BIOCHEMICAL FACTORS AFFECTING THE TIGHTNESS OF 5-FLUORODEOXYURIDYLATATE 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 1 class of binding sites for FdUMP with an apparent Kd of 3.1 .times. 10-11 M in the absence, and 3.5 .times. 10-10 M in the presence, of 80 mM potassium phosphate (Pi). The observed rate constant for FdUMP association .**GRAPHIC**. was dependent on the 5,10-CH2H4PteGlu concentration and attained a maximal value of 1.7 .times. 108 M-1 min-1 at a concentration of .apprx. 12 .mu.M 5,10-CH2H4PteGlu. Increasing the concentration of 5,10-CH2H4PteGlu decreased the apparent rate constant of FdUMP dissociation .**GRAPHIC**. although FdUMP had no effect on the rate of 5,10-CH2H4PteGlu dissociation. 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 FdUP 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 of 0.36 .mu.M compared to a Km of 2.8 .mu.M. The binding constant for Pi was 10 mM. The effects of dUMP plus Pi on decreasing the rate of FdUMP association were additive; the combined effects of dUMP and sub-optimal concentrations of 5,10-CH2H4PteGlu were not additive but multiplicative or greater. The levels of dUMP, Pi and 5,10-CH2H4PteGlu that reportedly are present in some cells and tissues theoretically could increase the Kd to > 10-6 M. These results could account for the coexistence of substantial levels of FdUMP and unbound thymidylate synthetase found in some cells and tissues. [Fd is a metabolite of FUra, a chemotherapeutic agent.].