Peroxynitrite-induced Nitration of Tyrosine Hydroxylase
2002; Elsevier BV; Volume: 277; Issue: 16 Linguagem: Inglês
10.1074/jbc.m200290200
ISSN1083-351X
AutoresDonald M. Kuhn, Mahdieh Sadidi, Xiuli Liu, Christian W. Kreipke, Timothy J. Geddes, Chad R. Borges, J. Throck Watson,
Tópico(s)Neutrophil, Myeloperoxidase and Oxidative Mechanisms
ResumoTyrosine hydroxylase (TH), the initial and rate-limiting enzyme in the biosynthesis of the neurotransmitter dopamine, is inactivated by peroxynitrite. The sites of peroxynitrite-induced tyrosine nitration in TH have been identified by matrix-assisted laser desorption time-of-flight mass spectrometry and tyrosine-scanning mutagenesis. V8 proteolytic fragments of nitrated TH were analyzed by matrix-assisted laser desorption time-of-flight mass spectrometry. A peptide of 3135.4 daltons, corresponding to residues V410-E436 of TH, showed peroxynitrite-induced mass shifts of +45, +90, and +135 daltons, reflecting nitration of one, two, or three tyrosines, respectively. These modifications were not evident in untreated TH. The tyrosine residues (positions 423, 428, and 432) within this peptide were mutated to phenylalanine to confirm the site(s) of nitration and assess the effects of mutation on TH activity. Single mutants expressed wild-type levels of TH catalytic activity and were inactivated by peroxynitrite while showing reduced (30–60%) levels of nitration. The double mutants Y423F,Y428F, Y423F,Y432F, and Y428F,Y432F showed trace amounts of tyrosine nitration (7–30% of control) after exposure to peroxynitrite, and the triple mutant Y423F,Y428F,Y432F was not a substrate for nitration, yet peroxynitrite significantly reduced the activity of each. When all tyrosine mutants were probed with PEO-maleimide activated biotin, a thiol-reactive reagent that specifically labels reduced cysteine residues in proteins, it was evident that peroxynitrite resulted in cysteine oxidation. These studies identify residues Tyr423, Tyr428, and Tyr432 as the sites of peroxynitrite-induced nitration in TH. No single tyrosine residue appears to be critical for TH catalytic function, and tyrosine nitration is neither necessary nor sufficient for peroxynitrite-induced inactivation. The loss of TH catalytic activity caused by peroxynitrite is associated instead with oxidation of cysteine residues. Tyrosine hydroxylase (TH), the initial and rate-limiting enzyme in the biosynthesis of the neurotransmitter dopamine, is inactivated by peroxynitrite. The sites of peroxynitrite-induced tyrosine nitration in TH have been identified by matrix-assisted laser desorption time-of-flight mass spectrometry and tyrosine-scanning mutagenesis. V8 proteolytic fragments of nitrated TH were analyzed by matrix-assisted laser desorption time-of-flight mass spectrometry. A peptide of 3135.4 daltons, corresponding to residues V410-E436 of TH, showed peroxynitrite-induced mass shifts of +45, +90, and +135 daltons, reflecting nitration of one, two, or three tyrosines, respectively. These modifications were not evident in untreated TH. The tyrosine residues (positions 423, 428, and 432) within this peptide were mutated to phenylalanine to confirm the site(s) of nitration and assess the effects of mutation on TH activity. Single mutants expressed wild-type levels of TH catalytic activity and were inactivated by peroxynitrite while showing reduced (30–60%) levels of nitration. The double mutants Y423F,Y428F, Y423F,Y432F, and Y428F,Y432F showed trace amounts of tyrosine nitration (7–30% of control) after exposure to peroxynitrite, and the triple mutant Y423F,Y428F,Y432F was not a substrate for nitration, yet peroxynitrite significantly reduced the activity of each. When all tyrosine mutants were probed with PEO-maleimide activated biotin, a thiol-reactive reagent that specifically labels reduced cysteine residues in proteins, it was evident that peroxynitrite resulted in cysteine oxidation. These studies identify residues Tyr423, Tyr428, and Tyr432 as the sites of peroxynitrite-induced nitration in TH. No single tyrosine residue appears to be critical for TH catalytic function, and tyrosine nitration is neither necessary nor sufficient for peroxynitrite-induced inactivation. The loss of TH catalytic activity caused by peroxynitrite is associated instead with oxidation of cysteine residues. Tyrosine hydroxylase (TH) 1The abbreviations used are: THtyrosine hydroxylaseONOO−peroxynitritePEOpolyethylene oxideDTNB5,5′-dithiobis-2-nitrobenzoic acidpCMBp-chloromercuribenzoic acid, PMAB, PEO-maleimide-activated biotinBIAMN-biotinoyl-N-(iodoacetyl)ethylene diamineHPLChigh performance liquid chromatographyMALDImatrix-assisted laser desorption-ionizationTOFtime-of-flightELISAenzyme-linked immunosorbent assayMSmass spectrometryis the initial and rate-limiting enzyme in the biosynthesis of the neurotransmitter dopamine. TH is inhibited by the dopamine neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in PC12 cells and in mice after in vivo treatment (1.Ara J. Przedborski S. Naini A.B. Jackson-Lewis V. Trifiletti R.R. Horwitz J. Ischiropoulos H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7659-7663Crossref PubMed Scopus (382) Google Scholar), suggesting that losses in TH activity that are seen in this model of Parkinson's disease may occur early in the process of dopamine neuronal degeneration. The mechanisms by which 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine damages dopamine neurons are complex and are thought to involve, at least in part, the production of peroxynitrite (ONOO−) (2.Blum D. Torch S. Lambeng N. Nissou M. Benabid A.L. Sadoul R. Verna J.M. Prog. Neurobiol. 2001; 65: 135-172Crossref PubMed Scopus (1011) Google Scholar). TH is inhibited by ONOO−in vitro (1.Ara J. Przedborski S. Naini A.B. Jackson-Lewis V. Trifiletti R.R. Horwitz J. Ischiropoulos H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7659-7663Crossref PubMed Scopus (382) Google Scholar, 3.Kuhn D.M. Aretha C.W. Geddes T.J. J. Neurosci. 1999; 19: 10289-10294Crossref PubMed Google Scholar) and in PC12 cells (4.Ischiropoulos H. Duran D. Horwitz J. J. Neurochem. 1995; 65: 2366-2372Crossref PubMed Scopus (79) Google Scholar). The ONOO−-induced inhibition of TH is associated with nitration of tyrosine residues (1.Ara J. Przedborski S. Naini A.B. Jackson-Lewis V. Trifiletti R.R. Horwitz J. Ischiropoulos H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7659-7663Crossref PubMed Scopus (382) Google Scholar) and oxidation of cysteine residues (3.Kuhn D.M. Aretha C.W. Geddes T.J. J. Neurosci. 1999; 19: 10289-10294Crossref PubMed Google Scholar), yet neither the identity of the modified residues in TH nor the relative contribution of these posttranslational modifications to loss of catalytic function is known. tyrosine hydroxylase peroxynitrite polyethylene oxide 5,5′-dithiobis-2-nitrobenzoic acid p-chloromercuribenzoic acid, PMAB, PEO-maleimide-activated biotin N-biotinoyl-N-(iodoacetyl)ethylene diamine high performance liquid chromatography matrix-assisted laser desorption-ionization time-of-flight enzyme-linked immunosorbent assay mass spectrometry Ischiropoulos and colleagues (1.Ara J. Przedborski S. Naini A.B. Jackson-Lewis V. Trifiletti R.R. Horwitz J. Ischiropoulos H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7659-7663Crossref PubMed Scopus (382) Google Scholar) first concluded that Tyr225 was the site in TH of ONOO−-induced nitration and attributed enzyme inhibition to this posttranslational modification. A more recent paper from the same group purports that Tyr423, not Tyr225, is the actual site mediating enzyme inactivation after nitration by ONOO−(5.Blanchard-Fillion B. Souza J.M. Friel T. Jiang G.C.T. Vrana K. Sharov V. Baron L. Schoneich C. Quijano C. Alvarez B. Radi R. Przedborski S. Fernado G.S. Horwitz J. Ischiropoulos H. J. Biol. Chem. 2001; 276: 46017-46023Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). A Y423F mutant of TH was extensively nitrated by high concentrations of ONOO−, but its catalytic activity was not inhibited (5.Blanchard-Fillion B. Souza J.M. Friel T. Jiang G.C.T. Vrana K. Sharov V. Baron L. Schoneich C. Quijano C. Alvarez B. Radi R. Przedborski S. Fernado G.S. Horwitz J. Ischiropoulos H. J. Biol. Chem. 2001; 276: 46017-46023Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). ONOO− is a powerful oxidant that can modify cysteine, tryptophan, methionine, and tyrosine residues in proteins. It can also cause lipid peroxidation and DNA damage and lead to mitochondrial dysfunction, effects that contribute to its cytotoxic potential (6.Groves J.T. Curr. Opin. Chem. Biol. 1999; 3: 226-235Crossref PubMed Scopus (177) Google Scholar, 7.Ducrocq C. Blanchard B. Pignatelli B. Ohshima H. Cell Mol. Life Sci. 1999; 55: 1068-1077Crossref PubMed Scopus (231) Google Scholar, 8.Squadrito G.L. Pryor W.A. Free Radic. Biol. Med. 1998; 25: 392-403Crossref PubMed Scopus (735) Google Scholar, 9.Lymar S.V. Hurst J.K. Chem. Res. Toxicol. 1998; 11: 714-715Crossref PubMed Scopus (60) Google Scholar, 10.Beckman J.S. Koppenol W.H. Am. J. Physiol. 1996; 271: C1424-C1437Crossref PubMed Google Scholar). Indeed, the ONOO−-induced nitration of free tyrosine or of tyrosine residues in proteins is used increasingly as a molecular marker of ONOO− participation in conditions that are associated with cell damage or disease states (11.Crow J.P. Beckman J.S. Adv. 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Chen Q. Vila M. Giasson B.I. Djaldatti R. Vukosavic S. Souza J.M. Jackson-Lewis V. Lee V.M. Ischiropoulos H. J. Neurochem. 2001; 76: 637-640Crossref PubMed Scopus (173) Google Scholar), in post-mortem tissue from individuals with Parkinson's disease (19.Good P.F. Hsu A. Werner P. Perl D.P. Olanow C.W. J. Neuropathol. Exp. Neurol. 1998; 57: 338-342Crossref PubMed Scopus (518) Google Scholar, 20.Torreilles F. Salman-Tabcheh S. Guerin M. Torreilles J. Brain Res. Brain Res. Rev. 1999; 30: 153-163Crossref PubMed Scopus (373) Google Scholar) suggests that ONOO−-induced tyrosine nitration plays a causative role in dopamine neuronal degeneration and in TH dysfunction. Based on the importance of TH to dopamine neuronal function, and considering the possibility that ONOO− causes the inhibition of TH as an early event in the process of dopamine neuronal degeneration (1.Ara J. Przedborski S. Naini A.B. Jackson-Lewis V. Trifiletti R.R. Horwitz J. Ischiropoulos H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7659-7663Crossref PubMed Scopus (382) Google Scholar, 5.Blanchard-Fillion B. Souza J.M. Friel T. Jiang G.C.T. Vrana K. Sharov V. Baron L. Schoneich C. Quijano C. Alvarez B. Radi R. Przedborski S. Fernado G.S. Horwitz J. Ischiropoulos H. J. Biol. Chem. 2001; 276: 46017-46023Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar), we sought to determine the sites at which TH is modified by ONOO−. MALDI-TOF mass spectrometry and tyrosine-scanning mutagenesis have identified tyrosines 423, 428, and 432 as the sites of ONOO−-induced nitration. Diethylenetriamine pentaacetic acid, 5,5′-dithiobis-2-nitrobenzoic acid (DTNB),p-chloromercuribenzoic acid (pCMB), Me2SO, glutathione, bradykinin, bovine pancreatic insulin, and α-cyano-4-hydroxycinnamic acid and glutathione-agarose were obtained from Sigma. Catalase and a monoclonal antibody against TH were products of Boehringer Mannheim. Thrombin and pGEX vectors were obtained from Amersham Biosciences. Protease V8 was purchased from Promega (Madison, WI). Guanidine hydrochloride was from Invitrogen (Carlsbad, CA). Tetrahydrobiopterin was purchased from Dr. Shircks Laboratories (Jona, Switzerland). A monoclonal antibody against nitrotyrosine was purchased from Cayman Chemical Company (Ann Arbor, MI), and horseradish peroxidase-linked goat anti-mouse IgGs were products of Cappel. Trypsin, PEO-maleimide-activated biotin (PMAB), and Immunopure TMB peroxidase kits were obtained from Pierce.N-biotinoyl-N-(iodoacetyl)ethylene diamine (BIAM) was purchased from Molecular Probes, Inc. (Eugene, OR). Enhanced chemiluminescence reagents were products of PerkinElmer Life Sciences, and Bio-Max MR film was from Eastman Kodak Co. Restriction endonucleases, T4 ligase, and T4 kinase were products of New England Biolabs. Acetonitrile and trifluoroacetic acid were HPLC grade, and all other reagents were obtained from commercial sources in the highest possible qualities. TH was cloned by reverse transcriptase-polymerase chain reaction and expressed as a glutathioneS-transferase fusion protein as previously described (3.Kuhn D.M. Aretha C.W. Geddes T.J. J. Neurosci. 1999; 19: 10289-10294Crossref PubMed Google Scholar,21.Kuhn D.M. Arthur Jr., R.E. Thomas D.M. Elferink L.A. J. Neurochem. 1999; 73: 1309-1317Crossref PubMed Scopus (164) Google Scholar). Tyr-to-Phe site-directed mutagenesis of TH was carried out for each of its 17 tyrosine residues (22.Grima B. Lamouroux A. Blanot F. Biguet N.F. Mallet J. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 617-621Crossref PubMed Scopus (486) Google Scholar) via splicing by overlap extension (23.Horton R.M. Ho S.N. Pullen J.K. Hunt H.D. Cai Z. Pease L.R. Methods Enzymol. 1993; 217: 270-279Crossref PubMed Scopus (432) Google Scholar). For selected tyrosine residues (see below), double and triple tyrosine mutants were also created. Automated nucleotide sequencing confirmed all mutations. Recombinant fusion proteins were purified by glutathione-agarose affinity chromatography, and the glutathioneS-transferase fusion tag was removed by thrombin cleavage, resulting in highly purified TH preparations (>95% pure). ONOO− was synthesized by the quenched-flow method of Beckman et al. (24.Beckman J.S. Chen J. Ischiropoulos H. Crow J.P. Methods Enzymol. 1994; 233: 229-240Crossref PubMed Scopus (971) Google Scholar), and its concentration was determined by the extinction coefficient ε302 = 1670m−1 cm−1. The hydrogen peroxide contamination of ONOO− solutions was removed by manganese dioxide chromatography and filtration (24.Beckman J.S. Chen J. Ischiropoulos H. Crow J.P. Methods Enzymol. 1994; 233: 229-240Crossref PubMed Scopus (971) Google Scholar). ONOO−(100–500 μm) was added to TH (10 μm with respect to the 60-kDa monomer) with vigorous mixing in 50 mm potassium phosphate buffer, pH 7.4, containing 100 μm diethylenetriamine pentaacetic acid, and incubations were carried out for 15 min at 30°C. The volume of ONOO−added to the enzyme samples was always less than 1% (v/v) and did not influence pH. Upon completion of incubation with ONOO−, samples were diluted 1:10 with 50 mm potassium phosphate, pH 6, and stored at 4°C. Residual TH activity was assayed according to the method of Lerner et al. (25.Lerner P. Nose P. Ames M.M. Lovenberg W. Neurochem. Res. 1978; 3: 641-651Crossref PubMed Scopus (39) Google Scholar). Protein concentrations were determined as described by Bradford (26.Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217544) Google Scholar). TH and ONOO−-treated TH (TH-ONOO−) were individually purified by reversed-phase HPLC. The HPLC system consisted of two Waters 6000 pumps (controlled by a personal computer) in line with a Spectroflow 757 UV detector set at 214 nm. A gradient of 20–65% acetonitrile in 0.1% trifluoroacetic acid was applied in a 60-min time period over a Vydac C18 (4.6 × 250 mm, 5-μm particle size, 300-Å pores) column. Fractions corresponding to the whole protein were collected and dried in a vacuum system. Seventy micrograms of TH and 70 μg of TH-ONOO− were individually reconstituted in 60 μl of 6 m guanidine hydrochloride in 50 mm Trizma hydrochloride buffer, pH 8.0. To this was added 330 μl of water and 330 μl of 1 mmCaCl2 in 50 mm Trizma hydrochloride, pH 7.6. Three micrograms of protease V8 (in 3 μl) was added to each sample, which was mixed and allowed to sit for 18 h at room temperature. Samples were then evaporated under vacuum to ∼200 μl followed by the addition of 1.6 μl of trifluoroacetic acid. Next, they were purified by C18 ZipTip (Millipore Corp., Bedford, MA) application and eluted into 5 μl of 0.1% trifluoroacetic acid/acetonitrile (1:1) saturated with α-cyano-4-hydroxycinnamic acid. One microliter was then spotted onto a gold-plated MALDI plate to which had been applied an ultrathin layer of α-cyano-4-hydroxycinnamic acid according to the method of Cadene and Chait (27.Cadene M. Chait B.T. Anal. Chem. 2000; 72: 5655-5658Crossref PubMed Scopus (152) Google Scholar). To obtain better MALDI-TOF MS ion counting statistics for the triply nitrated species, 40 μg of the V8 digest was separated by microbore HPLC, and fractions were collected corresponding to the nitrated species. Microbore HPLC conditions were as follows. Flow rate was set at 0.05 ml/min across a 150 × 1.0-mm column with the same sorbent as above. Solvent A was 0.1% trifluoroacetic acid in water containing 10 mm EDTA-free acid. Solvent B was 0.1% trifluoroacetic acid in 98:2 acetonitrile/water containing 10 mm EDTA free acid. A gradient of 0% B to 45% B was applied linearly over 60 min. The fraction corresponding to the triply nitrated species was dried under a vacuum and reconstituted in 1 μl of the above matrix solution, and 0.5 μl was spotted onto a MALDI plate. MALDI mass spectra were acquired on a Voyager DE-STR TOF mass spectrometer (PerkinElmer Life Sciences) equipped with a 337-nm nitrogen laser. In linear mode, the accelerating voltage was set to 20,000 V with grid voltage at 95%, guide wire turned off, and extraction delay time at 400 ns. In reflector mode, the accelerating voltage was set to 20,000 V with grid voltage at 76%, mirror voltage ratio at 1.12, guide wire at 0.05%, and extraction delay time at 310 ns. Time of flight to mass conversion was achieved with the use of external standards of bradykinin (monoisotopic calculated mass for [M + H]+ = 1060.57 Da; average mass for [M + H]+ = 1061.22 Da) and bovine pancreatic insulin (average calculated mass for [M + H]+= 5734.56 Da; average calculated mass for [M + 2H]2+ = 2867.78 Da). Following treatment with ONOO−, wild type TH and all Tyr-to-Phe mutants were analyzed for nitrotyrosine content by ELISA and Western blotting. ELISA was used in initial screens of TH tyrosine nitration because of its sensitivity and high sample throughput capability. Once Tyr-to-Phe mutants were identified that were reduced in the extent to which they were tyrosine-nitrated by ONOO−, these mutants were analyzed by Western blotting as well. ELISA assays were optimized to determine nitrotyrosine immunoreactivity in TH preparations. Control and ONOO−-treated TH samples containing 200 ng of protein were adsorbed overnight at 4 °C to 96-well Nunc-Immuno plates with Maxi-Sorp surfaces. Sample wells were washed three times with phosphate-buffered saline and then blocked with nonfat dry milk (5% w/v) for 2 h at room temperature. Plates were incubated overnight at 4 °C with a monoclonal antibody against nitrotyrosine (1:1000 dilution in nonfat dry milk). Following three washes with phosphate-buffered saline, wells were incubated with a horseradish peroxidase-coupled goat anti-mouse secondary antibody (1:10,000 dilution in nonfat dried milk) at room temperature for 2 h. Immunopure TMB peroxidase substrate was added to wells, and absorbance was read in a microtiter plate reader at 550 nm. Under these conditions, absorbance readings for nitrotyrosine immunoreactivity in TH were linear up to 500 ng of protein/well. All samples were applied to plates in triplicate. Untreated wild-type TH and ONOO−-nitrated TH samples were applied to wells throughout the plate as internal controls. For Western blotting, TH preparations were subjected to SDS-polyacrylamide gel electrophoresis on 10% gels according to Laemmli (28.Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar). Proteins were transferred to nitrocellulose, blocked in Tris-buffered saline containing Tween 20 (0.1% v/v) and 5% nonfat dry milk and probed with a monoclonal antibody specific for nitrotyrosine. After incubations with primary antibodies (diluted 1:2000), blots were incubated with goat anti-mouse secondary antibody conjugated with horseradish peroxidase (diluted 1:5000), and immunoreactive protein bands were visualized with enhanced chemiluminescence by exposure to Kodak Biomax MR film. Digital images of films were captured with a Sony CCD-IRIS/RGB color video camera, and relative pixel densities of protein bands were obtained. The effect of ONOO− on TH sulfhydryls was determined with the use of thiol-reactive biotinylation reagents as described by Kim et al. (29.Kim J.R. Yoon H.W. Kwon K.S. Lee S.R. Rhee S.G. Anal. Biochem. 2000; 283: 214-221Crossref PubMed Scopus (245) Google Scholar). PMAB and BIAM react selectively with reduced cysteines in proteins and do not react with cysteines that have been oxidized (29.Kim J.R. Yoon H.W. Kwon K.S. Lee S.R. Rhee S.G. Anal. Biochem. 2000; 283: 214-221Crossref PubMed Scopus (245) Google Scholar). These probes are not quantitative, but they allow a relative measure of the extent to which cysteine residues have been oxidized. Initial screening of the effects of ONOO− on the status of sulfhydryls in TH (wild-type and all mutants) was carried out with ELISA as described above for the determination of nitrotyrosine content of TH. Untreated or ONOO−-treated TH was diluted 1:2 with 100 mmTris-HCl, pH 6.5 or pH 8.5, for subsequent labeling with PMAB (50 μm) or BIAM (50 μm), respectively. Proteins were labeled for 60 min at room temperature in the dark, after which they were subjected to SDS-PAGE and blotting to nitrocellulose. Remaining samples were diluted 1:50 with phosphate-buffered saline, and 50 ng of protein was applied to 96-well plates. Blots and plates were then processed as described above with the exception that horseradish peroxidase-linked avidin (diluted 1:500 in nonfat dry milk) was used in place of a secondary antibody, and biotin reactivity was visualized by ECL. Absorbance readings for the biotinylated labels on 96-well plates were linear up to 200 ng per well. Wild-type TH (10 μm) was treated at 30 °C for 15 min with varying concentrations of the selective sulfhydryl reagents pCMB or DTNB as described above for ONOO−. Following treatment, TH samples were diluted 1:10 with 50 mm potassium phosphate, pH 6. Residual TH activity was assayed, or samples were probed with PMAB as described above. The V8 partial cleavage fragment containing amino acid residues 410–436 (VRAFDPDTAAVQPYQDQTYQPVYFVSE) was obtained in good yield and used to determine the nitration status of tyrosine residues 423, 428, and 432 after treatment of intact TH with ONOO−. The calculated average mass for [M + H]+ of the native peptide is 3136.40 Da; the observed peak at m/z3136.0 in linear mode and at m/z 3136.46 in reflector mode represented the protonated species. Treatment of TH with ONOO− produced four congeners of the protein consisting of 0–3 nitration events per molecule, with each nitro group adding 45 Da to the V8 fragment. Thus, peaks at m/z 3181.48,m/z 3226.88, and m/z3271.85 (Fig. 1) correspond to one, two, and three nitration events per protein molecule, respectively. Peaks occurring 16 and 32 units lower than those representing nitration correspond to products from prompt fragmentation caused by the immediate loss of an oxygen from a nitro group to form a nitroso species, followed by loss of a second oxygen possibly to form a nitrene or dehydroazepine species as outlined by Sarver et al. (30.Sarver A. Scheffler N.K. Shetlar M.D. Gibson B.W. J. Am. Soc. Mass Spectrom. 2001; 12: 439-448Crossref PubMed Scopus (134) Google Scholar). Untreated TH did not produce any peaks in the m/zrange corresponding to the nitrated peptides (data not shown). Two additional, unrelated peaks are present in the mass spectra of both the ONOO−-treated and untreated samples. The peak atm/z 3289 corresponds to V8 partial cleavage fragment Leu333–Glu363. The peak atm/z 3307 is unidentified but is not related to ONOO− treatment, since it is also present in the mass spectrum of the analogous V8 digestion fragment of untreated TH. The inset to Fig. 1 is an abbreviated segment of a better quality mass spectrum of only the triply nitrated species. To obtain the sample for this spectrum, another sample of ONOO−-treated TH was digested by V8, and the mixture was separated by HPLC, which allowed the differently nitrated peptides to be isolated and analyzed by MALDI-TOF. The peak atm/z 3271.4 in the inset mass spectrum in Fig. 1 corresponds to the triply nitrated species as shown in Fig. 1. Prompt fragmentation peaks are also seen in the inset at 16-unit multiples lower than the m/z 3271.4 peak (e.g. at m/z 3255.4 and 3239.8). The prompt fragmentation peaks appear larger for the triply nitrated species than for the doubly or singly nitrated species, because all three nitro groups in the triply nitrated species can undergo photodecomposition, leading to greater accumulation of lower mass species (i.e. increments of 16 Da). Each tyrosine residue within the 3135.4-Da peptide of TH was mutated to phenylalanine, and the effect of ONOO− on tyrosine nitration and enzyme activity expressed by these mutants was determined. Tyr225 was also mutated, because it was previously claimed to be the site of ONOO−-induced nitration (1.Ara J. Przedborski S. Naini A.B. Jackson-Lewis V. Trifiletti R.R. Horwitz J. Ischiropoulos H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7659-7663Crossref PubMed Scopus (382) Google Scholar). Fig. 2Apresents results with ONOO−-induced nitration of Y423F, Y428F, and Y432F as assessed by anti-nitrotyrosine immunoreactivity. The extent to which ONOO− caused nitration in these mutants was reduced by comparison with wild-type TH. Digital scans of the data in Fig. 2A indicated that Y423F nitration was reduced to 27% of control when normalized to the amount of TH protein in each sample (see Fig. 2B). Similarly, the nitration of Y428F and Y432F was reduced to 32 and 39% of control, respectively. All possible combinations of double tyrosine mutants were made among these tyrosines, and the ONOO−-induced nitration of these mutants is shown in Fig. 2A as well. It can be seen that nitration of Y423F/Y432F (6% of control), Y428F,Y432F (16% of control), and Y423F,Y428F (27% of control) was reduced more than seen with the single mutants. Finally, the triple tyrosine mutant Y423F,Y428F,Y432F was not nitrated by ONOO−(last lane of Fig. 2A). The removal of Tyr225 from TH resulted in a 20% increase in tyrosine nitration after ONOO− treatment. The effects of tyrosine mutagenesis on basal TH activity and on the extent to which ONOO− modified catalytic activity of the mutants are presented in Fig. 2C. Single or double mutations of Tyr423, Tyr428, and Tyr432 to phenylalanine were tolerated very well by the enzyme. All expressed levels of activity that were within 20% of wild-type TH, with the exceptions of Y423F,Y432F and Y428F,Y432F, whose basal levels of activity were 60 and 69% of wild type, respectively. The triple Y423F,Y428F,Y432F mutant showed somewhat more disruption of catalytic function, expressing basal levels of activity that were ∼20% of control. Fig. 2C also shows that ONOO− (100 μm) reduced the catalytic activity of wild type TH and each mutant, regardless of basal levels of activity. The effect of ONOO− on Y423F and Y428F was the same as its effect on wild-type enzyme, but the remaining mutants appeared to be more sensitive to inhibition. For example, ONOO− reduced the activity of Y432F to 20% of control, and the double mutants were inhibited by 80–85% as compared with a 50% inhibition of wild-type TH. The reduction in TH catalytic activity caused by ONOO−was significant for wild-type enzyme and for all mutants (p < 0.05, Bonferroni's test). Each of the remaining 14 tyrosines in TH, not described above for Fig. 2, was converted to phenylalanine individually, and the effects of ONOO− on the levels of enzyme activity and tyrosine nitration were determined. The results are presented in Table I. All Tyr-to-Phe mutants of TH retained catalytic activity, but some were sensitive to substitution. For example, the basal activities of Y200F and Y314F were about 40–45% of wild-type, and Y448F and Y463F expressed higher levels of activity than wild-type TH (35–40% increases). ONOO− treatment of each of these mutants caused a significant reduction in their activities. Y214F was most sensitive to inhibition, showing reductions in activity to 15% of control after ONOO− treatment. Y225F and Y371F were inhibited to 20% of control by ONOO−. The remaining mutants were inhibited by ONOO− to about the same extent as wild-type TH. Table I also shows that these same mutants were tyrosine-nitrated to the same extent as wild-type enzyme.Table ITyrosine-scanning mutagenesis of TH and the effects of ONOO−on catalytic activity and relative tyrosine nitrationTyrosine mutant1-aData for tyrosines 423, 428, and 432 are presented in Fig. 2 and are excluded from the table.TH activity1-bWild-type TH and all mutants (10 μm) were exposed to ONOO− (100 μm) for 15 min at 30 °C, after which samples were diluted 1:10 with 50 mm phosphate buffer, pH 6.0, and remaining enzyme activity was determined. TH activity is expressed as μmol mg−1min−1 and represents mean ± S.E. for 3–5 experiments run in duplicate.Tyrosine nitration1-cTyrosine
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