Enzyme thymidylate synthase

Fluorouracil (5-FU) acts as a false pyrimidine, inhibiting the formation of the DNA base thymidine.26,35 The main mechanism by which it accomplishes this is by inhibiting the enzyme thymidylate synthase, the rate-limiting step in thymidine formation. 5-FU first must be metabolized to its active metabolite (F-dUMP). Additionally, metabolites of 5-FU may incorporate into RNA, inhibiting its synthesis. 

Ribonucleotide reductase works on ribo-A, -U, -G, -C diphosphates to give the deoxynucleotide. The deoxyuridine, which is useless for RNA synthesis, is converted to deoxythymidine by the enzyme thymidylate synthase, which uses methylene tetrahydrofolate as a one-carbon donor. The odd thing here is that ribonucleotide reductase uses the UDP as a substrate to give the dUDP. This must then be hydrolyzed to the dUMP before thymidylate synthase will use it to make dTMP. Then the dTMP has to be kinased (phosphorylated) up to dTTP before DNA can be made. 

Freer, Calculation of solvation and binding free energy differences for folate-based inhibitors of the enzyme thymidylate synthase, J. Am. Chem. Soc. 114 10117 (1992). 

Fluorouracil (5-FU) is inactive in its parent form and requires activation via a complex series of enzymatic reactions to ribosyl and deoxyribosyl nucleotide metabolites. One of these metabolites, 5-fluoro-2 -deoxyuridine-5 -monophosphate (FdUMP), forms a covalently ternary complex with the enzyme thymidylate synthase and the reduced folate 5,10-methylenetetrahydrofolate, a reaction critical for the de novo synthesis of thymidylate. This results in inhibition of DNA synthesis through "thymineless death." 5-FU is converted to 5-fluorouridine-5 -triphosphate (FUTP), which is then incorporated into RNA, where it interferes with RNA processing and mRNA translation. 5-FU is also converted to 5-fluorodeoxyuridine-5 -triphosphate (FdUTP), which can be incorporated into cellular DNA, resulting in inhibition of DNA synthesis and function. Thus, the cytotoxicity of 5-FU is thought to be the result of combined effects on both DNA- and RNA-mediated events. 

Tetrahydrofolate cofactors participate in one-carbon transfer reactions. As described above in the section on vitamin B12, one of these essential reactions produces the dTMP needed for DNA synthesis. In this reaction, the enzyme thymidylate synthase catalyzes the transfer of the one-carbon unit of N 5,N 10-methylenetetrahydrofolate to deoxyuridine monophosphate (dUMP) to form dTMP (Figure 33-2, reaction 2). Unlike all of the other enzymatic reactions that utilize folate cofactors, in this reaction the cofactor is oxidized to dihydrofolate, and for each mole of dTMP produced, one mole of tetrahydrofolate is consumed. In rapidly proliferating tissues, considerable amounts of tetrahydrofolate can be consumed in this reaction, and continued DNA synthesis requires continued regeneration of tetrahydrofolate by reduction of dihydrofolate, catalyzed by the enzyme dihydrofolate reductase. The tetrahydrofolate thus produced can then reform the cofactor N 5,N 10-methylenetetrahydrofolate by the action of serine transhydroxy- methylase and thus allow for the continued synthesis of dTMP. The combined catalytic activities of dTMP synthase, dihydrofolate reductase, and serine transhydroxymethylase are often referred to as the dTMP synthesis cycle. Enzymes in the dTMP cycle are the targets of two anticancer drugs methotrexate inhibits dihydrofolate reductase, and a metabolite of 5-fluorouracil inhibits thymidylate synthase (see Chapter 55 Cancer Chemotherapy). 

In order to determine the relationship between protein structure and function and to create mutant enzymes with altered properties useful for biotechnology and cancer therapy, a directed evolution approach has been explored and novel proteins developed for Pol I DNA polymerase enzymes thymidylate synthase, thymidine kinase and 06-alkylguanine-DNA alkyltransferase. In every case the creation of a large variety of altered proteins has been achieved, and the emerging picture is that even highly conserved proteins can tolerate wide-spread amino acid changes at the active site with-... 

Random oligonucleotide mutagenesis was first applied to promotor sequences that regulate the production of enzymes in cells [21] and was the first method used to alter systematically the functions of enzymes by directed evolution [22], Based on our experience, we will focus on this approach and emphasize recent applications of this methodology to enzymes involved in DNA repair and synthesis, including DNA polymerase enzymes, thymidylate synthase, thymidine kinase, and 06-alkylguanine-DNA alkyltransferase. 

In the 1990s, it was found that tylophorine alkaloids inhibit several key targets for clinically important anticancer drugs, including the metabolic enzymes thymidylate synthase (TS) and dihydrofolate reductase [8, 94], TS catalyzes the reductive methylation of the substrate dUMP (2 -deoxyuridine 5 -monophosphate) to dTMP (2 -deoxythymidine 5 -monophosphate thymidylate) with concomitant conversion of the cofactor CH2THF (5,10-methylenetetrahydrofolate) to DHF (7,8-dihydrofolate) (see Equation 1). 

Fluoropropynyl-dUMP (23), a novel mechanism based inhibitor of the A A -methylenetetrahydrofolate-dependent enzyme thymidylate synthase, has been synthesised. This 5-fluoropropynyl-dUMP caused rapid, irreversible inactivation of thymidylate synthase both in the presence and in the absence of the cofactor. 

The answer is c. (Murray, pp 48-73. Scriver, pp 4571-4636. Sack, pp 3-17. Wilson, pp 287-317.) Since rapidly multiplying cancer cells are dependent upon the synthesis of deoxythymidilate (dTMP) from deoxy-uridylate (dUMP), a prime target in cancer therapy has been inhibition of dTMP synthesis. The anticancer drug fluorouracil is converted in vivo to fluorodeoxyuridylate (FdUMP), which is an analogue of dlJMP FdUMP irreversibly forms a covalent complex with the enzyme thymidylate synthase and its substrate N5,N10-methylene-tetrahydrofolate. This is a case of suicide inhibition, where an enzyme actually participates in the change of a substrate into a covalently linked inhibitor that irreversibly inhibits its catalytic activity. 

All cells, especially rapidly growing cells, must synthesize thymidylate (dTMP) for DNA synthesis. The difference between (T) and (U) is one methyl gronp at the carbon-5 position. Thymidylate is synthesized by the methyla-tion of uridylate (dUMP) in a reaction catalyzed by the enzyme thymidylate synthase. This reaction reqnires a methyl donor and a source of reducing eqnivalents, which are both provided by N, N °-methylene THF (Figure 3-2). For this reaction to continue, the regeneration of THF from dihydrofolate (DHF) must occur. 

Figure 3-2. Thymidylate synthesized by the methylation of uridylate (dUMP) in a reaction catalyzed by the enzyme thymidylate synthase.

Thymidylate (dTMP) is formed intracellularly either de novo, in a process of the C(5) methylation of 2 -deoxyuridylate (dUMP), catalyzed by the enzyme thymidylate synthase (TS), or as a product of thymidine salvage via phosphorylation, catalyzed by the enzyme thymidine kinase. The dUMP methylation reaction involves a concerted transfer and reduction of the one-carbon group of... 

Newer antifolates have been developed that are THF antagonists, which potendy inhibit the enzyme thymidylate synthase (TS). Again, cells exposed to increasing levels of the antifolates resulted in an increased resistance to these antifolates. In this circumstance the increased resistance was related to increased expression of TS and not DHFR. Similarly cells made resistant to classical TS inhibitors such as 5-fluorouracil were cross-resistant with the TS inhibitor antifolates. 

The synthesis of deoxyuridine, cytidine, deoxycytidine and thymidine nucleotides from UMP (Fig. 6.13) involves three reactions CTP synthetase, ribonucleotide reductase, and thymidylate synthase (80). The first enzyme converts UTP into CTP and the second catalyzes the conversion of CDP, UDP, ADP and GDP into their respective deoxyribonucleotides. The last enzyme, thymidylate synthase, catalyzes the reductive methylation of deoxyUMP at the C-5 position giving deoxyTMP. The human enzyme has been extensively studied as it is a target enzyme in cancer chemotherapy. Besides these three enzymes, two other enzymes are involved in pyrimidine nucleotide synthesis and interconversion. DeoxyCMP deaminase converts deoxyCMP into deoxyUMP and deoxyUTP triphosphatase converts deoxyUTP into deoxyUMP. Giardia lamblia, and Trichomonas vaginalis lack both ribonucleotide reductase and thymidylate synthase and... 

The drug fluorouracil is used as an anticancer agent. It irreversibly inactivates the enzyme thymidylate synthase. Explain how this treatment retards the growth of tumor tissue. Will the growth of normal cells be affected as well ... 

The antimetabolite, raltitrexed, is a folate analogue and is a potent and specific inhibitor of the enzyme thymidylate synthase. Inhibition of this enzyme ultimately interferes with the synthesis of deoxyribonucleic acid (DNA) leading to cell death. The intracellular polyglutamation of raltitrexed leads to the formation within cells of even more potent inhibitors of thymidylate synthase. Folate (methylene tetrahydrofolate) is a co-faetor required by thymidylate synthase and therefore theoretically folinic acid or folic acid may interfere with the aetion of raltitrexed. Clinieal interaction studies have not yet been undertaken to confirm these predieted inter-aetions. ... 

Enzymes with folate coenzymes in human metabolism (examples, 20 enzymes). Thymidylate-synthase (EC 2.1.1.45) serine-hydroxymethyl-transferase (EC 2.1.2.1) formiminoglutamate-formiminotrans-ferase (EC 2.1.2.5). 

M. R. Reddy, R. J. Bacquet, D. Zichi, D. A. Matthews, K. M. Welsh, T. R. Jones, and S. Freer, /. Am. Chem. Soc., 114, 10117 (1992). Calculation of Solvation and Binding Free Energy Differences for Folate-Based Inhibitors of the Enzyme Thymidylate Synthase. 

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