Biochemical factors affecting the tightness of 5-fluorodeoxyuridylate binding to human thymidylate synthetase

Ligand binding studies of thymidylate synthetase (5,10-methylenetetrahydrofolate: deoxyuridylate C-methyltransferase, EC 2.1.1.45) isolated from CCRF-CEM human lymphoblastic leukemia cells were conducted to examine the mechanism of 5-fluorodeoxyuridylate (FdUMP) binding to the human enzyme in the presence of L-1-(+)-5,10-methylenetetrahydrofolate (5,10-CH₂H₄PteGlu) and to assess biochemical factors which could account for decreased binding of FdUMP by the enzyme in cells and tissues. Scatchard plots showed one class of binding sites for FdUMP with an apparent dissociation constant (K_D) of 3.1 × 10^?11 M in the absence, and 3.5 × 10^?10 M in the presence, of 80 mM potassium phosphate (P_i). The observed rate constant for FdUMP association (k_on^obs) was dependent on the 5,10-CH₂H₄PteGlu concentration and attained a maximal value of 1.7 × 10⁸M^?1 min^?1 at a concentration of ca. 12μM 5,10-CH₂H₄PteGlu. Increasing the concentration of 5,10-CH₂H₄PteGlu decreased the apparent rate constant of FdUMP dissociation (k_off^obs), although FdUMP had no effect on the rate of 5,10-CH₂H₄PteGlu dissociation. These studies showed that CCRF-CEM thymidylate synthetase has an ordered mechanism of ligand binding and release, with the nucleotide binding first and dissociating last. Incubation of the enzyme-FdUMP-5,10-CH₂H₄PteGlu ternary complex with the substrate dUMP resulted in renewal of enzyme activity at about the same rate as that of FdUMP release. Nucleotides, deoxyuridine (dUrd), and polyoxyanions decreased the rate of FdUMP association but had no significant effect on the rate of FdUMP dissociation. dUMP was the most potent inhibitor of FdUMP binding found, with a binding constant determined from competition experiments of 0.36 μM compared to a Michaelis constant of 2.8 μM. The binding constant for p_i was determined similarly to be 10 mM. The effects of dUMP plus P_i, on decreasing the rate of FdUMP association were additive, whereas the combined effects of dUMP and sub-optimal concentrations of 5,10-CH₂H₄PteGlu were not additive, but multiplicative or greater. The levels of dUMP, P_i, and 5,10-CH₂H₄PteGlu that reportedly are present in some cells and tissues, theoretically could increase the K_Dto more than 10^?6M. These results could account for the coexistence of substantial levels of FdUMP and unbound thymidylate synthetase found in some cells and tissues.