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, Gayani S. Fernando, Joel Horwitz, Harry Ischiropoulos

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Abstract

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). Recent 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.

Original languageEnglish (US)
Pages (from-to)46017-46023
Number of pages7
JournalJournal of Biological Chemistry
Volume276
Issue number49
DOIs
StatePublished - Dec 7 2001

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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    Blanchard-Fillion, B., Souza, J. M., Friel, T., Jiang, G. C. T., Vrana, K., Sharov, V., Barrón, L., Schöneich, C., Quijano, C., Alvarez, B., Radi, R., Przedborski, S., Fernando, G. S., Horwitz, J., & Ischiropoulos, H. (2001). Nitration and Inactivation of Tyrosine Hydroxylase by Peroxynitrite. Journal of Biological Chemistry, 276(49), 46017-46023. https://doi.org/10.1074/jbc.M105564200