Substrate For Purine Nucleoside Phosphorylase Research Articles

Separate Evaluation of Fraction Absorbed and Intestinal Availability after Oral Administration of Drugs Based on the Measurement of Portal and Systemic Plasma Concentrations and Luminal Concentration.

There are several experimental methods to estimate the product of the fraction absorbed (Fa) and intestinal availability (Fg) in vivo after oral administration of drugs. Metabolic enzyme inhibitors are typically used to separate Fg from Fa·Fg. Since Fa·Fg can be regarded as Fa under metabolism-inhibited conditions, Fg can be isolated by dividing Fa·Fg by Fa. However, if the inhibition of intestinal metabolism is insufficient, Fa is overestimated, which results in an underestimation of Fg compared to the actual value. In this study, to avoid this problem, an experimental method for the separate estimation of Fa and Fg in rats without utilizing metabolic enzyme inhibitors was established. Buspirone, a CYP3A substrate, and ribavirin, a substrate of purine nucleoside phosphorylase and adenosine kinase, were selected as models. Following oral administration of the drugs with fluorescein isothiocyanate dextran 4000 (FD-4, an unabsorbable marker), Fa·Fg was pharmacokinetically calculated from portal and systemic plasma concentration-time profiles of model drugs and Fa was calculated from the difference in the ileal concentration profiles of the drugs and FD-4. Fg was evaluated by dividing Fa·Fg by Fa. Following oral administration, buspirone was not detected in any segment of the small intestine, indicating that the administered buspirone was completely absorbed. In addition, buspirone was extensively metabolized in enterocytes (Fg = 20.1). Ribavirin was primarily absorbed in the upper segment of the small intestine, and 64.4% of the ribavirin was absorbed before it reached the ileum. In addition, it was revealed that ribavirin was metabolized more extensively in the intestine than in the liver. Our method may be effective in quantitatively assessing Fa and Fg in vivo, which can help in the formulation design and prediction of drug-drug interactions.

Site-directed mutation of purine nucleoside phosphorylase for synthesis of 2'-deoxy-2'-fluoroadenosine

Nucleoside analogs are commonly used drugs for the treatment of cancer and viral infections. Purine nucleoside phosphorylase (PNP) is one of key enzymes required for the biosynthesis of 2’-deoxy-2’-fluoroadenosine. To improve PNP activity, two methods for selecting mutation sites were used based on molecular docking and dynamic simulations of PNP and various substrates. An efficient PNP mutant screening method was established. The variant E166 F/M167D 2 M had the highest activity and was selected for further characterization. The enzymatic activity and reaction rate of transglycosylation catalyzed by this variant were increased by 47.6 % and 38.8 % compared with the wild-type PNP, respectively. Two representative variants were used for analysis of conformational differences. We discovered that the flexibility of the random coil where Phe159 was located had a significant impact on the active center. In addition, the synthesis of 2’-deoxy-2’-fluoroadenosine was scaled up using 500 mL of phosphate buffer (100 mM, pH 7.0) containing 62.5 mM adenine, 25 mM 2’-deoxy-2’-fluorouridine, 3 mg thymidine phosphorylase (TP), and 3 mg E166 F/M167D 2 M. The concentration of the product reached 14.35 mM and the conversion rate reached 57.4 %. Thus, this process represents a promising approach for industrial production of 2’-deoxy-2’-fluoroadenosine.

Tricyclic Nucleobase Analogs and Their Ribosides as Substrates and Inhibitors of Purine-Nucleoside Phosphorylases III. Aminopurine Derivatives.

Etheno-derivatives of 2-aminopurine, 2-aminopurine riboside, and 7-deazaadenosine (tubercidine) were prepared and purified using standard methods. 2-Aminopurine reacted with aqueous chloroacetaldehyde to give two products, both exhibiting substrate activity towards bacterial (E. coli) purine-nucleoside phosphorylase (PNP) in the reverse (synthetic) pathway. The major product of the chemical synthesis, identified as 1,N2-etheno-2-aminopurine, reacted slowly, while the second, minor, but highly fluorescent product, reacted rapidly. NMR analysis allowed identification of the minor product as N2,3-etheno-2-aminopurine, and its ribosylation product as N2,3-etheno-2-aminopurine-N2-β-d-riboside. Ribosylation of 1,N2-etheno-2-aminopurine led to analogous N2-β-d-riboside of this base. Both enzymatically produced ribosides were readily phosphorolysed by bacterial PNP to the respective bases. The reaction of 2-aminopurine-N9-β -d-riboside with chloroacetaldehyde gave one major product, clearly distinct from that obtained from the enzymatic synthesis, which was not a substrate for PNP. A tri-cyclic 7-deazaadenosine (tubercidine) derivative was prepared in an analogous way and shown to be an effective inhibitor of the E. coli, but not of the mammalian enzyme. Fluorescent complexes of amino-purine analogs with E. coli PNP were observed.

Crystal structure of Escherichia coli purine nucleoside phosphorylase complexed with acyclovir.

Escherichia coli purine nucleoside phosphorylase (PNP), which catalyzes the reversible phosphorolysis of purine ribonucleosides, belongs to the family I hexameric PNPs. Owing to their key role in the purine salvage pathway, PNPs are attractive targets for drug design against some pathogens. Acyclovir (ACV) is an acyclic derivative of the PNP substrate guanosine and is used as an antiviral drug for the treatment of some human viral infections. The crystalline complex of E. coli PNP with acyclovir was prepared by co-crystallization in microgravity using counter-diffusion through a gel layer in a capillary. The structure of the E. coli PNP-ACV complex was solved at 2.32 Å resolution using the molecular-replacement method. The ACV molecule is observed in two conformations and sulfate ions were located in both the nucleoside-binding and phosphate-binding pockets of the enzyme. A comparison with the complexes of other hexameric and trimeric PNPs with ACV shows the similarity in acyclovir binding by these enzymes.

Enzymatic synthesis and phosphorolysis of 4(2)-thioxo- and 6(5)-azapyrimidine nucleosides by E. coli nucleoside phosphorylases.

The trans-2-deoxyribosylation of 4-thiouracil (4SUra) and 2-thiouracil (2SUra), as well as 6-azauracil, 6-azathymine and 6-aza-2-thiothymine was studied using dG and E. coli purine nucleoside phosphorylase (PNP) for the in situ generation of 2-deoxy-α-D-ribofuranose-1-phosphate (dRib-1P) followed by its coupling with the bases catalyzed by either E. coli thymidine (TP) or uridine (UP) phosphorylases. 4SUra revealed satisfactory substrate activity for UP and, unexpectedly, complete inertness for TP; no formation of 2’-deoxy-2-thiouridine (2SUd) was observed under analogous reaction conditions in the presence of UP and TP. On the contrary, 2SU, 2SUd, 4STd and 2STd are good substrates for both UP and TP; moreover, 2SU, 4STd and 2’-deoxy-5-azacytidine (Decitabine) are substrates for PNP and the phosphorolysis of the latter is reversible. Condensation of 2SUra and 5-azacytosine with dRib-1P (Ba salt) catalyzed by the accordant UP and PNP in Tris∙HCl buffer gave 2SUd and 2’-deoxy-5-azacytidine in 27% and 15% yields, respectively. 6-Azauracil and 6-azathymine showed good substrate properties for both TP and UP, whereas only TP recognizes 2-thio-6-azathymine as a substrate. 5-Phenyl and 5-tert-butyl derivatives of 6-azauracil and its 2-thioxo derivative were tested as substrates for UP and TP, and only 5-phenyl- and 5-tert-butyl-6-azauracils displayed very low substrate activity. The role of structural peculiarities and electronic properties in the substrate recognition by E. coli nucleoside phosphorylases is discussed.

Development of a new HPLC method using fluorescence detection without derivatization for determining purine nucleoside phosphorylase activity in human plasma.

Purine nucleoside phosphorylase (PNP) activity is involved in cell survival and function, since PNP is a key enzyme in the purine metabolic pathway where it catalyzes the phosphorolysis of the nucleosides to the corresponding nucleobases. Its dysfunction has been found in relevant pathological conditions (such as inflammation and cancer), so the detection of PNP activity in plasma could represent an attractive marker for early diagnosis or assessment of disease progression. Thus the aim of this study was to develop a simple, fast and sensitive HPLC method for the determination of PNP activity in plasma. The separation was achieved on a Phenomenex Kinetex PFP column using 0.1% formic acid in water and methanol as mobile phases in gradient elution mode at a flow rate of 1ml/min and purine compounds were detected using UV absorption and fluorescence. The analysis was fast since the run was achieved within 13min. This method improved the separation of the different purines, allowing the UV-based quantification of the natural PNP substrates (inosine and guanosine) or products (hypoxanthine and guanine) and its subsequent metabolic products (xanthine and uric acid) with a good precision and accuracy. The most interesting innovation is the simultaneous use of a fluorescence detector (excitation/emission wavelength of 260/375nm) that allowed the quantification of guanosine and guanine without derivatization. Compared with UV, the fluorescence detection improved the sensitivity for guanine detection by about 10-fold and abolished almost completely the baseline noise due to the presence of plasma in the enzymatic reaction mixture. Thus, the validated method allowed an excellent evaluation of PNP activity in plasma which could be useful as an indicator of several pathological conditions.

Two fluorogenic substrates for purine nucleoside phosphorylase, selective for mammalian and bacterial forms of the enzyme

Two nontypical nucleosides, 7-β-d-ribosyl-2,6-diamino-8-azapurine and 8-β-d-ribosyl-2,6-diamino-8-azapurine, have been found to exhibit moderately good, and selective, substrate properties toward calf and bacterial (Escherichia coli) forms of purine nucleoside phosphorylase (PNP). The former compound is effectively phosphorolysed by calf PNP and the latter by PNP from E. coli. Both compounds are fluorescent with λmax∼425 to 430nm, but the reaction product, 2,6-diamino-8-azapurine, emits in a different spectral region (λmax∼363nm) with nearly 40% yield, providing a strong fluorogenic effect at 350 to 360nm.

Chemoenzymatic method of 1,2,4-triazole nucleoside synthesis: Possibilities and limitations

The potentialities and restrictions of chemoenzymatic approach to the synthesis of new structural analogues of antiviral drug Ribavirin (1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide) have been determined. Syntheses of various amides of 1H-1,2,4-triazole-3-carboxylic acid and its 5-substituted analogues, prospective substrates of purine nucleoside phosphorylase (PNP), have been reported. The comparative effectiveness of the methods for obtaining amides aforementioned and also the methods for introducing functional groups to the C5 position of the heterocyclic system has been studied. New Ribavirin analogues bearing various substituents in the carboxamide group have been synthesized. The biotechnological method for the preparation of 1-beta-D-ribofuranosyl- 1,2,4-triazole-3-carbonitrile used as the intermediate in the synthesis of Viramidine, a contemporary Ribavirin analogue, has been developed.

A continuous spectrophotometric assay for mitogen-activated protein kinase kinases

We describe a convenient and simple continuous spectrophotometric method for the determination of mitogen-activated protein kinase (MAPK) kinase activity with its protein substrate. The assay relies on the measurement of phosphoprotein product generated in the first step of the MAPK kinase reaction. Dephosphorylation of the phosphoprotein is coupled to a MAPK phosphatase to generate phosphate, which is then used as the substrate of purine nucleoside phosphorylase to catalyze the N-glycosidic cleavage of 2-amino 6-mercapto 7-methyl purine ribonucleoside. Of the reaction products ribose 1-phosphate and 2-amino 6-mercapto 7-methylpurine, the latter has a high absorbance at 360 nm relative to the nucleoside and, hence, provides a spectrophotometric signal that can be continuously followed. In the presence of excess phosphatase, the phosphorylated protein substrate molecules undergo dephosphorylation almost immediately after their formation; the steady-state use of the resultant inorganic phosphate is a reflection of the constant initial velocity of the exchange reaction. The validity of this method has been confirmed by using it to measure the activities of MEK1 (MAPK/ERK kinase 1) and MKK6 (MAPK kinase 6) toward their physiological substrates. Our findings of the MAPK kinases in the current study provide evidence that the substrate binding affinities of this subfamily of protein kinases are at the submicromolar concentration.

Purine nucleoside phosphorylase activity and expression are upregulated in sites affected by periodontal disease

Purine nucleoside phosphorylase (PNP) is an enzyme that catalyzes the reversible phosphorolysis of purine nucleosides, playing a key role in the purine salvage pathway. Activated T cells seem to rely heavily on PNP to remain functionally active and are particularly sensitive to PNP deficiency. The role of PNP in periodontal tissues has not been characterized thus far. The aim of this study therefore was to assess the activity and expression of PNP in the gingival tissues of periodontitis patients. Ten patients consecutively admitted for treatment had their periodontal clinical variables recorded and their gingival crevicular fluid collected. After periodontal treatment the patients were seen once a month for plaque and bleeding control, and had their periodontal variables recorded and gingival crevicular fluid collected at 90 and 180 d. Purine nucleoside phosphorylase-specific activity was assessed using a spectrophotometer through the addition of the PNP substrate analog 2-amino-6mercapto-7-methyl purine riboside to the gingival crevicular fluid. In parallel, PNP expression was assessed by immunohistochemistry and real-time PCR in gingival biopsies and cell culture. Purine nucleoside phosphorylase activity was higher in the gingival crevicular fluid of periodontally diseased sites, which was positively correlated with improvements of the clinical variables. Treatment of periodontal disease induced a striking decrease of PNP activity in periodontally diseased sites. Expression of PNP was more pronounced in mononuclear cells and endothelial cells of the gingiva, and the mRNA levels were 5.7-fold higher in inflamed tissues compared with control samples. Purine nucleoside phosphorylase activity and expression are upregulated in periodontally diseased sites and can be detected in the gingival crevicular fluid.

Fluorescence emission properties of 8-azaxanthine and its N-alkyl derivatives: Excited-state proton transfer, and potential applications in enzymology

We have investigated the emission properties of 8-azaxanthine (8-azaXan) and its triazolo N-alkyl derivatives, with particular emphasis on N(8)-methyl-8-azaXan. In aqueous medium, both 8-azaXan and its 8-alkyl congener emit at 420 nm, with an unusually large Stokes’ shift, and decay times of 9 and 12 ns, respectively. With the parent 8-azaXan, emission is observed over a narrow pH range (2–6), where it is partially or predominantly in its neutral form in the ground state (p K a 4.9), whereas for 8-methyl-8-azaXan emission is observed in the pH range 2–12, hence for both the neutral and monoanionic species (p K a 7.2). Fluorescence excitation spectra are in accord with the absorption spectra at all pH values. In anhydrous methanol, 8-azaX, in its neutral form, emits at about 335 nm. In 10% aqueous methanol, acidified to pH ∼3, two emission bands are observed, at 420 and 335 nm. With the 8-methyl derivative, dual emission at 420 and ∼340 nm is observed in anhydrous methanol, but in isopropanol and acetonitrile the 420 band virtually disappears. It is concluded that the 420 nm emission band, ascribed to the monoanionic form of 8-methyl-8-azaXan, appears in neutral or acidic media as the result of rapid deprotonation of the N(3)–H in the excited states of both 8-azaXan and its 8-alkyl derivative. This leads to phototautomerism in the 8-azaXan monoanion which, in the ground state, is present in the form resulting from dissociation of the triazole proton, i.e., N(8)–H. This proton transfer is slowed down in non-aqueous solvents, where emission from the neutral molecules (335–340 nm) is observed. The foregoing interpretation is supported by determination, with the aid of the Foerster cycle, of the excited state p K * values, close to −0.5 for 8-methyl-8-azaXan. The strong emission of 8-azaXan and its N-alkyl derivatives should prove useful in studies on enzyme/ligand interactions, several examples of which are proposed. In particular, we report that 8-azaXan is a substrate for purine nucleoside phosphorylase II from E. coli, and show that the reaction can be monitored fluorimetrically.

Identification of a Subversive Substrate of Trichomonas vaginalis Purine Nucleoside Phosphorylase and the Crystal Structure of the Enzyme-Substrate Complex

Trichomonas vaginalis is an anaerobic protozoan parasite that causes trichomoniasis, a common sexually transmitted disease with worldwide impact. One of the pivotal enzymes in its purine salvage pathway, purine nucleoside phosphorylase (PNP), shows physical properties and substrate specificities similar to those of the high molecular mass bacterial PNPs but differing from those of human PNP. While carrying out studies to identify inhibitors of T. vaginalis PNP (TvPNP), we discovered that the nontoxic nucleoside analogue 2-fluoro-2'-deoxyadenosine (F-dAdo) is a "subversive substrate." Phosphorolysis by TvPNP of F-dAdo, which is not a substrate for human PNP, releases highly cytotoxic 2-fluoroadenine (F-Ade). In vitro studies showed that both F-dAdo and F-Ade exert strong inhibition of T. vaginalis growth with estimated IC(50) values of 106 and 84 nm, respectively, suggesting that F-dAdo might be useful as a potential chemotherapeutic agent against T. vaginalis. To understand the basis of TvPNP specificity, the structures of TvPNP complexed with F-dAdo, 2-fluoroadenosine, formycin A, adenosine, inosine, or 2'-deoxyinosine were determined by x-ray crystallography with resolutions ranging from 2.4 to 2.9 A. These studies showed that the quaternary structure, monomer fold, and active site are similar to those of Escherichia coli PNP. The principal active site difference is at Thr-156, which is alanine in E. coli PNP. In the complex of TvPNP with F-dAdo, Thr-156 causes the purine base to tilt and shift by 0.5 A as compared with the binding scheme of F-dAdo in E. coli PNP. The structures of the TvPNP complexes suggest opportunities for further improved subversive substrates beyond F-dAdo.

Purine Nucleoside Phosphorylase Activity in Rat Cerebrospinal Fluid

The sequential hydrolysis of purines is present in rat CSF and generates nucleosides as inosine and guanosine that are usual substrates for purine nucleoside phosphorylase (PNP). PNP catalyzes phosphorolysis of the purine nucleosides and deoxynucleosides releasing purine bases. Here we investigated the presence of PNP in CSF of rats using: i) a specific chromophoric analogue of nucleosides, 2-amino-6-mercapto-7-methylpurine ribonucleoside (MESG), and ii) an inhibitor of PNP activity, immucillin-H. Additionally, we performed a preliminary kinetic characterization (K(M): Henry-Michaelis-Menten constant; V: maximal velocity) for MESG and inorganic phosphate (Pi). The values of K(M) and V for MESG (n = 3, mean+/-SD) were 142.5+/-29.5 microM and 0.0102+/-0.0006 U mg(-1), respectively. For Pi (n=3, mean+/-SD), the K(M) values and V were 186.8+/-43.7 microM and 0.0104+/-0.0016 U mg(-1), respectively. The results indicated that PNP is present in rat CSF and provided a preliminary kinetic characterization.

Synthesis of 6-Aryloxy- and 6-Arylalkoxy-2-chloropurines and Their Interactions with Purine Nucleoside Phosphorylase from Escherichia coli

The phase transfer method was applied to perform the nucleophilic substitution of 2,6-dichloropurines by modified arylalkyl alcohol or phenols. Since under these conditions only the 6-halogen is exchanged, this method gives 2-chloro-6-aryloxy- and 2-chloro-6-arylalkoxy-purines. 2-Chloro-6-benzylthiopurine was synthesized by alkylation of 2-chloro-6-thiopurine with benzyl bromide. The stereoisomers of 2-chloro-6-(1-phenyl-1-ethoxy)purine were obtained from R- and S-enantiomers of sec.-phenylethylalcohol and 2,6-dichloropurine. All derivatives were tested for inhibition with purified hexameric E. coli purine nucleoside phosphorylase (PNP). For analogues showing IC50 < 10 microM, the type of inhibition and inhibition constants were determined. In all cases the experimental data were best described by the mixed-type inhibition model and the uncompetitive inhibition constant, Kiu, was found to be several-fold lower than the competitive inhibition constant, Kic. This effect seems to be due to the 6-aryloxy- or 6-arylalkoxy substituent, because a natural PNP substrate adenine, as well as 2-chloroadenine, show mixed type inhibition with almost the same inhibition constants Kiu and Kic. The most potent inhibition was observed for 6-benzylthio-2-chloro-, 6-benzyloxy-2-chloro-, 2-chloro-6-(2-phenyl-1-ethoxy), 2-chloro-6-(3-phenyl-1-propoxy)- and 2-chloro-6-ethoxypurines (Kiu = 0.4, 0.6, 1.4, 1.4 and 2.2 microM, respectively). The R-stereoisomer of 2-chloro-6-(1-pheny-1-ethoxy)purine has Kiu = 2.0 microM, whereas inhibition of its S counterpart is rather weak (IC50 > 12 microM). More rigid (e.g. phenoxy-), non-planar (cyclohexyloxy-), or more bulky (2,4,6-trimethylphenoxy-) substituents at position 6 of the purine base gave less potent inhibitors (IC50 = 26, 56 and > 100 microM, respectively). The derivatives are selective inhibitors of hexameric "high-molecular mass" PNPs because no inhibitory activity vs. trimeric Cellulomonas sp. PNP was detected. By establishing the ligand-dependent stabilization pattern of the E. coli PNP it was shown that the new derivatives, similarly as the natural purine bases, are able to form a dead-end ternary complex with the enzyme and orthophosphate. It was also shown that the derivatives are substrates in the reverse synthetic direction catalyzed by E. coli PNP.

Absorption and Intestinal Metabolism of Purine Dideoxynucleosides and an Adenosine Deaminase-Activated Prodrug of 2′,3′-Dideoxyinosine in the Mesenteric Vein Cannulated Rat Ileum

□ This study investigates the mechanisms of absorption and the role of intestinally localized purine salvage pathway enzymes on the ileal availabilities of 2′,3′-dideoxyinosine (ddI), a substrate for purine nucleoside phosphorylase (PNP); 2′-fluoro-2′,3′-dideoxyinosine (F-ddI), a non-PNP substrate; and 6-chloro-2′,3′-dideoxypurine (6-Cl- ddP), an adenosine deaminase (ADA) activated prodrug of ddI. The potential for increasing the intestinal availability of 6-Cl-ddP through the use of ADA inhibitors, namely, 2'-deoxycoformycin (DCF) and erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), is also explored. Drug permeability coefficients across the intestinal epithelium were determined in in situ perfusions in the mesenteric vein cannulated rat ileum based on both drug appearance in blood (Pblood) and disappearance from the lumen (Plumen) and their paracellular and transcellular components were estimated by comparison to the permeabilities of two paracellular markers, mannitol and urea. Values of Pblood for ddI were determined to be (1.1±0.3) × 10-6 cm/s, in close agreement with the value of (1.0±0.3) × 10-6 cm/s obtained for F-ddI, a PNP resistant analogue of ddI having virtually the same molecular size and lipophilicity as ddI. This indicates that PNP may not play an important role in the low intestinal absorption of ddI. The Pblood for 6-Cl-ddP, (19±2) × 10-6 cm/s, was 4.5-fold lower than Plumen, (84±12) × 10-6 cm/s, which means that 77±6% of 6-Cl-ddP was metabolized during its intestinal transport, thus qualitatively accounting for the low oral bioavailability (7%) of 6-Cl-ddP observed in vivo in rats. Extensive intracellular metabolism of 6-Cl-ddP by ADA was confirmed by the high concentrations of ddI found both in the intestinal lumen and blood during 6-Cl-ddP perfusions and by a rate of ddI appearance in blood which was ≈10-fold higher than ddI controls. Co-perfusion of the potent, hydrophilic ADA inhibitor DCF (K=0.0010.05 nM) with 6-Cl-ddP led to only partial inhibition of intestinal ADA, while complete inhibition was obtained using the less potent but more lipophilic inhibitor EHNA (Ki=1-20 nM). Hence, EHNA may be used to improve intestinal absorption of 6-Cl-ddP in vivo.

Purine Nucleoside Phosphorylase. 1. Structure−Function Studies

To probe the catalytic mechanism of human purine nucleoside phosphorylase (PNP), 13 active-site mutants were constructed and characterized by steady-state kinetics. In addition, microtiter plate assays were developed for both the phosphorolytic and synthetic reactions and used to determine the kinetic parameters of each mutant. Mutations in the purine binding site exhibited the largest effects on enzymatic activity with the Asn243Ala mutant resulting in a 1000-fold decrease in the kcat for inosine phosphorolysis. This result in combination with the crystallographic location of the Asn243 side chain suggested a potential transition state (TS) structure involving hydrogen bond donation by the carboxamido group of Asn243 to N7 of the purine base. Analogous to the oxyanion hole of serine proteases, this hydrogen bond was predicted to aid catalysis by preferentially stabilizing the TS as a consequence of the increase in negative charge on N7 that occurs during glycosidic bond cleavage and the associated increase in the N7-Asn243 hydrogen bond strength. Two residues in the phosphate binding site, namely His86 and Glu89, were also predicted to be catalytically important based on their alignment with phosphate in the X-ray structure and the 10-25-fold reduction in catalytic activity for the His86Ala and Glu89Ala mutants. In contrast, catalytic efficiencies for the Tyr88Phe and Lys244Ala mutants were comparable with wild-type, indicating that the hydrogen bonds predicted in the initial X-ray structure of PNP [Ealick, S. E., et al. (1990) J. Biol. Chem. 265, 1812-1820] were not essential for catalysis. These results provided the foundation for studies reported in the ensuing two manuscripts focused on the PNP catalytic mechanism [Erion, M. D., et al. (1997) Biochemistry 36, 11735-11748] and the use of mutagenesis to reverse the PNP substrate specificity from 6-oxopurines to 6-aminopurines [Stoeckler, J. D., et al. (1997) Biochemistry 36, 11749-11756].

Kinetics of phosphorolysis of 3-(beta-D-ribofuranosyl)adenine and 3-(beta-D-ribofuranosyl)hypoxanthine, non-conventional substrates of purine-nucleoside phosphorylase.

The properties of two non-conventional substrates of the calf-spleen and Escherichia coli purine nucleoside phosphorylases (PNP), 3-(beta-D-ribofuranosyl)adenine (RibfAde) and 3-(beta-D-ribofuranosyl)hypoxanthine (RibfHyp), are described. In contrast to Ado, RibfAde is a substrate for the mammalian enzyme. With the calf enzyme, the pseudo-first-order rate constants (Vmax/K(m)) for phosphorolysis of RibfAde and RibfHyp are 3% and 13%, respectively, that for phosphorolysis of Ino, while for E. coli PNP the corresponding values are 22% and 30%, respectively. The Michaelis constants (K(m)) for RibfAde were 800 microM (calf PNP) and 150 microM (E. coli PNP). For RibfHyp, the corresponding K(m) values were 220 microM and 260 microM. Two well-characterized inhibitors of calf spleen PNP [9-(2-fluoro-3,4-dihydroxybutyl)guanine] and E. coli PNP (formycin A) were found to inhibit phosphorolysis of RibfAde and RibfHyp with the same inhibition constants as for Ino. Moreover, the inhibition was competitive, which indicates that phosphorolysis of 3-beta-nucleosides occurs at the same active site as for the natural substrate Ino. In particular, the substrate properties of both 3-beta-nucleosides are consistent with their binding to the enzyme in the conformation anti to the imidazole ring about the glycosidic bond, which is superimposable on the structure of natural 9-beta-nucleosides in the conformation anti to the pyrimidine ring. The results are examined in relation to present concepts regarding the binding of substrates and inhibitors at the active site(s) of these enzymes.

Fluorescence emission properties of 8-azapurines and their nucleosides, and application to the kinetics of the reverse synthetic reaction of purine nucleoside phosphorylase

An extensive study has been made of the fluorescence emission properties of the neutral and ionic forms in aqueous medium of the azapurine nucleosides, 8-azaadenosine (8-azaAdo), 8-azainosine (8-azaIno), 8-azaguanosine (8-azaGuo), and their aglycons. The fluorescence of 8-azaGuo at pH 7 originates from its anionic species (p K a = 8.05, φ = 0.55), as is also the case for 8-azaIno (p Ka = 8.0, φ = 0.02), whereas 8-azaAdo is a strong emitter ( φ = 0.06) as the neutral species. By contrast the corresponding free 8-azapurines are only weakly fluorescent in aqueous medium, with the exception of 8-azaguanine (8-azaG). Examination of the emission properties of N-substituted 8-azaguanines demonstrated that the observed blue emission of the neutral form of 8-azaG ( φ = 0.05 to 0.33, dependent on λ exc) originates from a minor tautomer of the compound, the N(8)-H form, present to the extent of 10–15%; while the principal N(9)-H tautomer is virtually nonfluorescent. The 8-azapurines are substrates of purine nucleoside phosphorylase (PNP), leading to their irreversible conversion to the corresponding nucleosides in the synthetic pathway of this enzyme. The fluorescent properties of these compounds, together with spectrophotometric methods, were applied to determine the basic kinetic parameters for synthesis of 8-azapurine nucleosides by PNP from mammalian (calf spleen) and bacterial ( Escherichia coli) sources. The fluorimetric method was also used to determine the kinetic parameters for the second substrate, α- d-ribose 1-phosphate, and for the analytical titration of the latter in solution. The pH optimum of the reverse synthetic PNP reaction with 8-azapurines as substrates is below pH 7, due to their enhanced acidity in comparison with natural purines. The 8-azapurine nucleosides, but not their aglycons, are reasonably good inhibitors of phosphorylysis of Ino and Guo by E. coli PNP. The most effective is 8-azaIno ( K i ∼ 20 μM), also the only one to inhibit phosphorolysis by the calf spleen enzyme ( K i ∼ 40 μM). The nature of this inhibition is apparently uncompetitive.

A broadly applicable continuous spectrophotometric assay for measuring aminoacyl-tRNA synthetase activity.

We describe a convenient, simple and novel continuous spectrophotometric method for the determination of aminoacyl-tRNA synthetase activity. The assay relies upon the measurement of inorganic pyrophosphate generated in the first step of the aminoacylation of a tRNA. Pyrophosphate release is coupled to inorganic pyrophosphatase, to generate phosphate, which in turn is used as the substrate of purine nucleoside phosphorylase to catalyze the N-glycosidic cleavage of 2-amino 6-mercapto 7-methylpurine ribonucleoside. Of the reaction products, ribose 1-phosphate and 2-amino 6-mercapto 7-methylpurine, the latter has a high absorbance at 360 nm relative to the nucleoside and hence provides a spectrophotometric signal that can be continuously followed. The non-destructive nature of the spectrophotometric assay allowed the re-use of the tRNAs in question in successive experiments. The usefulness of this method was demonstrated for glutaminyl-tRNA synthetase (GlnRS) and tryptophanyl-tRNA synthetase. Initial velocities measured using this assay correlate closely with those assayed by quantitation of [3H]Gln-tRNA or [14C]Trp-tRNA formation respectively. In both cases amino acid transfer from the aminoacyl adenylate to the tRNA represents the rate determining step. In addition, aminoacyl adenylate formation by aspartyl-tRNA synthetase was followed and provided a more sensitive means of active site titration than existing techniques. Finally, this novel method was used to provide direct evidence for the cooperativity of tRNA and ATP binding to GlnRS.

Purine nucleoside phosphorylase: Inhibition by purine N(7)- and N(9)-acyclonucleosides; and substrate properties of 7-β- D-ribofuranosylguanine and 7-β- D-ribofuranosylhypoxanthine

A series of 10 N(9)- and N(9)-acyclonucleosides of guanine and 8-substituted guanines (8-Br, 8 8-SH, and 8-NH 2), and two N(7)-acyclonucleosides of hypoxanthine, were tested for their ability to inhibit purine nucleoside phosphorylase (PNP) (E.C. 2.4.2.1) from human erythrocytes and rabbit kidney. The acyclic chains contained a nitrogen in place of a carbon at the 3′, 4′ or 5′ position and, in one case, an ether oxygen at the 2′ position. Most striking was the finding that one of the N(7)-acyclonucleoside analogues, 7-[(1,3-dihydroxypropyl-2)amino]ethylguanine, proved to be a 3-fold more effective inhibitor than its corresponding N(9) counterpart, with K i = 5 vs 14 μM for the human enzyme and 0.7 vs 2.3 μM for the rabbit enzyme. Both analogues, as well as the others examined, inhibited phosphorolysis competitively with respect to nucleoside substrates (inosine with the human enzyme and guanosine with the rabbit enzyme). The foregoing logically led to the finding that the 7-β- D-ribosides of guanine (N 7Guo) and hypoxanthine (N 7Ino) were weak substrates of PNP from human erythrocytes, calf spleen and E. coli. With the human enzyme the pseudo-first-order rate constants ( V max/ K m ) for phosphorolysis of N 7Guo and N 7Ino were 0.08 and 0.02% that for Ino. The Michaelis constants ( K m for N 7Guo were 27 (calf PNP), 108 (human PNP) and 450 -gmM ( E. coli PNP). For N 7Ino the corresponding K m values were 1.52, 1.26 and 0.64 mM. Four previously well-characterized N(9)-acyclonucleoside inhibitors of calf spleen PNP were found to inhibit phosphoolysis of N 7Ino by the same enzyme 2–10-fold more effectively than the parent Ino. The overall results, along with the known excellent substrate properties of N(7)-alkyl-Guo and Ino (Bzowska et al. J Biol. Chem 263, 9212-9-9217, 1988), were examined in relation to present concepts regarding binding of substrates and inhibitors at the active site(s) of these enzymes.