 1997; 404: 216-220Crossref PubMed Scopus (64) Google Scholar, 10Schafer H.J. Haag-Kerwer A. Rausch T. Plant Mol. Biol. 1998; 37: 87-97Crossref PubMed Scopus (130) Google Scholar, 11Xiang C. Oliver D.J. Plant Cell. 1998; 10: 1539-1550Crossref PubMed Scopus (588) Google Scholar). Overexpression of E. coli GCL in plants improves tolerance to cadmium and arsenic, demonstrating the importance of this enzyme in heavy metal protection (12Zhu Y.L. Pilon-Smits E.A. Tarun A.S. Weber S.U. Jouanin L. Terry N. Plant Physiol. 1999; 121: 1169-1177Crossref PubMed Scopus (497) Google Scholar, 13Dhankher O.P. Li Y. Rosen B.P. Shi J. Salt D. Senecoff J.F. Sashti N.A. Meagher R.B. Nat. Biotechnol. 2002; 20: 1140-1145Crossref PubMed Scopus (449) Google Scholar). Bioinformatic analysis of the GCL genes from multiple species suggests that these sequences group into three families (14Copley S.D. Dhillon J.K. Genome Biol. 2002; 3: 25.1-25.16Crossref Google Scholar), 1) sequences from the γ-proteobacteria, such as Escherichia coli; 2) sequences from non-plant eukaryotes (mammals, Drosophila, and nematodes); and 3) sequences from plants (Arabidopsis) and α-proteobacteria (Rhizobium). Sequence comparisons within each family show similarity, but pairwise comparisons between groups display no statistically significant relationships (14Copley S.D. Dhillon J.K. Genome Biol. 2002; 3: 25.1-25.16Crossref Google Scholar). For example, A. thaliana GCL (AtGCL) shares less than 15% amino acid sequence identity with the members of other families (15May M.J. Leaver C.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10059-10063Crossref PubMed Scopus (122) Google Scholar). The differences among GCL sequences also reflect the functional properties of each family. Of the three GCL families, the enzymes from the non-plant eukaryotes are the best studied. The mammalian and Drosophila GCL consist of a 70-kDa catalytic or heavy subunit and a 30-kDa regulatory or light subunit (16Sekura R. Meister A. J. Biol. Chem. 1977; 252: 2599-2605Abstract Full Text PDF PubMed Google Scholar, 17Fraser J.A. Saunders R.D.C. McLellan L.I. J. Biol. Chem. 2002; 277: 1158-1165Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). The heavy subunit catalyzes the formation of γ-glutamylcysteine and is inhibited by glutathione (18Seelig G.F. Simondsen R.P. Meister A. J. Biol. Chem. 1984; 259: 9345-9347Abstract Full Text PDF PubMed Google Scholar), whereas the light subunit increases the affinity of the enzyme for glutamate and decreases the inhibitory effect of glutathione (19Huang C.S. Chang L.S. Anderson M.E. Meister A. J. Biol. Chem. 1993; 268: 19675-19680Abstract Full Text PDF PubMed Google Scholar). Interestingly, the GCL from Trypanosoma brucei and the mammalian catalytic subunit are related by 45% amino acid identity, but the T. brucei enzyme functions as a 77-kDa monomer with kinetic constants similar to the “activated” heterodimeric rat enzyme (20Lueder D.V. Phillips M.A. J. Biol. Chem. 1996; 271: 17485-17490Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). From the γ-proteobacteria family of GCL, the E. coli enzyme has been isolated and characterized as a functional 58-kDa monomeric protein (21Kelly B.S. Antholine W.E. Griffith O.W. J. Biol. Chem. 2002; 277: 50-58Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). L−Glutamate+L−Cysteine+ATP→GCLL−γ−glutamyl−L−cysteine+ADP+Pi(SCHEME 1) The plant GCL are largely unexamined at the molecular level. Expression cloning isolated a cDNA from Arabidopsis that was unrelated to the mammalian, E. coli, or yeast GCL but complemented a GCL-deficient E. coli strain (14Copley S.D. Dhillon J.K. Genome Biol. 2002; 3: 25.1-25.16Crossref Google Scholar). However, doubts about the specificity of the assay used to measure GCL activity in cell lysates and the limited sequence similarity with other GCL have challenged the identity of the AtGCL clone (4May M.J. Vernoux T. Leaver C. Van Montagu M. Inze D. J. Exp. Bot. 1998; 49: 649-667Google Scholar, 22Brunhold C. Rennenberg H. Prog. Bot. 1996; 58: 164-186Google Scholar). To characterize the biochemical properties of the putative AtGCL, recombinant enzyme was expressed in E. coli and purified to homogeneity. We demonstrate that the purified protein catalyzes the formation of γ-glutamylcysteine. Our analysis shows that the Arabidopsis enzyme shares some functional properties with the GCL from other species but is regulated differently than the GCL from either bacteria or non-plant eukaryotes. Materials—Integrated DNA Technologies, Inc. synthesized all oligo-nucleotides used in this study. The pGEM-T Easy vector was obtained from Promega. E. coli Rosetta (DE3) cells were from Novagen. Ni2+-nitrilotriacetic acid (NTA)-agarose was bought from Qiagen. Benzamidine-Sepharose and the HiLoad 26/60 Superdex-75 FPLC column were from Amersham Biosciences. All other reagents were purchased from Sigma-Aldrich and were of ACS reagent quality or better. Cloning and Generation of Expression Vectors—AtGCL (GenBank Z29490) (15May M.J. Leaver C.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10059-10063Crossref PubMed Scopus (122) Google Scholar) was amplified by PCR from an Arabidopsis cDNA library using 5′-dCCATGGCATGGCGCTGCTGTCTCAAGCAGG-3′ as the forward primer (NcoI site is underlined, the AtGCL start codon is in bold, and two E. coli optimized codons are in italic) and 5′-dTTATAGACACCTTTTGTTCACGTCCCATTTTC-3′ as the reverse primer (the putative AtGCL stop codon is in bold). The 1.6-kb PCR product was subcloned into the pGEM-T Easy vector (Promega). Automated nucleotide sequencing confirmed the fidelity of the PCR product (Washington University Sequencing Facility, St. Louis, MO). The pHIS8-AtGCL expression vector was constructed by digesting pGEM-T-AtGCL with NcoI and NotI and then ligating the fragment into a NcoI/NotI-digested pHIS8 vector (23Jez J.M. Ferrer J.L. Bowman M.E. Dixon R.A. Noel J.P. Biochemistry. 2000; 39: 890-902Crossref PubMed Scopus (284) Google Scholar). An expression construct of AtGCL with a truncated N terminus (pHIS8-AtGCLΔ85) was generated by PCR using the appropriate oligonucleotides. Expression in E. coli and Protein Purification—Expression constructs were transformed into E. coli Rosetta (DE3) cells. Transformed E. coli were grown at 37 °C in Terrific broth containing 50 μg ml-1 kanamycin and 34 μg ml-1 chloramphenicol until A600 nm ∼0.8. After induction with 1 mm isopropyl 1-thio-β-d-galactopyranoside, the cultures were grown at 18 °C for 6 h. Cells were pelleted by centrifugation and resuspended in lysis buffer (50 mm Tris (pH 8.0), 500 mm NaCl, 20 mm imidazole, 5 mm MgCl2, 10% (v/v) glycerol, and 1% (v/v) Tween 20). After sonication and centrifugation, the supernatant was passed over a Ni2+-NTA column previously equilibrated with lysis buffer. His-tagged protein was eluted with elution buffer (wash buffer containing 250 mm imidazole). Incubation with thrombin during overnight dialysis at 4 °C against wash buffer removed the His-tag. Dialyzed protein was reloaded on a Ni2+-NTA column, and the flow-through was depleted of thrombin using a benzamidine-Sepharose column. The flow-through of this step was dialyzed overnight against 30% (v/v) glycerol, 25 mm HEPES (pH 7.5), 5 mm MgCl2, and 100 mm NaCl then loaded onto a Superdex-75 FPLC column equilibrated in the same buffer without glycerol. Enzyme Assays—The activity of AtGCL was determined spectrophotometrically at 25 °C by measuring the rate of ADP formation using a coupled assay with pyruvate kinase and lactate dehydrogenase. A standard reaction mixture (0.5 ml) contained 100 mm MOPSO (pH 7.0), 150 mm NaCl, 20 mm MgCl2, 10 mm cysteine, 20 mm sodium glutamate, 5 mm disodium ATP, 2 mm sodium phosphoenolpyruvate, 0.2 mm NADH, 5 units of type III rabbit muscle pyruvate kinase, and 10 units of type II rabbit muscle lactate dehydrogenase. Reactions were initiated by adding AtGCL (50 μg). The rate of decrease in A340 nm was followed using a Cary Bio300 spectrophotometer. Steady-state kinetic parameters were determined by initial velocity experiments. Measurements of the kcat and Km values for glutamate (1-80 mm) were made at 20 mm ATP and 20 mm cysteine. Kinetic constants for cysteine (0.1-20 mm) were measured at 20 mm ATP and 80 mm glutamate. For determination of the kinetic constants for ATP (0.25-20 mm), 20 mm cysteine and 80 mm glutamate were used. Kinetic parameters were calculated to fit untransformed data to v = kcat[S]/(Km + [S]) using Kaleidagraph (Synergy Software). Inhibition and Inactivation Assays—For glutathione inhibition of AtGCLΔ85, initial velocities were determined spectrophotometrically using the standard assay system. Enzyme activity was determined after addition of glutathione (0-5 mm) to assay solutions containing either varied glutamate (1-40 mm) or cysteine (0.5-20 mm). Global fitting analysis was used to simultaneously fit all data to the equation for non-competitive inhibition, v = Vmax/((1 + [I]/Ki)(1 + Km/[S])) in SigmaPlot (Systat Software, Inc.). The time-dependent inactivation of AtGCLΔ85 by buthionine sulfoximine and cystamine was performed as follows. AtGCLΔ85 (125 μg) was incubated (37 °C) in 100 μl of 0.1 m MOPSO (pH 7.0) and 20 mm MgCl2 in the presence of either 0-50 mm cystamine or 10 mm ATP and 0-50 mm buthionine sulfoximine. All incubations were initiated by the addition of the inactivator. Aliquots (20 μl) were withdrawn from the incubation mixture and diluted into the standard assay system and then the enzymatic activity remaining was determined. All inactivation experiments were monitored relative to a control sample without inactivator, which is set to 100% activity at each time point. Inactivation data were plotted as log (% initial enzyme activity) versus time. Semilog plots were fitted to the equation -dE/dt = k[I], where the disappearance of enzyme activity over time is related to the concentration of inactivator (I), multiplied by k, a rate constant. This allowed a determination of the half-life for inactivation (t1/2) at each [I]. A Kitz-Wilson analysis of the data was used to generate the limiting constant for inactivation (kinact) and Ki by plotting t½ versus 1/[I] (24Kitz R. Wilson I.B. J. Biol. Chem. 1962; 237: 3235-3239Abstract Full Text PDF Google Scholar). Analysis of the Kinetic Mechanism—Analysis of the kinetic mechanism of AtGCLΔ85 used global curve fitting (25Brekken D.L. Phillips M.A. J. Biol. Chem. 1998; 273: 26317-26322Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 26Emanuele J.J. Jin H. Yanchunas J. Villafranca J.J. Biochemistry. 1997; 36: 7264-7271Crossref PubMed Scopus (55) Google Scholar). The reaction rates were measured as described above using a matrix of substrate concentrations (glutamate, 2-40 mm; ATP, 0.5-20 mm; cysteine, 1-20 mm). In this matrix, the rate measured for the concentration of one substrate is measured over the entire range of the other two substrates. SigmaPlot was used for curve fitting and modeling of the kinetic data to rapid equilibrium rate equations of the possible ter-reactant kinetic mechanisms (27Segel I.H. Enzyme Kinetics: Behavior and Analysis of Rapid Equilibrium and Steady-State Enzyme Systems. John Wiley & Sons, Inc., New York1975Google Scholar). Mass Spectrometry—An Applied Biosystems QSTAR XL hybrid quadrupole time-of-flight (TOF) mass spectrometry (MS) system equipped with a nanoelectrospray source (Protana XYZ manipulator) was used for an accurate molecular weight determination. The nanoelectrospray was generated from a PicoTip needle (New Objectives, Inc.) at 1200 volts. The sample flow rate was estimated to be 100 nl min-1. The instrument m/z response was calibrated with standards from the manufacturer to provide molecular mass measurement accuracy of <30 ppm for proteins up to 50 kDa and of <5 ppm for lower mass peptides. For detection of protein complex species, data were acquired initially over the range m/z 900-10000 and later over a narrow mass range with intense signals. The accumulation time was 1 s, for 300 cycles. The two declustering potential parameters and focusing potential, i.e. DP, DP2, and FP, were 100, 15, and 300, respectively. To maintain protein complexes in gas phase, the gas pressure in the collision cell was increased to 4.0 × 105 Torr for ion cooling. For MS/MS analysis, data were acquired using the information-dependent acquisition feature in the Analyst QS software. TOF and tandem MS data were acquired over m/z ranges of 300-2200 and 65-2000, respectively. Every spectrum was accumulated for 1 s and was followed by three-product ion spectra (each for 3 s). The DP, DP2, FP settings were 50, 10, and 200, respectively, and the collision energy was dependent on the m/z values of the ions. For identification of the reaction product by electrospray ionization (ESI)-TOF MS, scaled-up reactions (5-ml volume, 1 mg of protein) were performed using standard assay conditions. Reactions were quenched by an addition of 5% (v/v) 5-sulfosalicyclic acid. After centrifugation, the supernatant was evaporated to dryness and dissolved in water. Enzymatically synthesized γ-glutamylcysteine was compared with an authentic standard (Sigma-Aldrich). Expression and Purification of AtGCL—The reported nucleotide sequence (15May M.J. Leaver C.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10059-10063Crossref PubMed Scopus (122) Google Scholar) was used to design oligonucleotides for amplifying AtGCL from an Arabidopsis library. Several clones were identical to each other but had a single base pair deletion (T1470) compared with the published sequence that shifts the reading frame at the C terminus. The sequence obtained was identical to other GenBank™ entries for AtGCL, including that from genome sequencing (NP_194041). The C-terminal amino acid sequence of our AtGCL clone was 70% identical to other plant GCL sequences, suggesting that this is the correct sequence. Because the N-terminal region of AtGCL encodes a chloroplast transit signal (10Schafer H.J. Haag-Kerwer A. Rausch T. Plant Mol. Biol. 1998; 37: 87-97Crossref PubMed Scopus (130) Google Scholar), we generated a version of AtGCL (At-GCLΔ85) lacking the localization sequence based on comparison with the cytosolic GCL from Zea mays (28Gomez L.D. Vanacker H. Buchner P. Noctor G. Foyer C.H. Plant Physiol. 2004; 134: 1662-1671Crossref PubMed Scopus (94) Google Scholar). AtGCL and AtGCLΔ85 were overexpressed and purified by Ni2+-affinity and size-exclusion chromatography (Fig. 1). SDS-PAGE analysis of the purified proteins showed that AtGCL and AtGCLΔ85 migrated with molecular masses of 58 and 50 kDa, respectively, corresponding to their predicted masses. Full-length AtGCL was primarily insoluble, but ∼1 mg of pure soluble protein was obtained from an 8-liter growth. Removal of the plastid target sequence improved protein solubility, as purification of AtGCLΔ85 yielded 5 mg of pure protein liter-1 of culture. AtGCL catalyzed the ligation of glutamate to cysteine in the presence of ATP with a specific activity of 120 nmol min-1 mg protein-1. AtGCLΔ85 catalyzed the same reaction with a similar specific activity. Because the removal of the localization sequence improved solubility without altering enzymatic activity, we used AtGCLΔ85 for subsequent analysis. Substitution of α-aminobutyrate for cysteine in these reactions resulted in 20-fold reductions in specific activity even when high concentrations (up to 100 mm) were used. Therefore, cysteine was employed as a substrate for all further enzyme assays. Kinetic Analysis and Identification of the Reaction Product—Steady-state kinetic parameters (kcat and Km) for glutamate, cysteine, and ATP were determined for AtGCLΔ85 (Table I). The kcat and Km values were comparable with those reported for GCL purified from tobacco cell suspensions (29Hell R. Bergmann L. Planta. 1990; 180: 603-612Crossref PubMed Scopus (207) Google Scholar). The reaction rate of recombinant purified AtGCLΔ85 was 50-fold higher than the tobacco enzyme likely because of the greater purity of these samples. Compared with other characterized GCL (17Fraser J.A. Saunders R.D.C. McLellan L.I. J. Biol. Chem. 2002; 277: 1158-1165Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 19Huang C.S. Chang L.S. Anderson M.E. Meister A. J. Biol. Chem. 1993; 268: 19675-19680Abstract Full Text PDF PubMed Google Scholar, 20Lueder D.V. Phillips M.A. J. Biol. Chem. 1996; 271: 17485-17490Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 21Kelly B.S. Antholine W.E. Griffith O.W. J. Biol. Chem. 2002; 277: 50-58Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 30Board P.G. Smith J.E. Moore K. Ou D. Biochim. Biophys. Acta. 1980; 613: 534-541Crossref PubMed Scopus (14) Google Scholar, 31Davis J.S. Balinsky J.B. Harington J.S. Sheperd J.B. Biochem. J. 1973; 133: 667-678Crossref PubMed Scopus (33) Google Scholar, 32Hussein A.S. Walter R.D. Mol. Biochem. Parasitol. 1995; 72: 57-64Crossref PubMed Scopus (19) Google Scholar, 33Luersen K. Muller S. Hussein A. Liebau E. Walter R.D. Mol. Biochem. Parasitol. 2000; 11: 243-251Crossref Scopus (15) Google Scholar), AtGCL displays a Km value for glutamate 3-10-fold higher and a Km value for cysteine 0.5-10-fold different.Table ISteady-state kinetic parameters of AtGCLΔ85kcatKmkcat/Kmmin−1mmm−1 s−1Glutamate6.1 ± 0.29.1 ± 1.010.1Cysteine4.5 ± 0.11.6 ± 0.246.9ATP6.8 ± 0.22.7 ± 0.242.0 Open table in a new tab The reaction product of AtGCLΔ85 was analyzed by ESITOF mass spectrometry (Fig. 2). A TOF-MS survey scan showed a major component of m/z = 251.0894. The mass matches that of authentic γ-glutamylcysteine (MW 251.0841). Fragmentation of the precursor ion generated two major product ions corresponding to γ-glutamylcysteine cleaved at the peptide bond. The data demonstrated that the purified AtGCL catalyzed the production of γ-glutamylcysteine and is a GCL, even though it displays a low sequence homology with the GCL from other species. Inhibition and Inactivation of AtGCL—Glutathione inhibits the activity of GCL from non-plant eukaryotes and bacteria (8Richman P.G. Meister A. J. Biol. Chem. 1975; 250: 1422-1426Abstract Full Text PDF PubMed Google Scholar, 17Fraser J.A. Saunders R.D.C. McLellan L.I. J. Biol. Chem. 2002; 277: 1158-1165Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 19Huang C.S. Chang L.S. Anderson M.E. Meister A. J. Biol. Chem. 1993; 268: 19675-19680Abstract Full Text PDF PubMed Google Scholar, 20Lueder D.V. Phillips M.A. J. Biol. Chem. 1996; 271: 17485-17490Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 21Kelly B.S. Antholine W.E. Griffith O.W. J. Biol. Chem. 2002; 277: 50-58Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). To determine whether similar inhibition occurs in plants, we examined the effect of increasing glutathione levels on AtGCLΔ85 (Fig. 3). Glutathione was determined to be a non-competitive inhibitor of GCL activity versus both glutamate (Ki = 0.72 ± 0.05 mm) and cysteine (Ki = 2.21 ± 0.14 mm). These values fall within the range of inhibition constants (0.1-8.2 mm) reported for other GCL (8Richman P.G. Meister A. J. Biol. Chem. 1975; 250: 1422-1426Abstract Full Text PDF PubMed Google Scholar, 17Fraser J.A. Saunders R.D.C. McLellan L.I. J. Biol. Chem. 2002; 277: 1158-1165Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 19Huang C.S. Chang L.S. Anderson M.E. Meister A. J. Biol. Chem. 1993; 268: 19675-19680Abstract Full Text PDF PubMed Google Scholar, 20Lueder D.V. Phillips M.A. J. Biol. Chem. 1996; 271: 17485-17490Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 21Kelly B.S. Antholine W.E. Griffith O.W. J. Biol. Chem. 2002; 277: 50-58Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 30Board P.G. Smith J.E. Moore K. Ou D. Biochim. Biophys. Acta. 1980; 613: 534-541Crossref PubMed Scopus (14) Google Scholar, 31Davis J.S. Balinsky J.B. Harington J.S. Sheperd J.B. Biochem. J. 1973; 133: 667-678Crossref PubMed Scopus (33) Google Scholar, 32Hussein A.S. Walter R.D. Mol. Biochem. Parasitol. 1995; 72: 57-64Crossref PubMed Scopus (19) Google Scholar, 33Luersen K. Muller S. Hussein A. Liebau E. Walter R.D. Mol. Biochem. Parasitol. 2000; 11: 243-251Crossref Scopus (15) Google Scholar). Buthionine sulfoximine (34Griffith O.W. J. Biol. Chem. 1982; 257: 13704-13712Abstract Full Text PDF PubMed Google Scholar) and cystamine (35Lebo R.V. Kredich N.M. J. Biol. Chem. 1978; 253: 2615-2623Abstract Full Text PDF PubMed Google Scholar) inactivate GCL. GCL catalyzes the ATP-dependent phosphorylation of buthionine sulfoximine to form a γ-glutamylphosphate intermediate that mimics the transition state bound at the active site (34Griffith O.W. J. Biol. Chem. 1982; 257: 13704-13712Abstract Full Text PDF PubMed Google Scholar). Cystamine is a thiol-specific inactivator of GCL that targets a cysteine within the active site (35Lebo R.V. Kredich N.M. J. Biol. Chem. 1978; 253: 2615-2623Abstract Full Text PDF PubMed Google Scholar). We examined the inactivation of AtGCL by these compounds (Fig. 4). Both molecules inactivated AtGCLΔ85 with pseudo-first order kinetics (Fig. 4, A and C). Inactivation by buthionine sulfoximine displayed a kinact = 0.024 min-1 (t½ = 28.7 min) and a Ki = 1.2 mm (Fig. 4B). Buthionine sulfoximine did not inactivate AtGCLΔ85 in the absence of ATP or MgCl2, confirming that inactivation requires phosphorylation of the inactivator. Inactivation by cystamine showed a kinact = 0.028 min-1 (t½ = 24.5 min) and a Ki = 11 mm (Fig. 4D). Overall, inactivation of AtGCL by both molecules occurs more slowly and with increased Ki values than inactivation of the bacterial, mammalian, or nematode enzymes (20Lueder D.V. Phillips M.A. J. Biol. Chem. 1996; 271: 17485-17490Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 21Kelly B.S. Antholine W.E. Griffith O.W. J. Biol. Chem. 2002; 277: 50-58Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 33Luersen K. Muller S. Hussein A. Liebau E. Walter R.D. Mol. Biochem. Parasitol. 2000; 11: 243-251Crossref Scopus (15) Google Scholar, 35Lebo R.V. Kredich N.M. J. Biol. Chem. 1978; 253: 2615-2623Abstract Full Text PDF PubMed Google Scholar, 36Orlowski M. Meister A. J. Biol. Chem. 1971; 246: 7095-7105Abstract Full Text PDF PubMed Google Scholar). For example, E. coli GCL is inactivated by buthionine sulfoximine with kinact = 0.3 min-1 (t½ = 2.3 min) and Ki = 66 μm (21Kelly B.S. Antholine W.E. Griffith O.W. J. Biol. Chem. 2002; 277: 50-58Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar) and inactivation of rat GCL is even more rapid with kinact = 3.7 min-1 (t½ = 11 s) and Ki < 100 μm (34Griffith O.W. J. Biol. Chem. 1982; 257: 13704-13712Abstract Full Text PDF PubMed Google Scholar). Kinetic Mechanism of AtGCL—To determine the kinetic mechanism of AtGCL, we obtained a complete matrix of kinetic data for the three substrates (25Brekken D.L. Phillips M.A. J. Biol. Chem. 1998; 273: 26317-26322Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 26Emanuele J.J. Jin H. Yanchunas J. Villafranca J.J. Biochemistry. 1997; 36: 7264-7271Crossref PubMed Scopus (55) Google Scholar, 27Segel I.H. Enzyme Kinetics: Behavior and Analysis of Rapid Equilibrium and Steady-State Enzyme Systems. John Wiley & Sons, Inc., New York1975Google Scholar). Six families of data were generated in which one of the ligands is maintained at a saturating concentration whereas the other two substrates are varied. Kinetic data were first analyzed as double-reciprocal plots (Fig. 5). Because the plots for each family of data converged, potential p" @default.
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• W2050168058 date "2004-08-01" @default.
• W2050168058 modified "2023-10-18" @default.
• W2050168058 title "Arabidopsis thaliana Glutamate-Cysteine Ligase" @default.
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