Pyrimidine-nucleoside 5’-monophosphates

Nitrogenous base plus sugar moiety are called nucleosides. Ribonucleic acids (RNA) resemble DNA in that nucleoside monophosphates are joined through phosphodiester bonds. RNAs differ in that the sugars are p-D-ribose units and the pyrimidine uracil is found in place of thymine. Molecular structures and nomenclature for nitrogenous bases, nucleosides, and nucleotides are delineated in Table 2.2. 

The nucleotides are among the most complex metabolites. Nucleotide biosynthesis is elaborate and requires a high energy input (see p. 188). Understandably, therefore, bases and nucleotides are not completely degraded, but instead mostly recycled. This is particularly true of the purine bases adenine and guanine. In the animal organism, some 90% of these bases are converted back into nucleoside monophosphates by linkage with phosphori-bosyl diphosphate (PRPP) (enzymes [1] and [2]). The proportion of pyrimidine bases that are recycled is much smaller. 

Orengo, A. Maness, P. Pyrimidine nucleoside monophosphate kinase from rat liver and rat Novikoff ascites hepatoma (EC 2.7.4.14). Methods Enzy-mol., 51, 321-331 (1978)... 

Scott, E.M. Wright, R.C. Kinetics and equihbria of pyrimidine nucleoside monophosphate kinase from human erythrocytes. Biochim. Biophys. Acta, 571, 45-54 (1979)... 

Maness, P. Orengo, A. Activation of rat liver pyrimidine nucleoside monophosphate kinase. Biochim. Biophys. Acta, 429, 182-190 (1976)... 

Teng, Y.-S. Chen, S.-H. Scott, C.R. Human erythrocyte pyrimidine nucleoside monophosphate kinase. Partial purification and properties of two allelic gene products. J. Biol. Chem., 251, 4179-4183 (1976)... 

Seagrave, J. Reyes, P. Pyrimidine nucleoside monophosphate kinase from rat bone marrow cells chromatographic, electrophoretic, and sedimentation behavior of active and inactive enzyme forms. Arch. Biochem. Biophys., 247, 76-83 (1986)... 

Kohno, H. Kumagai, H. Tochikura, T. Purification and properties of pyrimidine nucleoside monophosphate kinase from baker s yeast. Agric. Biol. Chem., 47, 19-24 (1983)... 

Again we allow for competition between the proton and metal ion for the basic N(l) sites on purine and N(3) sites on pyrimidine 5 -nucleoside monophosphates to find for neutral solutions at pH 7.4 the sequence... 

Fig. 15-15 The de novo pyrimidine biosynthetic pathway. CAP, carbamoyl phosphate CA-asp, /V-carbamoyl-L-aspartate DHO, L-dihydroorotate Oro, orotate OMP, orotidine 5 -monophosphate. Enzymes (1) carbamoyl phosphate synthetase II (2) aspartate transcarbamoylase (3) dihydroorotase, (4) dihydroorotate dehydrogenase (5) orotate phosphoribosyltransferase (6) OMP decarboxylase (7) nucleoside monophosphate kinase (8) nucleoside diphosphate kinase (9) CTP synthetase.

The activity is found in erythrocytes, platelets, and lymphocytes, and determination of its value aids in diagnosis of some blood disorders. In this assay, which can readily be used for purine and pyrimidine 5 - and 3 -nucleotidase activities, the nucleoside monophosphate (the substrate) was separated from the nucleoside (the product) using ion-pair reversed-phase HPLC with a mobile phase of 5% methanol-5 mAf potassium dihydrogen phosphate 0.25 mAf 1-decanesulfonic acid was also added to the mobile phase. The elution was carried out at room temperature and the eluent monitored at 254 nm. 

How is the other major pyrimidine ribonucleotide, cytidine, formed It is synthesized from the uracil base of UMP, but UMP is converted into UTP before the synthesis can take place. Recall that the diphosphates and triphosphates are the active forms of nucleotides in biosynthesis and energy conversions. Nucleoside monophosphates are converted into nucleoside triphosphates in stages. First, nucleoside monophosphates are converted into diphosphates by specific nucleoside monophosphate kinases that utilize ATP as the phosphoryl-group donor (Section 9.4). For example, UMP is phosphorylated to UDP by UMP kinase. 

Pyrimidine Phosphoribosyl Transferase Pyrimidine base + PRPP —> Nucleoside monophosphate + PPi... 

Modified Celluloses AC-10 CM DEAE ECTEOLA PEI Antioxidants Inorganics, metal ions Nucleoside mono-, di-, and tri-phosphates Purines, pyrimidines, nucleosides Monophosphate nucleosides... 

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. 

S ATP + deoxyadenosine <1, 3, 5, 6> (<1> involved in biosynthesis of nucleoside monophosphates from preformed deoxyribonucleosides [4] <3> key anaboHc enzyme for activation of purine and pyrimidine deoxyriho-nucleosides as weU as cytosine arabinoside and other anti-tumour drugs [9] <5> involved in nucleoside metabolism [11] <6> dGK/dAK plays an essential role in generating the dexyribonucleotide precursors, dGTP and dATP, for DNA metabolism [12]) (Reversibility <1, 3, 5, 6> [4, 9, 11, 12]) [4, 9, 11, 12]... 

Nucleoside monophosphates are synthesized de novo in the course of Purine biosynthesis (see) and Pyrimidine biosynthesis (see). They are then phosphoryla-ted stepwise by the action of kinases, to produce nucleoside di- and triphosphates. The 2-deoxyribose moiety is formed by reduction of the ribose in ribonucleotides (see Ribonucleotide reductase). Reduction of free ribose to 2-deoxyribose does not occur in vivo. 

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). 

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). 

Pyrimidine 5 -nucleotidase (P5N) is a unique enzyme that was recognized from studies of families with relatively common hemolytic disorders. The enzyme catalyzes the hydrolytic dephosphorylation of pyrimidine 5 -nucleotides but not purine nucleotides. The role of this enzyme is to eliminate RNA and DNA degradation products from the cytosol during erythroid maturation by conversion of nucleotide monophosphates to diffusible nucleosides. P5N is inhibited by lead, and its activity is considered to be a good indicator of lead exposure (PI). 

The HPLC-MS/MS assay was also successfully applied to the measurement of UV-induced dimeric pyrimidine photoproducts [123, 124]. The latter lesions were released from DNA as modified dinucleoside monophosphates due to resistance of the intra-dimer phosphodiester group to the exonuclease activity during the hydrolysis step [125, 126]. The hydrolyzed photoproducts exhibit mass spectrometry and chromatographic features that allow simultaneous quantification of the three main classes of photolesions, namely cyclobutane dimers, (6-4) photoproducts, and Dewar valence isomers, for each of the four possible bipyrimidine sequences. It may be added that these analyses are coupled to UV detection of normal nucleosides in order to correct for the amount of DNA in the sample and obtain a precise ratio of oxidized bases or dimeric photoproducts to normal nucleosides. 

Trifluridine (Viroptic) is a fluorinated pyrimidine nucleoside that has in vitro activity against HSV-1 and HSV-2, vaccinia, and to a lesser extent, some adenoviruses. Activation of trifluridine requires its conversion to the 5 monophosphate form by cellular enzymes. Trifluridine monophosphate inhibits the conversion of deoxyuridine monophosphate (dUMP) to deoxythymidine monophosphate (dTMP) by thymidylate synthetase. In addition, it competes with deoxythymidine triphosphate (dTTP) for incorporation by both viral and cellular DNA polymerases. Trifluridine-resistant mutants have been found to have alterations in thymidylate synthetase specificity. 

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