The paper demonstrates thymidylate synthase, a target enzyme in chemotherapy, to undergo tyrosine nitration in vivo, identifies potential sites of this modification and its influence on enzyme properties.

Highly purified preparations of the enzyme, isolated from normal and tumor tissues, were found to be nitrated, as indicated by a specific reaction with anti-nitro-tyrosine antibodies, suggesting this modification to appear endogenously.

Each human, mouse and Ceanorhabditis elegans recombinant thymidylate synthase preparation, incubated in vitro in the presence of NaHCO3, NaNO2 and H2O2 at pH 7.5, underwent tyrosine nitration, leading to a marked decrease of catalytic potency, but remaining without influence on enzyme interactions with substrates and inhibitor (5-fluoro-dUMP). Nitration under the same conditions of model tripeptides, monitored by NMR spectroscopy, pointed to formation of nitro-species only for H-Gly-Tyr-Gly-OH and H-Gly-Phe-Gly-OH peptides, and NMR spectra for nitrated H-Gly-Tyr-Gly-OH peptide and nitrated protein were in a very good agreement. MS analysis of nitrated human and C. elegans proteins 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.