Salvage enzymes

Many yeasts are inhibited by 5-fluorocytosine and a block in the synthesis of 5-fluorouridylic acid by loss of cytosine deaminase or of nracil phosphoribosyltransferase is sufficient to cause resistance. Mntational loss of pyrimidine salvage enzymes has been frequently observed. 

Free purines can be salvaged and rebuilt into nucleotides. Genetic deficiencies in certain salvage enzymes cause serious disorders such as Lesch-Nyhan syndrome and ADA deficiency. 

It was demonstrated in 1978 by Cohn and Hu (l) and by Lowe and Sproat (2) that substitution of 0 for - -°0 in phosphates causes an upfield shift of approximately 0.02ppm/l80 atom on the 31p chemical shift. The magnitude of this isotopic shift varies with the environment and appears to be proportional to the P-0 bond order. As part of a continuing effort to elucidate the mechanism of action of purine salvage enzymes, we employed (3.) this... 

Comparative complement selection in bacteria enables screening for lead compounds targeted to a purine salvage enzyme of parasites. Antimicrob Agents Chemo-ther 39 620-625, 1995. 

Salvage pathway is a useful term to refer to that collection of biochemical reactions whose transformations result in the phosphorylation of purines. As a consequence of this phosphorylation, purines are not secreted by cells but, in fact, are returned to the cellular metabolic pool. One of these salvage enzymes is hypoxanthine-guanine phosphoribosyltransferase (HGPRTase),... 

Figure 13.11. Structure of 5-fluorouracil (a), its activation by pyrimidine salvage enzymes (b), and its tautomerism that promotes ambiguous base pairing (c). While 5-FU is not very efficiently incorporated into DNA, the same mechanism applies to 5-bromonracil, whichbecanse of its closer steric resemblance of thymidine is readily incorporated.

Free purine bases, derived from the turnover of nucleotides or from the diet, can be attached to PRPP to form purine nucleoside monophosphates, in a reaction analogous to the formation of orotidylate. Two salvage enzymes with different specificities recover purine bases. Adenine phosphorihosyltransferase catalyzes the formation of adenylate... 

Answer C. The purine antimetabolite 6-mercaptopurine is bioactivated in cancer cells by the purine salvage enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT). The most common form of resistance to 6-MP is a decrease in activity of this enzyme. Azathioprine, a drug used as an immunosuppressant, is closely related to 6-MP and also requires bioactivation to exert cytotoxic actions. 

Wang J, Neuhard J, Eriksson S. An Escherichia coli system expressing human deoxyribonucleoside salvage enzymes for evaluation of potential antiproliferative nucleoside analogues. Antimicrobial Agents Chemother 1998 42 2620-5. 

Several other enzymes known to be directly involved in the biogenesis of pyridine mononucleotides via salvage and recycling of pyridines and pyridine nucleosides are less ubiquitous than the three families of phosphoribosyltransferases described above. Flere, we briefly describe some of the best-studied salvage enzymes that are captured in Figure 2 and Table 1. 

Lack of ADA allows dATP to accumulate. High dATP levels inhibit production of the other dNTPs needed for DNA replication due to its effect on the regulation of ribonucleotide reductase. White blood cells, which must proliferate for an immune response to occur, and which have abundant salvage enzymes for making dATP, are most affected by lack of the enzyme. They are unable to proliferate, a necessary step for antibody production. 

Purine salvage enzymes - The drugs allopurinol (see here) and formycin B inhibit the action of cellular purine salvage enzymes. Thus, these drugs can be used to treat individuals infected by the parasitic protozans, Plasmodium, and Leishmania because these parasites lack the capacity for de novo purine synthesis (i.e., they depend entirely upon cellular purine salvage enzymes and bases provided by the host)... 

G. lamblia and salvage adenine and guanine by first converting them into their ribonucleosides which are in turn phosphorylated to their ribonucleotide monophosphates (Figs 6.5 and 6.6). Assays for the various salvage enzyme activities in E. histolytica and T. vaginalis have been performed and the results agree with the metabolic studies (4,19-21). 

Few detailed studies have been done on the purine salvage enzymes of procyclic African trypanosomes. Tb. gambiense has high levels of guanine deaminase and lacks adenine and adenosine deaminase activities (8). Tb. brucei, T.b. gambiense and T.b. rhodesiense convert allopurinol into aminopyrazolopyrimidine nucleotides and incorporates these into RNA (49). This indicates that HPRTase, succino-AMP synthetase, and succino-AMP lyase are present. At least three nucleoside cleavage activities are present (Berens, unpublished results) two are hydrolases, of which one is specific for purine ribonucleosides and the other is specific for purine deoxyribonucleosides. The third nucleoside cleavage activity is a methylthioadenosine/adenosine phosphorylase. The adenosine kinase is similar to that of L. donovani (Berens, unpublished results). 

Two salvage enzymes from Eimeria have been characterized. HGXPRTase is present in crude extracts of oocysts of E. tinella (11). Adenosine kinase is found in sporulated oocysts of three avian species of Eimeria (65). 

Plasmodium species. The mature erythrocyte does not have a requirement for pyrimidine nucleotides but it may contain low levels of de novo and salvage enzymes. 

Evidence for pyrimidine de novo biosynthesis has also been found in other trematodes. Its enzymes are present in Fasciola gigantica (81), Paragonimus ohirai (112) and Clonorchis sinensis (112). De novo biosynthesis was verified by demonstrating the incorporation of [ C]bicarbonate into the C-2 position of uracil in P. ohirai (112). The pyrimidine salvage enzymes, uridine kinase and thymidine kinase, are found in P. ohirai and C. sinensis extracts (112). Aspartate transcarbamoylase is found in Fasciola hepatica and Paramphistomum cervi extracts (113). 

There is good evidence for de novo pyrimidine synthesis in cestodes. Five of the six enzymes needed for UMP synthesis are present in Hymenolepis diminuta and aspartate transcarbamoylase activity has been found in Moniezia benedeni (81). Salvage of preformed pyrimidines by a cestode was first reported in Mesocestoides corti (70). Thymidine kinase is the only cestode H. diminuta) pyrimidine salvage enzyme that has been characterized (114). 

The purine requirement for this enzyme is unknown and it is likely that an AdoMet analog based on purine or methionine could affect both polyamine and methylation reactions. In trypanosomatids, the salvage enzyme methylthioadenosine (MTA) phosphorylase has been found to have a broad substrate requirement (Fig. 7.1)... 

Fig. 41.13. The purine nucleotide cycle. Using a combination of biosynthetic and salvage enzymes, the net effect is the conversion of aspartate to fumarate plus ammonia, with the fumarate playing an anaplerotic role in the muscle.

The degradation of the purine nucleotides (AMP and GMP) occurs mainly in the liver (Fig. 41.19). Salvage enzymes are used for most of these reactions. AMP is first deaminated to produce IMP (AMP deaminase). Then IMP and GMP are dephosphorylated (5 -nucleotidase), and the ribose is cleaved from the base by purine nucleoside phosphorylase. Hypoxanthine, the base produced by cleavage of IMP, is converted by xanthine oxidase to xanthine, and guanine is deaminated by... 

S ATP + 4-methyl-5-(2-hydroxyethyl)thiazole <1, 2> (<1> enzyme involved in biosynthesis of thiamine [1] <1> the bifunctioinal enzyme hydroxyethylthiazole kinase/thiamine-phosphate pyrophosphorylase catalyzes two sequential steps in the synthesis of thiamin monophosphate from hydroxyethylthiazole [2] <2> the enzyme is a salvage enzyme in the thiamin biosynthetic pathway and enables the cell to use recycled 4-methyl-5-j8-hydroxyethylthiazole as an alternative to its synthesis from 1-deoxy-o-xylulose-5-phosphate, cysteine, and tyrosine [3]) (Reversibility <1, 2> [1,2,3]) [1,2, 3]... 

Hypoxanthine-guanine phosphoribosyl transferase (HGPRT), a salvage enzyme of nucleotide metabolism, uses 5 -phosphoribosylpyrophosphate (PRPP) to convert hypox-... 

In HeLa ceils hydroxyurea is an efficient inhibitor of histone synthesis. This action requires protein synthesis and leads to rapid disappearance of cytoplasmatic histone mRNA The effect is not specific for hydroxyurea since suppression of DNA synthesis by arabino-cytosine or temperature-sensitive mutations yields analogous results. Similarly, the synthesis of some enzymes necessary for DNA replication and active in S-phase is altered by hydroxyurea. Increased activity of ribonucleotide reductase in HeLa and in hamster cells and of the salvage enzyme thymidine kinase in HeLa cells and KB cells has been observed, probably as a consequence of the increased fraction of cells in S-phase. Repression occurs for thymidine kinase in human lymphocytes and for ornithine decarboxylase in Chinese hamster fibroblasts whereas no or only slight effects were seen on ribonucleotide reductase in hamster fibroblasts , on thymidylate synthase in extracts from synchronous mouse cells " , and on DNA polymerase in rabbit kidney cells or HeLa cells . ... 

---