5,10-Methylenetetrahydrofolate

5,10-Methylenetetrahydrofolate is a donor of single-carbon units. 5,10-Methylenetetrahydrofolate can be made from tetrahydrofolate as follows:

Glycine + Tetrahydrofolate + NAD⁺ -> 5,10-Methylenetetrahydrofolate + CO2 + NH3 + NADH + H⁺

5-Fluorodeoxyuridine monophosphate (FdUMP) is a molecule that is a mechanism-based inhibitor. Irreversible binding of the substance to thymidylate synthase occurs only in the presence of 5,10-methylenetetrahydrofolate, a cofactor for the reaction catalyzed by thymidylate synthase (Figure 22.18). Crystallographic analysis of thymidylate synthase with dUMP and an analog of 5,10-methylenetetrahydrofolate (that could not be acted on by the enzyme) revealed that thymidylate synthase normally makes a transient covalent bond in the process of catalysis of the reaction. Apparently FdUMP's structure traps the enzyme-substrate covalent bond and prevents it from breaking down.