To which enzyme does 5-Fluorouracil (5-FU)'s metabolite bind and how does it interfere with DNA synthesis?

5-Fluorouracil Mechanism of Action: Thymidylate Synthase Inhibition

The active metabolite of 5-Fluorouracil, FdUMP (5-fluoro-2'-deoxyuridine-5'-monophosphate), binds to and inhibits thymidylate synthase (TS), thereby blocking the methylation of deoxyuridylic acid to thymidylic acid, which prevents DNA synthesis by creating a thymine deficiency that leads to unbalanced cell growth and death. 1

Molecular Mechanism

Primary Target Enzyme

• FdUMP forms a stable ternary complex with thymidylate synthase, the enzyme responsible for converting deoxyuridine monophosphate (dUMP) to deoxythymidine monophosphate (dTMP) 2, 1
• This binding blocks the methylation reaction that is essential for producing thymidine, one of the four nucleotides required for DNA synthesis 1

Interference with DNA Synthesis

Thymine Depletion Pathway:

• By inhibiting thymidylate synthase, FdUMP creates a critical shortage of thymidine triphosphate (TTP), which is essential for DNA replication 1
• This thymine deficiency provokes unbalanced growth and ultimately triggers cell death, with the most pronounced effects occurring in rapidly dividing cells that have higher metabolic demands 1

Direct DNA Incorporation:

• Beyond TS inhibition, 5-FU metabolites become directly incorporated into DNA itself, which has been underestimated historically but represents a predominant cause of cytotoxicity 3
• FdUMP incorporation into DNA causes both single-strand breaks (SSB) and double-strand breaks (DSB), leading to DNA damage and apoptosis 4

Secondary Mechanisms

RNA Interference

• 5-FU also inhibits RNA formation to a lesser extent, as fluorouracil metabolites can be incorporated into various RNA species including ribosomal RNA 1, 5
• This RNA incorporation creates additional cytotoxic effects beyond the primary DNA synthesis inhibition 5

Cell Cycle Effects

• FdUMP specifically causes G2/M cell cycle arrest, distinct from 5-FU itself which causes G1/S arrest 4
• This differential cell cycle modulation reflects the specific mechanism of TS inhibition versus other metabolic effects of the parent compound 4

Clinical Implications

Timing of Cytotoxic Effects

• The effects of DNA and RNA deprivation are most marked on rapidly proliferating cells that take up fluorouracil at higher rates 1
• FdUMP induces DNA strand breaks and apoptosis with a different temporal pattern than the parent 5-FU compound, though both ultimately produce similar clastogenic (chromosome-breaking) effects 4

DNA Repair Interactions

• The Smug1 DNA glycosylase can excise incorporated fluorouracil from DNA, which actually protects against cell killing and may contribute to drug resistance 3
• Base excision repair (BER) and mismatch repair (MMR) pathways participate in the cellular response to 5-FU treatment 6