Human erythrocytic purine nucleoside phosphorylase: reaction with sugar-modified nucleoside substrates

The kinetic parameters (Km and Vmax) of sugar-modified analogues of inosine and guanosine have been determined with human erythrocytic purine nucleoside phosphorylase (PNP). Steric alterations at the 2' and 3' positions greatly lessened or abolished substrate activity. However, the 5'-deoxy- and 2',5'-dideoxy-beta-D-ribofuranosyl and the alpha-L-lyxosyl analogues were good substrates, indicating that the 5'-hydroxyl and the orientation of the 5'-hydroxy-methyl group are not important for binding. The sugar phosphate analogue, 5-deoxyribose 1-phosphate, was synthesized from 5'-deoxyinosine with immobilized PNP, and its presence was verified by using it in the enzymic synthesis of 5'-deoxyguanosine. The adenosine versions of the 5'-modified analogues were also found to react with adenosine deaminase, albeit at less than 1% of Vmax.     

Hexamere purine nucleoside phosphorylase from Escherichia coli K-12. Kinetic analysis and mechanism of reaction

A kinetic analysis of the phosphorolytic reaction catalyzed by hexameric purine nucleoside phosphorylase II from E. coli K-12 in the presence and absence of reaction products was carried out. The results of the kinetic analysis are consistent with a rapid equilibrium random Bi-Bi mechanism, in which a dead-end ternary (enzyme.purine base.phosphate) complex is formed.     

Crystal structure of the purine nucleoside phosphorylase (PNP) from Cellulomonas sp. and its implication for the mechanism of trimeric PNPs

The three-dimensional structure of the trimeric purine nucleoside phosphorylase (PNP) from Cellulomonas sp. has been determined by X-ray crystallography. The binary complex of the enzyme with orthophosphate was crystallized in the orthorhombic space group P212121 with unit cell dimensions a=64.1 A, b=108.9 A, c=119.3 A and an enzymatically active trimer in the asymmetric unit. X-ray data were collected at 4 degrees C using synchrotron radiation (EMBL/DESY, Hamburg). The structure was solved by molecular replacement, with the calf spleen PNP structure as a model, and refined at 2.2 A resolution. The ternary "dead-end" complex of the enzyme with orthophosphate and 8-iodoguanine was obtained by soaking crystals of the binary orthophosphate complex with the very weak substrate 8-iodoguanosine. Data were collected at 100 K with CuKalpha radiation, and the three-dimensional structure refined at 2.4 A resolution. Although the sequence of the Cellulomonas PNP shares only 33 % identity with the calf spleen enzyme, and almost no identity with the hexameric Escherichia coli PNP, all three enzymes have many common structural features, viz. the nine-stranded central beta-sheet, the positions of the active centres, and the geometrical arrangement of the ligands in the active centres. Some similarities of the surrounding helices also prevail. In Cellulomonas PNP, each of the three active centres per trimer is occupied by orthophosphate, and by orthophosphate and base, respectively, and small structural differences between monomers A, B and C are observed. This supports cooperativity between subunits (non-identity of binding sites) rather than existence of more than one binding site per monomer, as previously suggested for binding of phosphate by mammalian PNPs. The phosphate binding site is located between two conserved beta- and gamma-turns and consists of Ser46, Arg103, His105, Gly135 and Ser223, and one or two water molecules. The guanine base is recognized by a zig-zag pattern of possible hydrogen bonds, as follows: guanine N-1...Glu204 O(epsilon1)...guanine NH2...Glu204 O(epsilon2). The exocyclic O6 of the base is bridged via a water molecule to Asn246 N(delta), which accounts for the inhibitory, but lack of substrate, activity of adenosine. An alternative molecular mechanism for catalysis by trimeric PNPs is proposed, in which the key catalytic role is played by Glu204 (Glu201 in the calf and human enzymes), while Asn246 (Asn243 in the mammalian enzymes) supports binding of 6-oxopurines rather than catalysis. This mechanism, in contrast to that previously suggested, is consistent with the excellent substrate properties of N-7 substituted nucleosides, the specificity of trimeric PNPs versus 6-oxopurine nucleosides and the reported kinetic properties of Glu201/Ala and Asn243/Ala point variants of human PNP.     

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.     

Open and closed conformation of the E. coli purine nucleoside phosphorylase active center and implications for the catalytic mechanism.

The crystal structure of the ternary complex of hexameric purine nucleoside phosphorylase (PNP) from Escherichia coli with formycin A derivatives and phosphate or sulphate ions is determined at 2.0 A resolution. The hexamer is found as a trimer of unsymmetric dimers, which are formed by pairs of monomers with active sites in different conformations. The conformational difference stems from a flexible helix (H8: 214-236), which is continuous in one conformer, and segmented in the other. With the continuous helix, the entry into the active site pocket is wide open, and the ligands are bound only loosely ("open" or "loose binding" conformation). By segmentation of the helix (H8: 214-219 and H8': 223-236, separated by a gamma-turn), the entry into the active site is partially closed, the pocket is narrowed and the ligands are bound much more tightly ("closed" or "tight binding" conformation). Furthermore, the side-chain of Arg217 is carried by the moving helix into the active site. This residue, conserved in all homologous PNPs, plays an important role in the proposed catalytic mechanism. In this mechanism, substrate binding takes place in the open, and and the catalytic action occurs in the closed conformation. Catalytic action involves protonation of the purine base at position N7 by the side-chain of Asp204, which is initially in the acid form. The proton transfer is triggered by the Arg217 side-chain which is moved by the conformation change into hydrogen bond distance to Asp204. The mechanism explains the broad specificity of E. coli PNP, which allows 6-amino as well as 6-oxo-nucleosides as substrates. The observation of two kinds of binding sites is fully in line with solution experiments which independently observe strong and weak binding sites for phosphate as well as for the nucleoside inhibitor.     

Hexameric purine nucleoside phosphorylase II from Escherichia coli K-12. Physico-chemical and catalytic properties and stabilization with substrates

Some properties of hexameric purine nucleoside phosphorylase II (EC 2.4.2.1) from Escherichia coli K-12 were studied. The enzyme obeys the Michaelis-Menten kinetics with respect to purine substrates (Km for inosine, deoxyinosine and hypoxanthine are equal to 492, 106 and 26.6 microM, respectively) and exhibits negative kinetic cooperativity towards phosphate and ribose-1-phosphate. The Hill coefficient is equal to approximately 0.5 for both substrates. Hexameric purine nucleoside phosphorylase II is not a metal-dependent enzyme; its activity is inhibited by Cu2+, Zn2+, Ni2+ and SO4(2-). The enzyme is the most stable at pH 6.0; it contains essential thiol groups. All substrates partly protect the enzyme against inactivation by 5.5'-dithiobis(2-nitrobenzoic acid) and heat-inactivation and, with the exception of phosphate-against inactivation by p-chloromercuribenzoate. Hypoxanthine, especially in combination with phosphate, afford the best protection against inactivation.     

Partial purification and properties of purine nucleoside phosphorylase from rabbit erythrocytes.

1. The partial purification of purine nucleoside phosphorylase from rabbit erythrocytes is described. 2. Analytical and preparative isoelectric focusing gave a pI value for the enzyme of 4.65. 3. Gel-chromatography and sucrose-density-gradient-centrifugation techniques gave estimates of the molecular weight in the range 75000-83000. 4. Lineweaver-Burk plots of kinetic data were non-linear at high inosine concentrations. Extrapolation of the linear part of such plots yielded a Km value for inosine of about 70 micrometer for the rabbit erythrocyte and liver enzymes. 5. A Hill interaction coefficient of 0.75 was obtained, suggesting negative co-operativity with respect to the binding of inosine. 6. Treatment of the enzyme with 5,5'-dithiobis-(2-nitrobenzoic acid) caused partial inactivation, and subsequent Lineweaver-Burk plots with inosine as substrate displayed complete linearity, with an increase in Km value for inosine to 200 micrometer. 7. Starch-gel electrophoresis did not reveal the presence of secondary isoenzymes; all tissue extracts examined gave electrophoretic patterns similar to those obtained with the partially purified enzyme from erythrocytes. 8. Results of hybridization studies with nucleoside phosphorylase from human foetal liver suggest that the rabbit enzyme is also a trimer.     

Liver purine nucleoside phosphorylase in Camelus dromedarius: purification and properties

1. Purine nucleoside phosphorylase (purine nucleoside:orthophosphate ribosyl transferase, EC 2.4.2.1) was purified to electrophoretic homogeneity from the liver of Camelus dromedarius. 2. The enzyme appears to be a dimer with a 44,000 subunit mol. wt and displays non-linear kinetics with concave downward curvature in double reciprocal plots with respect to both inosine and orthophosphate as variable substrates. 3. The effect of thiol compounds on the enzyme activity and of pH on kinetic parameters is reported.     

Thyroid purine nucleoside phosphorylase. II. Kinetic model by alternate substrate and inhibition studies.

Nucleoside analog inhibition studies have been conducted on thyroidal purine nucleoside phosphorylase (purine-nucleoside:orthophosphate ribosyltransferase, EC 2.4.2.1) which catalyzed an ordered bi-bi type mechanism where the first substrate is inorganic phosphate and the last product is ribose 1-phosphate. Heterocyclic- and carbohydrate-modified nucleoside inhibitors demonstrate mixed type inhibition suggesting such analogs show an affinity (Ki) for the free enzyme. A kinetic model is proposed which supports the observed inhibition patterns. These studies together with alternate substrate studies indicate that nucleoside binding requires a functional group capable of hydrogen bonding at the 6-position of the purine ring and that the orientation of the bound substrate may be syn. Proper geometry of the phosphate is dependent upon the 3'-substituent to the orientated below the furanose ring. The 5'-hydroxyl group is required for substrate activity. The proposed rate limiting step of the phosphorylase mechanism is the enzymatic protonation of the 7-N position of the nucleoside.     

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.     

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 λ_max ∼ 425 to 430 nm, but the reaction product, 2,6-diamino-8-azapurine, emits in a different spectral region (λ_max ∼ 363 nm) with nearly 40% yield, providing a strong fluorogenic effect at 350 to 360 nm.     

Structural basis for selective inhibition of purine nucleoside phosphorylase from Schistosoma mansoni: Kinetic and structural studies

Selectivity plays a crucial role in the design of enzyme inhibitors as novel antiparasitic agents, particularly in cases where the target enzyme is also present in the human host. Purine nucleoside phosphorylase from Schistosoma mansoni (SmPNP) is an attractive target for the discovery of potential antischistosomal agents. In the present work, kinetic studies were carried out in order to determine the inhibitory potency, mode of action and enzyme selectivity of a series of inhibitors of SmPNP. In addition, crystallographic studies provided important structural insights for rational inhibitor design, revealing consistent structural differences in the binding mode of the inhibitors in the active sites of the SmPNP and human PNP (HsPNP) structures. The molecular information gathered in this work should be useful for future medicinal chemistry efforts in the design of new inhibitors of SmPNP having increased affinity and selectivity.     

Crystal structure of calf spleen purine nucleoside phosphorylase with two full trimers in the asymmetric unit: important implications for the mechanism of catalysis

The crystal structure of the binary complex of trimeric purine nucleoside phosphorylase (PNP) from calf spleen with the acyclic nucleoside phosphonate inhibitor 2,6-diamino-(S)-9-[2-(phosphonomethoxy)propyl]purine ((S)-PMPDAP) is determined at 2.3A resolution in space group P2(1)2(1)2(1). Crystallization in this space group, which is observed for the first time with a calf spleen PNP crystal structure, is obtained in the presence of calcium atoms. In contrast to the previously described cubic space group P2(1)3, two independent trimers are observed in the asymmetric unit, hence possible differences between monomers forming the biologically active trimer could be detected, if present. Such differences would be expected due to third-of-the-sites binding documented for transition-state events and inhibitors. However, no differences are noted, and binding stoichiometry of three inhibitor molecules per enzyme trimer is observed in the crystal structure, and in the parallel solution studies using isothermal titration calorimetry and spectrofluorimetric titrations. Presence of phosphate was shown to modify binding stoichiometry of hypoxanthine. Therefore, the enzyme was also crystallized in space group P2(1)2(1)2(1) in the presence of (S)-PMPDAP and phosphate, and the resulting structure of the binary PNP/(S)-PMPDAP complex was refined at 2.05A resolution. No qualitative differences between complexes obtained with and without the presence of phosphate were detected, except for the hydrogen bond contact of Arg84 and a phosphonate group, which is observed only in the former complex in three out of six independent monomers. Possible hydrogen bonds observed in the enzyme complexed with (S)-PMPDAP, in particular a putative hydrogen bonding contact N(1)-H cdots, three dots, centered Glu201, indicate that the inhibitor binds in a tautomeric or ionic form in which position N(1) acts as a hydrogen bond donor. This points to a crucial role of this hydrogen bond in defining specificity of trimeric PNPs and is in line with the proposed mechanism of catalysis in which this contact helps to stabilize the negative charge that accumulates on O(6) of the purine base in the transition state. In the present crystal structure the loop between Thr60 and Ala65 was found in a different conformation than that observed in crystal structures of trimeric PNPs up to now. Due to this change a new wide entrance is opened into the active site pocket, which is otherwise buried in the interior of the protein. Hence, our present crystal structure provides no obvious indication for obligatory binding of one of the substrates before binding of a second one; it is rather consistent with random binding of substrates. All these results provide new data for clarifying the mechanism of catalysis and give reasons for the non-Michaelis kinetics of trimeric PNPs.     

Gene therapy of cancer: activation of nucleoside prodrugs with E. coli purine nucleoside phosphorylase.

During the last few years, many gene therapy strategies have been developed for various disease targets. The development of anticancer gene therapy strategies to selectively generate cytotoxic nucleoside or nucleotide analogs is an attractive goal. One such approach involves the delivery of herpes simplex virus thymidine kinase followed by the acyclic nucleoside analog ganciclovir. We have developed another gene therapy methodology for the treatment of cancer that has several significant attributes. Specifically, our approach involves the delivery of E. coli purine nucleoside phosphorylase, followed by treatment with a relatively non-toxic nucleoside prodrug that is cleaved by the enzyme to a toxic compound. This presentation describes the concept, details our search for suitable prodrugs, and summarizes the current biological data.     

Intron requirement for expression of the human purine nucleoside phosphorylase gene.

Abbreviated purine nucleoside phosphorylase (PNP) genes were engineered to determine the effect of introns on human PNP gene expression. PNP minigenes containing the first intron (complete or shortened from 2.9 kb down to 855 bp), the first two introns or all five PNP introns resulted in substantial human PNP isozyme expression after transient transfection of murine NIH 3T3 cells. Low level human PNP activity was observed after transfection with a PNP minigene containing the last three introns. An intronless PNP minigene construct containing the PNP cDNA fused to genomic flanking sequences resulted in undetectable human PNP activity. Heterogeneous, stable NIH 3T3 transfectants of intron-containing PNP minigenes (verified by Southern analysis), expressed high levels of PNP activity and contained appropriately processed 1.7 kb message visualized by northern analysis. Stable transfectants of the intronless PNP minigene (40-45 copies per haploid genome) contained no detectable human PNP isozyme or mRNA. Insertion of the 855 bp shortened intron 1 sequence in either orientation upstream or downstream of a chimeric PNP promoter-bacterial chloramphenicol acetyltransferase (CAT) gene resulted in a several-fold increase in CAT expression in comparison with the parental PNP-CAT construct. We conclude that human PNP gene expression at the mRNA and protein level is dependent on the presence of intronic sequences and that the level of PNP expression varies directly with the number of introns included. The disproportionately greatest effect of intron 1 can be explained by the presence of an enhancer-like element retained in the shortened 855 bp intron 1 sequence.     

Remote mutations and active site dynamics correlate with catalytic properties of purine nucleoside phosphorylase

It has been found that with mutation of two surface residues (Lys²² → Glu and His¹⁰⁴ → Arg) in human purine nucleoside phosphorylase (hPNP), there is an enhancement of catalytic activity in the chemical step. This is true although the mutations are quite remote from the active site, and there are no significant changes in crystallographic structure between the wild-type and mutant active sites. We propose that dynamic coupling from the remote residues to the catalytic site may play a role in catalysis, and it is this alteration in dynamics that causes an increase in the chemical step rate. Computational results indicate that the mutant exhibits stronger coupling between promotion of vibrations and the reaction coordinate than that found in native hPNP. Power spectra comparing native and mutant proteins show a correlation between the vibrations of Immucillin-G (ImmG):O5′…ImmG:N4′ and H257:Nδ…ImmG:O5′ consistent with a coupling of these motions. These modes are linked to the protein promoting vibrations. Stronger coupling of motions to the reaction coordinate increases the probability of reaching the transition state and thus lowers the activation free energy. This motion has been shown to contribute to catalysis. Coincident with the approach to the transition state, the sum of the distances of ImmG:O4′…ImmG:O5′…H257:Nδ became smaller, stabilizing the oxacarbenium ion formed at the transition state. Combined results from crystallography, mutational analysis, chemical kinetics, and computational analysis are consistent with dynamic compression playing a significant role in forming the transition state. Stronger coupling of these pairs is observed in the catalytically enhanced mutant enzyme. That motion and catalysis are enhanced by mutations remote from the catalytic site implicates dynamic coupling through the protein architecture as a component of catalysis in hPNP.     

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.     

Investigation of alpha-deuterium kinetic isotope effects on the purine nucleoside phosphorylase reaction by the equilibrium-perturbation technique.

1. alpha-Deuterium kinetic isotope effects on the phosphorolysis of inosine catalysed by Escherichia coli purine nucleoside phosphorylase were measured by the equilibrium-perturbation technique, by using the change in absorbance at 250 nm (approx. 20%). 2. Values of 2H(V/K) of 1.13(9) at pH 5.0, 1.10(5) at pH 6.1, 1.09(4) at pH 7.3, 1.08 at pH 8.4 and 1.16(4) at pH 9.4 were obtained. 3. These are compared with literature alpha-deuterium kinetic isotope effects for this and related reactions. 4. The equilibrium constant, defined as [inosine].[H2PO4-]/[hypoxanthine] [alpha-Rib f OPO3H-], is 46 at 25 degrees C. 5. N-3-beta-D-Ribofuranosylhypoxanthine, an impurity in chemically synthesized inosine, is a substrate.