Binding of 5-fluorodeoxyuridylate to thymidylate synthase in human colon adenocarcinoma xenografts

Abstract

The formation and stability of the covalent ternary complex formed between thymidylate synthase (E.C. 2.1.1.45), 5-fluoro 2′-deoxyuridylate (FdUMP) and 5,10-methylenetetrahydrofolate ( CH 2-H 4 PteGlu) has been examined in cytosols derived from xenografts of human colon adenocarcinomas. The rate of association ( k a ) for FdUMP was low being between 3.4 ± 0.9 and 10.2 ± 2.6 × 10 6 M −1 min −1 , with the lowest k a value being determined in cytosols from a tumor (HxELC 2) which has demonstrated some sensitivity to 5-fluoropyrimidines. Relative to reported k a values for human leukemic cells, the rate of association of FdUMP was 20- to 59-fold lower. This difference is not a consequence of FdUMP catabolism, or metabolism of CH 2-H 4 PteGlu. In cytosols the apparent K m values for dUMP (3.6–4.2 μ M) and [6RS]-CH 2-H 4 PteGlu (25–26.7 μ M) were similar to reported values for human enzyme. Data derived from cytosols were similar to those derived using affinity purified enzyme from HxVRC 5 colon adenocarcinoma xenografts. The net dissociation of [6- 3 H] FdUMP from the covalent ternary complex was 31–33 min in the absence of added CH 2-H 4 PteGlu, and the rate of dissociation was dependent upon the concentration of cofactor. The concentration of [6RS]-CH 2-H 4PteGlu required to stabilize ternary complex derived from HxELC 2 cytosols was slightly lower than that required for the same degree of stabilization of complex formed in cytosols from resistant tumors (HxGC 3, HxVRC 5). Addition of 5-CHO-H 4 PteGlu, 5-CH 3-H 4 PteGlu, H 2 PteGlu, and PteGlu did not stabilize the covalent complex, but H 4 PteGlu substituted for CH 2-H 4 PteGlu.