Thymidylate synthase-catalyzed, tetrahydrofolate-dependent self-inactivation by 5-FdUMP

Arch Biochem Biophys. 2019 Oct 15;674:108106. doi: 10.1016/ Epub 2019 Sep 11.


In view of previous crystallographic studies, N4-hydroxy-dCMP, a slow-binding thymidylate synthase inhibitor apparently caused "uncoupling" of the two thymidylate synthase-catalyzed reactions, including the N5,10-methylenetetrahydrofolate one-carbon group transfer and reduction, suggesting the enzyme's capacity to use tetrahydrofolate as a cofactor reducing the pyrimidine ring C(5) in the absence of the 5-methylene group. Testing the latter interpretation, a possibility was examined of a TS-catalyzed covalent self-modification/self-inactivation with certain pyrimidine deoxynucleotides, including 5-fluoro-dUMP and N4-hydroxy-dCMP, that would be promoted by tetrahydrofolate and accompanied with its parallel oxidation to dihydrofolate. Electrophoretic analysis showed mouse recombinant TS protein to form, in the presence of tetrahydrofolate, a covalently bound, electrophoretically separable 5-fluoro-dUMP-thymidylate synthase complex, similar to that produced in the presence of N5,10-methylenetetrahydrofolate. Further studies of the mouse enzyme binding with 5-fluoro-dUMP/N4-hydroxy-dCMP by TCA precipitation of the complex on filter paper showed it to be tetrahydrofolate-promoted, as well as to depend on both time in the range of minutes and the enzyme molecular activity, indicating thymidylate synthase-catalyzed reaction to be responsible for it. Furthermore, the tetrahydrofolate- and time-dependent, covalent binding by thymidylate synthase of each 5-fluoro-dUMP and N4-hydroxy-dCMP was shown to be accompanied by the enzyme inactivation, as well as spectrophotometrically confirmed dihydrofolate production, the latter demonstrated to depend on the reaction time, thymidylate synthase activity and temperature of the incubation mixture, further documenting its catalytic character.

Keywords: 5-Fluorodeoxyuridylate binding; Dihydrofolate production; Inactivation; Tetrahydrofolate-dependent; Thymidylate synthase.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Deoxycytidine Monophosphate / analogs & derivatives
  • Deoxycytidine Monophosphate / metabolism
  • Enzyme Inhibitors / metabolism
  • Fluorodeoxyuridylate / metabolism*
  • Folic Acid / analogs & derivatives
  • Folic Acid / metabolism
  • Mice
  • Protein Binding
  • Spectrophotometry, Ultraviolet
  • Tetrahydrofolates / metabolism*
  • Thymidylate Synthase / metabolism*


  • Enzyme Inhibitors
  • Tetrahydrofolates
  • Deoxycytidine Monophosphate
  • Fluorodeoxyuridylate
  • N-4-hydroxy-2'-deoxycytidylic acid
  • dihydrofolate
  • 5,6,7,8-tetrahydrofolic acid
  • Folic Acid
  • Thymidylate Synthase