Characteristics of thymidylate synthase purified from a human colon adenocarcinoma

Thymidylate synthase has been purified >4000-fold from a human colon adenocarcinoma maintained as a xenograft in immune-deprived mice. In this disease, the enzyme is an important target for the cytotoxic action of 5-fluorouracil, which is influenced by the reduced folate substrate CH₂-H₄PteGlu. Due to the importance of this interaction, and the existence in cells of folate species as polyglutamyl forms, the interaction of folylpolyglutamates with thymidylate synthase was examined. Polyglutamates of PteGlu were used as inhibitors, and the interaction of CH₂-H₄PteGlu polyglutamates as substrates or in an inhibitory ternary complex were also examined. Using PteGlu_1-7, K_i values were determined. A maximal 125-fold decrease in K_i was observed between PteGlui and PteGlu₄; further addition of up to three glutamyl residues did not result in an additional decrease in K_i. Despite the increased binding affinity of folylpolyglutamates for this enzyme, no change in the K_m values for either dUMP (3.6 μm) or CH₂-H₄PteGlu (4.3 μm) were detected when polyglutamates of [6R]CH₂-H₄PteGlu were used as substrates. Product inhibition studies demonstrated competitive inhibition between dTMP and dUMP in the presence of CH₂-H₄PteGlu₅. In addition, CH₂H₄PteGlu₄ stabilized an inhibitory ternary complex formed between FdUMP, thymidylate synthase, and CH₂-H₄PteGlu₄. Thus the data do not support a change in the order of substrate binding and product release upon polyglutamylation of CH₂-H₄PteGlu reported for non-human mammalian enzyme. This is the first study to characterize kinetically thymidylate synthase from a human colon adenocarcinoma.