Thymidine monophosphate formation

Kan, L.-S., Voituriez, L., and Cadet, J., The Dewar valence isomer of the (6-4) photoadduct of thymidylyl-(3 -5 )-thymidine monophosphate formation, alkaline lability and conformational properties, J. Photochem. PhotobioL B Biol., 12, 339, 1992. 

Thymidylate synthase (TS) is the enzyme that converts 2-deoxyuridine monophosphate into thymidine monophosphate. This is a key step in the biosynthesis of DNA. This enzymatic reaction of methylation involves the formation of a ternary complex between the substrate, the enzyme, and tetrahydrofolic acid (CH2FAH4). The catalytic cycle involves the dissociation of this complex and the elimination of FAH4. It is initiated by pulling out the proton H-5, thus generating an exocyclic methylene compound. As the release of a F" " ion is energetically forbidden, the fluorine atom that replaces the proton H-5 cannot be pulled out by the base. This leads to inhibition of the enzyme (Figure 7.2). 

Grachev SA, Kropachev EV, Litvyakova Gl (1983) Synthesis of 5-S-cysteamine-6-hydroxythymine and evidence of its formation in the y radiolysis of aqueous solutions of thymine and cyste-amine. Bull Acad Sci USSR, Chem Ser (Engl Trans) 1595-1600 Grachev SA, Kropachev EV, Litvyakova Gl (1986) Addition of cysteamine to thymine and thymidine monophosphate initiated by y-irradiation. Bull Acad Sci USSR, Chem Ser (Engl Trans) 10 2178-2184... 

Thymidine triphosphate (TTP) depletion. AZT and thymidine (T) compete with each other for phosphorylation by thymidine kinase into AZT-monophosphate (AZT-MP) and thymidine-monophosphate (TMP), respectively (Fig. 13) (Lynx and McKee 2006). AZT can therefore decrease the formation of TMP and TTP, whose relative deficiency can then slow mtDNA replication (Lynx and McKee 2006). Interestingly the administratimi of uridine in animals and perhaps also in humans can prevent AZT, ddC, and d4T toxicity (Walker and Venhoff 2005 Banasch et al. 2006). Uridine administration may provide an alternate route for TTP synthesis, thus preventing TTP depletimi and the impairment of mtDNA replication (Lynx and McKee 2006). Furthermore, the uridine-induced restoration of mtDNA levels and respiratory chain functirm could improve the activity of dihydroorotate dehydrogenase, a key mitochondrial enzyme involved in pyrimidine synthesis. Thus, a virtuous circle is initiated by uridine supplementation (Setzer et al. 2008). 

A method similar in principle to that described here has recently been employed by Cheng and Prusoff (1974) for the estimation of deoxythymidylate kinase activity. The separation of ( C) thymidine triphosphate from ( C) thymidine monophosphate was achieved by washing the latter from DEAE-paper disks pretreated with unlabelled monophosphate nucleotide by a solution composed of 4 M formic acid and 1 mM ammonium formate. 

Kornberg s work on the biosynthesis of deoxyribonucleic acid has shown that enzymes in Escherichia coli extracts catalyze the formation of the 5-triphosphates of 2-deoxyadenosine, 2-deoxyguanosine, 2-deoxycyti-dine, and thymidine from the corresponding monophosphates in the presence of adenosine 5-triphosphate, but fail to catalyze phosphorylation of deoxyuridine 5-phosphate this finding could explain why uracil is not a constituent of deoxyribonucleic acid. 

Capecitabine is a pyrimidine analog. It is an oral systemic prodrug that is enzymatically converted to 5-fluorouracil (5-FU). Healthy and tumor cells metabolize 5-FU to 5-fluoro-2-deoxyuridine monophosphate (FdUMP) and 5-flu-orouridine triphosphate (FUTP). These metabolites cause cell injury by two different mechanisms. First, they inhibit the formation of thymidine triphosphate, which is essential for the synthesis of DNA. Second, nuclear transcriptional enzymes can mistakenly incorporate FUTP during the synthesis of RNA. This metabolic error can interfere with RNA processing and protein synthesis. Capecitabine is indicated in the treatment of resistant metastatic breast cancer alone or in combination with docetaxel, and colorectal cancer. 

FIGURE 49 2 Conversion of acyclovir to acyclovir triphosphate leading to DNA chain termination. Acyclovir is converted to the monophosphate (MP) derivative by a herpes virus thymidine kinase. Acyclovir-MP is dien phosphory-lated to acyclovir-DP and acyclovir-TP by cellular enzymes. Uninfected cells convert very little or no drug to the phos-phorylated derivatives. Thus, acyclovir is selectively activated in cells infected with herpesviruses that code for appropriate thymidine kinases. Incorporation of acyclovir-MP from acyclovir-TP into the primer strand during viral DNA replication leads to chain termination and formation of an inactive complex with the viral DNA polymerase. (Adapted from Elion, 1982, with permission.)... 

Furthermore, the 3 -azido functional moiety [—= N = N] predominantly prevents the formation of a 5, 3 -phosphodiester bond and, therefore, AZT gives rise to DNA-chain termination effectively, thereby producing an incomplete proviral DNA substantially. Besides, AZT-monophosphate also executes competitive inhibition of the specific cellular thymidylate kinase, thereby lowering the intracellular levels of thymidine triphosphate. It has been reported that the point mutations at multiple sites prevailing in the reverse transcriptase invariably causes resistance that may ultimately lead to a rather lower degree of affinity for AZT. ... 

This question was resolved by demonstrations of the intermediate formation of dTDP by Grav and Smellie (38) and by Ives 39). Ives studied the phosphorylation of thymidylate in extracts of the Novikoff rat hepatoma, in which nucleoside diphosphate kinase activity was about three orders of magnitude higher than that of the other enzymes of thymine metabolism present (thymidine and thymidylate kinases, thymidylate synthetase, and thymidylate phosphatase). Ives showed that dTDP was an intermediate in dTTP formation by a method which was independent of the monophosphate and diphosphate kinase reactions. When ATP[7 P] was employed as the phosphorylating agent in the tumor extracts, the dTTP product was labeled in both y- and /3-phosphates when ATP[/3- P] was employed, dTTP was essentially unlabeled ... 

Preparation of Radioactive FdUMP. [ H]- and [ CJFUdR are commercially available and conveniently converted to the 5 -monophosphate using thymidine kinase found in the 20-40% (NH4) 2SO4 fraction of the cell-free supernatant from Escherichia coli B. A solution (1.0 ml) containing about 1 mAf [ H]- or [ CJFUdR, 7.5 mAf ATP, 7.5 mAf MgCU, 30 mAf KF, 0.5 mg of bovine serum albumin, 70 mAf Tris-HCl (pH 7.8), and approximately 0.1 mg of the crude thymidine kinase preparation is incubated at 37°. The reaction is monitored by application of aliquotes to a micro DEAE-cellulose column (0.5 X0 8 cm) and stepwise elution with 3 ml of 5 mAf ammonium formate (pH 4.5) to elute unreacted nucleoside and 3 ml of 100 mAf of the same buffer to elute the nucleotide radioactivity is determined in each fraction to ascertain the extent of conversion. After the reaction is 80-90% complete (usually 60 min), the mixture is diluted to 20 ml with water, and purified on a DEAE-cellulose column (1X5 cm) by the method of Rustum and Schwartz- ... 

In bleomycin-mediated degradation of DNA, the predominant pathway for base release has been shown to involve formation of radicals at C-4 of the nucleotides, the sugar produced being 2-deoxypentos-4-ulose.253 By examination of three adducts formed with the trapping agent 2-methyl-2-nitrosopropane, abstraction of hydrogen atoms from C-1 , C-4 and C-5 in thymidine 5 -monophosphate was detected in its reaction with hydroxyl... 

An enzyme partially purified from yeast acts upon the nucleotides of adenine, guanine, and uridine to carry out transphosphorylation (Eqs. 33) to 36)] 112-116). Nucleoside monophosphate kinases have been partially purified from E. coli, which catalyzes the formation of triphosphates of deoxyadenine, deoxyguanosine, thymidine, and deoxycytidine from their corresponding 5 -monophosphates, in the presence of ATP 116, 117). 

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