Purine nucleotides phosphorylation

The synthesis of purine nucleotides (1) starts from IMP. The base it contains, hypoxanthine, is converted in two steps each into adenine or guanine. The nucleoside monophosphates AMP and CMP that are formed are then phos-phorylated by nucleoside phosphate kinases to yield the diphosphates ADP and GDP, and these are finally phosphorylated into the triphosphates ATP and CTP. The nucleoside triphosphates serve as components for RNA, or function as coenzymes (see p. 106). Conversion of the ribonucleotides into deoxyribo-nucleotides occurs at the level of the diphosphates and is catalyzed by nucleoside diphosphate reductase (B). 

As the first committed step in the biosynthesis of AMP from IMP, AMPSase plays a central role in de novo purine nucleotide biosynthesis. A 6-phosphoryl-IMP intermediate appears to be formed during catalysis, and kinetic studies of E. coli AMPSase demonstrated that the substrates bind to the enzyme active sites randomly. With mammalian AMPSase, aspartate exhibits preferred binding to the E GTPTMP complex rather than to the free enzyme. Other kinetic data support the inference that Mg-aspartate complex formation occurs within the adenylosuccinate synthetase active site and that such a... 

These compounds inhibit synthesis of purine nucleotides, which are made up of purine bases and phosphorylated ribose. Both compounds must be transformed into nucleotides by adding a phosphoribosyl fragment. 

Restriction enzymes which cleave double-stranded DNA, making staggered cuts which leave the 5 -end of each strand extended. Double-stranded DNA with protruding 5 -ends is more efficiently phosphorylated by polynucleotide kinase than DNA with flush or recessed 5 -ends. The fore-shortened 3 -ends can also be labelled by extending them with 32P-labelled nucleoside triphosphates complementary to bases in the extended template strand using DNA polymerase. Y—pyrimidine nucleotide, R—purine nucleotide, N—any nucleotide. A complete list of commercially obtainable restriction endonucleases is given in Appendix III. 

Purine nucleotides are probably best made by phosphorylation of the corresponding nucleosides. In recent years phosphoryl chloride has proved most convenient for this purpose and may be used with an unprotected nucleoside to give yields of about 50% (B-78MI40903, p. 827). Yields of purine nucleotides up to 60% may also be obtained using a phosphotransferase and a suitable phosphate donor with the unprotected nucleoside. Typical preparations of this type using an enzyme from wheat shoots and p-nitrophenyl phosphate as a phosphate donor have been described (B-78MI40903, p. 955). 

By analogy with the purine nucleotides of ribosenucleic acid it seems probable that the phosphoryl group is situated at position (3) of the ribose chain, but no direct experimental evidence has yet appeared. Uridylic acid has been synthesized by the action of phosphorus oxychloride on uridine in the presence of barium hydroxide solution and by phosphorylation of 5-trityl-uridine but, unfortunately, these syntheses shed no light on the question. Furthermore, the fact that uridylic acid condenses with trityl chloride to give a trityl-uridylic acid does not prove its structure, though it suggests that position (5) of uridylic acid is free. This tentative conclusion is strengthened by the observation that neither of the pyrimidine nucleotides forms a complex with boric acid. 

See also Substrate Level Phosphorylation, Nucleotide Salvage Synthesis, De Novo Biosynthesis of Purine Nucleotides, Nucleotides, Guanine, G Proteins and Signal Transduction... 

Deoxyguanosine kinase is a mitochondrial enzyme catalyzing phosphorylation of deoxyguanosine in purine nucleotide salvage biosynthesis. 

Pyrimidine bases are normally salvaged by a two-step route. First, a relatively nonspecific pyrimidine nucleoside phosphorylase converts the pyrimidine bases to their respective nucleosides (Fig. 41.17). Notice that the preferred direction for this reaction is the reverse phosphorylase reaction, in which phosphate is being released and is not being used as a nucleophile to release the pyrimidine base from the nucleoside. The more specific nucleoside kinases then react with the nucleosides, forming nucleotides (Table 41.2). As with purines, further phosphorylation is carried out by increasingly more specific kinases. The nucleoside phosphorylase-nucleoside kinase route for synthesis of pyrimidine nucleoside monophosphates is relatively inefficient for salvage of pyrimidine bases because of the very low concentration of the bases in plasma and tissues. 

The purine nucleotides IMP and GMP are produced in a chemical process step by phosphorylation of the corresponding nucleosides, which are obtained by fermentation [278- 280]. The phosphorylation agent is phosphoryl chloride [281]. The traditional purine nucleoside production strains are mutants of B. amyloliq-uefaciens, in the older literature designated as B. subtUis K, but B. subtUis strains might also be used at industrial scale. 

The nucleoside monophosphates are converted to the triphosphates (the direct precursors of RNA) by two kinase reactions These kinases have a low specificity, and they catalyse the phosphorylation of nucleotides of adenine, guanine and the pyrimidines (Fig. 3). An alternative route for the synthesis of purine nucleotides is the Salvage pathway (see). 

Historically, the first route of purine nucleotide synthesis to be studied in detail was that involving the phosphorylation of adenosine by ATP. More recently, evidence has been presented for the existence in animal tissues of two other purine nucleoside kinases. The general reaction is... 

So far as is known, pyrimidine ribo- and deoxyribonucleotides are de-phosphorylated by the nucleotidases and phosphatases described in Chapter 10 as acting on purine nucleotides. Although a number of potential de-phosphorylating enzymes may be avaUable in most cells, the relative quantitative importance of each is not known. 

It has been reported recently that mycophenolic acid inhibits DNA synthesis by fibroblasts. This is due to an effect of the antibiotic on an early stage of the biosynthesis of purine nucleotides blocking the enzyme IMP-NAD oxidoreductase . Toyocamycin and tubercidin were found to cause accumulation in mammalian cells of the 45 S RNA precursor of the 28 S and 18 S ribosomal RNA. The antibiotic sangivamycin was shown to be phosphorylated by enzyme extracts from mouse liver. Sangivamycin triphosphate was shown to be a substrate for ribonucleotide reductase but not for deamination . ... 

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