Pyrimidine nucleotides Nucleotide , synthesis

Methotrexate One of a number of antifolate drugs. Methotrexate is an analog of folate which competitively inhibits dihydrofolate reductase. Since a plentiful supply of THF is required for ongoing synthesis of the pyrimidine nucleotide thymidylate, synthesis of this nucleotide is inhibited resulting in decreased DNA synthesis. 

Figure 34-7 summarizes the roles of the intermediates and enzymes of pyrimidine nucleotide biosynthesis. The catalyst for the initial reaction is cytosolic carbamoyl phosphate synthase II, a different enzyme from the mitochondrial carbamoyl phosphate synthase I of urea synthesis (Figure 29-9). Compartmentation thus provides two independent pools of carbamoyl phosphate. PRPP, an early participant in purine nucleotide synthesis (Figure 34-2), is a much later participant in pyrimidine biosynthesis.

While mammahan cells reutilize few free pyrimidines, salvage reactions convert the ribonucleosides uridine and cytidine and the deoxyribonucleosides thymidine and deoxycytidine to their respective nucleotides. ATP-dependent phosphoryltransferases (kinases) catalyze the phosphorylation of the nucleoside diphosphates 2 "-de-oxycytidine, 2 -deoxyguanosine, and 2 -deoxyadenosine to their corresponding nucleoside triphosphates. In addition, orotate phosphoribosyltransferase (reaction 5, Figure 34-7), an enzyme of pyrimidine nucleotide synthesis, salvages orotic acid by converting it to orotidine monophosphate (OMP). 

Since the end products of pyrimidine catabolism are highly water-soluble, pyrimidine overproduction results in few clinical signs or symptoms. In hypemricemia associated with severe overproduction of PRPP, there is overproduction of pyrimidine nucleotides and increased excretion of p-alanine. Since A, A -methyl-ene-tetrahydrofolate is required for thymidylate synthesis, disorders of folate and vitamin Bjj metabofism result in deficiencies of TMP. 

Twenty different amino acids are required for the synthesis of the cellular complement of proteins thus, there must be at least 20 distinct codons that make up the genetic code. Since there are only four different nucleotides in mRNA, each codon must consist of more than a single purine or pyrimidine nucleotide. Codons consisting of two nucleotides each could provide for only 16 (4 ) specific codons, whereas codons of three nucleotides could provide 64 4 ) specific codons. 

Not all analogues become active against cancer cells through incorporation into nucleic acid. Some analogues block the synthesis of normal purine and pyrimidine nucleotides for example, 8-azaguanine blocks guanosine monophosphate (GMP) synthesis and 6-mercaptopurine inhibits adenosine monophosphate (AMP) syn-thesis. 

The catalytically active RNA species (ribozymes) have been shown in recent years to undergo an unbelievable range of reactions. This led to the question as to whether they are also involved in nucleotide syntheses. Unrau and Bartel (1998) have reported successful nucleotide syntheses carried out using ribozymes (see Sect. 6.5). It was possible to isolate in vitro selected RNA which acted catalytically in the synthesis of a pyrimidine nucleotide it remains unclear whether these results are important for biogenesis. 

Another important vitamin is folate, which is required for purine and pyrimidine nucleotide synthesis. Since folate and its derivatives are generally lipo-phobic anions, they do not traverse biological membranes via simple diffusion but rather have to be taken up into the cells by specific transport processes... 

Reisner M, Carmeli S, Werman M, Sukenik A (2004) The cyanobacterial toxin cylindrospermopsin inhibits pyrimidine nucleotide synthesis and alters cholesterol distribution in mice. 

In many cells, the capacity for de novo synthesis to supply purines and pyrimidines is insufficient, and the salvage pathway is essential for adequate nucleotide synthesis. In patients with Lesch-Nyhan disease, an enzyme for purine salvage (hypoxanthine guanine phosphoribosyl pyrophosphate transferase, HPRT) is absent. People with this genetic deficiency have CNS deterioration, mental retardation, and spastic cerebral palsy associated with compulsive self-mutilation, Cells in the basal ganglia of the brain (fine motor control) normally have very high HPRT activity. These patients also all have hyperuricemia because purines cannot be salvaged. 

Figure 8.29 The initial reactions of glutamine metabolism in kidney, intestine and cells of the immune system. The initial reaction in all these tissues is the same, glutamine conversion to glutamate catalysed by glutaminase the next reactions are different depending on the function of the tissue or organ. In the kidney, glutamate dehydrogenase produces ammonia to buffer protons. In the intestine, the transamination produces alanine for release and then uptake and formation of glucose in the liver. In the immune cells, transamination produces aspartate which is essential for synthesis of pyrimidine nucleotides required for DNA synthesis otherwise it is released into the blood to be removed by the enterocytes in the small intestine or by cells in the liver.

Tumour cells also require glutamine as a fuel for energy generation and as a precursor for the synthesis of purine and pyrimidine nucleotides for DNA and RNA synthesis. The roles and importance of glutamine in tumour cells and possible competition between the cells for glutamine are discussed in Chapter 21. The pathway for the metabolism of glutamine is similar to that in the immune cells. 

DNA and RNA synthesis De novo formation of purine and pyrimidine nucleotide Nucleoside diphosphate reductase Thymidylate synthase Polymerase reactions Chapter 20... 

Figure 17.38 A simple diagram illustrating the two roles of glutamine (i) generation of ATP, via glutaminolysis, (ii) formation of purine and pyrimidine nucleotides, for the synthesis of nucleic acids in proliferating cells (Chapter 20). ( represents the carbon atoms of glutamine, one of which is released as CO2 and the others are converted to aspartate, via part of the Krebs cycle (Chapter 9). (N) represents the amide nitrogen of glutamine.

The two classes of nucleotide that must be synthesised are the pyrimidine and purine ribonucleotides for RNA synthesis and the deoxyribonucleotides for DNA synthesis. For the original sources of the nitrogen atoms in the bases of the pyrimidine and purine nucleotides, see Figure 20.7. The pathway for the synthesis of the pyrimidine nucleotides is... 

Figure 20.8 Summary of pathways for de novo synthesis of purine and pyrimidine nucleotides. C represents transfer of a single carbon atom (a one-carbon transfer). Details are provided in Appendix 20.1. IMP - inosine monophosphate. For thymi-dylate synthesis, see Figure 20.12a.

A different, simpler , pathway is involved in the synthesis of pyrimidine nucleotides. A pyrimidine base (orotate), is synthesised first. Then the ribose is added from 5-phosphoribosyl 1-pyrophosphate. The two precursors for the formation of orotate are carbamoylphosphate and aspartate, which form carbamoyl aspartate, catalysed by aspartate carbamoyltransferase. 

Figure 20.9 The positions in the pathway for de novo pyrimidine nucleotide synthesis where GLUCOSE provides the ribose molecule and GLUTAMINE provides nitrogen atoms. Glucose forms ribose 5-phosphate, via the pentose phosphate pathway (see chapter 6), which enters the pathway, after phosphorylation, as 5-phospho-ribosyl 1-pyrophosphate. Glutamine provides the nitrogen atom to synthesise carbamoylphos-phate (with formation of glutamate), and also to form cytidine triphosphate (CTP) from uridine triphosphate (UTP), catalysed by the enzyme CTP synthetase. It is the amide nitrogen of glutamine that is the nitrogen atom that is provided in these reactions.

Antimetabolites (inhibition of purine and pyrimidine nucleotide synthesis) Methotrexate Folic acid antagonist, inhibits tetrahydrofolate reductase and therefore dTMP synthesis 6-Mercaptopurine Interferes with purine synthesis 5-Fluorouracil Inhibits dTMP synthesis ... 

The fact that many agents which interrupt the synthesis of pyrimidine nucleotides from orotic acid in animals can also inhibit the growth of experimental neoplasms suggests a search for additional antimetabolites whose locus of action is in this metabolic sequence. Two in vitro biological screening systems were developed for this purpose [202—207]. From a study of systems with oxidative energy sources, 5-bromo-[208—209] (Villa), 5-chloro-[210] (Vlllb) and 5-diazo-orotic acid [211] (IX) were found to inhibit the conversion of orotic acid to the uridine nucleotides by 40—100 per cent [202]. 

A common intermediate for all the nucleotides is 5-phosphoribosyl-l-diphosphate (PRPP), produced by successive ATP-dependent phosphorylations of ribose. This has an a-diphosphate leaving group that can be displaced in Sn2 reactions. Similar Sn2 reactions have been seen in glycoside synthesis (see Section 12.4) and biosynthesis (see Box 12.4), and for the synthesis of aminosugars (see Section 12.9). For pyrimidine nucleotide biosynthesis, the nucleophile is the 1-nitrogen of uracil-6-carboxylic acid, usually called orotic acid. The product is the nucleotide orotidylic acid, which is subsequently decarboxylated to the now recognizable uridylic acid (UMP). 

De novo synthesis of purines and pyrimidines yields the monophosphates IMP and UMP, respectively (see p. 188). All other nucleotides and deoxynucleotides are synthesized from these two precursors. An overview of the pathways involved is presented here further details are given on p. 417. Nucleotide synthesis by recycling of bases (the salvage pathway) is discussed on p. 186. 

M. Nomura, S. Shuto, M. Tanaka, T. Sasaki, S. Mori, S. Shigeta, and A. Matsuda, Nucleosides and Nucleotides. 185. Synthesis and biological activities of 4 -a-C-branched-chain sugar pyrimidine nucleosides, J. Med. Chem., 42 (1999) 2901-2908. 

Pemetrexed is chemically similar to folic acid. It inhibits three enzymes used in purine and pyrimidine synthesis - thymidylate synthetase, dihydrofolate reductase, and glycinamide ribonucleotide formyl transferase. By inhibiting the formation of precursor purine and pyrimidine nucleotides, pemetrexed prevents the formation of DNA and RNA. In 2004 it was approved for treatment of malignant pleural mesothelioma and as a second-line agent for the treatment of non-small cell lung cancer. Adverse effects include gastrointestinal complaints, bone marrow suppression, alopecia, allergic and neurotoxic reactions. 

The pyrimidine antagonists inhibit the biosynthesis of pyrimidine nucleotides or interfere with vital cellular functions, such as the synthesis or function of nucleic acids. The analogues of deoxycytidine and thymidine that are used are inhibitors of DNA synthesis while 5-fluorouracil (5-FU) an analogue of uracil, is an inhibitor of both RNA function and of the synthesis of thymidylate (see Fig. 2). PALA (N-phosphonoacetyl-L-aspartate), an inhibitor of as-... 

Purine and pyrimidine nucleotides are essential for a vast number of biological processes such as the synthesis of RNA, DNA, phospholipids, glycogen, and the si-alylation and glycosylation of proteins. Both purines and pyrimidines can be synthesized de novo in mammalian cells through multistep processes. In addition to the de novo synthesis, purine nucleotides can also be synthesized via the salvage of... 

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