Pyrimidine-5’-nucleotidase specificity

The results obtained by electrophoresis and quantitative assay are complementary and support the hypothesis that there are two types of pyrimidine nucleotidase in human erythrocytes. The patient shows deficient activity with UMP, CMP and dCMP, the preferred substrates of rodent UMPH-1 and this agrees with the conclusion that rodent UMPH-1 and the human pyrimidine-specific nucleotidase identified by Valentine et al. (1) and purified by Torrance et al. (3) are homologous. 

Purines and pyrimidines in excess of cellular requirements can be degraded. The extent of degradation depends on the organism. Humans cannot degrade purines beyond uric acid because we lack the enzyme uricase, which splits the purine ring to form allantoin. In humans excess AMP is deaminated to IMP by the action of a specific deaminase. IMP is then hydrolyzed by 5 -nucleotidase to form inosine. Inosine and guanine are oxidized to urate as follows ... 

Pyrimidine-5 -nucleotidases are a group of enzymes dephos-phorylating pyrimidine nucleotides to the corresponding nucleosides. The pyrimidine bases diffuse out of the erythrocyte and the phosphates are retained. Pyrimidine phosphates are present on ribosomes of erythroblasts and reticulocytes, but there are normally no pyrimidines in mature RBCs. Two cytoplasmic forms of the enzyme were identified in the erythrocyte, P5 N-1 and P5 N-2. These enzymes are encoded by different genes and have different molecular properties and substrate specificities. Since there are no known disorders associated with deficiency of P5 N-2, this enzyme will not be further discussed here. 

FIG. 6.13 Mammalian pyrimidine salvage and interconversion pathways. Enzymes listed in Figs 6.13-6.17 are as follows (1) deoxyCMP deaminase (2) thymidylate synthase (3) ribonucleotide reductase (4) deoxyuridine triphosphatase (5) CTP synthetase (6) nucleotide kinase (7) deoxyTMP kinase (8) nucleotide diphosphokinase (9) non-specific phosphatase or nucleotidase (10) cytidine kinase (11) pyrimidine phos-phorylase or hydrolase (12) uracil PRTase (13) cytidine deaminase (14) thymidine kinase (15) cytidine phosphotransferase (16) uridine phosphotransferase (17) thymidine phosphotransferase (18) deoxyribo-nucleotide phosphotransferase (19) cytosine PRTase. 

The drug is a pyrimidine nucleoside antimetabolite which is cytotoxic to a plethora of cell-types. Precisely the induction of the enzyme nucleotidase into DNA inhibits polymerization via termination of strand synthesis. It is S-phase specific. 

In addition to the relatively unspecific P, some P show a high specificity, e.g. 5 -nucleotidase from snake venom and 3 -nucleotidase from rye, which catalyse the hydrolysis of 5 -nucleotides or 3 -nucleotides to their respective nucleosides (see Purine degradation, Pyrimidine degradation). 

Hereditary deficiency of erythrocyte pyrimidine 5 nucleotidase results in a chronic haemolytic anaemia. The red cells show basophilic stippling and contain a markedly increased content of nucleotides, 3-6 times greater than normal, and 65 to 80% of nucleotides are pyrimidine in type (Valentine, et al., 1974 Torrance and Whittaker, 1979). Pyrimidine 5 nucleotidase is unique amongst the 5 nucleotidases in its strict substrate specificity for pyrimidine nucleoside 5 monophosphates, its pH profile, and for its cytosolic localisation (Paglia and Valentine, 1975). 

In summary, it is proposed that the most hopeful prospect for therapy of this condition would be to search for a specific inhibitor of orotate transport across cell membranes. This is likely to affect the metabolism of pyrimidines in only two tissues, liver and erythrocytes, and, unless it gives rise to toxic accumulations of orotate in the liver, could help to reduce pyrimidine nucleotide levels in pyrimidine 5 nucleotidase deficient erythrocytes. A trial of a specific inhibitor of uridine kinase might also be appropriate, and the final choice of a therapeutic approach will depend on the demonstration of which of these two alternative pathways amenable to therapeutic intervention contributes most to erythrocyte pyrimidine nucleotide accumulation. 

Paglia, D.E., Valentine, W.N., 1975. Characteristics of a pyrimidine-specific 5 -nucleotidase in human erythrocytes. J. Biol. Chem. 250, 7973—7979. 

---