Tyrosine        nitration affects thymidylate synthase properties

Elżbieta Dąbrowska-Maś; Tomasz Frączyk; Tomasz Ruman; Karolina Radziszewska; Piotr Wilk; Joanna Cieśla; Zbigniew Zieliński; Agata Jurkiewicz; Barbara Gołos; Patrycja Wińska; Elżbieta Wałajtys-Rode; Andrzej Leś; Joanna Nizioł; Adam Jarmuła; Piotr Stefanowicz; Zbigniew Szewczuk; Wojciech Rode

doi:10.1039/C1OB06360J

Tyrosinenitration affects thymidylate synthase properties

Elżbieta Dąbrowska-Maś,†^a Tomasz Frączyk,†^a Tomasz Ruman,^b Karolina Radziszewska,^c Piotr Wilk,^a Joanna Cieśla,^a Zbigniew Zieliński,^a Agata Jurkiewicz,^d Barbara Gołos,^a Patrycja Wińska,^a Elżbieta Wałajtys-Rode,^e Andrzej Leś,^d Joanna Nizioł,^b Adam Jarmuła,^a Piotr Stefanowicz,^c Zbigniew Szewczuk^c and Wojciech Rode*^ab

Author affiliations

* Corresponding authors

^a Nencki Institute of Experimental Biology, Polish Academy of Sciences, Warszawa, Poland
E-mail: rode@nencki.gov.pl
Fax: (+48-22) 822 5342

^b Rzeszów University of Technology, Faculty of Chemistry, Rzeszów, Poland

^c Faculty of Chemistry, University of Wrocław, 14 F. Joliot-Curie Street, Wrocław, Poland

^d Faculty of Chemistry, University of Warsaw, 1 L. Pasteur Street, Warszawa, Poland

^e University of Information Technology and Management in Rzeszów, Chair of Cosmetology, 2 Sucharskiego Street, Rzeszów, Poland

Abstract

Highly purified preparations of thymidylate synthase, isolated from calf thymus, and L1210 parental and FdUrd-resistant cells, were found to be nitrated, as indicated by a specific reaction with anti-nitro-tyrosine antibodies, suggesting this modification to appear endogenously in normal and tumor tissues. Each human, mouse and Ceanorhabditis elegans recombinant TS preparation, incubated in vitro in the presence of NaHCO₃, NaNO₂ and H₂O₂ at pH 7.5, underwent tyrosine nitration, leading to a V_max^app 2-fold lower following nitration of 1 (with human or C. elegans TS) or 2 (with mouse TS) tyrosine residues per monomer. Enzyme interactions with dUMP, meTHF or 5-fluoro-dUMP were not distinctly influenced. Nitration under the same conditions of model tripeptides of a general formula H₂N-Gly-X-Gly-COOH (X = Phe, Tyr, Trp, Lys, Arg, His, Ser, Thr, Cys, Gly), monitored by NMR spectroscopy, showed formation of nitro-species only for H-Gly-Tyr-Gly-OH and H-Gly-Phe-Gly-OH peptides, the chemical shifts for nitrated H-Gly-Tyr-Gly-OH peptide being in a very good agreement with the strongest peak found in ¹⁵N-¹H HMBC spectrum of nitrated protein. MS analysis of nitrated human and C. elegansproteins revealed several thymidylate synthase-derived peptides containing nitro-tyrosine (at positions 33, 65, 135, 213, 230, 258 and 301 in the human enzyme) and oxidized cysteine (human protein Cys²¹⁰, with catalytically critical Cys¹⁹⁵ remaining apparently unmodified) residues.

Organic & Biomolecular Chemistry

Tyrosinenitration affects thymidylate synthase properties

Abstract

Article information

Download Citation

Permissions

Tyrosine nitration affects thymidylate synthase properties

Social activity

Search articles by author

Spotlight

Advertisements