Interaction of Escherichia coli purine nucleoside phosphorylase (PNP) with the cationic and zwitterionic forms of the fluorescent substrate N(7)-methylguanosine

Steady-state and time-resolved fluorescence spectroscopy, and enzyme kinetics, were applied to study the reaction of purine nucleoside phosphorylase (PNP) from Escherichia coli with its substrate N(7)-methylguanosine (m7Guo), which consists of an equilibrium mixture of cationic and zwitterionic forms (pK(a)=7.0), each with characteristic absorption and fluorescence spectra, over the pH range 6-9, where absorption and intrinsic fluorescence of the enzyme are virtually unchanged. The pH-dependence of kinetic constants for phosphorolysis of m7Guo were studied under condition where the population of the zwitterion varied from 10% to 100%. This demonstrated that, whereas the zwitterion is a 3- to 6-fold poorer substrate, if at all, than the cation for the mammalian enzymes, both ionic species are almost equally good substrates for E. coli PNP. The imidazole-ring-opened form of m7Guo is neither a substrate nor an inhibitor of phosphorolysis. Enzyme fluorescence quenching, and concomitant changes in absorption and fluorescence spectra of the two ionic species of m7Guo on binding, showed that both forms are bound by the enzyme, the affinity of the zwitterion being 3-fold lower than that of the cation. Binding of m7Guo is bimodal, i.e., an increase in ligand concentration leads to a decrease in the association constant of the enzyme-ligand complex, typical for negative cooperativity of enzyme-ligand binding, with a Hill constant <1. This is in striking contrast to interaction of the enzyme with the parent Guo, for which the association constant is independent of concentration. The weakly fluorescent N(7)-methylguanine (m7Gua), the product of phosphorolysis of m7Guo, is a competitive non-substrate inhibitor of phosphorolysis (K(i)=8+/-2 microM) and exhibits negative cooperativity on binding to the enzyme at pH 6.9. Quenching of enzyme emission by the ligands is a static process, inasmuch as the mean excited-state lifetime, =2.7 ns, is unchanged in the presence of the ligands, and the constants K(SV) may therefore be considered as the association constants for the enzyme-ligand complexes. In the pH range 9.5-11 there is an instantaneous reversible decrease in PNP emission of approximately 15%, corresponding to one of the six tyrosine residues per subunit readily accessible to solvent, and OH- ions. Relevance of the overall results to the mechanism of phosphorolysis, and binding of substrates/inhibitors is discussed.     

Kinetic properties of Cellulomonas sp. purine nucleoside phosphorylase with typical and non-typical substrates: implications for the reaction mechanism

Phosphorolysis catalyzed by Cellulomonas sp. PNP with typical nucleoside substrate, inosine (Ino), and non-typical 7-methylguanosine (m7Guo), with either nucleoside or phosphate (Pd) as the varied substrate, kinetics of the reverse synthetic reaction with guanine (Gua) and ribose-1-phosphate (R1P) as the varied substrates, and product inhibition patterns of synthetic and phosphorolytic reaction pathways were studied by steady-state kinetic methods. It is concluded that, like for mammalian trimeric PNP, complex kinetic characteristics observed for Cellulomonas enzyme results from simultaneous occurrence of three phenomena. These are sequential but random, not ordered binding of substrates, tight binding of one substrate purine bases, leading to the circumstances that for such substrates (products) rapid-equilibrium assumptions do not hold, and a dual role of Pi, a substrate, and also a reaction modifier that helps to release a tightly bound purine base.     

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.     

Purine nucleoside phosphorylase of rabbit liver. Mechanism of catalysis.

Initial velocity studies and product inhibition patterns for purine nucleoside phosphorylase from rabbit liver were examined in order to determine the predominant catalytic mechanism for the synthetic (forward) and phosphorolytic (reverse) reactions of the enzyme. Initial velocity studies in the absence of products gave intersecting or converging linear double reciprocal plots of the kinetic data for both the synthetic and phosphorolytic reactions of the enzyme. The observed kinetic pattern was consistent with a sequential mechanism, requiring that both substrates add to the enzyme before products may be released. The product inhibition patterns showed mutual competitive inhibition between guanine and guanosine as variable substrates and inhibitors. Ribose 1-phosphate and inorganic orthophosphate were also mutually competitive toward each other. Other combinations of substrates and products gave noncompetitive inhibition. Apparent inhibition constants calculated for guanine as competitive inhibitor and for ribose 1-phosphate as noncompetitive inhibitor of the enzyme, with guanosine as variable substrate, did not vary significantly with increasing concentrations of inorganic orthophosphate as fixed substrate. These results suggest that the mechanism was order and that substrates add to the enzyme in an obligatory order. Dead end inhibition studies carried out in the presence of the products guanine and ribose 1-phosphate, respectively, showed that the kinetically significant abortive ternary complexes of enzyme-guanine-inorganic orthophosphate (EQB) and enzyme-guanose-ribose 1-phosphate (EAP) are formed. The results of dead end inhibition studies are consistent with an obligatory order of substrate addition to the enzyme. The nucleoside or purine is probably the first substrate to form a binary complex with the enzyme, and with which inorganic orthophosphate or ribose 1-phosphate may interact as secondary substrates. The evidences presented in this investigation support an Ordered Theorell-Chance mechanism for the enzyme.     

KINETIC PROPERTIES OF CELLULOMONAS SP. PURINE NUCLEOSIDE PHOSPHORYLASE WITH TYPICAL AND NON-TYPICAL SUBSTRATES: IMPLICATIONS FOR THE REACTION MECHANISM

Phosphorolysis catalyzed by Cellulomonas sp. PNP with typical nucleoside substrate, inosine (Ino), and non-typical 7-methylguanosine (m⁷Guo), with either nucleoside or phosphate (P_i) as the varied substrate, kinetics of the reverse synthetic reaction with guanine (Gua) and ribose-1-phosphate (R1P) as the varied substrates, and product inhibition patterns of synthetic and phosphorolytic reaction pathways were studied by steady-state kinetic methods. It is concluded that, like for mammalian trimeric PNP, complex kinetic characteristics observed for Cellulomonas enzyme results from simultaneous occurrence of three phenomena. These are sequential but random, not ordered binding of substrates, tight binding of one substrate purine bases, leading to the circumstances that for such substrates (products) rapid-equilibrium assumptions do not hold, and a dual role of P_i, a substrate, and also a reaction modifier that helps to release a tightly bound purine base.     

Purine nucleoside phosphorylase. Kinetic mechanism of the enzyme from calf spleen.

Ribose 1-phosphate, phosphate, and acyclovir diphosphate quenched the fluorescence of purine nucleoside phosphorylase at pH 7.1 and 25 degrees C. The fluorescence of enzyme-bound guanine was similar to that of anionic guanine in ethanol. Guanine and ribose 1-phosphate bound to free enzyme, whereas inosine and guanosine were not bound to free enzyme in the absence of phosphate. Thus, synthesis proceeded by a random mechanism, and phosphorolysis proceeded by an ordered mechanism. Steady-state kinetic data for the phosphorolysis of 100 microM guanosine were fitted to a bifunctional kinetic model with catalytic rate constants of 22 and 1.3 s-1. The dissociation rate constants for guanine from the enzyme-guanine complex at high and low phosphate concentrations were similar to the catalytic rate constants. Fluorescence changes of the enzyme during phosphorolysis suggested that ribose 1-phosphate dissociated from the enzyme ribose 1-phosphate-guanine complex rapidly and that guanine dissociated from the enzyme-guanine complex slowly. The association and dissociation rate constants for acyclovir diphosphate, a potent inhibitor of the enzyme (Tuttle, J. V., and Krenitsky, T. A. (1984) J. Biol. Chem. 259, 4065-4069), were also dependent on phosphate concentration. The effects of phosphate are discussed in terms of a dual functional binding site for phosphate.     

Properties of two unusual, and fluorescent, substrates of purine-nucleoside phosphorylase: 7-methylguanosine and 7-methylinosine

The properties of two unusual substrates of calf spleen purine-nucleoside phosphorylase (purine-nucleoside:orthophosphate ribosyltransferase, EC 2.4.2.1), 7-methylguanosine and 7-methylinosine, are described. The corresponding bases, 7-methylguanine and 7-methylhypoxanthine, are neither substrates in the reverse, synthetic reaction, nor inhibitors of the phosphorolysis reaction. Both nucleosides exhibit fluorescence, which disappears on cleavage of the glycosidic bond, providing a new convenient procedure for continuous fluorimetric assay of enzymatic activity. For 7-methylguanosine at neutral pH and 25 degrees C, Vmax = 3.3 mumol/min per unit enzyme and Km = 14.7 microM, so that Vmax/Km = 22 X 10(-2)/min per unit as compared to 8 X 10(-2) for the commonly used substrate inosine. The permissible initial substrate concentration range is 5-100 microM. Enzyme activity may also be monitored spectrophotometrically. For 7-methylinosine, Vmax/Km is much lower, 2.4 X 10(-2), but its 10-fold higher fluorescence partially compensates for this, and permits the use of initial substrate concentrations in the range 1-500 microM. At neutral pH both substrates are mixtures of cationic and zwitterionic forms. Measurements of pH-dependence of kinetic constants indicated that the cationic forms are the preferred substrates, whereas the monoanion of inosine appears to be almost as good a substrate as the neutral form. With 7-methylguanosine as substrate, and monitoring of activity fluorimetrically and spectrophotometrically, inhibition constants were measured for several known inhibitors, and the results compared with those obtained with inosine as substrate, and with results reported for the enzyme from other sources.     

Purine nucleoside phosphorylase from Cellulomonas sp.: physicochemical properties and binding of substrates determined by ligand-dependent enhancement of enzyme intrinsic fluorescence,and by protective effects of ligands on thermal inactivation of the enzyme

Purine nucleoside phosphorylase (PNP) from Cellulomonas sp., homotrimeric in the crystalline state, is also a trimer in solution. Other features of the enzyme are typical for "low molecular mass" PNPs, except for its unusual stability at pH 11. Purine bases, alpha-D-ribose-1-phosphate (R1P) and phosphate enhance the intrinsic fluorescence of Cellulomonas PNP, and hence form binary complexes and induce conformational changes of the protein that alter the microenvironment of tryptophan residue(s). The effect due to guanine (Gua) binding is much higher than those caused by other ligands, suggesting that the enzyme preferentially binds a fluorescent, most probably rare tautomeric anionic form of Gua, further shown by comparison of emission properties of the PNP/Gua complex with that of Gua anion and its N-methyl derivatives. Guanosine (Guo) and inosine (Ino) at 100 microM concentration show little and no effect, respectively, on enzyme intrinsic fluorescence, but their protective effect against thermal inactivation of the enzyme points to their forming weak binary complexes with PNP. Binding of Gua, hypoxanthine (Hx) and R1P to the trimeric enzyme is described by one dissociation constant, K(d)=0.46 microM for Gua, 3.0 microM for Hx, and 60 microM for R1P. The binding stoichiometry for Gua (and probably Hx) is three ligand molecules per enzyme trimer. Effects of phosphate on the enzyme intrinsic fluorescence are due not only to binding, but also to an increase in ionic strength, as shown by titration with KCl. When corrected for effects of ionic strength, titration data with phosphate are most consistent with one dissociation constant, K(d)=270 microM, but existence of a very weak binding site with K(d)>50 mM could not be unequivocally ruled out. Binding of Gua to the PNP/phosphate binary complex is weaker (K(d)=1.7 microM) than to the free enzyme (K(d)=0.46 microM), suggesting that phosphate helps release the purine base in the catalytic process of phosphorolysis.The results indicate that nonlinear kinetic plots of initial velocity, typical for PNPs, including Cellulomonas PNP, are not, as generally assumed, due to cooperative interaction between monomers forming the trimer, but to a more complex kinetic mechanism than hitherto considered.     

Purine nucleoside phosphorylase. Inosine hydrolysis, tight binding of the hypoxanthine intermediate, and third-the-sites reactivity.

Purine nucleoside phosphorylase from calf spleen is a trimer which catalyzes the hydrolysis of inosine to hypoxanthine and ribose in the absence of inorganic phosphate. The reaction occurs with a turnover number of 1.3 x 10(-4) s-1 per catalytic site. Hydrolysis of enzyme-bound inosine occurs at a rate of 2.0 x 10(-3) s-1 to form a stable enzyme-hypoxanthine complex and free ribose. The enzyme hydrolyzes guanosine; however, a tightly-bound guanine complex could not be isolated. The complex with hypoxanthine is stable to gel filtration but can be dissociated by acid, base, or mild denaturing agents. Following gel filtration, the E.hypoxanthine complex dissociates at a rate of 1.9 x 10(-6) s-1 at 4 degrees C and 1.3 x 10(-4) s-1 at 30 degrees C. The dissociation constant for the tightly-bound complex of enzyme-hypoxanthine is estimated to be 1.3 x 10(-12) M at 30 degrees C on the basis of the dissociation rate. The stoichiometry of the reaction is 1 mol of hypoxanthine bound per trimer. The reaction is reversible since the same complex can be formed from enzyme and hypoxanthine. Addition of ribose 1-phosphate to the complex results in the formation of inosine without release of hypoxanthine. Thus, the complex is catalytically competent. Inorganic phosphate or arsenate prevents formation of the tightly-bound E.hypoxanthine complex from inosine or hypoxanthine. Direct binding studies with hypoxanthine in the presence of phosphate result in 3 mol of hypoxanthine bound per trimer with a dissociation constant of 1.6 microM. In the absence of phosphate, three hypoxanthines are bound, but higher hypoxanthine concentrations cause the release of two of the hypoxanthines with an apparent inhibition constant of 130 microM. The results establish that enzymatic contacts with the nucleoside alone are sufficient to destabilize the N-glycosidic bond. In the absence of phosphate, water attacks slowly, causing net hydrolysis. The hydrolytic reaction leaves hypoxanthine stranded at the catalytic site, tightly bound to the enzyme with a conformation related to the transition state. In the phosphorolysis reaction, ribose 1-phosphate causes relaxation of this conformation and rapid release of hypoxanthine.     

Methylthioinosine phosphorylase from Pseudomonas aeruginosa. Structure and annotation of a novel enzyme in quorum sensing

The PA3004 gene of Pseudomonas aeruginosa PAO1 was originally annotated as a 5'-methylthioadenosine phosphorylase (MTAP). However, the PA3004 encoded protein uses 5'-methylthioinosine (MTI) as a preferred substrate and represents the only known example of a specific MTI phosphorylase (MTIP). MTIP does not utilize 5'-methylthioadenosine (MTA). Inosine is a weak substrate with a k(cat)/K(m) value 290-fold less than MTI and is the second best substrate identified. The crystal structure of P. aeruginosa MTIP (PaMTIP) in complex with hypoxanthine was determined to 2.8 ? resolution and revealed a 3-fold symmetric homotrimer. The methylthioribose and phosphate binding regions of PaMTIP are similar to MTAPs, and the purine binding region is similar to that of purine nucleoside phosphorylases (PNPs). The catabolism of MTA in P. aeruginosa involves deamination to MTI and phosphorolysis to hypoxanthine (MTA → MTI → hypoxanthine). This pathway also exists in Plasmodium falciparum, where the purine nucleoside phosphorylase (PfPNP) acts on both inosine and MTI. Three tight-binding transition state analogue inhibitors of PaMTIP are identified with dissociation constants in the picomolar range. Inhibitor specificity suggests an early dissociative transition state for PaMTIP. Quorum sensing molecules are associated with MTA metabolism in bacterial pathogens suggesting PaMTIP as a potential therapeutic target.     

Effects of acyclovir and its metabolites on purine nucleoside phosphorylase

Acyclovir (9-(2-hydroxyethoxymethyl)guanine), the clinically useful antiherpetic agent, is an "acyclic" analogue of 2'-deoxyguanosine. Purine nucleoside phosphorylase partially purified from human erythrocytes did not catalyze detectable phosphorolysis of this drug or any of its metabolites (less than 0.07% of the rate with Guo). However, these compounds were competitive inhibitors of this enzyme with Ino as the variable substrate. Acyclovir per se was a relatively weak inhibitor. Its Ki value (91 microM) was much greater than that for its 8-hydroxy metabolite (Ki = 4.7 microM) but less than that for its carboxylic acid metabolite (9-carboxymethoxy-methylguanine) (K'i = 960 microM). The phosphorylated metabolites of acyclovir were more potent inhibitors than were their guanine nucleotide counterparts. At a phosphate concentration of 50 mM, the apparent Ki values for the mono- (120 microM), di- (0.51 microM), and tri (43 microM)-phosphate esters of acyclovir were 1/2, 1/1200, and 1/26 those for dGMP, dGDP, and dGTP, respectively. The concentration of phosphate did not markedly affect the Ki value of acyclovir but dramatically affected those of its phosphorylated metabolites and their nucleotide counterparts. Decreasing phosphate to a physiological concentration (1 mM) decreased the apparent Ki values for the mono-, di-, and triphosphate esters of acyclovir to 6.6, 0.0087, and 0.31 microM, respectively. Inhibition of the enzyme by acyclovir diphosphate was also influenced by pH. This metabolite of acyclovir is the most potent inhibitor of purine nucleoside phosphorylase reported to date. It has some features of a "multisubstrate" analogue inhibitor.     

Interactions of calf spleen purine nucleoside phosphorylase with 8-azaguanine, and a bisubstrate analogue inhibitor: implications for the reaction mechanism

Interactions of calf spleen purine nucleoside phosphorylase (PNP) with a non-typical substrate, 8-azaguanine (8-azaG), and a bisubstrate analogue inhibitor, 9-(2-phosphonylmethoxyethyl)-8-azaguanine (PME-azaG), were investigated by means of steady-state fluorescence spectroscopy. Both 8-azaG and PME-azaG form fluorescent complexes with the enzyme, and dissociation constants are comparable to the appropriate parameters (Km or Ki) obtained from kinetic measurements. PME-azaG inhibits both the phosphorolytic and synthetic pathway of the reaction in a competitive mode. The complex of 8-azaG with PNP is much weaker than the previously reported Gua-PNP complex, and its dissociation constant increases at pH > 7, where 8-azaG exists predominantly as the monoanion (pKa approximately 6.5). The fluorescence difference spectrum of the PNP/8-azaG complex points to participation of the N(7)H or/and N(8)H tautomers of the neutral substrate, and the 9-(2-phosphonylmethoxyethyl) derivative also exists as a neutral species in the complex with PNP. The latter conclusion is based on spectral characteristics of the PNP/PME-azaG complex, confirmed by fluorimetric determination of dissociation constants, which are virtually pH-independent in the range 6-7. These findings testify to involvement of the neutral purine molecule, and not its monoanion, as the substrate in the reverse, synthetic reaction. It is proposed that, in the reverse reaction pathway, the natural purine substrate is bound to the enzyme as the neutral N(7)H tautomer, which is responsible for the reported strong fluorescence of the guanine-PNP complex.     

Xanthosine and xanthine. Substrate properties with purine nucleoside phosphorylases, and relevance to other enzyme systems.

Substrate properties of xanthine (Xan) and xanthosine (Xao) for purine nucleoside phosphorylases (PNP) of mammalian origin have been reported previously, but only at a single arbitrarily selected pH and with no kinetic constants. Additionally, studies have not taken into account the fact that, at physiological pH, Xao (pKa = 5.7) is a monoanion, while Xan (pKa = 7.7) is an equilibrium mixture of the neutral and monoanionic forms. Furthermore the monoanionic forms, unlike those of guanosine (Guo) and inosine (Ino), and guanine (Gua) and hypoxanthine (Hx), are still 6-oxopurines. The optimum pH for PNP from human erythrocytes and calf spleen with both Xao and Xan is in the range 5-6, whereas those with Guo and Gua, and Ino and Hx, are in the range 7-8. The pH-dependence of substrate properties of Xao and Xan points to both neutral and anionic forms as substrates, with a marked preference for the neutral species. Both neutral and anionic forms of 6-thioxanthine (pKa = 6.5 +/- 0.1), but not of 2-thioxanthine (pKa = 5.9 +/- 0.1), are weaker substrates. Phosphorolysis of Xao to Xan by calf spleen PNP at pH 5.7 levels off at 83% conversion, due to equilibrium with the reverse synthetic pathway (equilibrium constant 0.05), and not by product inhibition. Replacement of Pi by arsenate led to complete arsenolysis of Xao. Kinetic parameters are reported for the phosphorolytic and reverse synthetic pathways at several selected pH values. Phosphorolysis of 200 micro m Xao by the human enzyme at pH 5.7 is inhibited by Guo (IC50 = 10 +/- 2 micro m), Hx (IC50 = 7 +/- 1 micro m) and Gua (IC50 = 4.0 +/- 0.2 micro m). With Gua, inhibition was shown to be competitive, with Ki = 2.0 +/- 0.3 micro m. By contrast, Xao and its products of phosphorolysis (Xan and R1P), were poor inhibitors of phosphorolysis of Guo, and Xan did not inhibit the reverse reaction with Gua. Possible modes of binding of the neutral and anionic forms of Xan and Xao by mammalian PNPs are proposed. Attention is directed to the fact that the structural properties of the neutral and ionic forms of XMP, Xao and Xan are also of key importance in many other enzyme systems, such as IMP dehydrogenase, some nucleic acid polymerases, biosynthesis of caffeine and phosphoribosyltransferases.     

Phosphorolytic and ribosyl transfer mechanisms of purified chicken liver purine nucleoside phosphorylase

Purified chicken liver purine nucleoside phosphorylase shows two ionizable groups at the active site whose pKa were near pH 6.9 and 8; the molecular weight (67,000-89,000) depends on the protein concentration. Initial velocity studies and product inhibition patterns were consistent with a random mechanism, which is rapid equilibrium in the phosphorolytic reaction with a dead-end complex, but not in the synthetic reaction. Free inorganic orthophosphate purine nucleoside phosphorylase (Sephadex G-100) catalyzes a pentosyl transfer reaction from inosine to guanine according to a random Bi, Bi mechanism.     

Functional Identification of the Hypoxanthine/Guanine Transporters YjcD and YgfQ and the Adenine Transporters PurP and YicO of Escherichia coli K-12

The evolutionarily broad family nucleobase-cation symporter-2 (NCS2) encompasses transporters that are conserved in binding site architecture but diverse in substrate selectivity. Putative purine transporters of this family fall into one of two homology clusters: COG2233, represented by well studied xanthine and/or uric acid permeases, and COG2252, consisting of transporters for adenine, guanine, and/or hypoxanthine that remain unknown with respect to structure-function relationships. We analyzed the COG2252 genes of Escherichia coli K-12 with homology modeling, functional overexpression, and mutagenesis and showed that they encode high affinity permeases for the uptake of adenine (PurP and YicO) or guanine and hypoxanthine (YjcD and YgfQ). The two pairs of paralogs differ clearly in their substrate and ligand preferences. Of 25 putative inhibitors tested, PurP and YicO recognize with low micromolar affinity N⁶-benzoyladenine, 2,6-diaminopurine, and purine, whereas YjcD and YgfQ recognize 1-methylguanine, 8-azaguanine, 6-thioguanine, and 6-mercaptopurine and do not recognize any of the PurP ligands. Furthermore, the permeases PurP and YjcD were subjected to site-directed mutagenesis at highly conserved sites of transmembrane segments 1, 3, 8, 9, and 10, which have been studied also in COG2233 homologs. Residues irreplaceable for uptake activity or crucial for substrate selectivity were found at positions occupied by similar role amino acids in the Escherichia coli xanthine- and uric acid-transporting homologs (XanQ and UacT, respectively) and predicted to be at or around the binding site. Our results support the contention that the distantly related transporters of COG2233 and COG2252 use topologically similar side chain determinants to dictate their function and the distinct purine selectivity profiles.     

Purine metabolism in microplasmodia of Physarum polycephalum.

The uptake and utilization of purine nucleosides and purines in microplasmodia of Physarum polycephalum were investigated. The results revealed a unique pattern, namely that exogenous purine nucleosides are readily taken up and metabolised, while free purine bases are hardly taken up. The pathways of incorporation have been elucidated in studies with whole cells and with cell-free extracts. The ribonucleosides (adenosine, inosine and guanosine) can be converted into ribonucleotides in two ways; either directly catalysed by a kinase or by a phosphorolytic cleavage to the free base (adenine, hypoxanthine and guanine respectively) which can then be activated by a purine phosphoribosyltransferase. Apparently the purine phosphoribosyltransferases do not react with exogenous purine bases. The deoxyribonucleosides (deoxyadenosine, deoxyinosine and deoxyguanosine) are also phosphorolysed by purine nucleoside phosphorylase to adenine, hypoxanthine and guanine respectively. A portion of deoxyadenosine is directly phosphorylated to dAMP. It appears that only a minor part of the soluble nucleotide pool can be synthesised from exogenous supplied nucleosides and that none of the deoxyribonucleosides specifically label DNA. There is no catabolism of the purine moiety. In agreement with the above findings, we have found that analoguees of purine nucleosides are more toxic than their corresponding purine base analogues.     

Ribose 1-phosphate and inosine activate uracil salvage in rat brain

The purpose of this study was to determine the mechanism by which inosine activates pyrimidine salvage in CNS. The levels of cerebral inosine, hypoxanthine, uridine, uracil, ribose 1-phosphate and inorganic phosphate were determined, to evaluate the Gibbs free energy changes (deltaG) of the reactions catalyzed by purine nucleoside phosphorylase and uridine phosphorylase, respectively. A deltaG value of 0.59 kcal/mol for the combined reaction inosine+uracil <==> uridine+hypoxanthine was obtained, suggesting that at least in anoxic brain the system may readily respond to metabolite fluctuations. If purine nucleoside phosphorolysis and uridine phosphorolysis are coupled to uridine phosphorylation, catalyzed by uridine kinase, whose activity is relatively high in brain, the three enzyme activities will constitute a pyrimidine salvage pathway in which ribose 1-phosphate plays a pivotal role. CTP, presumably the last product of the pathway, and, to a lesser extent, UTP, exert inhibition on rat brain uridine nucleotides salvage synthesis, most likely at the level of the kinase reaction. On the contrary ATP and GTP are specific phosphate donors.     

Comparative study of purine nucleoside phosphorylase from rabbit kidney, spleen, liver and embryos

Homogeneous preparations of purine nucleoside phosphorylase (EC 2.4.2.1) from rabbit kidney, spleen, liver and embryos were studied. The enzyme preparations do not differ in electrophoretic mobility. The molecular weight of the enzyme obtained from various sources was determined by gel filtration on Sephadex G-150 superfine and is about 90-92 kD. The enzyme subunits are identical in terms of molecular weight, as can be evidenced from sodium dodecyl sulfate polyacrylamide gel electrophoresis (Mr approximately 31 kD). The pH optima of these enzyme preparations for guanosine and xanthosine phosphorolysis are 6.2 and 5.7, respectively. The isoelectric point of purine nucleoside phosphorylase from rabbit kidney was determined in the presence of 9 M urea and is equal to 5.55. The enzyme is the most stable at pH 7.7; it is specific towards hypoxanthine and guanine nucleosides as well as towards xanthosine, but does not cleave adenine nucleosides. The Km values for guanosine and inosine are 1.4.10(-4) M and 1.2.10(-4) M, respectively. The enzyme does not catalyze the ribosyl transfer reaction in the absence of Pi.     

Leaving group activation by aromatic stacking: an alternative to general acid catalysis

General acid catalysis is a powerful and widely used strategy in enzymatic nucleophilic displacement reactions. For example, hydrolysis/phosphorolysis of the N-glycosidic bond in nucleosides and nucleotides commonly involves the protonation of the leaving nucleobase concomitant with nucleophilic attack. However, in the nucleoside hydrolase of the parasite Trypanosoma vivax, crystallographic and mutagenesis studies failed to identify a general acid. This enzyme binds the purine base of the substrate between the aromatic side-chains of Trp83 and Trp260. Here, we show via quantum chemical calculations that face-to-face stacking can raise the pKa of a heterocyclic aromatic compound by several units. Site-directed mutagenesis combined with substrate engineering demonstrates that Trp260 catalyzes the cleavage of the glycosidic bond by promoting the protonation of the purine base at N-7, hence functioning as an alternative to general acid catalysis.     

Purine nucleoside phosphorylase. Structure-activity relationships for substrate and inhibitor properties of N-1-, N-7-, and C-8-substituted analogues; differentiation of mammalian and bacterial enzymes with N-1-methylinosine and guanosine

The previous finding that 7-methylinosine (m7Ino) and 7-methylguanosine (m7Guo) are excellent, as well as fluorescent, substrates for calf spleen purine nucleoside phosphorylase has been extended to include a series of 7-alkylguanosines with higher alkyl groups (ethyl, propyl, butyl, isopropyl, isobutyl, benzyl). All of these are good substrates, with increases in Km compensated for by corresponding increases in Vmax, and excluding the ring N-7 as a binding site. Both m1Ino and m1Guo are neither substrates nor inhibitors of this enzyme, implicating the ring N-1 as a binding site. Included also are guanosine analogues with C-8 substituents (alpha-hydroxyisopropyl, methyl, bromo, chloro, amino) with known populations of syn and anti conformers about the glycosidic bond; while these did not unequivocally point to involvement of only one form, efficiency of phosphorolysis appeared to be correlated with a conformation in the anti region. The influence of various substituents is also considered in relation to steric and electronic effects. Kinetic parameters for all the foregoing have been determined fluorimetrically and/or spectrophotometrically from initial velocities and/or continuous monitoring of phosphorolysis including Ki values for inhibition by the base moieties. Furthermore, kinetic data demonstrated that, in striking contrast to the mammalian enzyme, both m1Ino and m1Guo are almost as good substrates as the parent nucleosides for a bacterial (Escherichia coli) enzyme, suggesting that the ring N-1 is not a binding site for the latter.