Agnieszka Bzowska

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.

Calf spleen purine nucleoside phosphorylase: purification, sequence and crystal structure of its complex with an N(7)-acycloguanosine inhibitor

Calf spleen purine nucleoside phosphorylase was purified to homogeneity and its amino acid sequence was determined. The complex of the enzyme with an N(7)‐acycloguanosine inhibitor crystallized in the cubic space group P213, with unit cell dimension and one monomer in the asymmetric crystal unit. The biologically active trimer is formed by the crystallographic three‐fold axis. The structure was solved by molecular replacement methods, using the model of the human erythrocyte enzyme, and refined at a resolution of 2.9 Å to an R‐factor of 0.21. The orientation of the inhibitor at the active site is examined in relation to the catalytic activity of the enzyme in the phosphorolysis of nucleosides.

Calf spleen purine nucleoside phosphorylase: complex kinetic mechanism, hydrolysis of 7-methylguanosine, and oligomeric state in solution

The active enzyme form was found to be a homotrimer, no active monomers were observed. Only in the presence of an extremely high orthophosphate concentration (0.5 M) or at a low enzyme concentration (0.2 microg/ml) with no ligands present a small fraction of the enzyme is probably in a dissociated and/or non-active form. The specific activity is invariant over a broad enzyme concentration range (0.017 microg/ml-0.29 mg/ml). At concentrations below 0.9 microg/ml and in the absence of ligands the enzyme tends to loose its catalytic activity, while in the presence of any substrate or at higher concentrations it was found to be active as a trimer. In the absence of phosphate the enzyme catalyses the hydrolysis of 7-methylguanosine (m7Guo) with a catalytic rate constant 1.3x10(-3) x s(-1) as compared with the rate of 38 s(-1) for the phosphorolysis of this nucleoside. The initial pre-steady-state phase of the phosphorolysis of m7Guo, 70 s(-1), is almost twice faster than the steady-state rate and indicates that the rate-limiting step is subsequent to the glycosidic bond cleavage. Complex kinetic behaviour with substrates of phosphorolytic direction (various nucleosides and orthophosphate) was observed; data for phosphate as the variable substrate with inosine and guanosine, but not with their 7-methyl counterparts, might be interpreted as two binding sites with different affinities, or as a negative cooperativity. However, the titration of the enzyme intrinsic fluorescence with 0.2 microM-30 mM phosphate is consistent with only one dissociation constant for phosphate, K(d)=220+/-120 microM. Protective effects of ligands on the thermal inactivation of the enzyme indicate that all substrates of the phosphorolytic and the synthetic reactions are able to form binary complexes with the calf spleen purine nucleoside phosphorylase. The purine bases, guanine and hypoxanthine, bind strongly with dissociation constants of about 0.1 microM, while all other ligands studied, including 7-methylguanine and 7-methylhypoxanthine, bind at least 3 orders of magnitude less potently. Binding of guanine and hypoxanthine is about 10-fold weakened by the presence of phosphate. These observations are best interpretable by the complex kinetic mechanism of the phosphorolytic reaction involving (i) random substrate binding, (ii) unusually slow, hence strongly rate-limiting, dissociation of the products guanine and hypoxanthine, but not 7-methylguanine and 7-methylhypoxanthine, and (iii) dual function of the phosphate binding site with phosphate acting as a substrate and as a modifier helping in the release of a purine base after glycosidic bond cleavage.

FLUORESCENCE OF TYROSINE AND TRYPTOPHAN IN PROTEINS USING ONE‐ AND TWO‐PHOTON EXCITATION

Abstract— We examined the emission spectra of tyrosine‐ and tryptophan‐containing proteins using one‐photon (270–310 nm) and two‐photon (565–610 nm) excitation. Emission spectra for two‐photon excitation of native and denatured human serum albumin and of three purine nucleoside phosphorylases indicated an absence of the tyrosine emission normally seen for one‐photon excitation below 290 nm. We examined the one‐photon and two‐photon excitation spectra of tyrosine‐tryptophan mixtures to determine the origin of selective excitation of the tryptophan residues. These results confirmed a short‐wavelength shift of the tyrosine two‐photon excitation spectrum relative to that of tryptophan, as recently reported by Rehms and Callis (1993) Chem. Phys. Lett. 208, 276–282.

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.

Calf spleen purine-nucleoside phosphorylase: crystal structure of the binary complex with a potent multisubstrate analogue inhibitor

Purine-nucleoside phosphorylase (PNP) deficiency in humans leads to inhibition of the T-cell response. Potent membrane-permeable inhibitors of this enzyme are therefore considered to be potential immunosuppressive agents. The binary complex of the trimeric calf spleen phosphorylase, which is highly homologous to human PNP, with the potent ground-state analogue inhibitor 9-(5,5-difluoro-5-phosphonopentyl)guanine (DFPP-G) was crystallized in the cubic space group P2(1)3, with unit-cell parameter a = 93.183 A and one monomer per asymmetric unit. High-resolution X-ray diffraction data were collected using synchrotron radiation (EMBL Outstation, DESY, Hamburg, station X13). The crystal structure was refined to a resolution of 2.2 A and R and Rfree values of 19.1 and 24.2%, respectively. The crystal structure confirms that DFPP-G acts as a multisubstrate analogue inhibitor as it binds to both nucleoside- and phosphate-binding sites. The structure also provides the answers to some questions regarding the substrate specificity and molecular mechanism of trimeric PNPs. The wide access to the active-site pocket that was observed in the reported structure as a result of the flexibility or disorder of two loops (residues 60-65 and 251-266) strongly supports the random binding of substrates. The putative hydrogen bonds identified in the base-binding site indicate that N1-H and not O6 of the purine base defines the specificity of trimeric PNPs. This is confirmed by the fact that the contact of guanine O6 with Asn243 Odelta1 is not a direct contact but is mediated by a water molecule. Participation of Arg84 in the binding of the phosphonate group experimentally verifies the previous suggestion [Blackburn & Kent (1986), J. Chem. Soc. Perkin Trans. I, pp. 913-917; Halazy et al. (1991), J. Am. Chem. Soc. 113, 315-317] that fluorination of alkylphosphonates yields compounds with properties that suitably resemble those of phosphate esters and in turn leads to optimized interactions of such analogues with the phosphate-binding site residues. DFPP-G shows a Ki(app) in the nanomolar range towards calf and human PNPs. To date, no high-resolution X-ray structures of these enzymes with such potent ground-state analogue inhibitors have been available in the Protein Data Bank. The present structure may thus be used in the rational structure-based design of new PNP inhibitors with potential medical applications.

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

Abstract 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 ≈ 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.

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.