Thymidilate synthesis

DHFR has been the object of intense research for the last few decades. The enzyme catalyses the NADPH-dependent reduction of 7,8-dihydrofolate to 5,6,7,8 tetrahydrofolate, a chemical which participates in the thymidilate synthesis cycle. Thus, the enzyme is crucial in the synthesis of thymidine monophosphate as well as in various one-carbon unit transfer reactions. 

Fig. 140. Evidence for the participation of DNA in the formation of crown galls. Both halves of the blade of a leaf of Bryophyllum daigremontianum are wounded and infected with Agrobacterium tumefaciens. The left half of the leaf is treated, in addition, with 5-fluorodeoxyuridine, a competitive inhibitor of thymidilate synthesis. On this side no tumors form, only a wound callus (modified from Bopp 1963).

Flucytosine is converted into the anti metabolite 5-fluorouracil that inhibits thymidilate synthetase, thereby disrupting DNA synthesis. It also interferes with protein synthesis by incorporation of fluorouracil into RNA in place of uracil. Although active against most Candida species, its spectrum of antifungal activity, overall, is narrow. Since resistance can develop rapidly it is usually coadministered with another agent and its main value is that it facilitates a reduction in the dose (and, presumably, the toxic effect) of amphotericin when co-prescribed in this way. The main adverse effects are marrow aplasia and hepatotoxicity. 

The concept that oestrogens stimulate cell proliferation directly arises mainly from the observation that physiological concentrations of oestrogens stimulate both the de novo and salvage pathways of DNA synthesis as well as inducing a number of enzymes intimately involved in DNA synthesis and including DNA polymerase, thymidine and uridine kinases, thymidilate synthetase and dihydrofolate reductase. There is evidence that some of these enzymes may be regulated at the transcrip-... 

In addition to the examples discussed above, the structures of many clinically and biologically important proteins have been determined. Some of these are amenable to immediate use in drug discovery efforts. These include enzymes such as dihydrofolate reductase (Jansy, 1988), and thymidilate synthase (Appelt et al, 1991) both of which are involved in the synthesis of DNA precursors. Their inhibition is a target in anticancer chemotherapies. Structures of other proteins such as cAMP-dependent protein kinase, acetylcholinesterase, and the glucocorticoid receptor add to the knowledge base that will open new avenues... 

The six one-carbon substituents of tetrahydrofolate. The oxidation state is the same in the one-carbon moiety of N -formyl, N -formyl, and N, N" -methenyl FH4. N -FormyI FH4 is required for de novo synthesis of purine nucleotides, whereas N, N -methylene FH4 is needed for formation of thymidilic acid. 

De novo synthesis of thymidilic acid (TMP) occurs exclusively by methylation of the C-5 of dUMP (Figure 27-28) by thymidylate synthase. The methylene group of, N -methylene FH4 is the source of the methyl group, and FH4 is oxidized to FH2. For sustained synthesis, FH4 must be regenerated by dihydrofolate reductase. Recall that deoxynucleotides are formed at the diphosphate level by ribonucleotide reductase thus, UDP is converted to dUDP, then to dUTP, dUMP is then generated mainly by dUTPase. 

With the aid of cytosine permease, flucytosine reaches the fungal cell where it is converted by cytosine deaminase into 5-fluorouracil [51-21-8]. Cytosine deaminase is not present in the host, which explains the low toxicity of 5-FC. 5-Fluorouracil is then phosphorylated and incorporated into RNA and may also be converted into 5-fluorodeoxyuridine monophosphate, which is a potent and specific inhibitor of thymidilate synthetase. As a result, no more thymidine nucleotides are formed, which in turn leads to a disturbance of the DNA-synthesis. These effects produce an inhibition of the protein synthesis and cell replication (1,23,24). 5-Fluorouracil cannot be used as an antimycotic. It is poorly absorbed by the fungus to begin with and is also toxic for mammalian cells. 

One-carbon metabolism is a series of biochemical reactions that transfer single methyl groups from one site to another, synthesize nucleotides (purines and thymidilate) for DNA synthesis and repair, and methylate DNA. 

Competitive Inhibition of enzymes. In the case of competitive inhibition, the structural analogs compete with the natural substrates for the active site of enzymes. The enzymes of nucleic acid, and protein, synthesis can also be inhibited in this way. Thus, the enzyme thymidilate synthetase is competitively inhibited by 5-fiuorodeoxyuridine, a derivative of 5-fluorouracil (Fig. 14). Thymidilate synthetase supplies d-thymidine-5 -phosphate (-thymidilate), a substance which is important to the synthesis of DNA. Inhibition of this enzyme thus brings DNA synthesis to a stop and, with it, all further development. We will discuss DNA synthesis and also thymidilate synthetase in more detail later (p. 165). 

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.

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