Folate pyrimidine biosynthesis

These are pyrimidine derivatives and are effective because of differences in susceptibility between the enzymes in humans and in the infective organism. Anticancer agents based on folic acid, e.g. methotrexate, inhibit dihydrofolate reductase, but they are less selective than the antimicrobial agents and rely on a stronger binding to the enzyme than the natural substrate has. They also block pyrimidine biosynthesis. Methotrexate treatment is potentially lethal to the patient, and is usually followed by rescue with folinic acid (A -formyl-tetrahydrofolic acid) to counteract the folate-antagonist action. The rationale is that folinic acid rescues normal cells more effectively than it does tumour cells. 

A5-Methyltetrahydrofolate is the methyl-group donor substrate for methionine synthase, which catalyzes the transfer of the five-methyl group to the sulfhydryl group of homocysteine. This and selected reactions of the other folate derivatives are outlined in figure 10.15, which emphasizes the important role tetrahydrofolate plays in nucleic acid biosynthesis by serving as the immediate source of one-carbon units in purine and pyrimidine biosynthesis. 

FIGURE 9.7 Pyrimidine biosynthetic pathway. The pathway of pyrimidine biosynthesis involves sbc steps and results in the production of uridine 58-monophosphate. Folate is not used in this pathway The pathway commences with the transfer of the amide nitrogen of glutamine to bicarbonate to produce caibamy I phosphate. This molecule then reacts with aspartate to form the beginnings of the siK-membered pyrimidine ring. 

Deficiency of folate or vitamin Bn can cause hematological changes similar to hereditary orotic aciduria. Folate is directly involved in thymidylic acid synthesis and indirectly involved in vitamin Bn synthesis. Orotic aciduria without the characteristic hematological abnormalities occurs in disorders of the urea cycle that lead to accumulation of carbamoyl phosphate in mitochondria (e.g., ornithine transcarbamoylase deficiency see Chapter 17). The carbamoyl phosphate exits from the mitochondria and augments cytosolic pyrimidine biosynthesis. Treatment with allopurinol or 6-azauridine also produces orotic aciduria as a result of inhibition of orotidine-5 phosphate decarboxylase by their metabolic products. 

Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning. A laboratory manual. Cold Spring Harbor Laboratory Press, New York, pp 1.25-1.31 Santi DV, Danenberg PV (1984) Folates in pyrimidine biosynthesis. In Blakley RL, Benkovic SJ (eds) Folates and pterines, vol 1. Wiley, New York, pp 345-398 Schertel C, Conradt B (2007) C. elegans orthologs of components of the RB tumor suppressor complex have distinct pro-apoptotic functions. Development 134 3691-3701 Schweitzer BI, Dicker AP, Bertino JR (1990) Dihydrofolate reductase as a chemotherapeutic target. FASEB J 4 2441-2452... 

A key enzyme in pyrimidine biosynthesis, thymidylate synthetase, catalyzes the reductive methylation of 2 -deoxyuridylate (dUMP) to thymidylate (dTMP) with the concomitant conversion of 5,10-methylene-H4-folate to 7,8-dihydrofolate. The cofactor serves both as a 1-carbon carrier and a reductant. There is substantive evidence based on direct and indirect studies with the inhibitor 5 -fluoro-2 -deoxyuridylate, which will not be reviewed here, that the dUMP is covalently bound to the enzyme, possibly through a thiol group [59]. The intermediate ternary complex is hypothesized to have the following structure (10), where the CH2 unit is attached through the N-5 of H4-folate. The ring opening of the 5,10-methylene-H4-folate is... 

Methotrexate, a folate antagonist, interferes vAth nucleic acid biosynthesis. Would you expect it to inhibit purine or pyrimidine biosynthesis or both processes Explain. 

In spite of the large number of folate derivatives, all the folate coenzymes have a similar function in metabolism. The folate coenzyme activates 1-carbon units (methyl, methylene, and formyl) and facilitates their transfer from one metabolite to another. A 1-carbon unit is transferred principally in amino acid conversion and in purine and pyrimidine biosynthesis. Many of the reactions involving folate coenzymes are discussed in other chapters, so these reactions are reviewed only briefly here. 

Mechanistic aspects of the action of folate-requiring enzymes involve one-carbon unit transfer at the oxidation level of formaldehyde, formate and methyl (78ACR314, 8OMI2I6OO) and are exemplified in pyrimidine and purine biosynthesis. A more complex mechanism has to be suggested for the methyl transfer from 5-methyl-THF (322) to homocysteine, since this transmethylation reaction is cobalamine-dependent to form methionine in E. coli. 

Very little interest in the subject was shown until the late 1960s when Roth (69JMC227) undertook studies in chemotherapy based on inhibition of folate biosynthesis and function by certain 2,4-diaminothieno[2,3-[Pg.194]

A review on the potent inhibitors of de novo pyrimidine and purine biosynthesis summarizes the developments in this field <90MI 718-04) and another report is concerned mainly with the synthetic approaches to the various types of inhibitors of folate-dependent enzymes [Pg.729]

The metabolism of folic acid involves reduction of the pterin ting to different forms of tetrahydrofolylglutamate. The reduction is catalyzed by dihydtofolate reductase and NADPH functions as a hydrogen donor. The metabolic roles of the folate coenzymes are to serve as acceptors or donors of one-carbon units in a variety of reactions. These one-carbon units exist in different oxidation states and include methanol, formaldehyde, and formate. The resulting tetrahydrofolylglutamate is an enzyme cofactor in amino acid metabolism and in the biosynthesis of purine and pyrimidines (10,96). The one-carbon unit is attached at either the N-5 or N-10 position. The activated one-carbon unit of 5,10-methylene-H folate (5) is a substrate of T-synthase, an important enzyme of growing cells. 5-10-Methylene-H folate (5) is reduced to 5-methyl-H,j folate (4) and is used in methionine biosynthesis. Alternatively, it can be oxidized to 10-formyl-H folate (7) for use in the purine biosynthetic pathway. 

Folate analogues, such as methotrexate (Figure 27-3), are folate antagonists. They block production of FH2 and FH4 by dihydrofolate reductase and lead to diminished purine biosynthesis (inhibition of reactions 3 and 9 in Figure 27-8). Methotrexate also affects metabolism of amino acids and pyrimidine (inhibition of thymidylate synthesis) and inhibits DNA, RNA, and protein synthesis. It is effective in the treatment of breast cancer, cancer of the head and neck, choriocarcinoma, osteogenic sarcoma, and acute forms of leukemia. High doses of methotrexate can be tolerated provided that the patient also receives folinic... 

Folic acid or the folate coenzyme [6] is a nutritional factor both for the parasites and the hosts. It exists in two forms, viz. dihydro- and tetrahydrofolic acids [4,5] which act as cofactors involved in the transfer of one carbon units like methyl, hydroxymethyl and formyl. The transfer of a one carbon unit is associated with de novo synthesis of purines, pyrimidines and amino acids. Mammals can not synthesize folate and, therefore, depend on preformed dietary folates, which are converted into dihydrofolate by folate reductase. Contrary to this, a number of protozoal parasites like plasmodia, trypanosomes and leishmania can not utilize exogenous folate. Consequently, they carry out a de novo biosynthesis of their necessary folate coenzymes [12]. The synthesis of various folates follows a sequence of reactions starting from 2-amino-4-hydroxy-6-hydroxymethyldihydropteridine (1), which is described in Chart 4 [13,14]. 

Dihydrofolate reductase (DHFR, EC 1.5.1.3) is an essential enzyme required for normal folate metabolism in prokaryotes and eukaryotes. Its role is to maintain necessary levels of tetrahydrofolate to support the biosynthesis of purines, pyrimidines and amino acids. Many compounds of pharmacological value, notably methotrexate and trimethoprim, vork by inhibition of DHFR. Their clinical importance justified the study of DHFR in the rapidly evolving field of enzymology. Today, there is a vast amount of published literature (ca. 1000 original research articles) on the broad subject of dihydrofolate reductase contributed by scientists from diverse disciplines. We have selected kinetic, structural, and computational studies that have advanced our understanding of the DHFR catalytic mechanism with special emphasis on the role of the enzyme-substrate complexes and protein motion in the catalytic efficiency achieved by this enzyme. 

What are called antifolate drugs pertain in general to blocking the biosynthesis of purines and pyrimidines, the heterocyclic bases used in the further synthesis of DNA and RNA, where folic acid is required as a coenzyme (or vitamin) for the enzyme dihydrofolate reductase. The previously mentioned compound called methotrexate or amethopterin (4-amino-A °-methyl folic acid), being a structural analog of folate or folic acid, locks up the enzyme dihydrofolate reductase, which in turn blocks the synthesis of a thymidine nucleotide necessary for cell division. 

Aminopterin 4-amino-4-deoxyfolic acid (see Vitamins, folic acid), a cytostatic agent used in the management of some caneers. It inhibits the enzyme dihy-drofolate reductase, which reduces the folate coenzymes required for Purine biosynthesis (see) and thymine production (see Pyrimidine biosynth is), and thus prevents DNA synthesis However, it is toxic to nondividing cells as well, and cannot be tolerated indefinitely. Methotrexate (amethopterin) has similar activity. 

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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