In the proposed mechanism, TS forms a covalent bond to the substrate dUMP through a 1,4-addition involving a cysteine nucleophile.
The substrate tetrahydrofolate donates a methyl group to the alpha carbon while reducing the new methyl on dUMP to form dTMP.

It has been proven that the imine formed through reaction with THF and dUMP is an intermediate in the reaction with dUMP through mutations in the structure of TS that inhibit the completion of the mechanism.
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.
The structure was deduced through x-ray crystallography of V316Am TS to illustrate full homodimer TS structure (Figure 1).
In addition, it showed possible interactions of the 175Arg and 174Arg between dimers.
These arginines are thought to stabilize the UMP structures within the active sites by creating hydrogen bonds to the phosphate group (Figure 2). [
Stroud and Finer-Moore]
5-FU is an inhibitor of TS.
Upon entering the cell, 5-fluorouracil (5-FU) is converted to a variety of active metabolites, intracellularly.
One such metabolite is FdUMP which differs from dUMP by a fluorine in place of a hydrogen on the alpha carbon.
FdUMP is able to inhibit TS by binding to the nucleotide-binding site of dUMP.
This competitive binding inhibits the normal function of dTMP synthesis from dUMP [Longley].
Thus the dUMP is unable to have an elimination reaction and complete the methyl donation from THF.

Figure 1.
This figure depicts the homodimer that is TS.
As you can see the orange and teal backbones never connect or intertwine, but there are side chains interactions between the dimers.
On the orange protein, you can visibly detect two long side chains that enter the teal protein (this is located within the yellow circle).
The other beige parts are side chains that interact within the active site.
Just below the yellow circle, you are able to see the same pattern of side chains and configuration.


 
Figure 2.
This figure shows the possible H-bond interactions between the arginines and the UMP in the active site of thymidylate synthase.
This can be seen by the faint lines between the blue tips and the red tips.
These arginines are used to hold the position of the UMP molecule so that the interaction may occur correctly.
The two arginines in the top right corner that are located next to each other on the back bone are actually from the other protein of this dimer enzyme.
This interaction is one of the many intermolecular forces that holds these two tertiary structures together.
The yellow stand in the top-middle region shows a sulfur bond that forms between a cysteine side chain and UMP.
This covalently holds the UMP within the active site until it is reacted to yield TMP.
In the proposed mechanism, TS forms a covalent bond to the substrate dUMP through a 1,4-addition involving a cysteine nucleophile. The substrate tetrahydrofolate donates a methyl group to the alpha carbon while reducing the new methyl on dUMP to form dTMP.
It has been proven that the imine formed through reaction with THF and dUMP is an intermediate in the reaction with dUMP through mutations in the structure of TS that inhibit the completion of the mechanism. 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. The structure was deduced through x-ray crystallography of V316Am TS to illustrate full homodimer TS structure (Figure 1). In addition, it showed possible interactions of the 175Arg and 174Arg between dimers. These arginines are thought to stabilize the UMP structures within the active sites by creating hydrogen bonds to the phosphate group (Figure 2). [Stroud and Finer-Moore]
5-FU is an inhibitor of TS. Upon entering the cell, 5-fluorouracil (5-FU) is converted to a variety of active metabolites, intracellularly. One such metabolite is FdUMP which differs from dUMP by a fluorine in place of a hydrogen on the alpha carbon. FdUMP is able to inhibit TS by binding to the nucleotide-binding site of dUMP. This competitive binding inhibits the normal function of dTMP synthesis from dUMP [Longley]. Thus the dUMP is unable to have an elimination reaction and complete the methyl donation from THF.
Figure 1. This figure depicts the homodimer that is TS. As you can see the orange and teal backbones never connect or intertwine, but there are side chains interactions between the dimers. On the orange protein, you can visibly detect two long side chains that enter the teal protein (this is located within the yellow circle). The other beige parts are side chains that interact within the active site. Just below the yellow circle, you are able to see the same pattern of side chains and configuration.

 
Figure 2. This figure shows the possible H-bond interactions between the arginines and the UMP in the active site of thymidylate synthase. This can be seen by the faint lines between the blue tips and the red tips. These arginines are used to hold the position of the UMP molecule so that the interaction may occur correctly. The two arginines in the top right corner that are located next to each other on the back bone are actually from the other protein of this dimer enzyme. This interaction is one of the many intermolecular forces that holds these two tertiary structures together. The yellow stand in the top-middle region shows a sulfur bond that forms between a cysteine side chain and UMP. This covalently holds the UMP within the active site until it is reacted to yield TMP.
Thymidylate synthase