Deoxyuridylate

Wataya Y, DV Santi (1975) Thymidylate synthetase catalyzed dehalogenation of 5-bromo- and 5-iodo-2 -deoxyuridylate. Biochem Biophys Res Commun 61 818-823. 

Thymine is found only in DNA but not in RNA. Deoxythymidylic acid is formed by methylation of deoxyuridylic acid this may be an indication that ribonucleosides were present on the young Earth before the deoxynucleosides. 

Crystal structure of Escherichia coli thymidylate synthase containing bound 5-fluoro-2 -deoxyuridylate and 10-propargyl-5,8-dideazafolate, J. Mol. Biol. 214 937 (1990). 

Ingraham H. Dickey L, Goulian M. DNA fragmentation and cytotoxicity from increased cellular deoxyuridylate. Biochemistry 1986 25 3225-3230. 

C. Deoxythymidylate (dTMP) is formed from 2 -deoxyuridylate (dUMP) in a one-carbon transfer by thymidylate synthetase (Figure 10-5). 

Figure 10-5. Conversion of deoxyuridylate (dUMP) to deoxythymidylate (dTMP) by thymidylate synthetase. The importance of folate coenzymes in this reaction is illustrated. NADPH + H provide the necessary reducing equivalents and serine is the source of one-carbon units present on N, N °-methylene tetrahydrofolate (THF).

Several possible mechanisms of resistance to 5-fluo-rouracU have been identified, including increased synthesis of the target enzyme, altered affinity of thymidy-late synthetase for FdUMP, depletion of enzymes (especially uridine kinase) that activate 5-fluorouracil to nucleotides, an increase in the pool of the normal metabolite deoxyuridylic acid (dUMP), and an increase in the rate of catabolism of 5-fluorouracil. 

A key step in DNA biosynthesis, that of conversion of deoxyuridylate (dUMP) to deoxythymidylate (dTMP), is catalyzed by thymidylate synthetase which uses (25) as cofactor. This reaction involves both the transfer of a one carbon unit at the formaldehyde level and hydride transfer (from C-6 of (25)) to produce 7,8-dihydrofolate (27) and dTMP... 

Figure 7.9 An outline of the synthesis of deoxythymidylate monophosphate (dTMP) from 2-deoxyuridylate monophosphate (dUMP). N5, N10-methylene-THF is the source of the methyl group in the conversion. Antifolates such as methotrexate inhibit DHFR...

Fluorodl is metabolized by the same route to 5-fluoro- — 2-deoxyuridylic acid (FUdRP)... 

U UDP UDPGA dUMP UdRP Uracil Uridine diphosphate Uridine diphosphate glucuronic acid Deoxyuridylate-5 -monophosphate Deoxyuridylic acid... 

Deoxyribose Deoxyuridine Deoxyuridylic acid deoxyuridine monophosphate (dUMP) Deoxyuridine diphosphate (dUDP) Deoxyuridine triphosphate (dUTP)... 

Figure 10.11 Biosynthesis of thymidylic acid from deoxyuridylic acid.

Fluorouracil [flure oh YOOR a sil] (5-FU), a pyrimidine analog, has a stable fluorine atom in place of a hydrogen atom at position 5 of the uracil ring. The fluorine interferes with the conversion of deoxyuridylic acid to thymidylic acid, thus depriving the cell of one of the essential precursors for DNA synthesis. 

Uracil, U Uridylic acid, UMP Deoxyuridylic acid, dUMP... 

Thymidylate synthetase catalyzes the conversion of deoxyuridylate to deoxy-thymidylate in a folate-dependent reaction. This enzyme is a target for many agents used in chemotherapy of cancer and treatment of infectious diseases. 

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. 

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. 

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