A second purine nucleoside phosphorylase in Escherichia coli K-12

Summary The presence of a second purine nucleoside phosphorylase in wild-type strains of E. coli K-12 after growth on xanthosine has been demonstrated. Like other purine nucleoside phosphorylases it is able to carry out both phosphorylosis and synthesis of purine deoxy- and ribonucleosides whilst pyrimidine nucleosides cannot act as substrates. In contrast to the well characterised purine nucleoside phosphorylase of E. coli K-12 (encoded by the deoD gene) this new enzyme could act on xanthosine and is hence called xanthosine phosphorylase. Studies of its substrate specificity showed that xanthosine phosphorylase, like the mammalian purine nucleoside phosphorylases, has no activity towards adenine and the corresponding nucleosides. Determinations of K _m and gel filtration behaviour was carried out on crude dialysed extracts. The presence of xanthosine phosphorylase enables E. coli to grow on xanthosine as carbon source. Xanthosine was the only compound found which induced xanthosine phosphorylase. No other known nucleoside catabolising enzyme was induced by xanthosine. The implications of non-linear induction kinetics of xanthosine phosphorylase is discussed.     

Purification and characterization of RihC, a xanthosine-inosine-uridine-adenosine-preferring hydrolase from Salmonella enterica serovar Typhimurium

Salmonella enterica serovar Typhimurium normally salvage nucleobases and nucleosides by the action of nucleoside phosphorylases and phosphoribosyltransferases. In contrast to Escherichia coli, which catabolizes xanthosine by xanthosine phosphorylase (xapA), Salmonella cannot grow on xanthosine as the sole carbon and energy source. By functional complementation, we have isolated a nucleoside hydrolase (rihC) that can complement a xapA deletion in E. coli and we have overexpressed, purified and characterized this hydrolase. RihC is a heat stable homotetrameric enzyme with a molecular weight of 135 kDa that can hydrolyze xanthosine, inosine, adenosine and uridine with similar catalytic efficiency (k(cat)/Km=1 to 4 x 10(4) M(-1)s(-1)). Cytidine and guanosine is hydrolyzed with approximately 10-fold lower efficiency (k(cat)/Km=0.7 to 1.2 x 10(3) M(-1)s(-1)) while RihC is unable to hydrolyze the deoxyribonucleosides thymidine and deoxyinosine. The Km for all nucleosides except adenosine is in the mM range. The pH optimum is different for inosine and xanthosine and the hydrolytic capacity (k(cat)/Km) is 5-fold higher for xanthosine than for inosine at pH 6.0 while they are similar at pH 7.2, indicating that RihC most likely prefers the neutral form of xanthosine.     

Development of a FIA system with immobilized enzymes for specific post-column detection of purine bases and their nucleosides separated by HPLC column.

A sensitive and highly selective method for the simultaneous determination of purine bases and their nucleosides is proposed. An amperometric flow-injection system with the two immobilized enzyme reactors (guanase immobilized reactor and purine nucleoside phosphorylase/xanthine oxidase co-immobilized reactor) is used as the specific post-column detection system of HPLC, to convert compounds separated by a reversed-phase. HPLC column to electroactive species (hydrogen peroxide and uric acid) which can be detected at a flow-through platinum electrode. The proposed detection system is specific for a group of purine bases and purine nucleosides and does not respond for purine nucleotides and pyrimidine bases. The linear determination ranges are from 10 pmol to 5 nmol for four purine bases (hypoxanthine, xanthine, guanine, and adenine) and four purine nucleosides (inosine, xanthosine, guanosine, and adenosine). The detection limits are 1.2-5.5 pmol.     

Isolation of the hexameric form of purine nucleoside phosphorylase from E. coli. Comparative study of trimeric and hexameric forms of the enzyme

The presence of two forms (high and low molecular weight ones) of purine nucleoside phosphorylase II (purine nucleoside: orthophosphate ribosyltransferase, EC 2.4.2.1) was demonstrated. The high molecular weight form of the enzyme was purified, and the properties of both forms were compared. The enzyme forms were shown to differ in their quaternary structure (trimeric and hexameric), molecular weight of the native enzyme and its subunits (85,000 and 28,000 for the trimer, 150,000 and 25,000 for the hexamer, respectively) as well as substrate specificity (the trimer is specific for all major purine nucleosides, while the hexamer does not cleave adenine nucleosides). Adenosine is a competitive inhibitor of the hexameric form with respect to deoxyguanosine (Ki = 1.16 X 10(-3) M); the Km value for deoxyguanosine is 9.85 X 10(-5) M. The isoelectric point for the both forms of the enzyme in the presence of 9 M urea is about 5.5. Both forms have a pH optimum of phosphorolytic activity between 6.5 and 7.0.     

Purification and characterization of purine nucleoside phosphorylase from developing embryos of Hyalomma dromedarii

Purine nucleoside phosphorylase from Hyalomma dromedarii, the camel tick, was purified to apparent homogeneity. A molecular weight of 56,000 - 58,000 was estimated for both the native and denatured enzyme, suggesting that the enzyme is monomeric. Unlike purine nucleoside phosphorylase preparations from other tissues, the H. dromedarii enzyme was unstable in the presence of beta-mercaptoethanol. The enzyme had a sharp pH optimum at pH 6.5. It catalyzed the phosphorolysis and arsenolysis of ribo- and deoxyribo-nucleosides of hypoxanthine and guanine, but not of adenine or pyrimidine nucleosides. The Km values of the enzyme at the optimal pH for inosine, deoxyinosine, guanosine, and deoxyguanosine were 0.31, 0.67, 0.55, and 0.33 mM, respectively. Inactivation and kinetic studies suggested that histidine and cysteine residues were essential for activity. The pKa values determined for catalytic ionizable groups were 6-7 and 8-9. The enzyme was completely inactivated by thiol reagents and reactivated by excess beta-mercaptoethanol. The enzyme was also susceptible to pH-dependent photooxidation in the presence of methylene blue, implicating histidine. Initial velocity studies showed an intersecting pattern of double-reciprocal plots of the data, consistent with a sequential mechanism.     

Purification and characterization of a novel nucleoside phosphorylase from a Klebsiella sp. and its use in the enzymatic production of adenine arabinoside

An adenosine-assimilating bacterium, Klebsiella sp. strain LF1202, inducibly formed a novel nucleoside phosphorylase which acted on both purine and pyrimidine nucleosides when the cells were cultured in medium containing adenosine as a sole source of carbon and nitrogen. The enzyme was purified (approximately 83-fold, with a 17% activity yield) to the homogeneous state by polyacrylamide gel electrophoresis. The molecular weight of the purified enzyme was calculated to be 125,000 by gel filtration of Sephadex G-200 column chromatography, although the enzyme migrated as a single protein band with a molecular weight of 25,000 on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis; thus, it was thought to consist of five identical subunits. Besides purine nucleosides (adenosine, inosine, and guanosine), the purified enzyme also acted on pyrimidine nucleosides such as uridine, 2'-deoxyuridine, and thymidine. The purified enzyme catalyzed the synthesis of adenine arabinoside, a selective antiviral pharmaceutic agent, from uridine arabinoside and adenine.     

Purification and characterization of a novel nucleoside phosphorylase from a Klebsiella sp. and its use in the enzymatic production of adenine arabinoside.

An adenosine-assimilating bacterium, Klebsiella sp. strain LF1202, inducibly formed a novel nucleoside phosphorylase which acted on both purine and pyrimidine nucleosides when the cells were cultured in medium containing adenosine as a sole source of carbon and nitrogen. The enzyme was purified (approximately 83-fold, with a 17% activity yield) to the homogeneous state by polyacrylamide gel electrophoresis. The molecular weight of the purified enzyme was calculated to be 125,000 by gel filtration of Sephadex G-200 column chromatography, although the enzyme migrated as a single protein band with a molecular weight of 25,000 on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis; thus, it was thought to consist of five identical subunits. Besides purine nucleosides (adenosine, inosine, and guanosine), the purified enzyme also acted on pyrimidine nucleosides such as uridine, 2'-deoxyuridine, and thymidine. The purified enzyme catalyzed the synthesis of adenine arabinoside, a selective antiviral pharmaceutic agent, from uridine arabinoside and adenine.     

Expression of human malaria parasite purine nucleoside phosphorylase in host enzyme-deficient erythrocyte culture. Enzyme characterization and identification of novel inhibitors

The intraerythrocytic human malaria parasite, Plasmodium falciparum, requires a source of hypoxanthine for nucleic acid synthesis and energy metabolism. Adenosine has been implicated as a major source for intraerythrocytic hypoxanthine production via deamination and phosphorolysis, utilizing adenosine deaminase and purine nucleoside phosphorylase, respectively. To study the expression and characteristics of human malaria purine nucleoside phosphorylase, P. falciparum was successfully cultured in purine nucleoside phosphorylase-deficient human erythrocytes to an 8% parasitemia level. Purine nucleoside phosphorylase activity was undetectable in the uninfected enzyme-deficient host red cells but after parasite infection rose to 1.5% of normal erythrocyte levels. The parasite purine nucleoside phosphorylase was not cross-reactive with antibody against human enzyme, exhibited a calculated native molecular weight of 147,000, and showed a single major electrophoretic form of pI 5.4 and substrate specificity for inosine, guanosine and deoxyguanosine but not xanthosine or adenosine. The Km values for substrates, inosine and guanosine, were 4-fold lower than that for the human erythrocyte enzyme. In these studies we have identified two novel potent inhibitors of both human erythrocyte and parasite purine nucleoside phosphorylase, 8-amino-5'-deoxy-5'-chloroguanosine and 8-amino-9-benzylguanine. These enzyme inhibitors may have some antimalarial potential by limiting hypoxanthine production in the parasite-infected erythrocyte.     

Characterization of the adenine nucleoside specific phosphorylase of Bacillus cereus

Adenosine phosphorylase, a purine nucleoside phosphorylase endowed with high specificity for adenine nucleosides, was purified 117-fold from vegetative forms of Bacillus cereus. The purification procedure included ammonium sulphate fractionation, pH 4 treatment, ion exchange chromatography on DEAE-Sephacel, gel filtration on Sephacryl S-300 HR and affinity chromatography on N(6)-adenosyl agarose. The enzyme shows a good stability to both temperature and pH. It appears to be a homohexamer of 164+/-5 kDa. Kinetic characterization confirmed the specificity of this phosphorylase for 6-aminopurine nucleosides. Adenosine was the preferred substrate for nucleoside phosphorolysis (k(cat)/K(m) 2.1x10(6) s(-1) M(-1)), followed by 2'-deoxyadenosine (k(cat)/K(m) 4.2x10(5) s(-1) M(-1)). Apparently, the low specificity of adenosine phosphorylase towards 6-oxopurine nucleosides is due to a slow catalytic rate rather than to poor substrate binding.     

Purification and properties of inosine-guanosine phosphorylase from Escherichia coli K-12.

A xanthosine-inducible enzyme, inosine-guanosine phosphorylase, has been partially purified from a strain of Escherichia coli K-12 lacking the deo-encoded purine nucleoside phosphorylase. Inosine-guanosine phosphorylase had a particle weight of 180 kilodaltons and was rapidly inactivated by p-chloromercuriphenylsulfonic acid (p-CMB). The enzyme was not protected from inactivation by inosine (Ino), 2'-deoxyinosine (dIno), hypoxanthine (Hyp), Pi, or alpha-D-ribose-1-phosphate (Rib-1-P). Incubating the inactive enzyme with dithiothreitol restored the catalytic activity. Reaction with p-CMB did not affect the particle weight. Inosine-guanosine phosphorylase was more sensitive to thermal inactivation than purine nucleoside phosphorylase. The half-life determined at 45 degrees C between pH 5 and 8 was 5 to 9 min. Phosphate (20 mM) stabilized the enzyme to thermal inactivation, while Ino (1 mM), dIno (1 mM), xanthosine (Xao) (1 mM), Rib-1-P (2 mM), or Hyp (0.05 mM) had no effect. However, Hyp at 1 mM did stabilize the enzyme. In addition, the combination of Pi (20 mM) and Hyp (0.05 mM) stabilized this enzyme to a greater extent than did Pi alone. Apparent activation energies of 11.5 kcal/mol and 7.9 kcal/mol were determined in the phosphorolytic and synthetic direction, respectively. The pH dependence of Ino cleavage or synthesis did not vary between 6 and 8. The substrate specificity, listed in decreasing order of efficiency (V/Km), was: 2'-deoxyguanosine, dIno, guanosine, Xao, Ino, 5'-dIno, and 2',3'-dideoxyinosine. Inosine-guanosine phosphorylase differed from the deo operon-encoded purine nucleoside phosphorylase in that neither adenosine, 2'-deoxyadenosine, nor hypoxanthine arabinoside were substrates or potent inhibitors. Moreover, the E. coli inosine-guanosine phosphorylase was antigenically distinct from the purine nucleoside phosphorylase since it did not react with any of 14 monoclonal antisera or a polyvalent antiserum raised against deo-encoded purine nucleoside phosphorylase.     

Purine nucleoside phosphorylase of the malarial parasite, Plasmodium lophurae

Purine nucleoside phosphorylase (EC 2.4.2.1, purine nucleoside:orthophosphate ribosyltransferase) was purified and characterized from the malarial parasite, Plasmodium lophurae, using a chromatofocusing (Pharmacia) column and a formycin B affinity column. The apparent isoelectric point of the native protein, as determined by chromatofocusing, was 6.80. By gel filtration and both native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the native enzyme appeared to be a pentamer with a native molecular weight of 125,300 and a subunit molecular weight of 23,900. The enzyme had a broad pH optimum, pH 5.5-7.5, with maximum activity at pH 6.0-6.5. The enzyme reaction was readily reversible with a Km for inosine of 33 microM and a Km for hypoxanthine of 82 microM. Thioinosine, guanosine, and guanine were also substrates for the plasmodial enzyme, but allopurinol and adenine were not. The parasite enzyme was competitively inhibited by formycin B (Ki = 0.39 microM). Formycin A, azaguanine, and 8-aminoguanosine were not inhibitors of the enzyme.     

Sugar-free growth of mammalian cells on some ribonucleosides but not on others

It was shown earlier that a variety of vertebrate cells could grow indefinitely in sugar-free medium supplemented with either uridine or cytidine at greater than or equal to 1 mM. In contrast, most purine nucleosides do not support sugar-free growth for one of the following reasons. The generation of ribose-1-P from nucleoside phosphorylase activity is necessary to provide all essential functions of sugar metabolism. Some nucleosides, e.g. xanthosine, did not support growth because they are poor substrates for this enzyme. De novo pyrimidine synthesis was inhibited greater than 80% by adenosine or high concentrations of inosine, e.g. 10 mM, which prevented growth on these nucleosides; in contrast, pyrimidine synthesis was inhibited only marginally on 1 mM inosine or guanosine, but normal growth was only seen on 1 mM inosine, not on guanosine. The inhibition of de novo adenine nucleotide synthesis prevented growth on guanosine, since guanine nucleotides could not be converted to adenine nucleotides. Guanine nucleotides were necessary for this inhibition of purine synthesis, since a mutant blocked in their synthesis grew normally on guanosine. De novo purine synthesis was severely inhibited by adenosine, inosine, or guanosine, but in contrast to guanosine, adenosine and inosine could provide all purine requirements by direct nucleotide conversions.     

Purification and characterization of human erythrocyte purine nucleoside phosphorylase and its subunits.

Purine nucleoside phosphorylase (EC 2.4.2.1; purine nucleoside:orthophosphate ribosyltransferase) from fresh human erythrocytes has been purified to homogeneity in two steps with an overall yield of 56%. The purification involves DEAE-Sephadex chromatography followed by affinity chromatography on a column of Sepharose/formycin B. This scheme is suitable for purification of the phosphorylase from as little as 0.1 ml of packed erythrocytes. The native enzyme appears to be a trimer with native molecular weight of 93,800 and the subunit molecular weight of 29,700 +/- 1,100. Two-dimensional gel electrophoresis of the purified enzyme under denaturing conditions revealed four major separable subunits (numbered 1 to 4) with the same molecular weight. The apparent isoelectric points of subunits 1 to 4 in 9.5 M urea are 6.63, 6.41, 6.29, and 6.20, respectively. The different subunits are likely the result of post-translational modification of the enzyme and provide an explanation of the complex native isoelectric focusing pattern of purine nucleoside phosphorylase from erythrocytes. Three of the four subunits are detectable in two-dimensional electrophoretic gels of crude hemolysates. Knowing the location of the subunits of purine nucleoside phosphorylase in a two-dimensional electropherogram allows one to characterize the purine nucleoside phosphorylase in crude cell extracts from individuals with variant or mutant purine nucleoside phosphorylase as demonstrated in a subsequent communication. Partial purification of the phosphorylase from 1 ml of erythrocytes on DEAE-Sephadex increases the sensitivity of detection of the subunits to the 0.3% level.     

Purine nucleoside phosphorylase from bovine liver.

1. Purine nucleoside phosphorylase (purine nucleoside:orthophosphate ribosyltransferase, E.C. 2.4.2.1) from liver of cattle, Bos taurus, was purified to homogeneity. Some properties of the enzymes from three different bovine tissues were compared and discussed. 2. The enzyme has a molecular weight of 83,000, a sedimentation coefficient of 5.3 S, a Stokes' radius of 3.71 nm, a frictional ratio of 1.30 and a subunit molecular weight of 30,000. 3. Optimal pH for xanthosine degradation is around 5.5, whereas a broad pH activity profile for inosine degradation was observed between 5.0 and 7.5. Lineweaver-Burk plots curved downward at high concentrations of substrates, inosine, phosphate and arsenate.     

Purine nucleoside phosphorylases purified from rat liver and Novikoff hepatoma cells.

Purine nucleoside phosphorylase (PNP) was purified from rat hepatoma cells and normal liver tissue utilizing the techniques of ammonium sulfate fractionation, heat treatment, ion-exchange and molecular exclusion chromatography, and polyacrylamide gel electrophoresis. Homogeneity was established by disc gel electrophoresis in the presence and absence of sodium dodecyl sulfate. Purified rat hepatoma and liver PNPs appeared to be identical with respect to subunit and native molecular weight, substrate specificity, heat stability, kinetics and antigenic identity. A native molecular weight of 84,000 was determined by gel filtration. A subunit molecular weight of 29,000 was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A single isoelectric point was observed at pH 5.8, and the pH optimum was 7.5. Inosine, guanosine, xanthosine, and 6-mercaptopurine riboside were substrates for the enzymes. The apparent K_m for both inosine and guanosine was about 1.0 × 10^?4m and for phosphate was 4.2 × 10^?4m. Hepatoma and liver PNP showed complete cross-reactivity using antiserum prepared against the liver enzyme.     

Nucleoside hydrolase from Crithidia fasciculata. Metabolic role, purification, specificity, and kinetic mechanism

Crithidia fasciculata cells grown on complex medium with added [8-14C, 5'-3H]inosine or [8-14C,5'-3H]adenosine metabolize greater than 50% of the salvaged nucleosides through a pathway involving N-glycoside bond cleavage. Cell extracts contain a substantial nucleoside hydrolase activity but an insignificant purine nucleoside phosphorylase. The nucleoside hydrolase has been purified 1000-fold to greater than 99% homogeneity from kilogram quantities of C. fasciculata. The enzyme is a tetramer of Mr 34,000 subunits to give an apparent holoenzyme Mr of 143,000 by gel filtration. All of the commonly occurring nucleosides are substrates. The Km values vary from 0.38 to 4.7 mM with purine nucleosides binding more tightly than the pyrimidines. Values of Vmax/Km vary from 3.4 x 10(3) M-1 s-1 to 1.7 x 10(5) M-1 s-1 with the pyrimidine nucleosides giving the larger values. The turnover rate for inosine is 32 s-1 at 30 degrees C. The kinetic mechanism with inosine as substrate is rapid equilibrium with random product release. The hydrolytic reaction can be reversed to give an experimental Keq of 106 M with H2O taken as unity. The product dissociation constants for ribose and hypoxanthine are 0.7 and 6.2 mM, respectively. Deoxynucleosides or 5'-substituted nucleosides are poor substrates or do not react, and are poor inhibitors of the enzyme. The enzyme discriminates against methanol attack from solvent during steady-state catalysis, indicating the participation of an enzyme-directed water nucleophile. The pH profile for inosine hydrolysis gives two apparent pKa values of 6.1 with decreasing Vmax/Km values below the pKa and a plateau at higher pH values. These effects are due to the pH sensitivity of the Vmax values, since Km is independent of pH. The pH profile implicates two negatively charged groups which stabilize a transition state with oxycarbonium character.     

Monomeric purine nucleoside phosphorylase from rabbit liver. Purification and characterization.

Rabbit liver purine nucleoside phosphorylase (purine nucleoside: orthophosphate ribosyltransferase EC 2.4.2.1.) was purified to homogeneity by column chromatography and ammonium sulfate fractionation. Homogeneity was established by disc gel electrophoresis in presence and absence of sodium dodecyl sulfate, and isoelectric focusing. Molecular weights of 46,000 and 39,000 were determined, respectively, by gel filtration and by sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis. Product inhibition was observed with guanine and hypoxanthine as strong competitive inhibitors for the enzymatic phosphorolysis of guanosine. Respective Kis calculated were 1.25 x 10(-5) M for guanine and 2.5 x 10(-5) M for hypoxanthine. Ribose 1-phosphate, another product of the reaction, gave noncompetitive inhibition with guanosine as variable substrate, and an inhibition constant of 3.61 x 10(-4) M was calculated. The protection of essential --SH groups on the enzyme, by 2-mercaptoethanol or dithiothreitol, was necessary for the maintenance of enzyme activity. Noncompetitive inhibition was observed for p-chloromercuribenzoate with an inhibition constant of 5.68 x 10(-6)M. Complete reversal of this inhibition by an excess of 2-mercaptoethanol or dithiothreitol was demonstrated. In the presence of methylene blue, the enzyme showed a high sensitivity to photooxidation and a dependence of photoinactivation on pH, strongly implicating histidine as the susceptible group at the active site of the enzyme. The pKa values determined for ionizable groups of the active site of the enzyme were near pH 5.5 and pH 8.5 The chemical and kinetic evidences suggest that histidine and cysteine may be essential for catalysis. Inorganic orthophosphate (Km 1.54 x 10(-2) M) was an obligatory anion requirement, and arsenate substituted for phosphate with comparable results. Guanosine (Km 5.00 x 10(-5) M), deoxyguanosine (Km 1.00 x 10(-4)M) and inosine (Km 1.33 x 10(-4)M), were substrates for enzymatic phosphorolysis. Xanthosine was an extremely poor substrate, and adenosine was not phosphorylyzed at 20-fold excess of the homogeneous enzyme. Guanine (Km 1.82 x 10(-5)M),ribose 1-phosphate (Km 1.34 x 10(-4) M) and hypoxanthine were substrates for the reverse reaction, namely, the enzymatic synthesis of nucleosides. The initial velocity studies of the saturation of the enzyme with guanosine, at various fixed concentrations of inorganic orthophosphate, suggest a sequential bireactant catalytic mechanism for the enzyme.     

Purification and characterization of purine nucleoside phosphorylase from Proteus vulgaris.

Purine nucleoside phosphorylase was isolated and purified from cell extracts of Proteus vulgaris recovered from spoiling cod fish (Gadus morhua). The molecular weight and isoelectric point of the enzyme were 120,000 +/- 2,000 and pH 6.8. The Michaelis constant for inosine as substrate was 3.9 x 10(-5). Guanosine also served as a substrate (Km = 2.9 x 10(-5). However, the enzyme was incapable of phosphorylizing adenosine. Adenosine proved to be useful as a competitive inhibitor and was used as a ligand for affinity chromatography of purine nucleoside phosphorylase following initial purification steps of gel filtration and ion-exchange chromatography.     

Purification and characterization of purine nucleoside phosphorylase from Proteus vulgaris

Purine nucleoside phosphorylase was isolated and purified from cell extracts of Proteus vulgaris recovered from spoiling cod fish (Gadus morhua). The molecular weight and isoelectric point of the enzyme were 120,000 +/- 2,000 and pH 6.8. The Michaelis constant for inosine as substrate was 3.9 x 10(-5). Guanosine also served as a substrate (Km = 2.9 x 10(-5). However, the enzyme was incapable of phosphorylizing adenosine. Adenosine proved to be useful as a competitive inhibitor and was used as a ligand for affinity chromatography of purine nucleoside phosphorylase following initial purification steps of gel filtration and ion-exchange chromatography.     

Purine nucleoside synthesis, an efficient method employing nucleoside phosphorylases

An improved method for the enzymatic synthesis of purine nucleosides is described. Pyrimidine nucleosides were used as pentosyl donors and two phosphorylases were used as catalysts. One of the enzymes, either uridine phosphorylase (Urd Pase) or thymidine phosphorylase (dThd Pase), catalyzed the phosphorolysis of the pentosyl donor. The other enzyme, purine nucleoside phosphorylase (PN Pase), catalyzed the synthesis of the product nucleoside by utilizing the pentose 1-phosphate ester generated from the phosphorolysis of the pyrimidine nucleoside. Urd Pase, dThd Pase, and PN Pase were separated from each other in extracts of Escherichia coli by titration with calcium phosphate gel. Each enzyme was further purified by ion-exchange chromatography. Factors that affect the stability of these catalysts were studied. The pH optima for the stability of Urd Pase, dThd Pase, and PN Pase were 7.6, 6.5, and 7.4, respectively. The order of relative heat stability was Urd Pase greater than PN Pase greater than dThd Pase. The stability of each enzyme increased with increasing enzyme concentration. This dependence was strongest with dThd Pase and weakest with Urd Pase. Of the substrates tested, the most potent stabilizers of Urd Pase, dThd Pase, and PN Pase were uridine, 2'-deoxyribose 1-phosphate, and ribose 1-phosphate, respectively. Some general guidelines for optimization of yields are given. In a model reaction, optimal product formation was obtained at low phosphate concentrations. As examples of the efficiency of the method, the 2'-deoxyribonucleoside of 6-(dimethylamino)purine and the ribonucleoside of 2-amino-6-chloropurine were prepared in yields of 81 and 76%, respectively.