Purine Riboside Research Articles

Importance of specific purine amino and hydroxyl groups for efficient cleavage by a hammerhead ribozyme.

Eight modified ribozymes of 19 residues have been prepared with individual purine amino or hydroxyl groups excised. The modified ribozymes were chemically synthesized with the substitution of a single 2'-deoxyadenosine, 2'-deoxyguanosine, inosine, or purine riboside for residues G10, A11, G13, or A14. Five of the modified ribozymes cleaved the 24-mer substrate with little change in rate as monitored by simple first-order kinetics. However, deletion of the 2-amino group at G10 (replacement with inosine) or deletion of either of the 2'-hydroxyls at G10 or G13 (replacement with 2'-deoxyguanosine) resulted in ribozymes with a drastic decrease in cleavage efficiency. Increasing the concentration of the Mg2+ cofactor from 10 mM to 50 mM significantly enhanced cleavage efficiency by these three derivatives. Steady-state kinetic assays for these three ribozymes indicated that the modifications result in both an increase in Km and a decrease in kcat. These results suggest that the exocyclic amino group at-G10 and the hydroxyls at G10 and G13 are important both for ribozyme-substrate binding and for the Mg(2+)-catalyzed cleavage reaction.

The rate of formation of transition-state analogs in the active site of adenosine deaminase is encounter-controlled: implications for the mechanism

We have previously shown that purine riboside, when bound to adenosine deaminase, forms a complex in which C-6 of the purine is tetrahedral [Kurz, L. C., & Frieden, C. (1987) Biochemistry 26, 8450]. We now report the rates of formation of enzyme-inhibitor complexes of two types, those which do and those which do not form such tetrahedral intermediates. In both cases, the rates are encounter-controlled since the progress curves for formation of the complexes are well-described by a simple second-order approach to equilibrium and the rate constants show an inverse solvent viscosity dependence. Assuming that the formation of the intermediate-analogue complex is preceded by an initial ground-state analogue complex, the lifetime of that ground-state complex must be less than approximately 20 microseconds. All of the enzyme-inhibitor complexes studied share three characteristics: (1) the complexes generate large UV-difference spectra; (2) a substantial solvent isotope effect is found on the enzyme's affinity for the inhibitors; and (3) a new signal appears in the CD spectra of the complexes. Two of the nucleosides studied, 1-deazapurine riboside and 1-deaza-adenosine, form complexes which appear to mimic a ground-state rather than a reactive intermediate when bound to adenosine deaminase. We find that the values for the association rate constants for those inhibitors which form intermediate analogues are very similar to that for adenosine. The presence of a significant solvent isotope effect on the affinity of all inhibitors is attributable in part to a large transfer isotope effect on the free ligand and in part to an effect on the bound ligand. This complicates use of the solvent isotope effect in applications of the multiple isotope method for estimating intrinsic isotope effects and commitment factors.

Kinetic properties of the common electrophoretic variants of human S-adenosylhomocysteine hydrolase (AHCY): the effect of four nucleoside analogue inhibitors.

Red blood cell S-adenosylhomocysteine hydrolase (AHCY) from individuals of 1, 2-1 and 3-1 phenotypes was partially purified and Km and Vmax determined in the absence and in the presence of the following inhibitors: 3-deaza-adenosine (DZA), 3-deaza-aristeromycin (DZAry), 2-chloro adenosine (2-Cl-ado) and purine riboside (or nebularine). The three phenotypes 1, 2-1, 3-1 showed similar Km (32.58, 39.22 and 34.84 microM respectively), but the ratio Km/Vmax was statistically different. DZA and DZAry appeared to be strong competitive inhibitors. The AHCY 1 phenotype was more resistant to their action, while the 3-1 variant was more sensitive. 2-Cl-ado and purine riboside were weaker inhibitors; the type of inhibition varied among the three phenotypes, but, again, the AHCY 1 phenotype was less sensitive than the other two.

Antitumor properties of 2(1H)-pyrimidinone riboside (zebularine) and its fluorinated analogs

2(1H)-Pyrimidinone riboside (zebularine, 1b) and its 5-fluoro (6b) and 2'-ara-fluoro (7b) analogues have been synthesized and evaluated in vivo as antitumor agents. Zebularine provides increase in life span (ILS) values of ca. 70% against intraperitoneal (ip) murine B16 melanoma and 50% against P388 leukemia. This compound is active when administered either ip or orally against ip or subcutaneously implanted L1210 leukemia, producing ILS values of about 100% at an optimum dose of 400 mg/kg. 1b is also active (60% ILS) against ara-C-resistant L1210. The analogous unsubstituted purine riboside nebularine (2) has modest activity against P388 leukemia (60% ILS). While 2'-ara-fluorozebularine (7b) is only marginally active (40% ILS) at high doses against L1210 leukemia, 5-fluoro analogue 6b is more active than zebularine and is ca. 100 times more potent. Although the activity of 6b is about the same as that of 1b against P388 leukemia, greater potency also is realized in this model. Zebularine is a strong inhibitor of cytidine deaminase, but in contrast to tetrahydrouridine, 1b is acid-stable. In an attempt to use this property to advantage in oral administration, 1b and ara-C have been orally coadministered to mice with ip L1210 leukemia. When zebularine is given in divided doses, up to a 2-fold increase in activity is realized, relative to treatment with the same dose of ara-C alone.

Adenosine deaminase in the adductor muscle of the scallop, Patinopecten yessoensis

1. 1. The scallop enzyme was separated by DE52 ion-exchange chromatography into two forms with the same mol. wt of 38,000 and similar characteristics. 2. 2. The enzyme was inactivated in the absence of dithiothreitol and complete reactivation was achieved by adding the agent within a critical storage period. 3. 3. The apparent values of pK m and V max sensitively increased as ionic strength was raised to 250 mM and phosphate and sodium ions elevated the former value with a further increase of the ionic strength. 4. 4. The apparent activation energies for the α (V max /K m and β (V max ) parameters of both the forms were approximately 5 and 8 kcal/mol, respectively. 5. 5. The enzyme deaminated 2′-, 3′-deoxyadenosine and 2′,3′-isopropylidene adenosine but did not deaminate 5′-deoxyadenosine, α-adenosine and adenine nucleotides. 6. 6. The affinity for inosine was much lowered with a high K i value. Adenine and purine riboside inhibited the enzyme competitively, and coformycin was a tight, slow binding inhibitor.

Purification of Adenosine Deaminase from Chicken-Egg Yolk by Affinity Column Chromatography

Adenosine deaminase (adenosine aminohydrolase; E.C. 3.5.4.4) has been purified 4686-fold from egg yolk. The procedure developed was used to isolate the enzyme from eight chicken eggs. An easily prepared affinity column employing purine riboside was used as the final step in the purification. The method developed permits the rapid isolation and a high recovery of the protein. The specific activity of the enzyme preparation obtained is 81.4 mU/mg.

Properties of Purine Nucleoside Phosphorylase (PNP) of Mammalian and Bacterial Origin

Purine nucleoside phosphorylase (PNP), from calf spleen, human erythrocytes and E. coli have been examined with regard to structural requirements of substrates and inhibitors. Kinetic parameters (Km, Vmax/Km) for a variety of N(1) and/or N(7)-methylated analogues of guanosine, inosine and adenosine have been evaluated for all three enzymes. The substrate and/or inhibitor properties of purine riboside, 1,6-dihydropurine riboside, some deazapurine nucleosides: 3-deaza- and 7-deazainosine, 1,3-dideazapurine riboside (ribobenzimidazole), and a variety of acyclonucleosides, have been determined with mammalian and bacterial enzymes. Overall results indicate distinct similarities of kinetic properties and structural requirements of the two mammalian enzymes, although there are some differences as well. The N(1) and O6 of the purine ring are necessary for substrate-inhibitor activity and constitute a binding site for the mammalian (but not the bacterial) enzymes. Moreover, nucleosides lacking the N(3) undergo phosphorolysis and those lacking N(7) are inhibitors (but not substrates). Methylation of the ring N(7) leads to two overlapping effects: labilization of the glycosidic bond, and impediment to protonation at this site by the enzyme, a postulated prerequisite for enzymatic phosphorolysis. It is proposed that a histidine interacts with N(1) as a donor and O6 as an acceptor. Alternatively N(1)-H and C(2)-NH2 may serve as donors for hydrogen bonds with a glutamate residue. The less specific E. coli enzyme phosphorolyses all purine ring modified nucleosides but 7-deazainosine which is only an inhibitor. On the other hand, the bacterial enzyme exhibits decreased activity towards N(7)-methylated nucleosides and lack of affinity for a majority of the tested acyclonucleoside inhibitors of the mammalian enzymes. The foregoing results underline the fundamental differences between mammalian and bacterial enzymes, including variations in the binding sites for the purine ring.

Preparative purification of adenosine deaminase from human erythrocytes by affinity chromatography

The purification of adenosine deaminase from human erythrocytes is reported. By means of classical procedures and by using affinity chromatography as the last step, the enzyme is purified 760 000-fold with a yield of 32%. The affinity resin is composed of purine riboside (nebularine) linked to Sepharose CL6B. Since the compound has no leaving group at the C-6 position the affinity gel is stable and the chromatography can be repeated several times (up to fifteen times in eight months). Purine riboside was chosen because its potency as a reversible inhibitor of adenosine deaminase is greater than that of inosine (a low-affinity inhibitor), but lower than that of erythro-9-(2-hydroxy-3-nonyl)adenine (a high-affinity inhibitor).

Purine nucleoside phosphorylase: Isolation and characterization

Purine nucleoside phosphorylase (PNP) from mouse leukemia cells L1210 was purified to homogeneity by a combination of ion exchange and affinity chromatography using AE-Sepharose 4B and 9-(p-succinylaminobenzyl)hypoxanthine as the matrix and the ligand, respectively. The native enzyme has a molecular weight of 104 000 and consists of three subunits of equal molecular weight of 34 000. The results of isoelectric focusing showed that the enzyme is considerably microheterogeneous over the pI-range 4.0-5.8 and most likely consists of eight isozymes. The temperature and pH-optimum of phosphorolysis, purine nucleoside synthesis and also of transribosylation is identical, namely 55 °C and pH 7.4. The transribosylation reaction proceeds in the presence of phosphate only. The following Km-values (μmol l-1) were determined for phosphorolysis: inosine 40, 2'-deoxyinosine 47, guanosine 27, 2'-deoxyguanosine 32. The Km-values (μmol l-1) of purine riboside and deoxyriboside synthesis are lower than the values for phosphorolysis (hypoxanthine 18 and 34, resp., guanine 8 and 11, resp.). An affinity lower by one order shows PNP for (-D-ribose-1-phosphate, (-D-2-deoxyribose-1-phosphate (Km = 200 μmol l-1 in both cases) and phosphate (Km = 805 μmol l-1). The substrate specificity of the enzyme was also studied: positions N(1), C(2) and C(8) are decisive for the binding of the substrate (purine nucleoside).

Time-resolved fluorescence spectroscopy of human adenosine deaminase: effects of enzyme inhibitors on protein conformation

Adenosine deaminase, a purine salvage enzyme essential for immune competence, was studied by time-resolved fluorescence spectroscopy. The heterogeneous emission from this four-tryptophan protein was separated into three lifetime components: tau 1 = 1 ns and tau 2 = 2.2 ns an emission maximum at about 330 nm and tau 3 = 6.3 ns with emission maximum at about 340 nm. Solvent accessibility of the tryptophan emission was probed with polar and nonpolar fluorescence quenchers. Acrylamide, iodide, and trichloroethanol quenched emission from all three components. Acrylamide quenching caused a blue shift in the decay-associated spectrum of component 3. The ground-state analogue enzyme inhibitor purine riboside quenched emission associated with component 2 whereas the transition-state analogue inhibitor deoxycoformycin quenched emission from both components 2 and 3. The quenching due to inhibitor binding had no effect on the lifetimes or emission maxima of the decay-associated spectra. These observations can be explained by a simple model of four tryptophan environments. Quenching studies of the enzyme-inhibitor complexes indicate that adenosine deaminase undergoes different protein conformation changes upon binding of ground- and transition-state analogue inhibitors. The results are consistent with localized structural alterations in the enzyme.

8-Substituted Purine Ribosides: Synthesis and Biological Activity

Abstract The syntheses of a series of 8-substituted purine ribofuranosides are described. Reductive deaminalion of 8-bromoadenosine triacetate (1) provides the key intermediate (2) for subsequent displacement reactions. Four compounds show significant activity in two cell culture Screens.

Adenosine deaminase converts purine riboside into an analogue of a reactive intermediate: a 13C NMR and kinetic study.

The 13C NMR spectra of [2-13C]- and [6-13C]purine ribosides have been obtained free in solution and bound to the active site of adenosine deaminase. The positions of the resonances of the bound ligand are shifted relative to those of the free ligand as follows: C-2, -3.7 ppm; C-6, -73.1 ppm. The binary complexes are in slow exchange with free purine riboside on the NMR time scale, and the dissociation rate constant is estimated to be 13.5 s-1 from the slow exchange broadening of the free signal. In aqueous solution, protonation of purine riboside at N-1 results in changes in 13C chemical shift relative to those of the free base as follows: C-2, -4.9 ppm; C-6, -7.9 ppm. The changes in chemical shift that occur when purine riboside binds to the enzyme indicate that the hybridization of C-6 changes from sp2 to sp3 in the binary complex with formation of a new bond to oxygen or sulfur. A change in C-2 hybridization can be eliminated as can protonation at N-1 as the sole cause of the chemical shift changes. The kinetic constants for the adenosine deaminase catalyzed hydrolysis of 6-chloro- and 6-fluoropurine riboside have been compared, and the reactivity order implies that carbon-halogen bond breaking does not occur in the rate-determining step. These observations support a mechanism for the enzyme in which formation of a tetrahedral intermediate is the most difficult chemical step. Enzymic stabilization of this intermediate may be an important catalytic strategy used by the enzyme to lower the standard free energy of the preceding transition state.

Adenosine deaminase: viscosity studies and the mechanism of binding of substrate and of ground- and transition-state analogue inhibitors.

We have studied the effects of viscosogenic agents, sucrose and ficoll, on (1) the hydrolysis of adenosine and of 6-methoxypurine riboside catalyzed by adenosine deaminase and (2) the rates of association and dissociation of ground-state and transition-state analogue inhibitors. For adenosine, Vmax/Km is found to be inversely proportional to the relative viscosity with sucrose, an agent affecting the microscopic viscosity, while no effect is found with ficoll, an agent affecting the macroscopic viscosity. Viscosogenic agents have no effect on the kinetic constants for 6-methoxypurine riboside. Thus, the bimolecular rate constant, Vmax/Km = 11.2 +/- 0.8 microM-1 s-1, for the reaction with adenosine is found to be at the encounter-controlled limit while that for the reaction with the poor substrate 6-methoxypurine riboside, 0.040 +/- 0.004 microM-1 s-1, is limited by some other process. Viscosity-dependent processes do not make a significant (less than 10%) contribution to Vmax. The dissociation constants for inhibitors are unaffected by viscosity. The ground-state analogue inhibitor purine riboside appears to bind at a rate comparable to that of adenosine. However, the slower rates of association (0.16-2.5 microM-1 s-1) and dissociation (5 X 10(-6) to 12 s-1) of transition-state analogue inhibitors are affected by the viscosity of the medium to approximately the same extent as the encounter-controlled rates of association and dissociation of adenosine.(ABSTRACT TRUNCATED AT 250 WORDS)

Kinetic characteristics and binding process of substrate analogs to the adenosine deaminase in the marine mussel, mytilus edulis

1. 1. The purified mussel enzyme deaminated several adenosine analogs with different K m and relative V max values. Affinity for adenine was similar to that for adenosine but the deamination rate was extremely slow. 2. 2. Purine riboside was competitive, coformycin was a tight, slow binding inhibitor, and inhibition by both these compounds was pH-dependent. 3. 3. Inosine, hypoxanthine, guanosine and 6-mercapto-purine riboside were slightly inhibitory. 4. 4. The results suggested that initial binding of the substrate was guided by the adenine moiety followed by a stereospecific steering due to a ribose-dependent distortion in the complex to facilitate nucleophilic attack at C-6.

Affinity difference of adenosine deaminases for the purine riboside-epoxyactivated Sepharose 6B column

1. 1. The small-size adenosine deaminases ( M r = 35,000 and 43,000 ) in calf intestinal mucosa, frog liver and scallop adductor muscle and the large-size deaminase ( M r = 100,000 ) in frog liver probably formed by adding a conversion protein to the small enzyme, were tightly bound to the purine riboside affinity column. 2. 2. Binding of the other large-size enzymes ( M r = 140,000 ) in the midgut gland of scallop and mussel to the column was not successful. 3. 3. It seems that the binding difference does not depend on a change in affinity between active site and ligand but rather on the stereospecific positioning of active site in the enzyme molecules.

Germination of Bacillus cereus spores induced by purine ribosides and their analogs: effects of modification of base and sugar moieties of purine nucleosides on germination-inducing activity.

Purine riboside and some of its analogs were tested for their ability to induce germination of Bacillus cereus T spores. Hypoxanthine and adenine showed no germination-inducing activity either in the present or absence of D-ribose or its phosphorylated derivatives. Purine riboside and 18 analogs with modified purine base were all able to induce germination of the spores to various extents. In contrast to this, the requirement for the sugar moiety in the purine riboside appeared to be more stringent. Only those nucleosides that contained either D-ribose or deoxy-D-ribose, and certain species of azole derivatives such as 5-aminoimidazole-4-carboxamide covalently linked to the C(1') of the sugar actively induced germination.

Inhibition of herpes simplex virus DNA polymerase by purine ribonucleoside monophosphates.

Purine ribonucleoside monophosphates were found to inhibit chain elongation catalyzed by herpes simplex virus (HSV) DNA polymerase when DNA template-primer concentrations were rate-limiting. Inhibition was fully competitive with DNA template-primer during chain elongation; however, DNA polymerase-associated exonuclease activity was inhibited noncompetitively with respect to DNA. Combinations of 5'-GMP and phosphonoformate were kinetically mutually exclusive in dual inhibitor studies. Pyrimidine nucleoside monophosphates and deoxynucleoside monophosphates were less inhibitory than purine riboside monophosphates. The monophosphates of 9-beta-D-arabinofuranosyladenine, Virazole (1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide), 9-(2-hydroxyethoxymethyl)guanine, and 9-(1,3-dihydroxy-2-propoxymethyl)guanine exerted little or no inhibition. In contrast to HSV DNA polymerase, human DNA polymerase alpha was not inhibited by purine ribonucleoside monophosphates. These studies suggest the possibility of a physiological role of purine ribonucleoside monophosphates as regulators of herpesvirus DNA synthesis and a new approach to developing selective anti-herpesvirus compounds.

Na++ K++ 2 Cl− Cotransport in Animal Cells—Its Role in Volume Regulationa

Cell membranes of various vertebrate cells catalyze a Na+ + K+ + 2Cl- cotransport specifically inhibitable by furosemide and other high ceiling diuretics. The energetics of this process is not elucidated unequivocally. It was clearly shown that cotransport is no ATP-consuming process. We assume that transport is secondary active functionally coupled to the operation of the electrogenic Na+-K+ pump. The role of this transport system in transepithelial ion movement is that it serves as flux amplifier, doubling from 6 to 12 the number of osmotically active particles transported per ATP hydrolyzed. In concert with Na+-K+ pump, cotransport provokes net uptake of KCl into the cell and therefore cellular swelling. This process is regulated by a feedback control system for cell volume; if actual volume reaches reference value, cotransport is switched off to prevent further swelling. How cell volume is measured is not known, nor is the nature of the signal generated to switch cotransport from the operating to the nonoperating state or vice versa. cAMP-level or intracellular Ca2+ play no role as signals or as part of the volume-sensoring mechanism. Theophylline, other alkylxanthines, and some purine ribosides influence cotransport indirectly by reducing reference volume. The role of cytoskeleton in volume regulation is obscure. While high Concentrations of cytochalasin B and of colchicin do not influence cell volume, it is reduced by vinblastine and also by lectins, for example concanavalin A. Volume reduction is accompanied by reduction in cellular KCl content. The observation that during hypertonic incubation protein synthesis is inhibited can be traced back to a correlation between cell volume and protein synthesis and not to elevation of osmolarity per se. Reduction in cell volume under isotonic conditions by varying K+ and/or Cl- concentration or by furosemide inhibition of cotransport is strongly correlated to inhibition of protein synthesis. The reason for this correlation is not yet clarified. Not all cells showing furosemide-sensitive cotransport are able to regulate it, for example lymphocytes. For mammalian erythrocytes drastic species differences exist; while cells from man, rabbit, rat, and mouse all show cotransport, only cells from rat (and mouse?) are able to regulate cotransport.

Specific replacement of functional groups of uridine-33 in yeast phenylalanine transfer ribonucleic acid.

Functional groups of the highly conserved uridine at position 33 in the anticodon loop of yeast tRNAPhe were altered by a synthetic protocol that replaces U-33 with any desired nucleotide and leaves all other nucleotides of the tRNA intact. The U-33-substituted tRNAs were prepared in an eight-step protocol that begins with partial cleavage of tRNAPhe at U-33 by ribonuclease A. By use of the combined half-molecules as substrate, U-33 was removed from the 5' half-molecule in three steps and then replaced by using RNA ligase to add the desired nucleoside 3',5'-bisphosphate. Each position 33 substituted 5' half-molecule was isolated and annealed to the original 3' half-molecule from the ribonuclease A digestion. The two halves were then rejoined in three steps to give a full-size tRNAPhe variant. This protocol should be applicable to other RNA molecules where a nucleotide substitution is desired at the 5' side of an available unique cleavage site. Seven substituted tRNAPheS containing uridine, pseudouridine, 3-methyluridine, 2'-O-methyluridine, cytidine, deoxycytidine, and purine riboside at position 33 were assayed for aminoacylation with yeast phenylalanyl-tRNA synthetase. Each of the seven tRNAs aminoacylated normally. Thus, unlike the adjacent guanine residue at position 34, U-33 is not involved in the interaction between yeast tRNAPhe and yeast phenylalanyl-tRNA synthetase.

Enzymatic synthesis of nucleoside antibiotics. Part V. Properties of nucleoside phosphorylase from Enterobacter aerogenes.

The properties of uridine Phosphorylase (UPase) and purine nucleoside Phosphorylase (PNPase) at high temperature were investigated. Both enzymes were found to be distributed in a wide range of bacteria and were partially purified from Enterobacter aerogenes AJ 11125 by heat treatment, ammonium sulfate fractionation and column chromatographies onDEAE-cellulose and Sephadex G-150. The UPase was purified 109-fold, and it showed an optimum pH of 8.5 and optimum temperature of 65°C, and activity toward uridine, 2′-deoxyuridine, thymidine and uracil arabinoside but not cytidine. The Km values of UPase for uridine were 0.7 mm at 40°C and 1.8 mm at 60°C. The PNPase was purified 83-fold, and it showed an optimum pH of 6.8 and optimum temperature of 60°C, and significant activity toward purine arabinosides as well as purine ribosides. The Km values of PNPase for inosine were 0.8 mm at 40°C and 2.2 mm at 60°C.