Nitration and Inactivation of Tyrosine Hydroxylase by Peroxynitrite

Béatrice Blanchard-Fillion, José M. Souza, Thomas Friel, George C. T. Jiang, Kent Vrana, Victor Sharov, Lorena Barrón, Christian Schöneich, Celia Quijano, Beatriz Alvarez, Rafael Radi, Serge Przedborski (+3 others)
2001 Journal of Biological Chemistry  
Tyrosine hydroxylase (TH) is modified by nitration after exposure of mice to 1-methyl-4-phenyl-1,2,3,6-tetrahydrophenylpyridine. The temporal association of tyrosine nitration with inactivation of TH activity in vitro suggests that this covalent post-translational modification is responsible for the in vivo loss of TH function (Ara, J., Przedborski, S., Naini, A. B., Jackson-Lewis, V., Trifiletti, R. R., Horwitz, J., and Ischiropoulos, H. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 7659 -7663).
more » ... ecent data showed that cysteine oxidation rather than tyrosine nitration is responsible for TH inactivation after peroxynitrite exposure in vitro (Kuhn, D. M., Aretha, C. W., and Geddes, T. J. (1999) J. Neurosci. 19, 10289 -10294). However, re-examination of the reaction of peroxynitrite with purified TH failed to produce cysteine oxidation but resulted in a concentration-dependent increase in tyrosine nitration and inactivation. Cysteine oxidation is only observed after partial unfolding of the protein. Tyrosine residue 423 and to lesser extent tyrosine residues 428 and 432 are modified by nitration. Mutation of Tyr 423 to Phe resulted in decreased nitration as compared with wild type protein without loss of activity. Stopped-flow experiments reveal a second order rate constant of (3.8 ؎ 0.9) ؋ 10 3 M ؊1 s ؊1 at pH 7.4 and 25°C for the reaction of peroxynitrite with TH. Collectively, the data indicate that peroxynitrite reacts with the metal center of the protein and results primarily in the nitration of tyrosine residue 423, which is responsible for the inactivation of TH. Tyrosine hydroxylase (TH) 1 (EC is a non-heme iron, tetrahydrobiopterin-dependent protein that catalyzes the conversion of tyrosine to L-dihydroxyphenylalanine (L-DOPA) and represents the rate-limiting step in the biosynthesis of catecholamines (1). Loss of ability to synthesize catecholamines is an important step in the development of Parkinson's disease (PD) and other neurodegenerative diseases (2-6). Early loss of TH activity followed by a decline in TH protein is thought to contribute to the dopamine deficiency and phenotypic expression in PD and the MPTP mouse model (4). Tyrosine hydroxylase is a selective target for nitration following administration of the parkinsonian toxin MPTP to mice and following exposure of PC12 cells to either peroxynitrite or 1-methyl-4-phenylpyridiniun ion (7). Nitration of one or more tyrosine residues of TH was temporally associated with loss of enzymatic activity. The magnitude of inactivation was proportional to the number of TH molecules that were nitrated in PC12 cells. In the mouse striatum, the tyrosine nitration-mediated loss in TH activity parallels the decline in dopamine levels whereas the levels of TH protein remain unchanged for the first 6 h post-MPTP injection (7). However, a recent report indicated that exposure of recombinant purified TH to peroxynitrite in vitro results not only in nitration of tyrosine residues but also in the formation of covalently linked dimers and oxidation of cysteine residues (8). The same report also indicated that cysteine oxidation rather than tyrosine nitration is responsible for the loss of TH enzymatic activity (8). Cysteine, methionine, tryptophan, and tyrosine appear to be the principal amino acids in proteins modified by peroxynitrite in vitro (9 -14). To resolve the apparent differences, the reaction of peroxynitrite with recombinant purified rat TH in vitro was re-examined, and no evidence of cysteine oxidation was found. Oxidation of one cysteine residue per molecule of TH was observed only at high peroxynitrite concentrations, and three cysteine residues were oxidized in partially unfolded protein. Amino acid analysis failed to show any * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
doi:10.1074/jbc.m105564200 pmid:11590168 fatcat:jx4qtuqlcnetbhyq5lf4w6f3he