Peroxynitrite Inactivates Tryptophan Hydroxylase via Sulfhydryl Oxidation

Abstract

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 (Me 2 SO, mannitol), and hydrogen peroxide (catalase). Assuming simple competition kinetics between peroxynitrite scavengers and the enzyme, a second-order rate constant of 3.4 ؋ 10 4 M ؊1 s ؊1 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.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...

Peroxynitrite Irreversibly Inactivates the Human Xenobioticmetabolizing Enzyme Arylamine N-Acetyltransferase 1 (NAT1) in Human Breast Cancer Cells

“…4). This value is similar to the k inact reported for other peroxynitrite-inactivated enzymes, such as tryptophan hydroxylase (k inact ϭ 3.4 ϫ 10 4 M Ϫ1 ⅐s Ϫ1 ) (35), and indicates that the inactivation of human NAT1 by peroxynitrite occurs rapidly.…”

“…Thus, as demonstrated for other peroxynitritesensitive enzymes (28, 35, 50, 54), peroxynitrite irreversibly inactivated NAT1. Our data also suggest that peroxynitrite oxidized the sulfhydryl groups of NAT1 sulfhydryl beyond the sulfenic acid, as shown in other cases (35).…”

“…Incubation of the enzyme with SIN1 in the presence of high concentrations of reducing agents (5 mM GSH or 5 mM DTT) protected NAT1 only partially against inactivation. DTT protected against inactivation more efficiently than GSH, as reported for other enzymes (35). At a final GSH concentration of 5 mM (corresponding to 20-fold excess of GSH over SIN1), ϳ60% residual NAT1 activity was obtained.…”

“…** These authors contributed equally to this paper and are to whom correspondence may be addressed. such as creatine kinase (34), tryptophan hydroxylase (35), caspases (36), and phosphatases (37). XMEs, such as cytochromes P450 (38,39) and glutathione S-transferase (40), have also been reported to be irreversibly inactivated by peroxynitrite.…”

Endogenous Nitration of Iron Regulatory Protein-1 (IRP-1) in Nitric Oxide-producing Murine Macrophages

“…In Vitro Treatment with a Bolus of Peroxynitrite-Cytosols (100 g) of control cells were treated with increasing concentrations of peroxynitrite added as a bolus in 50 mM phosphate buffer, pH 7.5, containing 100 mM sodium bicarbonate to favor formation of the nitrosoperoxocarboxylate intermediate, which causes more nitration and less sulfhydryl oxidation (17). In some experiments, cytosols were pretreated with increasing concentrations of cis-aconitate prior to incubation with a bolus of peroxynitrite.…”