Tyrosine nitration affects thymidylate synthase properties.

Area of Science:

• Biochemistry
• Enzymology
• Post-translational modifications

Background:

• Thymidylate synthase (TS) is a critical enzyme in DNA synthesis.
• Tyrosine nitration is a post-translational modification with implications in cellular function and disease.
• Previous studies have not fully elucidated the endogenous occurrence and functional impact of TS nitration.

Purpose of the Study:

• To investigate the endogenous occurrence of thymidylate synthase (TS) nitration in normal and tumor tissues.
• To characterize the in vitro nitration of recombinant TS from different species and its effect on enzyme kinetics.
• To identify specific nitrated residues and assess the impact on enzyme-substrate interactions.

Main Methods:

• Purification of TS from calf thymus and L1210 cells.
• In vitro nitration of recombinant human, mouse, and C. elegans TS using sodium nitrite and hydrogen peroxide.
• Enzyme kinetics assays (Vmax, Km) with various substrates and cofactors.
• Nuclear Magnetic Resonance (NMR) spectroscopy and Mass Spectrometry (MS) for identifying nitrated residues and modified peptides.

Main Results:

• Endogenous nitration of TS was detected in purified preparations from normal and tumor cells.
• In vitro nitration of recombinant TS reduced apparent Vmax by approximately twofold, affecting 1-2 tyrosine residues per monomer.
• Nitration did not significantly alter enzyme interactions with dUMP, meTHF, or 5-fluoro-dUMP.
• MS analysis identified specific nitrated tyrosine residues in human and C. elegans TS, with oxidized cysteine also observed.

Conclusions:

• Tyrosine nitration of thymidylate synthase (TS) occurs endogenously in mammalian tissues.
• In vitro nitration reduces TS catalytic activity (Vmax) by modifying specific tyrosine residues.
• The catalytic site and substrate binding appear largely unaffected by this modification, suggesting potential roles in enzyme regulation or signaling.