Purine nucleoside phosphorylase specificity

For the alternative production of nucleosides, Zuffi and Monciardini have reported a process by a transglycosylation reaction catalyzed by uridine phosphor-ylase and purine nucleoside phosphorylase. Specifically, their invention generally relates to a process for immobilizing cells and to the use of a resin for immobilizing cells, and the process is exemplified by the production of 306 from l-p-o-arabino-furanosyluracil 315 and 314 (Scheme 12.82). Similar biotransformations for the preparation of 306 and 307 from 314 and arabinofuranosyluracil have been reported by Farina et al. (using Enterobacter aerogenes), and Hummel-Marquardt et al. (using esterases (e.g., pig liver esterase) or lipases). ... 

The review articles by Schramm (1998, 2003) provide a number of examples of the successful application of this protocol to the design of enzyme-specific transition state-like inhibitors. Among these, the transition state inhibitors of human purine nucleoside phosphorylase (PNP) are particularly interesting from a medicinal chemistry perspective, as examples of these compounds have entered human clinical trials for the treatment of T-cell cancers and autoimmune disorders. 

Shames, and S. E. Ealick, Purine nucleoside phosphorylase. 3. Reversal of purine base specificity by site-directed mutagenesis, Biochemistry 36 11725 (1997). 

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 ... 

Fluorescence and phosphorescence emission spectroscopy were employed to study the interaction of E. coli purine nucleoside phosphorylase (PNP) with its specific inhibitor, FA. The results show, for the first time, the application of phosphorescence spectroscopy to the identification of the tautomeric form of the inhibitor bound by the enzyme <2004MI377>. 

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. 

Purine nucleosides are cleaved by the action of purine nucleoside phosphorylase with the liberation of ribose 1-phosphate (Kl, PI). The enzyme is apparently specific for purines. The material from erythrocytes catalyzes the phosphorolysis of purine but not pyrimidine nucleosides (T6.) Purine phosphorylase activity is found widespread in nature and in many animal tissues (FIO). Friedkin and Kalckar investigated an enzyme capable of cleaving purine deoxynucleosides to the aglycone and deoxy-ribose 1-phosphate. They concluded that the enzyme was identical to that which splits purine ribonucleosides (F8, F9). This enzyme is capable of degrading inosine, xanthosine, and guanosine to forms readily attacked by other enzymes. In so doing, it permits living cells to retain the ribose and deoxyribose moieties. 

Deoxyribonucleoside phosphorolysis is catalyzed by purine nucleoside phosphorylase, thymidine phosphorylase, and to some extent by uridine phosphorylase the specificity of animal uridine phosphorylases differs with the cells of origin (see Chapter 12). 

The purine nucleoside phosphorylase activity of animal tissues cleaves both ribo- and deoxyribonucleosides of 6-oxypurines. For example, the the purine nucleoside phosphorylase of human erythrocytes has been highly purified and crystallized by Parks and co-workers 2) this enzyme will cleave the ribo- and deoxyribonucleosides of guanine and hypoxan-thine. Zimmerman et al. (3) have shown that purine nucleoside phosphorylase of several animal tissues has a low intrinsic activity toward adenine in the presence of ribose 1-phosphate. The cleavage of deoxyadenosine by highly purified preparations of the animal enzyme has not been reported but by analogy with adenosine, one might expect it also to be a poor substrate. The specificities of the purine nucleoside phosphorylases of E. coli and S. typhimurium differ from that of the animal enzyme in that adenosine and deoxyadenosine are readily phosphorolyzed H-6). 

Since specificity of the nucleoside phosphorylases varies, enzymes that catalyze the formation of the nucleosides of the so-called fraudulent purine bases, 6-mercaptopurine (purine-6(lH)-thione) and 8-aza-guanine (5-amino-t -triazolo[ 4,5-d]pyrimidin-7(6if)-one), have been found, and gram quantities of 8-azaguanosine have been isolated. ... 

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. 

Amino-4-imidazole carboxamide ribotide, a precursor only two steps removed (formylation and cycli-zation) from inosinic acid, can be synthesized by the direct condensation of the imidazole with 5-phosphori-bosyl pyrophosphate. The enzyme catalyzing this reaction was purified from an acetone powder of beef liver. The same enzyme (AMP pyrophosphorylase) catalyzes the condensation of adenine, guanine, and hypoxan-thine. Nucleoside phosphorylase is an enzyme that catalyzes the formation of a ribose nucleoside from a purine base and ribose-1-phosphate. Guanine, adenine, xanthine, hypoxanthine, 2,6-diaminopurine, and aminoimidazole carboxamide are known to be converted to their respective nucleosides by such a mechanism. In the presence of a specific kinase and ATP, the nucleoside is then phosphorylated to the corresponding nucleotide. 

Nucleoside phosphorylase activity is widely distributed, having been reported in yeast, E. coli, L. casei, and other sources. A nucleosidase specific for uridine has been purified several hundredfold from E. coli, using sonic vibration, ammonium sulfate, and gel steps.It does not attack cytidine or the purine nucleosides. 

The mutant strain (S0 405) lacks the enzyme adenylosuccinate lyase, required for novo purine biosynthesis as well as for the conversion of s-AMP to AMP. This strain therefore has a specific requirement for an adenine compound and a general purine requirement. As the strain lacks nucleoside phosphorylase and adenosine deaminase the pathways for nucleoside utilization via free bases are ruled out. 

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). 

Fig. 23.1. Pyrimidine pathways Pathways for the de novo synthesis, interconversion, and breakdown of pyrimidine ribonucleotides, indicating their metabolic importance as the essential precursors of the pyrimidine sugars and, with purines, of DNA and RNA. Note that in contrast to purines salvage takes place at the nucleoside not the base level in human cells and pyrimidine metabolism normally lacks any detectable end-product. The importance of this network is highlighted by the variety of clinical symptoms associated with the possible enzyme defects indicated. 23.10, Uridine monophosphate synthase (UMPS), 23.11a, uridine monophosphate hydrolase 1 (UMPHl), 23.12, thymidine phosphorylase (TP), 23.13, dihydropyrimidine dehydrogenase (DPD), 23.14, dihydropyrimidine amidohydrolase (DHP), 23.15, y -ureidopropionase (UP) (23.11b, UMPH superactivity specific to fibroblasts is not shown). CP, carbamoyl phosphate. The pathological metabolites used as specific markers in differential diagnosis are highlighted...

---