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J. Biol. Chem., Vol. 277, Issue 16, 14336-14342, April 19, 2002

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Peroxynitrite-induced Nitration of Tyrosine Hydroxylase

IDENTIFICATION OF TYROSINES 423, 428, AND 432 AS SITES OF MODIFICATION BY MATRIX-ASSISTED LASER DESORPTION IONIZATION TIME-OF-FLIGHT MASS SPECTROMETRY AND TYROSINE-SCANNING MUTAGENESIS^*

Donald M. Kuhn^§¶, Mahdieh Sadidi, Xiuli Liu, Christian Kreipke, Timothy Geddes^¶, Chad Borges^**, and J. Throck Watson^**

From the  Department of Psychiatry and Behavioral Neurosciences, ^§ Center for Molecular Medicine and Genetics, Wayne State University School of Medicine, and ^¶ John D. Dingell Veterans Affairs Medical Center, Detroit, Michigan 48201 and the ^** Department of Biochemistry, Michigan State University, East Lansing, Michigan 48824

Received for publication, January 10, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Tyrosine hydroxylase (TH)¹ is 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), 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). TH is inhibited by ONOO in vitro (1, 3) and in PC12 cells (4). The ONOO-induced inhibition of TH is associated with nitration of tyrosine residues (1) and oxidation of cysteine residues (3), 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.

Ischiropoulos and colleagues (1) 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). A Y423F mutant of TH was extensively nitrated by high concentrations of ONOO, but its catalytic activity was not inhibited (5).

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 (6-10). 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 (11-15). Increased tyrosine nitration of proteins, including the Lewy body constituent -synuclein (16-18), in post-mortem tissue from individuals with Parkinson's disease (19, 20) 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, 5), 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.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- Diethylenetriamine pentaacetic acid, 5,5'-dithiobis-2-nitrobenzoic acid (DTNB), p-chloromercuribenzoic acid (pCMB), Me₂SO, glutathione, bradykinin, bovine pancreatic insulin, and -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.

Preparation of TH, Site-directed Mutagenesis, and Treatment with ONOO-- TH was cloned by reverse transcriptase-polymerase chain reaction and expressed as a glutathione S-transferase fusion protein as previously described (3, 21). Tyr-to-Phe site-directed mutagenesis of TH was carried out for each of its 17 tyrosine residues (22) via splicing by overlap extension (23). 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 glutathione S-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), and its concentration was determined by the extinction coefficient ₃₀₂ = 1670 M¹ cm¹. The hydrogen peroxide contamination of ONOO solutions was removed by manganese dioxide chromatography and filtration (24). 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). Protein concentrations were determined as described by Bradford (26).

MALDI-TOF Mass Spectrometry-- 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 mM CaCl₂ 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 -cyano-4-hydroxycinnamic acid. One microliter was then spotted onto a gold-plated MALDI plate to which had been applied an ultrathin layer of -cyano-4-hydroxycinnamic acid according to the method of Cadene and Chait (27). 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).

Analysis of ONOO-induced Tyrosine Nitration-- 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). 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.

Analysis of TH Sulfhydryl Status after ONOO Treatment-- The effect of ONOO on TH sulfhydryls was determined with the use of thiol-reactive biotinylation reagents as described by Kim et al. (29). PMAB and BIAM react selectively with reduced cysteines in proteins and do not react with cysteines that have been oxidized (29). 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 mM Tris-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.

Treatment of TH with Sulfhydryl-selective Reagents-- 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.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

MALDI-TOF Mass Spectrometry of ONOO-treated TH-- 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/z 3136.0 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/z 3271.85 (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). Untreated TH did not produce any peaks in the m/z range 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 at m/z 3289 corresponds to V8 partial cleavage fragment Leu³³³-Glu³⁶³. The peak at m/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.

View larger version (23K): [in this window] [in a new window]   Fig. 1.   Reflectron positive ion MALDI-TOF MS spectrum of a V8 proteolytic digest of TH. TH was nitrated with ONOO as described under "Experimental Procedures" and subjected to V8 proteolytic digestion. Analysis of the unseparated digest mixture by MALDI-TOF MS yielded an ion of m/z of 3136.41, representing amino acid residues Val410-Glu436 of TH. This proteolytic fragment of TH contains residues Tyr423, Tyr428, and Tyr432. Peaks at m/z values of 3181.48, 3226.88, and 3271.85 in the mass spectrum correspond to derivatives of this proteolytic fragment by nitration of one, two, or three tyrosine residues. Peaks corresponding to photodecomposition product ions 16 and 32 units lower than the peak for the nitrated species were also observed, representing 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 forming either a nitrene or dehydroazepine species. The inset shows a MALDI-TOF mass spectrum of an HPLC fraction containing the V8 proteolytic fragment of interest, more clearly showing the triply nitrated species at m/z 3271.4. Peaks 16-unit multiples lower than that of the triply nitrated species represent prompt fragmentation peaks as described for the mass spectrum in the primary figure. None of these peaks indicative of nitration was apparent in the mass spectrum of V8 proteolytic digests 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 at m/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).

Site-directed Mutagenesis of Tyrosines 423, 428, and 432-- 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). Fig. 2A presents 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).

View larger version (23K): [in this window] [in a new window]   Fig. 2.   Effects of ONOO on Tyr-to-Phe mutants of TH. Wild-type (WT) TH or single, double, or triple mutants (10 µM) of the indicated tyrosine residues were treated with ONOO (100 µM) and subjected to immunoblotting with an antibody against nitrotyrosine (A), immunoblotting with an antibody against TH (B), or determinations of catalytic activity (C). Blots in A and B were scanned with a CCD video camera, and the relative pixel densities were used to normalize levels of tyrosine nitration with the amount of TH protein in each lane. These experiments were repeated five times, and produced the same results. TH catalytic activity in C is expressed as percentage of the untreated control for each mutant. Results represent means ± S.E. of 4-6 independent experiments carried out in duplicate. Where indicated (*), basal levels of TH activity were significantly lower than wild-type enzyme in certain tyrosine mutants (p < 0.05, Bonferroni's test). The effect of ONOO on the activity of all forms of TH was significant (p < 0.01, Bonferroni's test).

Tyrosine-scanning Mutagenesis of TH-- 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.

View this table: [in this window] [in a new window]   Table I Tyrosine-scanning mutagenesis of TH and the effects of ONOO on catalytic activity and relative tyrosine nitration

Effects of ONOO on Cysteine Residues in Wild-type TH and Phenylalanine Mutants of Tyrosines 423, 428, and 432-- Wild-type TH and the mutants of tyrosines 225, 423, 428, and 432 (single, double, and triple mutants) were treated with ONOO (100 µM) and probed with the highly selective thiol reactants PMAB or BIAM, as described by Kim et al. (29). These biotinylated reagents only label reduced cysteines in proteins, and reductions in labeling are an index of cysteine oxidation (29). Fig. 3 shows that wild-type TH and each of the indicated tyrosine mutants were extensively labeled by PMAB under control conditions. After treatment of all proteins with ONOO, the extent of PMAB labeling was substantially reduced. Digital scans of the blot in Fig. 3 showed that the relative reduction in labeling of each ONOO-treated mutant varied between 60 and 80% by comparison with the respective controls. The same results were obtained if labeling of these same mutants was carried out with BIAM (data not shown). All remaining Tyr-to-Phe mutants of TH, listed in Table I, were also probed with PMAB after ONOO treatment using the ELISA format. The results with these mutants were the same as described above, showing that ONOO caused large reductions in PMAB labeling, indicative of cysteine oxidation (data not shown).

View larger version (29K): [in this window] [in a new window]   Fig. 3.   Effects of ONOO on PMAB Labeling of Cysteine Residues in TH. Wild-type TH or the indicated tyrosine mutants (10 µM) were treated with ONOO (100 µM). Untreated enzyme served as controls for each form of TH. Samples were labeled with the thiol-sensitive probe PMAB and subjected to SDS-PAGE and blotting to nitrocellulose. Blots were subsequently probed with avidin-linked horseradish peroxidase, and PMAB-labeled proteins were visualized with enhanced chemiluminescence as described under "Experimental Procedures." A, results using single tyrosine mutants; B, results using double and triple tyrosine mutants of TH. Each panel contains wild-type (WT) TH as a control. The addition of ONOO is indicated above each panel (, control; +, ONOO).

Effects of Selective Sulfhydryl Reagents on TH Activity-- Various sulfhydryl reagents that are highly specific in their reactivity with cysteines and that have no reactivity with tyrosine residues (31) were tested for their effects on TH activity and cysteine status of the enzyme. In view of the data of Fig. 3, showing that Tyr⁴²³, Tyr⁴²⁸, and Tyr⁴³² mutants did not differ from wild-type TH in the extent to which ONOO reduced cysteine labeling with PMAB, only results with wild-type TH are presented. Fig. 4 shows that DTNB (500 µM) reduced TH activity to 20% of control, and pCMB (500 µM) reduced enzyme activity to 50% of control. The effects of pCMB and DTNB on TH activity were significant (p < 0.01, Bonferroni's test). The inset to Fig. 4 shows that concentrations of pCMB and DTBN that inhibited TH catalytic activity reduced PMAB labeling of the protein. Digital scans indicated that pCMB reduced PMAB labeling of TH by 80%, whereas DTNB caused a 50% reduction in labeling.

View larger version (15K): [in this window] [in a new window]   Fig. 4.   Effects of sulfhydryl reagents on TH activity and PMAB-mediated cysteine labeling. Wild-type TH was treated with pCMB or DTNB (500 µM for each), and the effects on catalytic activity were determined. Results are expressed as percentage of control of untreated TH and represent means ± S.E. of six independent experiments carried out in duplicate. The inset presents the results of PMAB labeling of cysteine residues in TH after treatment with pCMB or DTNB.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

TH is the initial and rate-limiting enzyme in dopamine biosynthesis, and, as such, alterations in its activity will have corresponding effects on the availability of dopamine for release into synaptic activity. TH is inhibited by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in vivo through a process that is thought to involve ONOO-induced nitration of tyrosine residues in the enzyme (1, 5). This finding could offer insight into early biochemical processes that contribute to neuronal dopamine deficiencies. ONOO-induced nitration of TH and other proteins may be an early manifestation of oxidative stress in dopamine neurons brought on by drugs or diseases that are associated with toxicity and damage. The mechanism by which TH is inactivated by ONOO is not fully understood. Nitration of tyrosine residues is but one element of the reactivity associated with ONOO. In fact, ONOO is extremely reactive with cysteine (32, 33) and has been shown to target this residue with much higher probability than it nitrates tyrosines within the same protein (34). ONOO causes extensive oxidation of cysteine residues in TH, and we have attributed inactivation of the enzyme to sulfhydryl oxidation, not tyrosine nitration (3).

Identification of the sites in TH that are modified by ONOO would not only contribute to a better understanding of TH catalysis but could help identify site-specific posttranslational modifications that are early markers of neuronal damage. This goal has proved difficult with respect to TH. For example, Ara et al. (1) concluded that Tyr²²⁵ was the site of ONOO-induced nitration in TH. More recently, this same group concluded that Tyr⁴²³, not Tyr²²⁵, is the site that mediates ONOO-induced inactivation (5). A Y423F mutant of TH was extensively nitrated by ONOO, but, paradoxically, catalytic activity was not reduced. We have used a combination of MALDI-TOF mass spectrometry and tyrosine-scanning mutagenesis to identify the tyrosine residues in TH that are nitrated by ONOO.

Analysis of V8 protease-cleaved nitrated TH by MALDI-TOF MS identified a fragment having a [M + H]⁺ molecular mass of 3135.4 Da, corresponding to amino acid residues Val⁴¹⁰-Glu⁴³⁶. The mass spectrum also shows three less intense peaks that were shifted +45, +90, and +135 units from the major peak, corresponding to nitration of one, two, or three tyrosines, respectively, within this V8 protease-cleaved TH peptide. These peaks were not observed in the mass spectrum of untreated TH. Additional confirmation of these multiple nitrations can be drawn from the appearance of peaks 16 and 32 units lower than those representing nitration. These 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 a second loss of oxygen to probably either a nitrene or dehydroazepine species (30). To confirm MALDI-TOF findings and assess the impact of nitration of these residues on TH activity, tyrosines 423, 428, and 432 were mutated to phenylalanine individually and in all possible combinations of double and triple substitutions. This approach revealed several interesting results with regard to tyrosine nitration of the enzyme. First, ONOO-induced nitration was substantially reduced in each single mutant (30-60% reductions). Second, all double mutants (i.e. Y423F,Y428F, Y423F,Y432F, and Y428F,Y432F) showed less nitration than the single mutants in response to ONOO. The extent of reduction in nitration of the single mutants was retained in the respective double mutants, reaching levels of only 10-30% of control. Third, the triple mutant was not nitrated by ONOO. Thus, the findings with site-directed mutagenesis are in agreement with those from analyses by MALDI-TOF MS that TH is nitrated at as many as three tyrosines when treated with a 10-fold excess of ONOO. We could not find any evidence that Tyr²²⁵ was a site of ONOO-induced nitration, despite the conclusions of Ara et al. (1) that this residue represented the site in TH of ONOO-induced nitration.

The conservative substitution of tyrosines 423, 428, and 432 with phenylalanine had only minor impact on TH activity in the single and double mutants. Y423F and Y428F expressed wild-type levels of TH activity, and the activity Y432F was ~20% lower than wild-type. Y423/428F, Y423/432F, and Y428/432F expressed basal levels of activity that ranged from 60 to 80% of wild type. The differences in activity between the latter two double mutants and wild-type TH were statistically significant. The triple mutant expressed much lower levels of TH enzyme activity (~20% of wild type) by comparison with all other forms of the enzyme. Regardless of the basal levels of activity, all tyrosine mutants of TH were inhibited by ONOO, and it appeared that some mutants were actually more sensitive to inhibition than wild-type enzyme. Taken together, these data indicate that the progressive removal from TH of tyrosines that are sites of nitration neither completely disrupts catalytic function nor prevents enzyme inactivation by ONOO. The remaining 14 tyrosines in TH were mutated to phenylalanine individually with the goal of identifying additional nitration sites that might not be accounted for by MALDI-TOF MS (e.g. peptide fragments that did not desorb from the MALDI plate). It was observed that almost all single Tyr-to-Phe mutants of TH retained substantial levels of catalytic activity, all were substrates for nitration by ONOO, and all were inhibited to similar extents by treatment with ONOO. The triple mutant, however, was not nitrated, and in combination with the MALDI-TOF MS data, substantial evidence is provided to conclude that TH is nitrated by ONOO at Tyr⁴²³, Tyr⁴²⁸, and Tyr⁴³².

The observation that ONOO inhibited TH tyrosine mutants in the face of diminishing tyrosine nitration prompted consideration of alternative mechanisms. Previous work from our laboratory showed that ONOO lowered the number of DTNB-reactive cysteine residues in TH in parallel with losses in catalytic activity (3). These results are consistent with ONOO serving as a powerful cysteine oxidant (33, 34). Nevertheless, Ishiropoulos and colleagues (1, 5) could not find evidence of ONOO-induced cysteine modification in TH. We probed ONOO-treated TH with the thiol-reactive compounds PMAB and BIAM, reagents that are known to react with reduced cysteines (29). We hypothesized that ONOO would reduce labeling if it oxidized cysteines in TH. This is, in fact, what was observed. PMAB labeling of wild-type TH and of all Tyr-to-Phe mutants was lowered substantially by comparison with that of untreated controls. While not quantitative, the results with PMAB agree with our previous results using DTNB titration of reduced cysteines (3) and show that ONOO reacts with cysteine residues in the enzyme. TH was also sensitive to inhibition by thiol-sensitive reagents pCMB and DNTB. These compounds, like ONOO, inhibited TH catalytic function and significantly reduced PMAB labeling of the enzyme. Evidence from another line of investigation establishes that TH activity can be disrupted when its cysteines are modified. Dopamine-quinones inactivate TH by binding to cysteine residues (21). Modification of cysteines by quinones was substantiated by the loss of DTNB reactivity from TH and by the appearance of cysteinyl-dopamine. The extent of cysteine modification in TH was predictive of the losses in catalytic activity in these studies (21). Therefore, TH is readily inhibited by various thiol-reactive reagents, including ONOO.

Tyrosine-scanning mutagenesis of TH does not single out any particular tyrosine residue as being absolutely essential for TH catalytic function. Furthermore, it does not appear that tyrosine residues play a significant role in TH catalysis. Our results agree well with those of Daubner et al. (35), who showed that mutation of Tyr³⁷¹ to phenylalanine did not disrupt TH catalytic function. Similarly, a Y325F mutant of phenylalanine hydroxylase has normal levels of activity (36). Tyr³²⁵ in phenylalanine hydroxylase corresponds to Tyr³⁷¹ in TH. Tyr³⁷¹ is near the catalytic site of TH (37), where it is in one of four -helices that form a large hydrophobic pocket involved in pterin cofactor binding (35, 38, 39), yet it tolerates mutagenesis to phenylalanine very well. This is not that surprising when considering that this hydrophobic pocket accommodates numerous bulky substitutions at the C-6 position of the pterin cofactor (38, 39). Tyr⁴²³ and Tyr⁴²⁸ lie within one of two loops, the other of which is composed of residues 290-296, that reach over the active site opening of the TH (37, 38). The conservative substitution of these residues with phenylalanine, one at a time, has little impact on TH activity. However, double mutations among these tyrosines do lower catalytic activity slightly, and a triple mutant expresses only 20% of the activity of wild-type enzyme. It is possible that removal of the hydroxyl group from two of the tyrosine residues in this loop changes its orientation with respect to the other loop, hindering access of the substrates to the active site.

The present results have identified Tyr⁴²³, Tyr⁴²⁸, and Tyr⁴³² in TH as the sites of nitration by ONOO. Single, double, and triple tyrosine mutants of TH retained catalytic function and were inhibited by ONOO, while showing little if any tyrosine nitration. In the face of diminished levels of tyrosine nitration, cysteine modification was apparent in all tyrosine mutants of TH after treatment with ONOO. Tyrosine nitration is neither necessary nor sufficient to explain the inhibition of TH by ONOO, and evidence points increasingly to cysteines as determinants of TH catalytic function and as targets for modification by reactive nitrogen species like ONOO.

	FOOTNOTES

^* This work was supported by National Institute on Drug Abuse Grant DA 10756, the Joe Young, Sr. Psychiatric Research Fund of the Department of Psychiatry and Behavioral Neurosciences, and a Veterans Affairs Merit Award.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: 2125 Scott Hall, 540 E. Canfield, Wayne State University School of Medicine, Detroit, MI 48201. Tel./Fax: 313-577-9737; E-mail: donald.kuhn@wayne.edu.

Published, JBC Papers in Press, February 7, 2002, DOI 10.1074/jbc.M200290200

	ABBREVIATIONS

The abbreviations used are: TH, tyrosine hydroxylase; ONOO, peroxynitrite; PEO, polyethylene oxide; DTNB, 5,5'-dithiobis-2-nitrobenzoic acid; pCMB, p-chloromercuribenzoic acid, PMAB, PEO-maleimide-activated biotin; BIAM, N-biotinoyl-N-(iodoacetyl)ethylene diamine; HPLC, high performance liquid chromatography; MALDI, matrix-assisted laser desorption-ionization; TOF, time-of-flight; ELISA, enzyme-linked immunosorbent assay; MS, mass spectrometry.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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