Model chemical studies of thymidylate synthase. Nucleophilic addition of hydroxylamine to uracil and 5-fluorouracil

To explain the reaction of 5-fluoro-2'-deoxyuridine-5'-monophosphate towards Thymidylate Synthase (TS), a chemical model formed by hydroxylamine and 5-fluorouracil (FU) or uracil (U) was suggested. The reaction mechanism would involve a nucleophilic attack of the nitrogen of the free base to C-6 of the uracil ring; the reaction is catalyzed by the cationic form of the base. The kinetic constants, the activation energies, the entropies of activation for the forward and the reverse reactions and the equilibrium constants for the reactions of FU and U with hydroxylamine were measured. The value of the activation energy for the forward reaction of FU with hydroxylamine is 1.95 Kcal/mol lower than with U. The value of the equilibrium constant for the reaction of FU with hydroxylamine is 23 times higher than that of U at 25 degrees C. Therefore the product of the first reaction is the most stable one. The reactivity of FU towards hydroxylamine is, in its complex, higher with respect to the reactivity of U in the same conditions, because the reaction proceeds largely towards higher Keg values; the activation energy of the forward reaction is lower for FU and the entropy of activation too. Thus indicating that the activated complex is more ordered than that of U. The results indicate that the increased reactivity and stability of the complex with FU in the model reaction may account for the competition observed in the biological reaction.