[6][7] The following reaction is catalyzed by thymidylate synthase: By means of reductive methylation, deoxyuridine monophosphate (dUMP) and N5,N10-methylene tetrahydrofolate are together used to form dTMP, yielding dihydrofolate as a secondary product.

This provides the sole de novo pathway for production of dTMP and is the only enzyme in folate metabolism in which the 5,10-methylenetetrahydrofolate is oxidised during one-carbon transfer.

LSF and Thymidylate synthase plays significant role in Liver Cancer proliferation and progression and Drug resistance.

The most widely used inhibitor is 5-fluorouracil (5-FU) and its metabolized form 5-fluorodeoxyuridine monophosphate (5-FdUMP), which acts as an antimetabolite that irreversibly inhibits TS by competitive binding.

[13] However, due to a low level of 5-FU found in many patients, it has been discovered that in combination with leucovorin (LV), 5-FU has greater success in down regulating tumor progression mechanisms and increasing immune system activity.

[14] Experimentally, it has been shown that low levels of TS expression leads to a better response to 5-FU and higher success rates and survival of colon and liver cancer patients.

[§ 1] In the proposed mechanism, TS forms a covalent bond to the substrate dUMP through a 1,4-addition involving a cysteine nucleophile.

V316Am TS, a mutant with deletion of C terminal valines from both subunits, allows the catalysis of dehalogenation of BrdUMP preceding the mechanism described above and the covalent bond to THF and dUMP.

The mutant TS is unable to accomplish the C-terminal conformational change needed to break covalent bonds to form dTMP, thus showing the proposed mechanism to be true.

These arginines are thought to stabilize the UMP structures within the active sites by creating hydrogen bonds to the phosphate group (Figure 2).

The yellow stand in the top-middle region shows a sulfur bond that forms between a cysteine side chain and UMP.