Thymidine monophosphate dTMP

We first applied Tethering to thymidylate synthase (TS). This enzyme converts de-oxyuridine monophosphate (dUMP) to thymidine monophosphate (dTMP), an activity essential for DNA synthesis. The cancer drug 5-fluorouracil irreversibly inhibits TS, and a selective inhibitor of a non-human form of the enzyme could yield a new antibiotic or antifungal drug [23]. 

Since DNA contains thymine (5-methyluracil) as a major base instead of uracil, the synthesis of thymidine monophosphate (dTMP, or thymidylate) is essential to provide dTTP (thymidine triphosphate), which is needed for DNA replication together with dATP, dGTP, and dCTP. 

Thymine Deoxyribose Thymidine Thymidylic acid thymidine monophosphate (dTMP) Thymidine diphosphate (dTDP) Thymidine triphosphate (dTTP)... 

Purines, pyrimidines and their nucleosides and nucleoside triphosphates are synthesized in the cytoplasm. At this stage the antifolate drugs (sulphonamides and dihydrofolate reductase inhibitors) act by interfering with the synthesis and recycling of the co-factor dihydrofolic acid (DHF). Thymidylic acid (2-deoxy-thymidine monophosphate, dTMP) is an essential nucleotide precursor of DNA synthesis. It is produced by the enzyme thymidylate synthetase by transfer of a methyl group from tetrahydrofolic acid (THF) to the uracil base on uridylic acid (2-deoxyuridine monophosphate, dUMP) (Fig. 12.5). THF is converted to DHF in this process and must be reverted to THF by the enzyme dihydrofolate reductase (DHFR) before... 

Fig, 2. Nucleotidase isozymes in erythrocytes from the patient with pyrimidine 5 -nucleotidase deficiency ( ) and five healthy controls (1-5) detected using cytidine monophosphate (CMP), thymidine monophosphate (dTMP), deoxycytidine monophosphate (dCMP) as substrates. 

In 1995, Horie et al. described a polymorphic tandem repeat found in the 5 -un-translated region of the thymidylate synthase gene [70]. Thymidylate synthase (TS TYMS) catalyzes the intracellular transfer of a methyl group to deoxyuridine-5-monophosphate (dUMP) to form deoxythymidine-5-monophosphate (dTMP), which is anabolized in cells to the triphosphate (dTTP). This pathway is the only de- novo source of thymidine, an essential precursor for DNA synthesis and repair. The methyl donor for this reaction is the folate cofactor 5,10-methylenetetrahydro-folate (CH2-THF) (Figure 24.4). 

R-PO3 ) (O) 4-nitrophenyl phosphate (NPhp ), phenyl phosphate (php ), uridine S -monophosphate (UMp ), D-ribose 5-monophosphate (RibMp ), thymidine [-l-(2-deoxy-13-D-ribofuranosyl)thymine] 5 -monophosphate (dTMP ), n-butyl phosphate (Bup ), methanephosphonate (MeP ) and ethanephosphonate (EtP ) (from left to right). The least-squares lines (eq. (11)) are drawn through the corresponding 8 data sets (O) taken from ref [38] for the phosphate monoesters and from ref [5] for the phosphonates. The points due to the equilibrium constants for the M VPA systems are based on the values listed In Tables (column 4) and 2 (columns 4 or S). The vertical broken lines emphasize the stability differences from the reference lines they equal log as defined In eq. (12) for the M(PA) complexes. All the plotted equilibrium constants refer to... 

The de novo pathway to 2 -deoxythymidine monophosphate (dTMP) synthesis first requires the use of dUMP (2 -deoxyuridine-5 -monophosphate) from the metabolism of either UDP or CDP (cytidine diphosphate). The hydrolysis of dUTP (2 -deoxyuridine-5 -triphosphate) to dUMP and subsequent methylation at C-5 by the action of thymidylate synthase, using A, A i°-methylenetetrahydrofolate (THF) as the methyl donor, generate dTMP (Figure 6.54). The latter is subsequently phosphorylated to deoxy-thymidine triphosphate (dTTP) used in DNA synthesis and repair. 

Fractionation of extracts from calf thymus for kinase activity toward deoxyribonucleoside monophosphates has revealed the presence of at least four separate enzymes the kinase activities for dAMP, dGMP, dCMP, and dTMP are separate entities in this tissue 31). Each has been partly purified and examination of substrate specificities showed that the kinases for deoxyadenylate and deoxyguanylate also phosphorylate the ribosyl homologues, adenylate and guanylate, respectively. The deoxycytidylate kinase accepts as substrates both cytidylate and uridylate, but will not phosphor> late deoxyuridylate. The calf thymus thymidine monophosphate... 

Thymidine monophosphate (deoxythymidine monophosphate [dTMP]) is generated from deoxyuridine monophosphate (dUMP). The methyl group added to the ring is derived from the CIO methylene unit of tetrahydrofolate as described in Chapter 12 (Scheme 12.9), and reduction is brought about by nicotine adenine dinucleotide (NADH, NAD+). A representation of the process is provided in Scheme 14.12. 

Fig. 14. Effect of 5-fluorouracil (5-FU) and 5-fluorodeoxyuridine (5-FdUMP) on the synthesis of RNA and DNA. 5-FU is incorporated into RNA whereas 5-FdUMP inhibits thymidilate synthetase competitively and blocks DNA synthesis. The conversion of thymidine (dTr) into thymidilate (dTMP) by means of thymidine kinase is only a subsidary routine to thymidilate which can become important, however, under certain conditions such as in developing pollen (page 185). U = uridine triphosphate, Tr = thymidine, TMP = thymidine monophosphate = thymidilate, TDP = thymidine diphosphate, TTP = thymidine triphosphate, 5-F = 5-fluoro, d = deoxy, 1 = thymidine kinase, 2 = thymidilate synthetase.

All NRTIs, as exemplified for AZT (Fig. 7), act in a similar fashion following their uptake by the cells, they are phosphorylated successively to their 5 -monophosphate, 5 -diphosphate, and 5 -triphosphate form (De Clercq 2002). Unlike the first phosphorylation step in the metabolic pathway of the acyclic guanosine analogues (see above), which is carried out by a virus-encoded enzyme (thymidine kinase), the first as well as the subsequent phosphorylations of the 2, 3 -dideoxynucleosides are carried out by cellular enzymes, that is, a 2 -deoxynucleoside (e.g., dThd) kinase, a 2 -deoxynucleotide (e.g., dTMP) kinase, and a (2 -deoxy)nucleoside 5 -diphosphate (NDP) kinase. 

The Ts used for the biosynthesis of DNA are synthesized from Us by thymidylate synthase, an enzyme that requires N, lV -methylene-THF as a coenzyme. The actual substrate is dUMP (2 -deoxyuridine 5 -monophosphate) and the product is dTMP (2 -deoxy thymidine 5 -monophosphate). 

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