HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kuhn, D. M. Articles by Geddes, T. J. Search for Related Content PubMed PubMed Citation Articles by Kuhn, D. M. Articles by Geddes, T. J. Social Bookmarking                      What's this?

J Biol Chem, Vol. 274, Issue 42, 29726-29732, October 15, 1999

Peroxynitrite Inactivates Tryptophan Hydroxylase via Sulfhydryl Oxidation
COINCIDENT NITRATION OF ENZYME TYROSYL RESIDUES HAS MINIMAL IMPACT ON CATALYTIC ACTIVITY^*

Donald M. Kuhn^§¶ and Timothy J. Geddes

From the  Department of Psychiatry and Behavioral Neurosciences and ^§ Center for Molecular Medicine and Genetics, Wayne State University School of Medicine, Detroit, Michigan 48201

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Tryptophan hydroxylase, the initial and rate-limiting enzyme in serotonin biosynthesis, is inactivated by peroxynitrite in a concentration-dependent manner. This effect is prevented by molecules that react directly with peroxynitrite such as dithiothreitol, cysteine, glutathione, methionine, tryptophan, and uric acid but not by scavengers of superoxide (superoxide dismutase), hydroxyl radical (Me₂SO, mannitol), and hydrogen peroxide (catalase). Assuming simple competition kinetics between peroxynitrite scavengers and the enzyme, a second-order rate constant of 3.4 × 10⁴ M¹ s¹ at 25 °C and pH 7.4 was estimated. The peroxynitrite-induced loss of enzyme activity was accompanied by a concentration-dependent oxidation of protein sulfhydryl groups. Peroxynitrite-modified tryptophan hydroxylase was resistant to reduction by arsenite, borohydride, and dithiothreitol, suggesting that sulfhydryls were oxidized beyond sulfenic acid. Peroxynitrite also caused the nitration of tyrosyl residues in tryptophan hydroxylase, with a maximal modification of 3.8 tyrosines/monomer. Sodium bicarbonate protected tryptophan hydroxylase from peroxynitrite-induced inactivation and lessened the extent of sulfhydryl oxidation while causing a 2-fold increase in tyrosine nitration. Tetranitromethane, which oxidizes sulfhydryls at pH 6 or 8, but which nitrates tyrosyl residues at pH 8 only, inhibited tryptophan hydroxylase equally at either pH. Acetylation of tyrosyl residues with N-acetylimidazole did not alter tryptophan hydroxylase activity. These data suggest that peroxynitrite inactivates tryptophan hydroxylase via sulfhydryl oxidation. Modification of tyrosyl residues by peroxynitrite plays a relatively minor role in the inhibition of tryptophan hydroxylase catalytic activity.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Tryptophan hydroxylase (TPH,¹ EC 1.14.16.4; L-tryptophan,tetrahydrobiopterin:oxygen oxidoreductase (5-hydroxylating)) is the initial and rate-limiting enzyme in the biosynthesis of the neurotransmitter serotonin. The physiological roles for serotonin are diverse and include modulation of sleep, thermoregulation, and food intake (1). From a clinical perspective, alterations in serotonin function have been implicated in several neuropsychiatric disorders such as depression, obsessive-compulsive disorder, and suicide (2). Alterations in TPH activity produce corresponding changes in the synaptic levels of serotonin (3), suggesting that TPH can have influences on serotonin neurochemical function that extend well beyond its role in fulfilling the first step in serotonin synthesis from tryptophan. Drugs that cause long term changes in TPH activity could well change serotonin synaptic function to the detriment of normal physiological and behavioral function. For example, the neurotoxic amphetamines methamphetamine and 3,4-methylenedioxymethamphetamine (MDMA, "Ecstasy") significantly reduce TPH activity and serotonin levels (4). Long term abuse of these drugs produces behavioral and psychiatric conditions indicative of diminished serotonin function (5, 6). Because the effects of these drugs include permanent neuronal damage in humans (7, 8), great emphasis has been directed at achieving a better understanding of their toxic mechanisms of action.

Nitric oxide (NO) has been implicated in a variety of toxic neuronal effects, including those associated with methamphetamine and MDMA (9, 10), and TPH is known to be inactivated by NO (11-13). Apart from exerting direct effects of its own, NO reacts with other radical species such as superoxide to produce the peroxynitrite anion (ONOO; see Ref. 14). This possibility is also relevant to the neurotoxic amphetamines because their damaging effects on neurons can involve the superoxide radical (15-17). ONOO may be more toxic to cells and proteins than either of its precursors NO and superoxide radical, and ONOO has been implicated in neuronal toxicity (18-19) and neurodegenerative diseases (20-23). Effects on proteins that were initially attributed to NO have now been shown to occur via the action of ONOO arising from NO/superoxide interactions (24, 25).

TPH is a member of the monooxygenase superfamily of enzymes that includes tyrosine hydroxylase (TH) and phenylalanine hydroxylase (26). It was shown recently that the dopamine neurotoxin MPTP inactivates TH via an ONOO-mediated nitration of enzyme tyrosyl residues (27). Recalling that the neurotoxic properties of MPTP have also been linked to NO (28), we were prompted to examine the possibility that ONOO could influence TPH activity. We report presently that ONOO inactivates TPH and causes the nitration of enzyme tyrosyl residues. However, in contradiction to the effect of ONOO on TH (27), the ONOO-induced oxidation of enzyme sulfhydryl groups, not tyrosine nitration, mediates TPH inactivation.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- Tryptophan, DTT, superoxide dismutase, DTPA, DTNB, N-acetylimidazole, methionine, cysteine, Me₂SO, dithionite, glutathione, glutathione-agarose, sodium arsenite, sodium bicarbonate, sodium borohydride, and uric acid were obtained from Sigma. Catalase was a product of Roche Molecular Biochemicals. Tetranitromethane was purchased from Aldrich. Isopropyl--D-thiogalactopyranoside was obtained from Gold Biotechnologies. Thrombin and pGEX vectors were obtained from Amersham Pharmacia Biotech. Tetrahydrobiopterin was purchased from Dr. Shircks' Laboratories (Jona, Switzerland). A monoclonal antibody against nitrotyrosine was purchased from Cayman Chemical Co. (Ann Arbor, MI), and horseradish peroxidase-linked goat anti-mouse IgGs were products of Cappel. Enhanced chemiluminescence reagents were products of DuPont NEN, and Bio-Max MR film was from Eastman Kodak Co. Restriction endonucleases, T4 ligase, and T4 kinase were products of New England Biolabs. All other reagents were obtained from commercial sources in the highest possible qualities.

Cloning to TPH and Assay of Enzymatic Activity-- TPH was cloned and expressed as a glutathione S-transferase fusion protein as described previously (13, 29, 30). Both wild-type enzyme and a deletional mutant constituting the catalytic domain of TPH (amino acids 99-444) were expressed in BL-21 (Escherichia coli) cells. The catalytic core of TPH retains the essential catalytic properties of the wild-type enzyme and has proved useful in mechanistic studies of the catalytic properties of the enzyme (13, 29, 30). Cells transformed with the plasmid bearing the TPH cDNA were grown overnight at 37 °C and induced with 0.1 mM isopropyl--D-thiogalactopyranoside for 2 h at 30 °C. Bacteria were washed with 10% glycerol and resuspended in 0.1 volume of 50 mM Tris-HCl, pH 7.5. After sonication and centrifugation (40,000 × g) to sediment insoluble material, the supernatants were adsorbed on GSH-agarose for 60 min at 4 °C. Affinity beads with immobilized TPH were washed three times with 50 volumes of 50 mM Tris-HCl, pH 7.5, at 4 °C, and TPH was removed from the glutathione S-transferase fusion tag by digestion of bead-bound protein with thrombin protease (10 units of protease/mg of protein) at room temperature for 2 h. The cleaved TPH protein was separated from GSH affinity beads by filtration through glass wool. TPH activity was assayed by measuring the formation of 5-hydroxytryptophan from tryptophan as described previously (13, 29). The levels of protein were measured by the method of Bradford (31).

Synthesis of ONOO and Treatment of TPH-- ONOO was prepared fresh daily from acidified nitrite and hydrogen peroxide as described by Beckman et al. (32). The ONOO concentration was measured as an increase in absorbance at 302 nm (₃₀₂ = 1700 M¹ cm¹). ONOO solutions were chromatographed over small columns of granular manganese dioxide to remove hydrogen peroxide contamination. The removal of the peroxide was confirmed by assay of chromatographed ONOO preparations for hydrogen peroxide by the method of Matsubara et al. (33). Enzyme preparations were exposed to ONOO in the presence of 50 mM potassium phosphate buffer, pH 7.4, and 100 µM DTPA for 15 min at 25 °C. ONOO was added to enzyme preparations as a small volume bolus with a Hamilton syringe during vigorous mixing. After exposure to ONOO, enzyme solutions were diluted 5-fold with 50 mM phosphate buffer, pH 7.5. Residual TPH activity was immediately determined. The pH of all enzyme solutions was checked carefully to ensure that the alkaline ONOO did not cause it to vary above 7.4 during exposure. Agents tested for the ability to protect TPH from the effects ONOO (see below) were added 5 min prior to ONOO and remained present throughout exposure. Controls for ONOO addition to enzyme solutions included 1.2 N NaOH or ONOO preparations allowed to decompose at room temperature in 50 mM Tris-HCl, pH 7.4. Decomposition of ONOO was monitored as decreases in absorbance at 302 nm.

Titration of TPH Sulfhydryls with DTNB-- The reactivity of sulfhydryl groups in TPH was determined by titration with DTNB by the method of Ellman (34), as modified by Riddles et al. (35). TPH was treated with varying concentrations of ONOO as described above, and after dialysis against phosphate-buffered saline, protein (5-7 µM) was denatured with 6 M guanidine HCl and reacted with DTNB (200 µM) in phosphate-buffered saline at room temperature for 90 min. The reaction was monitored as an increase in absorbance at 412 nm. The concentration of sulfhydryl groups was calculated using ₄₁₂ = 13,700 M¹ cm¹ (35).

Nitration of Tyrosyl Residues-- TPH was treated with varying concentrations of ONOO, and after dialysis against phosphate-buffered saline, proteins were exposed to SDS-polyacrylamide gel electrophoresis and electroblotting to nitrocellulose (36). Nitrated tyrosines were detected with the use of a 3-nitrotyrosine-specific monoclonal antibody (1:1000 dilution). The presence of nitrotyrosines in TPH was visualized with enhanced chemiluminescence. As controls, blots of ONOO-modified TPH were treated with 100 mM dithionite to reduce nitrotyrosines to aminotyrosines (37), or TPH preparations treated with decomposed ONOO were tested for immunoreactivity with the nitrotyrosine antibody. The extent of tyrosine nitration was also measured spectrophotometrically as increases in absorbance at 430 nm (₄₃₀ = 4400 M¹ cm¹) as described by Crow and Ischiropoulos (38).

Treatment of TPH with Tetranitromethane-- TPH was exposed to varying concentrations of tetranitromethane (in ethanol) at either pH 6 or pH 8. TPH was diluted into the appropriate buffer (50 mM potassium phosphate buffer for pH 6 or 8 conditions), and tetranitromethane was added and allowed to react with the protein for 15 min at 30 °C. Enzyme samples were diluted 5-fold with 50 mM potassium phosphate buffer, pH 7.4, and assayed for residual activity at pH 7.4 as described. Tetranitromethane-modified tryptophan hydroxylase was also tested for sulfhydryl oxidation or for tyrosine nitration as described above.

Other Methods-- TPH was treated with dopamine-quinone (39) or NO (11, 12) as described previously. In some experiments, the tyrosine-acetylating reagent N-acetylimidazole was tested for effects on TPH activity. The oxidation state of sulfhydryl groups in ONOO-treated TPH was determined as described by Radi et al. (40).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Effects of ONOO on TPH Activity-- The catalytic core deletion mutant of recombinant TPH was treated with ONOO, and the results are presented in Fig. 1. TPH was tested at concentrations of 0.625, 1.25, 5, and 10 µM over the same concentration range of ONOO. The data show that TPH is inactivated by ONOO in a manner that depends on the concentrations of both reactants. The IC₅₀ for inhibition of enzyme activity was approximately 15 or 200 µM ONOO when the enzyme concentration was 0.625 or 10 µM, respectively. Decomposed ONOO did not have an effect on TPH activity. Full-length TPH was also tested for its response to ONOO and did not differ from the catalytic core deletion mutant (data not shown). The main effect of ONOO concentration was significant for each enzyme concentration (p < 0.01, ANOVA). Because the catalytic core form of TPH retains the essential catalytic properties of native TPH (13, 29), all subsequent experiments used this recombinant enzyme preparation.

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Inactivation of TPH by ONOO. TPH (, 0.625; , 1.25; , 5; or , 10 µM) was incubated with the indicated concentrations of ONOO in 50 mM potassium phosphate buffer, pH 7.5, containing 100 µM DTPA for 15 min at 25 °C. Enzyme samples were diluted 5-fold, and the remaining TPH activity was assayed immediately. The addition of decomposed ONOO was also tested in the presence of 1.25 µM TPH (). The results represent the means of 3-4 independent experiments carried out in duplicate. S.E. are omitted for clarity and were never more than 10% of the mean.

Protection of TPH from ONOO-induced Inactivation-- Various reagents were tested for the ability to prevent ONOO-induced inactivation of TPH, and the results are presented in Table I. It can be seen that reagents that react directly with ONOO, such as DTT, cysteine, GSH, methionine, tryptophan, and uric acid, provided significant protection of the enzyme from inactivation. The hydroxyl radical scavenger Me₂SO, the superoxide radical scavenger superoxide dismutase, and the hydrogen peroxide scavenger catalase did not protect TPH from ONOO-induced inactivation (Table I). None of the reagents tested for protection had effects on TPH activity in the absence of ONOO.

View this table: [in this window] [in a new window]   Table I Protection of TPH from ONOO-induced inactivation TPH (1.25 µM) was incubated with ONOO for 15 min at 25 °C. Agents tested for protection were added 5 min prior to ONOO. Enzyme samples were diluted 5-fold with 50 mM potassium phosphate buffer, pH 7.4, and assayed for residual catalytic activity. None of the reagents tested for protection had an effect on TPH activity in the absence of ONOO.

Kinetics of ONOO Reaction with TPH-- By modeling simple competition kinetics for the protection of various proteins from ONOO by different concentrations of GSH and methionine (41), TPH was tested for inhibition by ONOO in the presence of varying concentrations of GSH and methionine, and the results are shown in Fig. 2. The present calculations assumed rate constants of k = 740 M¹ s¹ for GSH (42) and k = 150 M¹ s¹ for methionine (43), at 25 °C and pH 7.4. Increasing concentrations of both GSH and methionine provided increasing levels of protection of TPH from inactivation by ONOO. It was estimated that the rate constant of the ONOO reaction with TPH was 3.4 × 10⁴ M¹ s¹.

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Rate constant between TPH and ONOO. TPH (0.625 µM) was incubated for 15 min at 25 °C with increasing concentrations of methionine or GSH in 50 mM potassium phosphate buffer, pH 7.5, containing 100 µM DTPA in the presence of 50 µM ONOO. Enzyme samples were then diluted 5-fold and assayed for remaining TPH activity. Fi represents the fraction of protection of TPH in the presence of ONOO scavengers (S); ks represents the rate constants for the reaction of ONOO with the scavengers, and kD represents the rate constant for the reaction of ONOO with TPH. The plot of Fi[TPH]/(1  Fi)ks versus [S] results in a straight line with a slope of 1/kD, allowing the determination of kD (40, 41, 55). The results represent the means of 3 independent experiments each for GSH and methionine carried out in duplicate.

Effects of ONOO on Sulfhydryl Groups in TPH-- TPH (5-7 µM) was treated with the same concentration range of ONOO shown to inactivate catalytic activity, and the effects on sulfhydryl reactivity were determined by titration with DTBN under denaturing conditions. The data in Fig. 3 show that ONOO caused a concentration-dependent loss of sulfhydryls from TPH that paralleled the loss of catalytic activity. Untreated TPH contained nine titratable sulfhydryl groups per monomer, consistent with predictions from the nucleotide sequence of the catalytic core deletional mutant (44). Increasing concentrations of ONOO resulted in the progressive loss of reactive sulfhydryl groups such that at a concentration of 0.1 mM ONOO, the number of reactive sulfhydryl groups was reduced to 2.6 mol/mol TPH subunit. ONOO concentrations of 200-500 µM caused the complete loss of DTNB-reactive sulfhydryl groups from the treated enzyme and completely inactivated catalytic activity. These results are consistent with ONOO being a strong sulfhydryl oxidant and confirm that TPH is very sensitive to inactivation by sulfhydryl oxidation (45). The oxidation status of TPH sulfhydryl groups after ONOO treatment was tested by reacting the modified enzyme with sodium arsenite, sodium borohydride, and DTT. Borohydride and DTT reduce disulfides, and arsenite reduces sulfenic acids to the RSH (40). The data in Table II show that ONOO-modified sulfhydryl groups in TPH were resistant to reduction by DTT, borohydride, and arsenite and suggest that ONOO is oxidizing enzyme sulfhydryl groups beyond sulfenic acid.

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Effect of ONOO on TPH activity and sulfhydryl groups. TPH (1.25 µM) was exposed to the indicated concentrations of ONOO in the presence of 50 mM potassium phosphate buffer, pH 7.5, containing 100 µM DTPA for 15 min at 25 °C. Aliquots of the enzyme were removed, diluted 5-fold, and assayed for remaining TPH activity. The remainder of the enzyme was denatured with 6 M guanidine HCl, and enzyme sulfhydryl groups were titrated with 200 µM DTNB. The concentration of sulfhydryl groups was determined using 412 = 13,700 M1 cm1, and the results are expressed as number of sulfhydryl (SH) groups per TPH monomer. The results represent the means ± S.E. of three independent experiments carried out in duplicate.

View this table: [in this window] [in a new window]   Table II Reduction of ONOO-oxidized TPH sulfhydryls with arsenite, borohydride, and DTT TPH (5 µM) was treated with 0.2 mM ONOO in 50 mM potassium phosphate buffer, pH 7.4, containing 100 µM DTPA for 15 min at 25 °C. Immediately following ONOO treatment, TPH was incubated with 37 mM sodium arsenite, 26 mM sodium borohydride, or 10 mM DTT for 45 min at 37 °C as described by Radi et al. (40). Samples were dialyzed against phosphate-buffered saline for 60 min at room temperature and then assayed for sulfhydryl content. Data represent the means ± S.E. for four independent experiments carried out in duplicate.

ONOO-induced Nitration of Tyrosyl Residues in TPH-- TPH was treated with the same concentration range of ONOO shown to inactivate catalytic activity (Fig. 1), and the effects on tyrosine nitration are presented in Fig. 4. The data show that ONOO causes a concentration-dependent increase in tyrosine nitration. At a cumulative concentration of 2 mM, ONOO caused the nitration of approximately 3.8 tyrosyl residues/TPH monomer. The IC₅₀ concentration of ONOO for inhibition of enzyme activity (15 µM) resulted in the nitration of approximately 0.2 tyrosyl residues/TPH monomer. The concentration of ONOO that completely inactivated TPH activity (500 µM) caused the nitration of 2 tyrosyl residues/TPH monomer. TPH modified by ONOO was also examined for nitration by reaction with a monoclonal antibody against 3-nitrotyrosine, and the results are presented in the inset of Fig. 4. The results agree with the spectrophotometric studies and show that nitrotyrosine immunoreactivity was increased in a concentration-dependent manner by ONOO. Immunoreactivity on blots was restricted to the 40-kDa TPH band. Evidence of higher molecular weight species was not observed, suggesting that the formation of dityrosines in TPH after ONOO treatment was not occurring. The specificity of the nitrotyrosine antibody was tested by incubating blots of nitrated TPH with dithionite to reduce nitrotyrosines to aminotyrosines prior to antibody addition. As predicted (37), the antibody no longer recognized ONOO-treated TPH (data not shown). The nitrotyrosine antibody was also unreactive with TPH treated with decomposed ONOO (data not shown). Agents shown to prevent ONOO-induced inactivation of enzyme catalytic activity (Table I) were also tested for their effects on tyrosine nitration. It was observed that each protective reagent prevented tyrosine nitration. Finally, Me₂SO, superoxide dismutase, and catalase, which did not protect TPH from ONOO-induced inactivation, were without effect on the ONOO-induced nitration of tyrosines in TPH (data not shown).

View larger version (22K): [in this window] [in a new window]   Fig. 4.   Effect of ONOO on nitration of tyrosyl residues in TPH. TPH (5 µM) was incubated with the indicated concentrations of ONOO in the presence of 50 mM potassium phosphate buffer, pH 7.5, containing 100 µM DTPA for 15 min at 25 °C. ONOO was added in 100 µM increments with vigorous mixing, with 2-min intervals between each addition. Aliquots of the enzyme were removed and diluted 5-fold, and the remaining TPH activity was determined. The remaining enzyme was measured spectrophotometrically at 430 nm, and the extent of tyrosine nitration was calculated using 430 = 4400 M1 cm1 (38). The results represent the means ± S.E. of 3-4 independent experiments carried out in duplicate and are expressed as number of tyrosines nitrated per TPH monomer. The inset shows the determination of tyrosine nitration with a monoclonal antibody against nitrotyrosine after treatment of TPH (1.25 µM) with ONOO in concentrations of 0 mM (lane 1), 0.01 mM (lane 2), 0.02 mM (lane 3), 0.05 mM (lane 4), 0.1 mM (lane 5), 0.2 mM (lane 6), 0.5 mM (lane 7), and 1.0 mM (lane 8). Molecular weight markers are shown to the left of the nitrotyrosine blot.

Effects of Sodium Bicarbonate on ONOO-induced Alterations in TPH-- Carbon dioxide reacts rapidly with ONOO to produce the nitrosoperoxocarboxylate (ONOOCO₂) adduct (46-50). The ONOOCO₂ adduct is a better tyrosine nitrating reagent than ONOO and has been shown to enhance protein tyrosine nitration while causing less sulfhydryl oxidation by comparison to ONOO (46, 50-52). The effects of sodium bicarbonate (10 mM) on the ONOO-induced modification of TPH were tested, and the results are presented in Table III. Bicarbonate significantly protected TPH from ONOO-induced catalytic inactivation and lessened the extent of sulfhydryl oxidation. At the same time, bicarbonate significantly increased ONOO-induced tyrosine nitration in TPH. Bicarbonate had no effect on TPH in the absence of ONOO (data not shown).

View this table: [in this window] [in a new window]   Table III Effects of sodium bicarbonate on the ONOO-induced modification of TPH TPH was treated with 0.2 mM ONOO in 50 mM potassium phosphate buffer, pH 7.4, containing 100 µM DTPA for 15 min at 25 °C. Sodium bicarbonate was added at a concentration of 10 mM and was present throughout the incubation of TPH with ONOO. After exposure of TPH to the various incubation conditions, aliquots were removed for determination of remaining catalytic activity, remaining sulfhydryls, and tyrosine nitration as described. Data represent the means ± S.E. for three independent experiments carried out in duplicate.

Effects of Tetranitromethane on TPH-- In view of ONOO reactivity with most amino acid residues (46), particularly cysteines and tyrosines, and considering that virtually all tyrosine nitrating reagents are also strong sulfhydryl oxidants, tetranitromethane was tested for its effects on TPH in an attempt to discern whether modification of tyrosyl or cysteinyl residues was responsible for enzyme inhibition. Tetranitromethane is useful in this regard because it nitrates tyrosyl residues with relative selectivity at pH 8, and not at pH 6, whereas it oxidizes sulfhydryls at both pH 6 and 8 (53, 54). It was hypothesized that if tetranitromethane was more inhibitory at pH 8 as compared with pH 6, the pH-dependent nitrating activity of the reagent would be responsible. The results in Fig. 5 show that tetranitromethane caused a concentration-dependent inactivation of TPH at pH 6 and at pH 8. The overall effect of tetranitromethane was statistically significant (p < 0.001 by ANOVA), but the effect of pH was not (p < 0.5 by ANOVA). The effect of tetranitromethane on tyrosine nitration is shown in the inset to Fig. 5. It can be seen that higher concentrations of tetranitromethane were needed to detect nitration (as compared with inhibition of enzyme activity). Tetranitromethane caused a substantial increase in tyrosine nitration of TPH over the concentration range of 50-200 µM at pH 8. The nitrating effects of tetranitromethane were very weak at pH 6, showing tyrosine nitration of TPH that was only 4.6% of the nitration seen at pH 8 (measured as relative pixel density by image analysis) at a concentration of 200 µM tetranitromethane. Finally, tetranitromethane oxidized sulfhydryl groups at pH 6 and pH 8. The total number of sulfhydryl groups per monomer was reduced by tetranitromethane (100 µM) from 9 in the untreated enzyme to 1.9 at pH 6 and to 2.3 at pH 8. 

View larger version (22K): [in this window] [in a new window]   Fig. 5.   Effects of tetranitromethane on TPH activity and tyrosine nitration. TPH (5 µM) was mixed with 50 mM potassium phosphate buffer at pH 6 or pH 8 and then incubated with the indicated concentrations of tetranitromethane in the presence of 100 µM DTPA for 15 min at 25 °C. Aliquots were removed and diluted 5-fold with 50 mM potassium phosphate buffer, pH 7.5, and TPH was assayed for residual activity. Results represent the means ± S.E. of 6 independent experiments run in duplicate at each pH. The inset shows the determination of tyrosine nitration with a monoclonal antibody against nitrotyrosine after treatment with tetranitromethane. Note that the concentrations of tetranitromethane used for tyrosine nitration (inset) were higher than those used for inhibition of enzyme activity. Tetranitromethane concentrations used for measures of tyrosine nitration in the inset were 0 mM (lane 1), 0.05 mM (lane 2), 0.1 mM (lane 3), and 0.2 mM (lane 4) for both panels. Molecular weight markers are shown to the left of the nitrotyrosine blots. The extent of protein nitration by 200 µM tetranitromethane at pH 6 was 4.6% that seen at pH 8 (lane 4 in each panel, determined as relative pixel density from image analysis).

Effects of Other Group-specific Reagents on TPH Activity-- N-Acetylimidazole, a mild tyrosine-acetylating agent (55, 56), was tested for its effects on TPH activity, and the results are presented in Fig. 6. It can be seen that N-acetylimidazole did not inhibit TPH catalytic activity at any concentration tested (up to 20 mM). In fact, at lower concentrations, N-acetylimidazole caused a small activation of TPH. N-Acetylimidazole did not cause the nitration of tyrosyl residues in TPH as measured by immunoblotting with a nitrotyrosine monoclonal antibody (data not shown). N-Acetylimidazole did show evidence of modifying tyrosines in TPH by causing a decrease in tyrosine absorbance at 278 nm. Attempts to quantify tyrosine modification by reversal of acetylation with hydroxylamine (56) were not possible because of enzyme aggregation.

View larger version (11K): [in this window] [in a new window]   Fig. 6.   Effects of N-acetylimidazole on TPH activity. TPH (5 µM) was incubated with the indicated concentrations of N-acetylimidazole in the presence of 50 mM potassium phosphate buffer, pH 7.5, containing 100 µM DTPA for 15 min at 25 °C. Aliquots were removed and diluted 5-fold, and the remaining TPH activity was determined. The results represent the means ± S.E. of 3 experiments assayed in duplicate.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

TPH is quite sensitive to inactivation by the ONOO anion. The approximate IC₅₀ of ONOO for enzyme inhibition was 15 µM at an enzyme concentration of 0.625 µM. It has been estimated that the bolus addition of 0.25 mM ONOO is roughly equivalent to a steady-state level of 1.0 µM maintained for 7 min (32, 40). Considering that ONOO can be formed in vivo by the diffusion-limited reaction of NO and superoxide, TPH could well be a target for ONOO in vivo (see below). TPH is known to be an extremely labile enzyme (45), and reagents like NO or quinones, which target cysteinyl residues, are also potent inhibitors of the enzyme (11-13, 39). ONOO is a powerful sulfhydryl oxidant (40), and this property alone leads to the prediction that it would cause the inactivation of TPH. However, ONOO can also nitrate tyrosyl residues in proteins (57), and this property of ONOO has been implicated as the mechanism by which the dopamine neurotoxin MPTP inhibits TH activity in a model of Parkinson's disease (27). Based on the structural and functional homologies among the monooxygenase family of enzymes, and considering that ONOO could play a role in the amphetamine-induced inhibition of TPH, it was important to determine if TPH contained catalytically important tyrosyl residues as appears to be the case for TH (27).

Various agents were tested for the ability to protect TPH from ONOO-induced inactivation. DTT, GSH, cysteine, methionine, tryptophan, and uric acid were very effective in this regard, consistent with their known reactivities with ONOO (46, 58-60). It does not appear that the effect of ONOO on TPH activity is mediated by radicals or reactive oxygen species because scavengers of hydroxyl radical (Me₂SO), hydrogen peroxide (catalase), or superoxide (superoxide dismutase) did not protect the enzyme from inactivation. The effect of tryptophan to protect TPH from inactivation by ONOO could indicate that the TPH substrate is protective via its occupation of the enzyme-active site, but this possibility is hard to substantiate in view of the direct reactivity of tryptophan with ONOO (46). Tryptophan is very effective in protecting TPH from inactivation caused by the sulfhydryl reagents N-ethylmaleimide and DTNB (45), and these results are consistent with the location of a reactive sulfhydryl at the enzyme-active site. By assuming that the inactivation of TPH by ONOO was the result of simple competition kinetics between the two reactants, it was possible to calculate an apparent second-order rate constant of 3.4 × 10⁴ M¹ s¹ by including cysteine or methionine as competing reagents. The rate constants of ONOO reaction with cysteine and methionine are well known (41-43) in this regard. TPH was found to be moderately reactive with ONOO. Other enzymes, such as glyceraldehyde-3-phosphate dehydrogenase, are remarkably sensitive to inactivation by ONOO (61). TPH is about 1 order of magnitude lower in reactivity with ONOO by comparison to glyceraldehyde-3-phosphate dehydrogenase. TPH, on the other hand, is similar to numerous other enzymes (e.g. aconitase, alcohol dehydrogenase, aconitase) that are inactivated by ONOO with similar reaction kinetics (25, 34, 62, 63).

The effects of ONOO on TPH tyrosyl residues were determined to examine whether catalytically important tyrosines were mediating the loss of enzyme activity. Indeed, a good correlation was observed between inactivation of TPH and nitration of tyrosines measured spectrophotometrically or immunochemically with a nitrotyrosine-specific monoclonal antibody. Increasing concentrations of ONOO led to greater inactivation of TPH activity as well as increasing nitration of tyrosyl residues. ONOO nitrated 3.8 tyrosines per enzyme subunit at a cumulative concentration of 2 mM. The catalytic core deletion mutant of TPH contains a total of 15 tyrosyl residues (44), so it appears that the secondary structure of the protein and the charge of ONOO act in concert to limit accessibility of ONOO to tyrosyl residues within TPH. The IC₅₀ for inactivation of TPH by ONOO was approximately 15 µM, and at this concentration, it was calculated that approximately 0.2 tyrosyl residues/monomer were nitrated.

ONOO is a powerful sulfhydryl oxidant (42), and TPH is very sensitive to inactivation by a variety of sulfhydryl reagents (11, 12, 39, 45). Therefore, the effects of ONOO on sulfhydryl groups in TPH were determined by DTNB titration. The nucleotide sequence of the catalytic domain of TPH (residues 99-444) predicts that each subunit of TPH contains 9 cysteinyl residues (44). Titration of untreated TPH with DTNB produced results that were consistent with this number. These results also indicate that all cysteine residues within the catalytic domain deletion mutant of TPH are accessible to solvent. Increasing concentrations of ONOO led to corresponding decreases in the number of titratable sulfhydryl groups in the enzyme. At the IC₅₀ for enzyme inhibition (15 µM), ONOO oxidized approximately 6 of the total of 9 cysteinyl residues in TPH. Concentrations of ONOO that completely inhibited TPH catalytic activity (200-500 µM) caused the loss of all DTNB-reactive sulfhydryl groups. It is possible that ONOO modification of TPH could cause a structural change in the enzyme that could bury sulfhydryl groups. However, when ONOO-treated TPH was denatured with 6 M guanidine HCl, the enzyme was still devoid of DTNB-reactive sulfhydryl groups. These results indicate that ONOO oxidizes all accessible sulfhydryl groups in TPH. The extent of the ONOO-induced oxidation of TPH sulfhydryl groups was also apparent from attempts to reduce them after ONOO-induced modification. Oxidized sulfhydryl groups in TPH were resistant to reduction by DTT, borohydride, and arsenite after treatment with ONOO, indicating that enzyme sulfhydryls were oxidized beyond sulfenic acid (40).

Results from studies of ONOO on TPH alone cannot discern whether the inactivation of TPH is mediated by sulfhydryl oxidation or tyrosine nitration. Unfortunately, from a mechanistic point of view, many tyrosine-nitrating reagents are also strong sulfhydryl oxidants (e.g. ONOO and 4-chloro-7-nitrobenzofurazan), making it difficult to assess the role of tyrosyl residues selectively in TPH catalysis. Therefore, bicarbonate was tested for its effects on the ONOO-induced modification of TPH. Carbon dioxide is well known for its ability to enhance ONOO-induced protein tyrosine nitration and decrease sulfhydryl oxidation (46-52). It was observed that bicarbonate protected TPH from the ONOO-induced loss of catalytic activity and lessened the extent of sulfhydryl oxidation. Under the same conditions, bicarbonate significantly increased the ONOO-induced nitration of tyrosines in the enzyme. TPH was also treated with tetranitromethane, a reagent that oxidizes sulfhydryls at pH 6 and 8 but which causes tyrosine nitration only at pH 8 (53, 54). The results showed that TPH was very sensitive to inactivation by tetranitromethane, but inhibition by this reagent was the same at pH 6 and pH 8. The pH dependence of tyrosine nitration by tetranitromethane supported catalytic studies with TPH. It was observed that tetranitromethane caused increasing nitration of tyrosyl residues in TPH at pH 8, but very little nitration was observed at pH 6. Tetranitromethane caused extensive sulfhydryl oxidation that was independent of pH, reducing TPH sulfhydryls from 9 to 2 at pH 6 or pH 8. These results with bicarbonate and tetranitromethane do not support a substantial role for tyrosine nitration in the inactivation of TPH, although we cannot rule out the possibility that ONOO, ONOOCO₂, and tetranitromethane are nitrating entirely distinct sets of tyrosine residues in TPH. Finally, N-acetylimidazole, a mild, tyrosine-acetylating reagent, was tested for its effects on enzyme activity. TPH was not altered by N-acetylimidazole in concentrations up to 20 mM. Taken together, these data suggest that tyrosyl residues in TPH do not appear to play an influential role in its catalytic function.

TPH is a non-heme iron protein that can be inhibited by agents known to chelate iron (39). Therefore, it is possible that ONOO is inhibiting TPH via attack at an active site Fe(II). This possibility is diminished by two observations: first, all incubations of TPH with ONOO, ONOOCO₂, and tetranitromethane included the iron chelator DTPA (32); second, recombinant TPH is expressed in bacteria as an apoenzyme, essentially free of iron that could serve as a target for ONOO.

The present findings with TPH are surprising in at least one regard. TH was recently reported to be inactivated by MPTP via a process that involved ONOO-induced nitration of tyrosyl residues (27). This finding is interesting because it reveals a potential mechanism by which a dopamine neurotoxin acts upon this important enzyme. In addition, the drug-induced posttranslational modification of TH leaves a footprint of ONOO (or other reactive nitrating species) action in the enzyme that could be detected by in vitro analysis. Given the sequence and functional homologies shared between TH and TPH (26), we predicted that TPH would also be inactivated by ONOO via modification of catalytically important tyrosines. Such an effect would be important and useful in investigations of the mechanisms of action of the neurotoxic amphetamines, whose effects include inactivation of TPH and damage to serotonin neurons (4, 7). However, nitration of TPH by ONOO was not indicative of catalytic function. On the other hand, several results point to sulfhydryls as important determinants of TPH catalytic activity. First, the kinetic and protection studies showed that sulfhydryl reductants are very effective in preventing ONOO-induced inactivation of TPH. Second, ONOO caused an extensive oxidation of enzyme sulfhydryl groups, and loss of TPH activity paralleled the loss of sulfhydryl reactivity more closely than nitration of tyrosyl residues. Third, bicarbonate protected TPH from ONOO-induced loss of catalytic activity and sulfhydryl oxidation while, at the same time, causing increased tyrosine nitration. Finally, the inhibitory effects on TPH of tetranitromethane were the same at pH 6 and pH 8. The tyrosine nitrating properties of tetranitromethane are evident at pH 8, not at pH 6, whereas it oxidizes sulfhydryls equally at either pH. Because tetranitromethane was no more inhibitory at pH 8 than at pH 6, it appears that the pH-dependent nitration of tyrosines (in addition to sulfhydryl oxidation) did not contribute to TPH inactivation.

These results reaffirm the importance of cysteinyl residues in TPH catalytic function (13, 45). The precise roles of cysteines in TPH catalysis are not known, and with 10 cysteinyls/monomer in the full-length enzyme (44), elucidation of their roles will be difficult. Our results also establish that TPH can be nitrated at tyrosyl residues by ONOO. These findings will be useful to establish if drugs like methamphetamine and MDMA, which are known to inactivate TPH and cause damage to serotonin neurons in vivo (4), leave an ONOO (or other reactive nitrogen species) footprint in TPH in the form of nitrotyrosines. Finally, the present results with TPH have led us to reevaluate the ONOO-induced inactivation of TH to determine if these closely related enzymes differ in the manner by which tyrosyl residues influence their respective catalytic activities.

	ACKNOWLEDGEMENT

We thank Dr. Bruce A. Freeman for advice on peroxynitrite and thoughtful comments on the data in this paper.

	FOOTNOTES

^* This work was supported by National Institute on Drug Abuse Research Grant DA10756.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 reprints and correspondence should be addressed: 2125 Scott Hall, 540 E. Canfield, Detroit, MI 48201. Tel./Fax: 313-577-9737; E-mail: donald.kuhn@wayne.edu.

	ABBREVIATIONS

The abbreviations used are: TPH, tryptophan hydroxylase; DTT, dithiothreitol; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; MDMA, 3,4-methylenedioxymethamphetamine; NO, nitric oxide, ONOO, peroxynitrite; TH, tyrosine hydroxylase; DTNB, 5,5'-dithiobis(2-nitrobenzoic acid); DTPA, diethylenetriaminepentaacetic acid; ANOVA, analysis of variance.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES