Human Glutathione Transferase P1-1 and Nitric Oxide Carriers
2001; Elsevier BV; Volume: 276; Issue: 45 Linguagem: Inglês
10.1074/jbc.m102344200
ISSN1083-351X
AutoresMario Lo Bello, Marzia Nuccetelli, Anna Maria Caccuri, Lorenzo Stella, Michael W. Parker, Jamie Rossjohn, William J. McKinstry, Alessia Francesca Mozzi, Giorgio Federici, Francesca Polizio, Jens Z. Pedersen, Giorgio Ricci,
Tópico(s)Genomics, phytochemicals, and oxidative stress
ResumoS-Nitrosoglutathione and the dinitrosyl-diglutathionyl iron complex are involved in the storage and transport of NO in biological systems. Their interactions with the human glutathione transferase P1-1 may reveal an additional physiological role for this enzyme. In the absence of GSH,S-nitrosoglutathione causes rapid and stable S-nitrosylation of both the Cys47 and Cys101 residues. Ion spray ionization-mass spectrometry ruled out the possibility of S-glutathionylation and confirms the occurrence of a poly-S-nitrosylation in GST P1-1. S-Nitrosylation of Cys47 lowers the affinity 10-fold for GSH, but this negative effect is minimized by a half-site reactivity mechanism that protects one Cys47/dimer from nitrosylation. Thus, glutathione transferase P1-1, retaining most of its original activity, may act as a NO carrier protein when GSH depletion occurs in the cell. The dinitrosyl-diglutathionyl iron complex, which is formed by S-nitrosoglutathione decomposition in the presence of physiological concentrations of GSH and traces of ferrous ions, binds with extraordinary affinity to one active site of this dimeric enzyme (Ki < 10−12m) and triggers negative cooperativity in the vacant subunit (Ki = 10−9m). The complex bound to the enzyme is stable for hours, whereas in the free form and at low concentrations, its life time is only a few minutes. ESR and molecular modeling studies provide a reasonable explanation of this strong interaction, suggesting that Tyr7 and enzyme-bound GSH could be involved in the coordination of the iron atom. All of the observed findings suggest that glutathione transferase P1-1, by means of an intersubunit communication, may act as a NO carrier under different cellular conditions while maintaining its well known detoxificating activity toward dangerous compounds. S-Nitrosoglutathione and the dinitrosyl-diglutathionyl iron complex are involved in the storage and transport of NO in biological systems. Their interactions with the human glutathione transferase P1-1 may reveal an additional physiological role for this enzyme. In the absence of GSH,S-nitrosoglutathione causes rapid and stable S-nitrosylation of both the Cys47 and Cys101 residues. Ion spray ionization-mass spectrometry ruled out the possibility of S-glutathionylation and confirms the occurrence of a poly-S-nitrosylation in GST P1-1. S-Nitrosylation of Cys47 lowers the affinity 10-fold for GSH, but this negative effect is minimized by a half-site reactivity mechanism that protects one Cys47/dimer from nitrosylation. Thus, glutathione transferase P1-1, retaining most of its original activity, may act as a NO carrier protein when GSH depletion occurs in the cell. The dinitrosyl-diglutathionyl iron complex, which is formed by S-nitrosoglutathione decomposition in the presence of physiological concentrations of GSH and traces of ferrous ions, binds with extraordinary affinity to one active site of this dimeric enzyme (Ki < 10−12m) and triggers negative cooperativity in the vacant subunit (Ki = 10−9m). The complex bound to the enzyme is stable for hours, whereas in the free form and at low concentrations, its life time is only a few minutes. ESR and molecular modeling studies provide a reasonable explanation of this strong interaction, suggesting that Tyr7 and enzyme-bound GSH could be involved in the coordination of the iron atom. All of the observed findings suggest that glutathione transferase P1-1, by means of an intersubunit communication, may act as a NO carrier under different cellular conditions while maintaining its well known detoxificating activity toward dangerous compounds. glutathione S-transferase 1-chloro-2,4-dinitrobenzene dinitrosyl-diglutathionyl-iron complex(es) dinitrosyl-glutathionyl-iron complex(es) dinitrosyl-iron complex(es) electron spin resonance S-nitrosoglutathione Glutathione transferases (EC 2.5.1.18) (GSTs)1 are a superfamily of enzymes involved in the detoxication of the cell against toxic and carcinogenic compounds (1Jakoby W.B. Habig W.H. Jakoby W.B. Enzymatic Basis of Detoxification. 2. Academic Press, New York1980: 63-94Google Scholar). The human cytosolic GSTs are dimeric proteins grouped into at least eight gene-independent classes (Alpha, Kappa, Mu, Omega, Pi, Sigma, Theta, and Zeta) on the basis of their amino acid sequence, substrate specificity, and immunological properties (2Mannervik B. Ålin P. Guthenberg C. Jensson H. Tahir M.K. Warholm M. Jörnvall H. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 7202-7206Crossref PubMed Scopus (1092) Google Scholar, 3Meyer D.J. Coles B. Pemble S.E. Gilmore K.S. Fraser G.M. Ketterer B. Biochem. J. 1991; 274: 409-414Crossref PubMed Scopus (754) Google Scholar, 4Buetler T.M. Eaton D.L. Environ. Carcinog. Ecotoxicol. Rev. 1992; 10: 181-200Crossref Scopus (175) Google Scholar, 5Meyer D.J. Thomas M.R. Biochem. J. 1995; 311: 739-742Crossref PubMed Scopus (143) Google Scholar, 6Pemble S.E. Wardle A.F. Taylor J.B. Biochem. J. 1996; 319: 749-754Crossref PubMed Scopus (266) Google Scholar, 7Board P.G. Baker R.T. Chelvanayagam G. Jermiin L.S. Biochem. 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EMBO J. 1995; 14: 2133-2143Crossref PubMed Scopus (218) Google Scholar). Each subunit contains a very similar binding site for GSH (G-site) and a second one for the hydrophobic co-substrate (H-site). Slight structural differences at the H-site confers a certain degree of substrate selectivity. For a more detailed review on the molecular properties of GSTs see Ref. 13Armstrong R.N. Chem. Res. Toxicol. 1997; 10: 2-18Crossref PubMed Scopus (984) Google Scholar. The physiological role of GSTs does not appear unequivocal. A well known function of GSTs is to promote the conjugation of the sulfur atom of glutathione to an electrophilic center of endogenous and exogenous toxic compounds, thereby increasing their solubility and excretion (1Jakoby W.B. Habig W.H. Jakoby W.B. Enzymatic Basis of Detoxification. 2. Academic Press, New York1980: 63-94Google Scholar). The Alpha class also displays a peroxidase activity with organic peroxides (14Mannervik B. Chem. Scripta. 1986; 26: 281-284Google Scholar). Because of the considerable level of expression of GSTs in many tissues and their property to bind a number of large hydrophobic compounds, a possible role of "ligandins" (ligand carriers) has been also proposed (15Litwach G. Ketterer B. Arias I.M. Nature. 1971; 234: 466-477Crossref PubMed Scopus (417) Google Scholar). Recent advances suggest a role for GST P1-1 in the regulation of Jun kinase protein (a stress-activated protein that phosphorylates c-Jun) (16Adler V. Yin Z. Fuchs S.Y. Benezra M. Rosario L. Tew K.D. Pincus M., R. Ardana M. Henderson C.J. Wolf C.R. Davis R.J. Ronai Z. EMBO J. 1999; 18: 1321-1334Crossref PubMed Scopus (975) Google Scholar) and as a "tissue" transglutaminase-specific substrate in neural cells committed to apoptosis (17Piredda L. Farrace M.G. Lo Bello M. Malorni W. Melino G. Petruzzelli R. Piacentini M. FASEB J. 1999; 13: 355-364Crossref PubMed Scopus (97) Google Scholar). It has also been reported that certain GSTs (class Theta) may inhibit the proapoptotic action of Bax (18Kampranis S.C. Damianova R. Atallah M. Toby G. Kondi G. Tsichlis P.N. Mahkris A.M. J. Biol. Chem. 2000; 275: 29207-29216Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar), and the most recently discovered class Omega, GST O1-1, modulates calcium channels, thus protecting mammalian cells from apoptosis induced by Ca2+ mobilization (19Dulhunty A. Gage P. Curtis S. Chelvanayagam G. Board P. J. Biol. Chem. 2001; 276: 3319-3323Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar). Thus, GSTs can be considered as multi-functional enzymes devoted to various aspects of cell defense. Notwithstanding this versatility, GSTs have never been considered to be involved in the complex mechanisms of detoxification or storage of nitric oxide. NO is formed in organisms from endogenous or exogenous sources and triggers a number of cellular responses, including the regulation of blood pressure, the relaxation of smooth muscle, and the modulation of immunity (20Moncada S. J. Hypertens. 1994; 12: 35-39Google Scholar, 21Loscalzo J. Welch G. Prog. Cardiovasc. Dis. 1995; 38: 87-104Crossref PubMed Scopus (515) Google Scholar). In the central nervous system, NO is a neuronal messenger and is possibly responsible for the development of various diseases (22Moncada S. Palmer R.M.J. Higgs E.A. Pharmacol. Rev. 1991; 43: 109-142PubMed Google Scholar, 23Tamir S. Tannenbaum S.R. Biochim. Biophys. Acta. 1996; 1288: 31-36Crossref PubMed Scopus (259) Google Scholar, 24Mey C. Curr. Med. Res. Opin. 1998; 14: 187-202Crossref PubMed Scopus (10) Google Scholar, 25Rubanyi G.M. Rubanyi G.M. Endothelial Cell Research Series. 5. Berlex Biosciences, Richmond, CA1999Google Scholar). NO has a very short life time in the cell, and its stabilization and transport are promoted by specific NO carriers of low molecular mass, which are formed by the interaction of nitric oxide with ferrous ions (dinitrosyl-iron complexes (DNIC)) (26Vanin A.F. Stukan R.A. Manukhina E.B. Biochim. Biophys. Acta. 1996; 1295: 5-12Crossref PubMed Scopus (86) Google Scholar, 27Vedernikov Y.P. Mordvintcev P.I. Malenkova I.V. Vanin A.F. Eur. J. Pharmacol. 1992; 211: 313-317Crossref PubMed Scopus (95) Google Scholar, 28Ueno T. Yoshimura T. Jpn. J. Pharmacol. 2000; 82: 95-101Crossref PubMed Scopus (49) Google Scholar) or with GSH (S-nitrosoglutathione (GSNO)) (29Hogg N. Singh R.J. Kalyanaraman B. FEBS Lett. 1996; 382: 223-228Crossref PubMed Scopus (250) Google Scholar, 30Kluge I. Gutteck-Amsler U. Zollinger M. Do K.Q. J. Neurochem. 1997; 69: 2599-2607Crossref PubMed Scopus (160) Google Scholar, 31Myers P.R. Minor Jr., R.L. Guerra Jr., R. Bates J.N. Harrison D. G Nature. 1990; 345: 161-163Crossref PubMed Scopus (854) Google Scholar, 32Stamler J.S. Cell. 1994; 78: 931-936Abstract Full Text PDF PubMed Scopus (1646) Google Scholar). DNIC bind to proteins like albumin forming paramagnetic NO-Fe-protein complexes that can be detected by ESR spectroscopy (33Boese M. Mordvintcev P.I. Vanin A.F. Busse R. Mulsch A. J. Biol. Chem. 1995; 272: 29244-29249Abstract Full Text Full Text PDF Scopus (216) Google Scholar). Similar complexes, known as 2.03 complexes, because of the g value of their characteristic ESR spectra, have been observed in cells and tissues exposed to NO, but nothing is known about the proteins involved (34Lancaster Jr., J.R. Hibbs Jr., J.B. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1223-1227Crossref PubMed Scopus (492) Google Scholar, 35Pellat C. Henry Y. Drapier J.-C. Biochem. Biophys. Res. Commun. 1990; 166: 119-125Crossref PubMed Scopus (167) Google Scholar, 36Drapier J.-C. Pellat C. Henry Y. J. Biol. Chem. 1991; 266: 10162-10167Abstract Full Text PDF PubMed Google Scholar, 37Lancaster Jr., J.R. Langrehr J.M. Bergonia H.A. Murase N. Simmons N. Hoffman R.A. J. Biol. Chem. 1992; 267: 10994-10998Abstract Full Text PDF PubMed Google Scholar). Recently, kinetic studies showed that both GSNO and dinitrosyl-diglutathionyl-iron complex (DNDGIC) are competitive inhibitors of GSTs, and they have been proposed as activity modulators of these enzymes (38Clark A.G. Debnam P. Biochem. Pharmacol. 1988; 37: 3199-3201Crossref PubMed Scopus (24) Google Scholar, 39Keese M.A. Bose M. Mulsch A. Shirmer R.H. Becker K. Biochem. Pharmacol. 1997; 54: 1307-1313Crossref PubMed Scopus (65) Google Scholar). Starting from these preliminary findings, we have now investigated the interaction of these compounds with GST P1-1, using ESR spectroscopy, site-directed mutagenesis, mass spectrometry, and molecular modeling. Our results reveal a surprising and sophisticated mode of interaction suggestive of a new role for this enzyme in the cell as a NO reservoir or NO scavenger protein. The plasmid pGST-1, producing large amounts of recombinant wild-type GST P1-1 in the cytoplasm of Escherichia coli, has been described previously (40Battistoni A. Mazzetti A.P. Petruzzelli R. Muramatzu M. Ricci G. Federici G. Lo Bello M. Protein Expression Purif. 1995; 6: 579-587Crossref PubMed Scopus (41) Google Scholar). The expression plasmid p18Seq-1, reported previously (41Lo Bello M. Battistoni A. Mazzetti A.P. Board P.G. Muramatzu M. Federici G. Ricci G. J. Biol. Chem. 1995; 270: 1249-1253Abstract Full Text Full Text PDF PubMed Google Scholar), was used to generate the single-stranded DNA template to be used for site-directed mutagenesis of Cys47 and Cys101 residues, according to the method described by Kunkel et al. (42Kunkel T.A. Bebenek K. McClary J. Methods Enzymol. 1990; 204: 125-139Crossref Scopus (639) Google Scholar) with minor modifications. Native and mutant GST P1-1 enzymes were produced as described previously (40Battistoni A. Mazzetti A.P. Petruzzelli R. Muramatzu M. Ricci G. Federici G. Lo Bello M. Protein Expression Purif. 1995; 6: 579-587Crossref PubMed Scopus (41) Google Scholar, 41Lo Bello M. Battistoni A. Mazzetti A.P. Board P.G. Muramatzu M. Federici G. Ricci G. J. Biol. Chem. 1995; 270: 1249-1253Abstract Full Text Full Text PDF PubMed Google Scholar). Briefly, TOP 10 E. coli cells harboring plasmid pGST-1 or plasmid expressing Cys47 and Cys101 mutant enzymes (pGST-A47 and pGST-A101) were grown in LB medium containing 100 μg/ml ampicillin and 50 μg/ml streptomycin. The synthesis of GST was induced by the addition of 0.2 mmisopropyl-1-thio-β-galactopyranoside when the absorbance at 600 nm was 0.5. Eighteen hours after induction, the cells were harvested by centrifugation and lysed as described previously (40Battistoni A. Mazzetti A.P. Petruzzelli R. Muramatzu M. Ricci G. Federici G. Lo Bello M. Protein Expression Purif. 1995; 6: 579-587Crossref PubMed Scopus (41) Google Scholar). Native and GST mutant enzymes were purified by affinity chromatography on immobilized glutathione (43Simmons P.C. Van der Jagt D.L. Anal. Biochem. 1977; 82: 334-341Crossref PubMed Scopus (441) Google Scholar). After affinity purification, the native and the mutant enzymes (C47A and C101A) were homogeneous as judged by SDS-polyacrylamide gel electrophoresis (44Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (212382) Google Scholar). Protein concentration was determined by the method of Lowry et al. (45Lowry O.H. Rosebrough N.J. Farr A.L. Randall R. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar). The enzymatic activities were determined spectrophotometrically at 25 °C with 1-chloro-2,4-dinitrobenzene (CDNB), as co-substrate, following the product formation at 340 nm, ε = 9600 m−1 cm−1 (46Habig W.H. Jakoby W.B. Methods Enzymol. 1981; 77: 398-405Crossref PubMed Scopus (2160) Google Scholar). Spectrophotometric measurements were performed in a double beam Uvicon 940 spectrophotometer (Kontron Instruments) equipped with a thermostatted cuvette compartment. Initial rates were measured at 0.1-s intervals for a total period of 12 s after a lag time of 5 s. Enzymatic rates were corrected for the spontaneous reaction. Apparent kinetic parameters kcat and K mCDNB were determined in 0.1 m potassium phosphate buffer, pH 6.5, and 0.1 mm EDTA, containing a fixed concentration of GSH (10 mm) and variable concentrations of CDNB (0.1–2 mm). The collected data were fitted to the Michaelis-Menten equation by nonlinear regression analysis using the GraphPad Prism (GraphPad Software, San Diego, CA). In a more accurate analysis, kinetic data were also fitted to Equation 1, which considers two distinct enzyme populations characterized by different Km values for GSH.v=Vmax1[S]Km1+[S]+Vmax2[S]Km2+[S]Eq. 1 The apparent K mGSH was also determined at fixed CDNB concentration (1 mm) and variable GSH concentrations (from 0.02 to 20 mm). Kinetic parameters reported in this paper represent the mean of at least three different experimental data sets. GSNO was prepared as described previously (47Park J.W. Biochem. Biophys. Res. Commun. 1988; 152: 916-920Crossref PubMed Scopus (111) Google Scholar). Briefly, a few drops of HCl were added to a solution containing equimolar amounts of GSH and sodium nitrite until pH 1.5 was reached. After standing for 5 min at room temperature, the red GSNO was neutralized with NaOH. GSNO displays an absorption maximum of 750m−1 cm−1 at 332 nm and appears to be stable for a few days at room temperature. Appropriate aliquots of freshly synthesized compound were stored at −80 °C and used when necessary, after checking their absorbance at 332 nm. Dinitrosyl-diglutathionyl-iron complex was synthesized according to the following procedure. Suitable amounts of ferrous ions (FeSO4, ranging from 10 to 50 μm) were added to a mixture containing 20 mmGSH and 2 mm GS-NO in 0.1 m phosphate buffer, pH 7.4, and 25 °C. The synthesis of the complex was completed in the first 15–20 min and gives an extinction coefficient of 3000m−1 cm−1 at 403 nm. Dinitrosyl-dicysteinyl-iron complex, under slightly different conditions, displays an identical extinction coefficient at 395 nm (48Mulsh A. Mordvintcev P. Vanin A.F. Busse R. FEBS Lett. 1991; 294: 252-256Crossref PubMed Scopus (150) Google Scholar). GST P1-1 or Cys mutant enzymes (ranging from 0.03 to 1 mg/ml) were incubated with 2 mm GSNO in 0.1 m phosphate buffer, pH 7.4, at 25 °C. At various times aliquots were taken and assayed for enzymatic activity with CDNB as co-substrate. In a different set of experiments, 20 mm GSH was added to the mixture containing the above concentrations of GSNO and protein with or without 1 mm EDTA. Difference spectra were performed with a double beam Uvicon 940 spectrophotometer (Kontron Instruments) equipped with a thermostatted cuvette compartment. Spectral data of free and bound DNDGIC complex were recorded in the range 290–500 nm at 25 °C at pH 7.4 using quartz cuvettes (1-cm light path). Inhibition of GST P1-1 by DNDGIC was measured by incubating variable amounts of DNDGIC (from 0.5 to 9 μm) with 0.1 mg/ml (4.4 μm of active sites) of GST P1-1. After 2 min 10-μl aliquots (final concentration of GST P1-1 is 44 nm active sites) were assayed for activity at pH 6.5 (final volume, 1 ml) in the presence of 10 mm GSH and 1 mm CDNB. Thus, the final concentration of DNDGIC in the activity sample ranged from 5 to 90 nm. In a second experimental set, GST P1-1 (44 nm of active sites) was incubated at pH 7.4 with variable amounts of DNDGIC (from 0.1 to 22 μm). After 2 min the pH was adjusted to 6.5, the GSH concentration was brought to a final concentration of 10 mm, and 1 mm CDNB was then added for activity measurement. ESR spectra were recorded with an ESP300 instrument (Bruker, Karlsruhe, Germany) operating at X-band frequency. Low temperature measurements (100 K) were made using a standard TE102-type cavity with samples in 3-mm-inner diameter quartz tubes, whereas experiments performed at room temperature were made using a high sensitivity TM110-type cavity with samples contained in 1.10-mm-inner diameter capillary tubes. ESR spectra were recorded at 20 mW microwave power and 1 G modulation; for resolution of the hyperfine structure some spectra were recorded using 0.2 G modulation. GST P1-1 (final concentration, 2.0 mg/ml) was incubated with 2 mm GSNO, under the conditions reported above, for 15 min. Thereafter, it was passed through a Sephadex G-25 column, equilibrated with distilled water, containing 0.1 mm EDTA, to remove any excess of reagent and used soon for mass spectrometry studies. A sample of unmodified human GST P1-1 was equilibrated in the same manner, before mass spectroscopic analysis. The samples were diluted with 99% acetonitrile to give the final solution of acetonitrile/water 50% + formic acid 0.2% (v/v). Mass spectrometry spectra were collected in continuous flow mode by connecting the built-in infusion pump directly to the ion spray probe. Molecular masses of both unmodified and modified GST P1-1 enzymes were determined using a Triple Quadrupole liquid chromatography/mass spectrometry/mass spectrometry mass spectrometer Applied Biosystem Sciex API 365 (Concord, Canada). The data were acquired and processed using Mass Chrom 1.2 (Applied Biosystem Sciex, Concord, Ontario, Canada), including BioMultiView 1.3.1 for mathematical transformation of ion spray spectra to true mass scale. Molecular modeling was performed on a Silicon Graphics O2 work station using the software package O (49Jones T.A. Zou J.-Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13036) Google Scholar). The model was based on the 1.9 Å resolution crystal structure of human class Pi GST P1-1 in complex with GSH (50Oakley A.J. Lo Bello M. Battistoni A. Ricci G. Rossjohn J. Villar H.O. Parker M.W. J. Mol. Biol. 1997; 274: 84-100Crossref PubMed Scopus (156) Google Scholar). A model of DNGIC was constructed assuming tetrahedral geometry and metal-ligand distances based on measurements from small molecule crystal structures (51Harding M.M. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 1432-1443Crossref PubMed Scopus (237) Google Scholar), with the topology and parameters files for the DNGIC complex obtained using XPLO2D and refined using crystallography and NMR system (52Brünger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.-S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (17024) Google Scholar). It is not clear whether the complex adopts a tetrahedral or octahedral (with two water ligands) configuration when bound to the enzyme. However, either configuration will fit into the active site, and the same conclusions can be drawn from either model. The DNGIC model was docked into the active site of the enzyme assuming that the GSH ligand of DNGIC occupied the same site as the GSH ligand in the crystal structure of the enzyme-GSH complex. It was immediately noted that the hydroxyl moiety of Tyr7 was in covalent bonding distance of the iron atom. A model of the complex covalently bound via Tyr7 was constructed. The resultant model was energy minimized with CNS (52Brünger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.-S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (17024) Google Scholar). Initially harmonic restraints of 10 kcal/mol were applied to the protein atoms, but subsequently the restraints were removed in the final stages of minimization. The final energy minimized model exhibited no significant structural differences with the crystal structure of the enzyme. Graphic representation was produced by the computer program MOLSCRIPT (53Kraulis P.J. J. Appl. Crystallogr. 1991; 24: 946-950Crossref Google Scholar). GSNO is a competitive inhibitor for GST P1-1 (38Clark A.G. Debnam P. Biochem. Pharmacol. 1988; 37: 3199-3201Crossref PubMed Scopus (24) Google Scholar, 39Keese M.A. Bose M. Mulsch A. Shirmer R.H. Becker K. Biochem. Pharmacol. 1997; 54: 1307-1313Crossref PubMed Scopus (65) Google Scholar), but in the absence of GSH this compound also causes a time-dependent partial inactivation of the enzyme. After 10 min of incubation, the activity is reduced to about 70% of its initial value and remains unchanged even after 1 h of incubation (Fig. 1). The presence of 1 mm EDTA in the incubation mixture does not affect significantly the extent and the rate of inactivation. Treatment of the inactivated enzyme with 100 mm1,4-dithiothreitol restores completely the original activity. The modified enzyme does not follow strictly Michaelian kinetics and shows an apparent Km value for GSH of 0.45 mm, which is three times higher than in the native enzyme and accounts completely for the observed loss of activity, using the standard GSH concentration of 1 mm (46Habig W.H. Jakoby W.B. Methods Enzymol. 1981; 77: 398-405Crossref PubMed Scopus (2160) Google Scholar). Cys47 and Cys101, the most reactive among the four cysteines of each subunit (41Lo Bello M. Battistoni A. Mazzetti A.P. Board P.G. Muramatzu M. Federici G. Ricci G. J. Biol. Chem. 1995; 270: 1249-1253Abstract Full Text Full Text PDF PubMed Google Scholar, 54Ricci G. Del Boccio G. Pennelli A. Lo Bello M. Petruzzelli R. Caccuri A.M. Barra D. Federici G. J. Biol. Chem. 1991; 266: 21409-21415Abstract Full Text PDF PubMed Google Scholar), are possible targets for the reversible modification by GSNO. Titration of the protein sulfhydryls after 1 h of GSNO (2 mm, pH 7.4) treatment shows that three sulfhydryl groups/dimer have been lost. The UV spectrum of the modified enzyme (Fig. 1, inset) shows a maximum at 332 nm, which corresponds to that found for the nitroso-thiol species (47Park J.W. Biochem. Biophys. Res. Commun. 1988; 152: 916-920Crossref PubMed Scopus (111) Google Scholar). The amount of the protein CysNO, calculated on the basis of an extinction value of 750m−1 cm−1 at 332 nm (47Park J.W. Biochem. Biophys. Res. Commun. 1988; 152: 916-920Crossref PubMed Scopus (111) Google Scholar), is 1.6 mol/subunit. When GSNO is reacted for 1 h with the C47S mutant enzyme, no inactivation was observed, and two cysteines/dimer are nitrosylated. Conversely, the C101A mutant only yields 1 mol of CysNO/dimer. Thus, it appears that Cys47 and Cys101 act as primary targets for a specific S-nitrosylation by GSNO, but only the modification of Cys47 decreases the enzyme activity. Furthermore, when this residue is nitrosylated in one subunit, the second in the adjacent subunit becomes less accessible. In fact, the kinetic data of the modified enzyme can be fitted more convincingly to a kinetic scheme involving two enzyme populations with different Kmvalues for GSH (see Equation 1). The best fit gives Vmax1 =Vmax2 and Kmvalues for GSH of 0.2 mm and 1.2 mm, respectively. This result is also consistent with two subunits with different affinities in the same dimeric population. The observed half-site reactivity of Cys47 compensates for the lowering of the affinity for GSH (like that observed in the modified subunit) and could reflect a self-preservation role of enzyme activity. This behavior is not unusual for GST P1-1 and is reminiscent of the nonequivalent reactivity of the two subunits of the horse GST P1-1 in their reaction with sulfhydryl reagents (55Ricci G. Del Boccio G. Pennelli A. Whitehead E.P. Federici G. J. Biol. Chem. 1989; 264: 5462-5467Abstract Full Text PDF PubMed Google Scholar). To gain direct evidence of S-nitrosylation of human GST P1-1 we have carried out ion spray ionization-mass spectrometry of the modified GST P1-1, and the results are shown in Fig. 2. Ion spray mass spectra of the unmodified protein (Fig. 2 a) showed two main peaks corresponding to the molecular masses of 23,226 and 23,356 Da, respectively. The former molecular mass corresponds to that calculated from its cDNA, whereas the latter is due to the failed removal of N-terminal methionine, and both are consistent with data reported previously (40Battistoni A. Mazzetti A.P. Petruzzelli R. Muramatzu M. Ricci G. Federici G. Lo Bello M. Protein Expression Purif. 1995; 6: 579-587Crossref PubMed Scopus (41) Google Scholar, 56Mitchell A. Zheng J. Hammock B. Lo Bello M. Jones D. Biochemistry. 1998; 37: 6752-6759Crossref PubMed Scopus (25) Google Scholar). In the Fig. 2 b, we observe two series of peaks with mass increases of 30 ± 1 Da, in addition to the unmodified proteins (23,226 and 23,356 Da, respectively), suggesting nitrosylation of GST P1-1. In particular, it appears that the first species (23,226 Da) exhibits molecular masses of M+ +30 and M+ + 60, respectively; the second species (23,356 Da) shows the same mass increases of +30 ± 1 and +60 ± 1 Da and, to lesser extent, a third peak with a mass increase of + 90 ± 1 Da. Thus, in accordance with the data reported above, these spectra demonstrate the simultaneous presence of, at least, the mono- and di-nitrosylated subunits, whereas the expected mass of about M+ +305, for glutathionylated GST P1-1, was not found. The presence of a third peak with a mass increase of + 90 ± 1 Da in the species with N-terminal methionine requires further investigation because it may be due also to a methionine oxidation. Further mass spectrometry studies on Cys mutant enzymes will be made to unravel the mechanism of GST P1-1 modification. It is well known that saturating amounts of GSH protect from the chemical modifications caused by thiol reagents. Cys47, located at the end of the mobile helix-2, is exposed to the solvent only in the apoenzyme, whereas it is buried in the holoenzyme (55Ricci G. Del Boccio G. Pennelli A. Whitehead E.P. Federici G. J. Biol. Chem. 1989; 264: 5462-5467Abstract Full Text PDF PubMed Google Scholar, 57Del Boccio G. Pennelli A. Whitehead E.P. Lo Bello M. Petruzzelli R. Federici G. Ricci G. J. Biol. Chem. 1991; 266: 13777-13782Abstract Full Text PDF PubMed Google Scholar, 58Stella L. Nicotra M. Ricci G. Rosato N. Di Iorio E.E
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