Thymidylic acid, complex with

With the normal substrate the next step would involve a hydride shift to the methylene of the folate and a proton abstraction from C-5, leading to thymidylic acid. Here, however, removal of an F from C-5 is impossible. The result is an irreversible blockade of the enzyme by a ternary covalent complex (Fig. 4-13). The synthesis of thymidylic acid (Fig. 

TK), 5-FU is activated to 5-fluorodeoxyuridine monophosphate (5-FdUMP). Potent inhibition of thymidylate synthase (TS) by 5-FdUMP is considered critical for 5-FU cytotoxicity. TS catalyzes the rate-limiting step of DNA synthesis, such as the conversion of dUMP into dTMP. Optimal TS function requires the formation of a covalent ternary complex consisting of TS, the folate cofactor 5,10-methylenetetra-hydrofolate (CH2THF), and 5-FdUMP. Inadequate cellular levels of 5,10-methyle-netetrahydrofolate reduce the stability of the ternary complex and consequently the inhibition of TS by 5-FdUMP. For this reason, 5-FU is administered in association with folinic acid, a precursor of 5,10-methylenetetrahydrofolate [40]. 

Site of action 5-FU per se is devoid of antineoplastic activity and must be converted to the corresponding deoxynucleotide (5-FdUMP, Figure 38.9), which competes with deoxyuridine monophosphate (dUMP) for thymidylate synthetase. 5-FdUMP acts as a pseudosubstrate and is entrapped with the enzyme and its N5,N10-methylene tetrahydrofolic acid coenzyme in a ternary complex that cannot proceed to products. DNA synthesis decreases due to lack of thymidine, leading to imbalanced cell growth and cell death. [Note Leucovorin is given with 5-FU because the reduced folate coenzyme is required in the thymidylate synthetase reaction. Lack of sufficient coenzyme reduces the effectiveness of the antipyrimidine.] 5-FU is also incorporated into RNA and low levels have been detected in DNA. 

It had been known for some time > that FdUMP is an extremely potent inhibitor of thymidylate synthetase, but the nature of inhibition was the topic of considerable controversy. Since the 6-position of 1-sub-stituted 5-fluorouracils is quite susceptible toward nucleophilic attack, it was suspected that FdUMP might exert its inhibitory effect by reaction with the proposed nucleophilic catalyst of thymidylate synthetase. It is now well established that, in the presence of CH,-H4folate, 5-fluoro-2 -deoxyuridylate (FdUMP) behaves as a quasi-substrate for thymidylate synthetase " and is, in effect, an affinity labeling agent for the enzyme. Whereas FdUMP binds relatively poorly to free enzyme, in the presence of the cofactor, CHa-Hifolate, a covalent bond is formed between an amino acid residue of the enzyme and the 6-position of the nucleotide to give the complex depicted in Fig. 1. Although covalent bonds are involved in linking the components of the complex, the reaction is slowly reversible. Nevertheless, the complex is sufficiently stable Ka 10" M) to permit isolation and characterization. 

---