# Thymidylate synthase

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Identifiers
, HST422, TMS, TS, thymidylate synthetase
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SpeciesHumanMouse
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thymidylate synthase
Thymidylate synthase homodimer, Human
Identifiers
EC no. 2.1.1.45
CAS no. 9031-61-2
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Gene Ontology AmiGO / QuickGO
Thymidylate synthase
Identifiers
SymbolThymidylat_synt
Pfam PF00303
InterPro IPR000398
PROSITE PDOC00086
SCOP2 1tys / SCOPe / SUPFAM

Thymidylate synthase (TS) (EC 2.1.1.45) [5] is an enzyme that catalyzes the conversion of deoxyuridine monophosphate (dUMP) to deoxythymidine monophosphate (dTMP). Thymidine is one of the nucleotides in DNA. With inhibition of TS, an imbalance of deoxynucleotides and increased levels of dUMP arise. Both cause DNA damage. [6] [7]

## Function

The following reaction is catalyzed by thymidylate synthase:

5,10-methylenetetrahydrofolate + dUMP ${\displaystyle \rightleftharpoons }$ dihydrofolate + dTMP

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. [8] The enzyme is essential for regulating the balanced supply of the 4 DNA precursors in normal DNA replication: defects in the enzyme activity affecting the regulation process cause various biological and genetic abnormalities, such as thymineless death. [9] The enzyme is an important target for certain chemotherapeutic drugs. Thymidylate synthase is an enzyme of about 30 to 35 kDa in most species except in protozoan and plants where it exists as a bifunctional enzyme that includes a dihydrofolate reductase domain. [8] A cysteine residue is involved in the catalytic mechanism (it covalently binds the 5,6-dihydro-dUMP intermediate). The sequence around the active site of this enzyme is conserved from phages to vertebrates.

Thymidylate synthase is induced by a transcription factor LSF/TFCP2 and LSF is an oncogene in hepatocellular carcinoma. LSF and Thymidylate synthase plays significant role in Liver Cancer proliferation and progression and Drug resistance. [10]

## Clinical significance

Thymidylate synthase (TS) plays a crucial role in the early stages of DNA biosynthesis. [11] DNA damage or deletion occur on a daily basis as a result of both endogenous and environmental factors. Such environmental factors include ultraviolet damage and cigarette smoke that contain a variety of carcinogens. [12] Therefore, synthesis and insertion of healthy DNA is vital for normal body functions and avoidance of cancerous activity. In addition, inhibition in synthesis of important nucleotides necessary for cell growth is important. For this reason, TS has become an important target for cancer treatment by means of chemotherapy. The sensitivity of TS to succumb to TS inhibitors is a key part to its success as treatment for colorectal, pancreatic, ovarian, gastric, and breast cancers. [11]

## Using TS as a drug target

The use of TS inhibitors has become a main focus of using TS as a drug target. The most widely used inhibitor is 5-fluorouracil (5-FU), which acts as an antimetabolite that irreversibly inhibits TS by competitive binding. [13] [14] 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. [15]

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. [11] However, additional experiments have merely stated that levels of TS may be associated with stage of disease, cell proliferation and tumor differentiation for those with lung adenocarcinoma but low levels are not necessarily indicators of high success. Expression levels of TS mRNA may be helpful in predicting the malignant potential of certain cancerous cells, thus improving cancer treatment targets and yielding higher survival rates among cancer patients [Hashimoto].

TS's relation to the cell cycle also contributes to its use in cancer treatment. Several cell-cycle dependent kinases and transcription factors influence TS levels in the cell cycle that increase its activity during the S phase but decrease its activity while cells are no longer proliferating. [11] In an auto-regulatory manner, TS not only controls its own translation but that of other proteins such as p53 that through mutation is the root of much tumor growth. Through its translation, TS has a varying expression in cancer cells and tumors, which leads to early cell death. [14]

## Interactive pathway map

Click on genes, proteins and metabolites below to link to respective articles. [§ 1]

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|alt=Fluorouracil (5-FU) Activity edit]]
Fluorouracil (5-FU) Activity edit
1. The interactive pathway map can be edited at WikiPathways: "FluoropyrimidineActivity_WP1601".

## Mechanism description

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. [16]

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][ citation needed ] 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].[ citation needed ] 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.

## Related Research Articles

Dihydrofolate reductase, or DHFR, is an enzyme that reduces dihydrofolic acid to tetrahydrofolic acid, using NADPH as electron donor, which can be converted to the kinds of tetrahydrofolate cofactors used in 1-carbon transfer chemistry. In humans, the DHFR enzyme is encoded by the DHFR gene. It is found in the q11→q22 region of chromosome 5. Bacterial species possess distinct DHFR enzymes, but mammalian DHFRs are highly similar.

The enzyme Uridine monophosphate synthase catalyses the formation of uridine monophosphate (UMP), an energy-carrying molecule in many important biosynthetic pathways. In humans, the gene that codes for this enzyme is located on the long arm of chromosome 3 (3q13).

A salvage pathway is a pathway in which a biological product is produced from intermediates in the degradative pathway of its own or a similar substance. The term often refers to nucleotide salvage in particular, in which nucleotides are synthesized from intermediates in their degradative pathway.

Folinic acid, also known as leucovorin, is a medication used to decrease the toxic effects of methotrexate and pyrimethamine. It is also used in combination with 5-fluorouracil to treat colorectal cancer and pancreatic cancer, may be used to treat folate deficiency that results in anemia, and methanol poisoning. It is taken by mouth, injection into a muscle, or injection into a vein.

Fluorouracil (5-FU), sold under the brand name Adrucil among others, is a cytotoxic chemotherapy medication used to treat cancer. By intravenous injection it is used for treatment of colorectal cancer, oesophageal cancer, stomach cancer, pancreatic cancer, breast cancer, and cervical cancer. As a cream it is used for actinic keratosis, basal cell carcinoma, and skin warts.

An antimetabolite is a chemical that inhibits the use of a metabolite, which is another chemical that is part of normal metabolism. Such substances are often similar in structure to the metabolite that they interfere with, such as the antifolates that interfere with the use of folic acid; thus, competitive inhibition can occur, and the presence of antimetabolites can have toxic effects on cells, such as halting cell growth and cell division, so these compounds are used as chemotherapy for cancer.

In biochemistry, suicide inhibition, also known as suicide inactivation or mechanism-based inhibition, is an irreversible form of enzyme inhibition that occurs when an enzyme binds a substrate analog and forms an irreversible complex with it through a covalent bond during the normal catalysis reaction. The inhibitor binds to the active site where it is modified by the enzyme to produce a reactive group that reacts irreversibly to form a stable inhibitor-enzyme complex. This usually uses a prosthetic group or a coenzyme, forming electrophilic alpha and beta unsaturated carbonyl compounds and imines.

HAT Medium is a selection medium for mammalian cell culture, which relies on the combination of aminopterin, a drug that acts as a powerful folate metabolism inhibitor by inhibiting dihydrofolate reductase, with hypoxanthine and thymidine which are intermediates in DNA synthesis. The trick is that aminopterin blocks DNA de novo synthesis, which is absolutely required for cell division to proceed, but hypoxanthine and thymidine provide cells with the raw material to evade the blockage, provided that they have the right enzymes, which means having functioning copies of the genes that encode them.

Thromboxane A synthase 1 , also known as TBXAS1, is a cytochrome P450 enzyme that, in humans, is encoded by the TBXAS1 gene.

Tissue alpha-L-fucosidase is an enzyme that in humans is encoded by the FUCA1 gene.

Raltitrexed is an antimetabolite drug used in cancer chemotherapy. It is an inhibitor of thymidylate synthase, and is manufactured by AstraZeneca.

5,10-Methylenetetrahydrofolate (N5,N10-Methylenetetrahydrofolate; 5,10-CH2-THF) is cofactor in several biochemical reactions. It exists in nature as the diastereoisomer [6R]-5,10-methylene-THF.

In enzymology, a thymidylate synthase (FAD) (EC 2.1.1.148) is an enzyme that catalyzes the chemical reaction

DUTP pyrophosphatase, also known as DUT, is an enzyme which in humans is encoded by the DUT gene on chromosome 15.

Thymidylate kinase also known as deoxythymidylate kinase or dTMP kinase is an enzyme that in humans is encoded by the DTYMK gene. and belongs to thymidylate kinase family of proteins.

Uridine phosphorylase 1 is an enzyme that in humans is encoded by the UPP1 gene.

Pyrimidine analogues are antimetabolites which mimic the structure of metabolic pyrimidines.

Thymidylate synthase inhibitors are chemical agents which inhibit the enzyme thymidylate synthase and have potential as an anticancer chemotherapy. This inhibition prevents the methylation of C5 of deoxyuridine monophosphate (dUMP) thereby inhibiting the synthesis of deoxythymidine monophosphate (dTMP). The downstream effect is promotion of cell death because cells would not be able to properly undergo DNA synthesis if they are lacking dTMP, a necessary precursor to dTTP. Five agents were in clinical trials in 2002: raltitrexed, pemetrexed, nolatrexed, ZD9331, and GS7904L.

Trifluridine/tipiracil, sold under the brand name Lonsurf, is a fixed-dose combination medication that is used as a third- or fourth-line treatment of metastatic colorectal cancer or gastric cancer, after chemotherapy and targeted therapeutics have failed. It is a combination of two active pharmaceutical ingredients: trifluridine, a nucleoside analog, and tipiracil, a thymidine phosphorylase inhibitor. Tipiracil prevents rapid metabolism of trifluridine, increasing the bioavailability of trifluridine.

Fluorodeoxyuridylate, also known as FdUMP, 5-fluoro-2'-deoxyuridylate, and 5-fluoro-2'-deoxyuridine 5'-monophosphate, is a molecule formed in vivo from 5-fluorouracil and 5-fluorodeoxyuridine.

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