Ewa Kulikowska

Purine nucleoside phosphorylases: properties, functions, and clinical aspects

The ubiquitous purine nucleoside phosphorylases (PNPs) play a key role in the purine salvage pathway, and PNP deficiency in humans leads to an impairment of T-cell function, usually with no apparent effects on B-cell function. This review updates the properties of the enzymes from eukaryotes and a wide range of prokaryotes, including a tentative classification of the enzymes from various sources, based on three-dimensional structures in the solid state, subunit composition, amino acid sequences, and substrate specificities. Attention is drawn to the compelling need of quantitative experimental data on subunit composition in solution, binding constants, and stoichiometry of binding; order of ligand binding and release; and its possible relevance to the complex kinetics exhibited with some substrates. Mutations responsible for PNP deficiency are described, as well as clinical methods, including gene therapy, for corrections of this usually fatal disease. Substrate discrimination between enzymes from different sources is also being profited from for development of tumour-directed gene therapy. Detailed accounts are presented of design of potent inhibitors, largely nucleosides and acyclonucleosides, their phosphates and phosphonates, particularly of the human erythrocyte enzyme, some with Ki values in nanomolar and picomolar range, intended for induction of the immunodeficient state for clinical applications, such as prevention of host-versus-graft response in organ transplantations. Methods of assay of PNP activity are reviewed. Also described are applications of PNP from various sources as tools for the enzymatic synthesis of otherwise inaccessible therapeutic nucleoside analogues, as coupling enzymes for assays of orthophosphate in biological systems in the micromolar and submicromolar ranges, and for coupled assays of other enzyme systems.

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.

Formycins A and B and some analogues: selective inhibitors of bacterial (Escherichia coli) purine nucleoside phosphorylase

Formycin B (FB), a moderate inhibitor (Ki approximately 100 microM) of mammalian purine nucleoside phosphorylase (PNP), and formycin A (FA), which is totally inactive vs. the mammalian enzyme, are both effective inhibitors of the bacterial (Escherichia coli) enzyme (Ki approximately 5 microM). Examination of a series of N-methyl analogues of FA and FB led to the finding that N(6)-methyl-FA, virtually inactive vs. the mammalian enzyme, is the most potent inhibitor of E. coli purine nucleoside phosphorylase (Ki approximately 0.3 uM) at neutral pH. Inhibition is competitive not only with respect to Ino, but also relative to 7-methyl-Guo and 7-methyl-Ado, as substrates. Both oxoformycins A and B are relatively poor inhibitors. For the most potent inhibitor, N(6)-methyl-FA, it was shown that the enzyme preferentially binds the neutral, and not the cationic, form. In accordance with this the neutral, but not the cationic form, of the structurally related N(1)-methyl-Ado was found to be an excellent substrate. Reported data on tautomerism of formycins were profited from, and extended, to infer which tautomeric species and ionic forms are the active inhibitors. A commercially available (Sigma) bacterial PNP, of unknown origin, was shown to differ from the E. coli enzyme by its inability to phosphorylase Ado; this enzyme was also resistant to FA and FB. These findings have been extended to provide a detailed comparison of the substrate/inhibitor properties of PNP from various microorganisms.

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 enzym es. 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 acyclonu cleosides, 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 proton ation at this site by the enzyme, a postulated prerequisite for enzymatic phosphorolysis. It is proposed that a histidine interacts with N(1) as a don or and O6 as an acceptor. Alternatively N(1)−H and C(2)−NH2, may serve as donors for hydrogen bonds with a glutam ate 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 acyclonu cleoside 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.

Interactions of Calf Spleen Purine Nucleoside Phosphorylase with Antiviral Acyclic Nucleoside Phosphonate Inhibitors: Kinetics and Emission Studies

Association between calf spleen purine nucleoside phosphorylase and a series of phosphonylalkoxyalkyl derivatives of purine bases was studied by inhibition kinetics and fluorimetric titrations. Dissociation constants, determined by fluorimetric titration in phosphate-free conditions, were lower than inhibition constants in 1 mM phosphate, and inhibition was still weaker in 50 mM phosphate, in accord with the postulated bisubstrate analogue character of this class of inhibitors.

Antiviral Acyclic Nucleoside Phosphonate Analogues as Inhibitors of Purine Nucleoside Phosphorylase

Purine nucleoside phosphorylase (PNP, E.C.2.4.2.1) catalyses reversible phosphoro-lysis of purine ribo- and deoxyribonucleosides. Mammalian phosphorylases are specific for 6-ketopurines while some bacterial PNPs (e.g. from E. coli and S. typhimurium) have very broad specificty.l–4 Genetic deficiency of PNP leads to a loss of cellular immunity.5 The potential clinical applications of PNP inhibitors include treatment of T-cell leukemias, suppression of the host-vs-graft response in organ transplantation and potentation of the chemotherapeutic action of PNP-cleavable nucleosides.6

Linear Free Energy Relationships for N(7)-Substituted Guanosines as Substrates of Calf Spleen Purine Nucleoside Phosphorylase. Possible Role of N(7)-Protonation as an Intermediary in Phosphorolysis

Abstract Quantitative structure-activity relationships (QSAR) for a series of N(7)-substituted guanosines as substrates for calf spleen purine nucleoside phosphorylase (PNP) were developed, and compared with those for acid hydrolysis of these analogues. There is no correlation between the rates for enzymatic phosphorolysis and acid hydrolysis, indicating that for the enzymatic reaction labilization of the glycosidic bond is not the only, nor the predominant, effect of N(7)-substitution. Multiple regression analysis of the enzymatic process revealed that optimal substrate properties (minimal Michaelis constant) are associated with the Taft electronic constant equal zero and a substituent size, parametrized by the Taft steric constant, smaller than that for a methyl group. These results support the hypothesis of protonation of the N(7)-position of the base by the enzyme as a catalytic mechanism for calf spleen PNP. Attention is drawn to the postulated similar mechanism of action of other purine N-glycosidases, including plant antiviral proteins which function as RNAN-glycosidases, and possibly some DNA N-glycosidases which function as repair enzymes

A NON-TYPICAL CYCLOBUTANE DIMER PHOTOPROD FROM 5-ETHYLOROTATE‡

Abstract— One of the two major photoproducts resulting from the irradiation of 5‐ethylorotate in neutral aqueous medium has been identified as an unusual cyclobutane photodimer formed from the 5.6 bond of the parent 5‐ethylorotate and the exocyclicC–5‐Cx bond of 5‐ethylidenehydroorotate the initial photoproduct of 5‐ethylorotate [Kulikowska et al., Z. Naturforsch. 31c, 514 (1976)]. Irradiation at 254 nm in neutral aqueous medium causes the heterodimer to photodissociate to the two parent monomers with a quantum yield of 0.28. The heterodimer also hydrolyzes to the parent monomers in aqueous base at room temperature or by heating in the solid state. A scheme for the major photochemical transformation of 5‐ethylorotate is presented.

Structure and properties of a trans–anti photodimer of 5-methylorotate

5-Methylorotate is relatively radiation resistant in aqueous fluid medium, but readily photodimerizes in an ice matrix. Rapid formation of such a matrix made possible the preparative isolation of the photodimer in good yield. The potassium salt of the photodimer crystallizes as the hexahydrate, C12H10N4O8K2•6H2O. The crystals are triclinic with space group a = 8.139(3), b = 9.759(3), c = 7.398(3) A, a = 100.28(7), β = 74.22(5), γ = 108.67(7)°, and V = 533.0 A3, Z = 1. The structure was solved by Patterson and direct methods; refinement by block-diagonal least-squares converged at R = 0.041 for all 1671 observed reflections. The pyrimidine rings of the centrosymmetric photodimer are arranged in trans–anti configuration across the planar cyclobutane ring. The potassium ion is seven-coordinated. In aqueous medium the photodimer exhibits a pKa of 12.8 due to dissociation of the ring N(3) hydrogen. Irradiation in aqueous medium at 254 nm leads to quantitative regeneration of the monomer with a quantum yield of...

Substrate/Inhibitor Properties of Tumour Purine Nucleoside Phosphorylase

Abstract Substrate/inhibitor properties of purine nucleoside phosphorylase (PNP), isolated from human lung and kidney tumour tissues, have been characterised and compared with those of the enzyme from the corresponding normal organs.