Purine salvages

The free bases of the purines can be salvaged to spare de novo synthesis. The only hard thing is remembering what the names stand for. HGPRTase is hypoxanthine-guanine phosphoribosyltransferase, and it makes both IMP and GMP. A separate enzyme exists for the salvage of adenine. The salvage pathways are included in Fig. 19-1. 

Deoxynucleotides for DNA synthesis are made at the nucleoside diphosphate level and then have to he phosphorylated up to the triphosphate using a kinase and ATP. The reducing equivalents for the reaction come from a small protein, thioredoxin, that contains an active site with two cysteine residues. Upon reduchon of the rihose to the 2 -deoxyri-hose, the thioredoxin is oxidized to the disulfide. The thioredoxin(SS) made during the reaction is recycled hy reduchon with NADPH by the enzyme thioredoxin reductase. 

Ribonucleotide reductase works on ribo-A, -U, -G, -C diphosphates to give the deoxynucleohde. The deoxyuridine, which is useless for RNA synthesis, is converted to deoxythymidine by the enzyme thymidylate synthase, which uses methylene tetrahydrofolate as a one-carbon donor. The odd thing here is that ribonucleotide reductase uses the UDP as a substrate to give the dUDP. This must then be hydrolyzed to the dUMP before thymidylate synthase will use it to make dTMP. Then the dTMP has to be kinased (phosphorylated) up to dTTP before DNA can be made. 

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Pentostatin (deoxycoformycin Fig. 4) is a purine isolated from cultures of Streptomyces antibioticus. Its mode of action involves inhibition of adenosine deaminase, which plays a key role in purine salvage pathways and DNA synthesis. As a consequence, deoxyadenosine triphosphate (dATP) is accumulated, which is highly toxic to lymphocytes. This is associated with augmented susceptibility to apoptosis, particularly in T cells. 

Guanine Phosphoribosyl Transferase. Guanine phosphoribosyl transferase (GPRT) is one of the enzymes of the purine salvage pathway, which is needed by protozoa because they lack the ability to synthesize purine nucleotides. 

Lesch-Nyhan syndrome, an overproduction hyperuricemia characterized by frequent episodes of uric acid hthiasis and a bizarre syndrome of self-mutilation, reflects a defect in hypoxanthme-guanine phosphoribo-syl transferase, an enzyme of purine salvage (Figure 34—4). The accompanying rise in intracellular PRPP results in purine overproduction. Mutations that decrease or abohsh hypoxanthine-guanine phosphoribosyltrans-ferase activity include deletions, frameshift mutations, base substitutions, and aberrant mRNA splicing. 

Xi H, BL Schneider, L Reitzer (2000) Purine catabolism in Escherichia coli and function of xanthine dehydrogenase in purine salvage J Bacterial 182 5332-5341. 

Purine Synthesis Purine Salvage Deoxynucleotides Purine Degradation... 

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. 

This enzyme [EC 2.4.2.8] (also known as hypoxanthine phosphoribosyltransferase, IMP pyrophosphorylase, transphosphoribosidase, and guanine phosphoribosyltransferase) catalyzes the purine salvage reaction of hypoxanthine with 5-phospho-a-D-ribose 1-diphosphate to... 

This rational approach to drug design has been adopted in developing a specific inhibitor of the human cellular enzyme, purine nucleoside phosphorylase (PNP). PNP functions in the purine salvage pathway, catalysing the reversible reaction shown below ... 

Mycophenolate mofetil (MMF, CellCept) is an ester prodrug of mycophenolic acid (MPA), a Penicillium-de-rived immunosuppressive agent (see Chapter 57) that blocks de novo purine synthesis by noncompetitively inhibiting the enzyme inosine monophosphate dehydrogenase. MPA preferentially suppresses the proliferation of cells, such as T and B lymphocytes, that lack the purine salvage pathway and must synthesize de novo... 

N-Ribohydrolases have been found to be involved in novel pathways of purine salvage in protozoan parasites as well as in nucleic acid repair, and exhibit other interesting biological activities [178]. In order to investigate the molecular electrostatic potential surface of the enzyme from the trypanosome Crithidia fasci-culata, several l,4-dideoxy-l,4-imino-D-ribitol derivatives were synthesized as nucleoside analogues and their inhibitory powers were tested [179,180]. In the course of this work, l,4-dideoxy-l,4-imino-l(S)-phenyl-D-ribitol (97) was found to inhibit this enzyme with K 30 nmol/1. 

The enzyme has been isolated from both eukaryotic and prokaryotic organisms [2] and functions in the purine salvage pathway [1,3]. Purine nucleoside phosphorylase isolated from human erythrocytes is specific for the 6-oxypurines and many of their analogs [4] while PNPs from other organisms vary in their specificity [5]. The human enzyme is a trimer with identical subunits and a total molecular mass of about 97,000 daltons [6,7]. Each subunit contains 289 amino acid residues. 

Figure 25-17 Some purine salvage pathways and related reactions. Green lines indicate biosynthetic pathways.

The role of hypoxanthine-guanine phosphoribosyltransferase in purine salvage has been confirmed by the abnormally high excretion of purines (as uric acid) in humans who lack hypoxanthine-guanine phosphoribosyltransferase. Studies of purine metabolism in cultures of cells from patients with this hereditary disorder also support this conclusion. 

Adenine phosphoribosyltransferase catalyzes the conversion of adenine to AMP in many tissues, by a reaction similar to that of hypoxanthine-guanine phosphoribosyltransferase, but is quite distinct from the latter. It plays a minor role in purine salvage since adenine is not a significant product of purine nucleotide catabolism (see below). The function of this enzyme seems to be to scavenge small amounts of adenine that are produced during intestinal digestion of nucleic acids or in the metabolism of 5 -deoxy-5 -methylthioadenosine, a product of polyamine synthesis. 

Manfredi, J. P., and E. W. Holmes, Purine salvage pathways in myocardium. Ann. Rev. Physiol. 47 691-705, 1985. Although this review applies specifically to salvage in heart muscle, it is an excellent summary of purine salvage path-... 

Purine and pyrimidine nucleotides are essential components of many biochemical molecules, from DNA and RNA to ATP and NAD. In recent years, the pyrimidine and especially the purine metabolism of parasitic helminths have been investigated extensively, mainly because they are different from the pathways in the mammalian host such that they have potential as targets for chemotherapeutic attack. For a review of purine and pyrimidine pathways in parasitic helminths and protozoa, see Berens et al. (1995). Although parasitic helminths do not synthesize purines de novo, but obtain them from the host, they do possess elaborate purine salvage pathways for a more economical management of this resource. Pyrimidines, on the other hand, are synthesized de novo by all parasitic flat-worms studied so far and, as with mammalian... 

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. 

The most recent application of RPLC to the analysis of enzymes has been reported by Halfpenny and Brown (HI). An assay for purine nucleoside phosphorylase, a key mediator in the purine salvage pathway, has been developed and optimal conditions for the analysis determined. Figure 20 illustrates the simultaneous separation of the substrate, inosine, and products, uric acid and hypoxanthine. In another analysis. Halfpenny and Brown (H2) developed an assay for hypoxanthine-guanine phos-phoribosyltransferase. Deficiency of this enzyme has been associated with Lesch-Nyhan syndrome as well as primary gout. The activity of the enzyme is determined by measurement of the decrease of the substrate, hypoxanthine, and increase in the product, inosine-5 -monophosphoric acid. A major advantage of using HPLC for enzyme assays is that the simultaneous measurement of both substrate and product reduces the error due to interference from competing enzymes. 

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