Peroxynitrite-induced Nitration of Tyrosine Hydroxylase

Abstract

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 timeof-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 Tyr 423 , Tyr 428 , and Tyr 432 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) 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 Ϫ ) (2). TH is inhibited by ONOO Ϫ in vitro (1, 3) and in PC12 cells (4). The ONOO Ϫ -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 Tyr 225 was the site in ...

Dopamine Prevents Nitration of Tyrosine Hydroxylase by Peroxynitrite and Nitrogen Dioxide

“…In agreement with previous studies (33), ONOO Ϫ lowered PMAB labeling of TH as shown in Fig. 5 (middle lane of each nitration condition), an indication of cysteine modification.…”

“…Ϫ caused a concentration-dependent inactivation of TH activity as previously reported (33,34), and, at a concentration of 100 M, TH activity was reduced to 50% of control. Fig.…”

“…The recombinant protein was purified by glutathione-agarose affinity chromatography and the glutathione Stransferase fusion tag was removed by thrombin cleavage, resulting in a highly purified TH preparation (Ͼ95% pure) as previously described (25,33,34). TH catalytic activity was assessed by the tritium release method as described by Lerner et al (35).…”

“…Ϫ and NO 2 -The effect of ONOO Ϫ and NO 2 with or without catechols on TH cysteine residues was determined with the use of the thiol-reactive biotinylation reagent PMAB as described previously (33). PMAB reacts selectively with reduced cysteines in proteins and does not react with cysteines that have been oxidized.…”

“…methamphetamine) on DA nerve endings have also been ascribed, in part, to ONOO Ϫ (6 -8). Methamphetamine and MPTP each cause reductions in TH activity in vivo (9,47), and ONOO Ϫ inhibits TH in vitro (9,33,34). Based on these findings, it is possible that ONOO Ϫ mediates the detrimental effects of MPTP and the neurotoxic amphetamines on DA neurons.…”