Uracil thymidylate from

Uracil, produced by the pyrimidine synthesis pathway, is not a component of DNA. Rather, DNA contains thymine, a methylated analog of uracil. Another step is required to generate thymidylate from uracil. Thymidylate synthase catalyzes this finishing touch deoxyuridylate (dUMP) is methylated to thymidylate (TMP). As will be discussed in Chapter 27. the methylation of this nucleotide facilitates the identification of DNA damage for repair and, hence, helps preserve the integrity of the genetic information stored in DNA. The methyl donor in this reaction is N, N methylenetetrahydrofolate rather than -S-adenosylmethionine. 

Thymine derivatives - 5-[7V-(2-Amino-4-hydroxy-6-methyl-5-pyrimidinyl-propyl)-p-carboxyanilinomethyl] uracil (XXXIII) was synthesized for study as a possible intermediate in the enzymatic synthesis of thymidylate. It is active as an enzyme inhibitor against thymidylate synthetase isolated from E. coli [298]. Certain thymine derivatives containing a 2-thioimidazole moiety (XXXIV, R = alkyl) inhibit growth of Ehrlich ascites carcinoma (fluid form) in mice [299]. 

The catalytic mechanism of the pyrimidine-5 methylation in nucleic acids is more complex as it involves covalent catalysis. The mechanism is common for numerous DNA/RNA cytosine and uracil MTases as well as for thymidylate synthase (although the latter uses tetrahydrofolic acid as the methyl donor) and has been studied in detail in several systems (50). Here, the cytosine-5 methylation in DNA is presented as an example (see Fig. 4a). The C5-position of cytosine, which is part of an aromatic ring, does not carry sufficient nucleophilicity for a direct methyl group transfer. The continuity of the aromatic system is disrupted by a nucleophilic attack of thiolate (from a conserved... 

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... 

Consistent with the metabolic data, there is no dihydrofolate reductase/thymidylate synthase activity (75). Thymidine is salvaged by a phosphotransferase. Uracil PRTase, uridine phosphorylase, cytidine deaminase and uridine and cytidine phosphotransferases were found. The major pyrimidine salvaged is uracil via its PRTase. The lack of incorporation of salvaged uracil into DNA and the lack of thymidylate synthase indicates that this parasite cannot synthesize TMP from UMP. It is dependent on salvage for its thymidine requirements. This parasite possesses a hydroxyurea-resistant ribonucleotide reductase and can synthesize deoxycytidine nucleotides from cytidine nucleotides. 

The heterocyclic bases in RNA are adenine, guanine, cytosine, and uracil (A, G, C, and U) the heterocyclic bases in DNA are adenine, guanine, cytosine, and thymine (A, G, C, and T). In other words, the heterocyclic bases in RNA and DNA are the same, except that RNA contains U s, whereas DNA contains T s (Sections 27.1 and 27.14). The T s used for the biosynthesis of DNA are synthesized from U s by thymidylate synthase, an enzyme that requires A, A °-methylene-THF as a coenzyme. Even though the only structural difference between a U and a T is a methyl group, a T is synthesized by first transferring a methylene group to a U. 

FU, floxuridine (5-fluoro-2 -deoxyuridine, or 5-FUdR), aTK/idoxuridine (5-iodo-deoxyuridine) (see Chapter 49). lododeoxyuridine behaves as an analog of thymidine, and its primary biological action results from its phosphorylation and ultimate incorporation into DNA in place of thymidylate. In 5-FU, the smaller fluorine allows the molecule to mimic uracil biochemically. However, the fluorine-carbon bond is much tighter than that of C—H and prevents the methyla-tion of the 5 position of 5-FU by TS. Instead, in the presence of the physiological cofactor 5,... 

Formation of a covalent adduct with an enzymatic nucleophile increases the reactivity of the substrate, which facilitates a reaction that is distinct from adduct formation. Examples of this include the thymidylate synthetase, in which the sulfhydryl of an active site Cys adds to C6 of the uracil. This breaks the aromaticity and adds electron density to increase the nucleophilicity of C5, thereby facilitating transfer of a methylene equivalent from tetrahydrofolate (Carreras and Santi, 1995). 

The methylation of dUMP by thymidylate synthetase is mediated by tetrahydrofolate and required for the formation of dTMP. Its loss by mutation creates a thymine-requiring auxotroph [127-129], Another alternate path for the synthesis of dTTP has been revealed by the use of the multimutational cytidine auxotrophs described above. Differential labeling of cytidine and uracil precursors in both E. coli [126] and Salmonella [125] shows that dTTP arises independently from either. Indeed, the contribution from cytidine is three to four times that from uracil. It is suggested that the second path involves a methylation of dCTP, then a deamination to give dTTP. One wonders why this pathway is not available in the thymine-requiring mutants which lack TMP synthetase. One explanation is that TMP synthetase may also function for the methylation of dCTP however, this has not yet been found to serve as an alternate substrate [130]. Two pathways may indeed exist in B. suhtilis, as indicated by the fact that two separate mutations are needed to obtain a thymine-requiring mutant [131]. 

The megaloblast differs from the normoblast in its increased amount of cytoplasmic RNA. The DNA and the nuclear RNA are apparently unchanged, and therefore the thymine-uracil ratio is decreased. From such observations, it was concluded that the megalo-blasts are red blood cell precursors whose interphase is abnormally prolonged because of interference with DNA synthesis. On the basis of the alteration of thymine-uracil ratios, it was assumed that the metabolic block would be located on the passage from deoxyuri-dylic acid to thymidylic acid. Such conclusions are unwarranted at present because the role of vitamin B12 in the methylation of thymidine is not established. Furthermore, it is not certain that cytoplasmic RNA acts as a reservoir for DNA as the above hypothesis implies. 

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