Deoxyuridylate formation

The enzyme assay contained in a total volume of 250 /xL 50 m M Tris-HCl buffer (pH 7.8), 50 mM 2-mercaptoethanol, 5 mil/ formaldehyde, and 1 mM methylenetetrahydrofolate. The reaction was initiated by adding 25 /xL of 1 mM deoxyuridylate. After 30 minutes at 37°C, the reaction was stopped by addition of 250 /xL of ice-cold 1 M perchloric acid. After 30 minutes in an ice bath, the reaction mixture was centrifuged and the resulting supernate was neutralized with 0.1 volume of 10 M KOH containing 1 M KH2PO4. The supernate obtained by centrifugation was injected onto the HPLC column. Formation of deoxythymidylate was linear with time up to 30 minutes, and with protein in the range of 80 to 720 /xg. 

Fluorouracil is a pyrimidine antimetabolite. The metabolism of fluorouracil in the anabolic pathway blocks the methylation reaction of deoxyuridylic acid to chymidylic acid. In this manner, fluorouracil interferes with the synthesis of DNA and to a lesser extent inhibits the formation of RNA. It is indicated in colon, rectum, breast, gastric, and pancreatic carcinoma (injection) multiple actinic or solar keratoses, and superficial basal-cell carcinoma (topical). 

The a- and p-L-fucosyl esters of UMP and dTMP have been prepared as anomeric mixtures by treating the nucleotides with orthoester (15) in DMF. In the absence of methylenetetrahydrofolate cofactor, thymidylate synthetase has been observed to catalyse nucleophilic addition reactions at the ethynyl moiety of 5-ethynyl-2 -deoxyuridylate. On the basis of isotope effects and other kinetic studies, it has been suggested that addition of a thiol group of the enzyme at C-6 of the nucleotides results in formation of the allenic intermediate (16), followed by addition of 2-mercaptoethanol, morpholine, or other nucleophiles at the jp-hybridized carbon of the allene, and subsequent elimination of the enzyme. 

Studies on the mechanism of the bacterial thymidylate synthetase reaction employing tritiated H4-folate showed that a hydrogen atom of 5,10-methylene H4-folate was transferred to the thymine methyl group and suggested the formation of a methylene bridge between folate and deoxyuridylate (9). 

Deoxycytidylate deaminase is evidently absent from a number of bacterial species, includii E. coli, S. typhimvrium, and B. subtilts. Accordingly, in these cells the deoxycytidine phosphates do not contribute to the synthesis of the thymidine phosphates in the manner described above for animal cells. However, enzymes catalyzing the deamination of dCTP have been demonstrated in E. ccU and S. typhimvrium the latter enzyme has been partly purified and shown to deaminate dCTP, but not CTP, dCDP, CDP, eytidylate, nor deoxycytidine (26). The formation of dUTP by this enzyme, followed by the action of the specific dUTP pyrophosphatase, would consitutue a route for the formation of deoxyuridylate ... 

In summary, the biochemical function of folate coenzymes is to transfer and use these one-carbon units in a variety of essential reactions (Figure 2), including de novo purine biosynthesis (formylation of glycinamide ribonucleotide and 5-amino-4-imidazole carboxamide ribonucleotide), pyrimidine nucleotide biosynthesis (methylation of deoxyuridylic acid to thy-midylic acid), amino-acid interconversions (the interconversion of serine to glycine, catabolism of histidine to glutamic acid, and conversion of homocysteine to methionine (which also requires vitamin B12)), and the generation and use of formate. 

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