Escherichia coli purine nucleoside phosphorylase II, the product of the xapA gene

Purine nucleoside phosphorylases (PNPs, E. C. 2.4.2.1) use orthophosphate to cleave the N-glycosidic bond of beta-(deoxy)ribonucleosides to yield alpha-(deoxy)ribose 1-phosphate and the free purine base. Escherichia coli PNP-II, the product of the xapA gene, is similar to trimeric PNPs in sequence, but has been reported to migrate as a hexamer and to accept xanthosine with comparable efficiency to guanosine and inosine, the usual physiological substrates for trimeric PNPs. Here, we present a detailed biochemical characterization and the crystal structure of E.coli PNP-II. In three different crystal forms, PNP-II trimers dimerize, leading to a subunit arrangement that is qualitatively different from the "trimer of dimers" arrangement of conventional high molecular mass PNPs. Crystal structures are compatible with similar binding modes for guanine and xanthine, with a preference for the neutral over the monoanionic form of xanthine. A single amino acid exchange, tyrosine 191 to leucine, is sufficient to convert E.coli PNP-II into an enzyme with the specificity of conventional trimeric PNPs, but the reciprocal mutation in human PNP, valine 195 to tyrosine, does not elicit xanthosine phosphorylase activity in the human enzyme.     

Crystal structure of calf spleen purine nucleoside phosphorylase in a complex with multisubstrate analogue inhibitor with 2,6-diaminopurine aglycone

The crystal structure at 2.05 A resolution of calf spleen PNP complexed with stoichiometric concentration of acyclic nucleoside phosphonate inhibitor, 2,6-diamino-(S)-9-[2-(phosphonomethoxy)propyl]purine, in a new space group P2(1)2(1)2(1) which contains two full trimers in the asymmetric crystal unit is described.     

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.     

Fluorescence studies of calf spleen purine nucleoside phosphorylase (PNP) complexes with guanine and 9-deazaguanine

Interactions of trimeric calf spleen purine nucleoside phosphorylase (PNP) with guanine (Gua) and its analogue, 9-deazaguanine (9-deaza-Gua), were studied by means of the steady-state fluorescence. The aim was to test the hypothesis that the enzyme stabilizes the anionic form of purine, inferred previously from the unusual increase of fluorescence observed after binding of guanine by calf spleen PNP. We have found that the dissociation constants obtained form titration experiments are in fact pH-independent in the range 7.0-10.25 for both PNP/Gua and PNP/9-deaza-Gua complexes. In particular, at pH 7.0 we found Kd = 0.12 +/- 0.02 micro M for Gua and 0.16 +/- 0.01 micro M for 9-deaza-Gua, while at the conditions where there is more than 40% of the anionic form the respective values were Kd = 0.15 +/- 0.01 micro M for Gua (pH 9.0) and 0.25 +/- 0.02 micro M for 9-deaza-Gua (pH 10.25). Hence, the enzyme does not prefer binding of anionic forms of these ligands in respect to the neutral ones. This result questions the involvement of the anionic forms in the reaction catalyzed by trimeric PNPs, and contradicts the hypothesis of a strong hydrogen bond formation between the enzyme Asn 243 residue and the purine N7 position.     

Fluorescence studies of calf spleen purine nucleoside phosphorylase (PNP) complexes with guanine and 9-deazaguanine

Interactions of trimeric calf spleen purine nucleoside phosphorylase (PNP) with guanine (Gua) and its analogue, 9-deazaguanine (9-deaza-Gua), were studied by means of the steady-state fluorescence. The aim was to test the hypothesis that the enzyme stabilizes the anionic form of purine, inferred previously from the unusual increase of fluorescence observed after binding of guanine by calf spleen PNP. We have found that the dissociation constants obtained form titration experiments are in fact pH-independent in the range 7.0-10.25 for both PNP/Gua and PNP/9-deaza-Gua complexes. In particular, at pH 7.0 we found K _d = 0.12 ± 0.02 μ M for Gua and 0.16 ± 0.01 μ M for 9-deaza-Gua, while at the conditions where there is more than 40% of the anionic form the respective values were K _d = 0.15 ± 0.01 μ M for Gua (pH 9.0) and 0.25 ± 0.02 μ M for 9-deaza-Gua (pH 10.25). Hence, the enzyme does not prefer binding of anionic forms of these ligands in respect to the neutral ones. This result questions the involvement of the anionic forms in the reaction catalyzed by trimeric PNPs, and contradicts the hypothesis of a strong hydrogen bond formation between the enzyme Asn 243 residue and the purine N(7) position.     

Crystal Structure of Calf Spleen Purine Nucleoside Phosphorylase in a Complex with Multisubstrate Analogue Inhibitor with 2,6-Diaminopurine Aglycone

Abstract

The crystal structure at 2.05 Å resolution of calf spleen PNP complexed with stoichiometric concentration of acyclic nucleoside phosphonate inhibitor, 2,6-diamino-(S)-9-[2-(phosphonomethoxy)propyl]purine, in a new space group P2₁2₁2₁ which contains two full trimers in the asymmetric crystal unit is described.     

Structures of purine nucleoside phosphorylase from Mycobacterium tuberculosis in complexes with immucillin-H and its pieces.

A structural genomics comparison of purine nucleoside phosphorylases (PNPs) indicated that the enzyme encoded by Mycobacterium tuberculosis (TB-PNP) resembles the mammalian trimeric structure rather than the bacterial hexameric PNPs. The crystal structure of M. tuberculosis PNP in complex with the transition-state analogue immucillin-H (ImmH) and inorganic phosphate was solved at 1.75 A resolution and confirms the trimeric structure. Binding of the inhibitor occurs independently at the three catalytic sites, unlike mammalian PNPs which demonstrate negative cooperativity in ImmH binding. Reduced subunit interface contacts for TB-PNP, compared to the mammalian enzymes, correlate with the loss of the cooperative inhibitor binding. Mammalian and TB-PNPs both exhibit slow-onset inhibition and picomolar dissociation constants for ImmH. The structure supports a catalytic mechanism of reactant destabilization by neighboring group electrostatic interactions, transition-state stabilization, and leaving group activation. Despite an overall amino acid sequence identity of 33% between bovine and TB-PNPs and almost complete conservation in active site residues, one catalytic site difference suggests a strategy for the design of transition-state analogues with specificity for TB-PNP. The structure of TB-PNP was also solved to 2.0 A with 9-deazahypoxanthine (9dHX), iminoribitol (IR), and PO(4) to reconstruct the ImmH complex with its separate components. One subunit of the trimer has 9dHX, IR, and PO(4) bound, while the remaining two subunits contain only 9dHX. In the filled subunit, 9dHX retains the contacts found in the ImmH complex. However, the region of IR that corresponds to the oxocarbenium ion is translocated in the direction of the reaction coordinate, and the nucleophilic phosphate rotates away from the IR group. Loose packing of the pieces of ImmH in the catalytic site establishes that covalent connectivity in ImmH is required to achieve the tightly bound complex.     

Cloning, expression, purification, and some properties of calf purine nucleoside phosphorylase

Calf spleen purine nucleoside phosphorylase (PNP) is considered a model enzyme for the trimeric PNPs subfamily. PCR amplification of the calf phosphorylase from the calf spleen library, cloning, overexpression of the recombinant PNP, its enzymatic activity and interactions with typical ligands of mammalian wild type PNP are described. Relative activity of the recombinant phosphorylase versus several substrates is similar to the respective values obtained for the enzyme isolated from calf spleen. As for the nonrecombinant calf PNP, the unusual fluorescence properties of the PNP/guanine complex were observed and characterized.     

Cloning,Expression, Purification,and Some Properties of Calf Purine Nucleoside Phosphorylase

Calf spleen purine nucleoside phosphorylase (PNP) is considered a model enzyme for the trimeric PNPs subfamily. PCR amplification of the calf phosphorylase from the calf spleen library, cloning, overexpression of the recombinant PNP, its enzymatic activity and interactions with typical ligands of mammalian wild type PNP are described. Relative activity of the recombinant phosphorylase versus several substrates is similar to the respective values obtained for the enzyme isolated from calf spleen. As for the nonrecombinant calf PNP, the unusual fluorescence properties of the PNP/guanine complex were observed and characterized.     

Crystal structure of purine nucleoside phosphorylase from Thermus thermophilus

The purine nucleoside phosphorylase from Thermus thermophilus crystallized in space group P4(3)2(1)2 with the unit cell dimensions a = 131.9 A and c = 169.9 A and one biologically active hexamer in the asymmetric unit. The structure was solved by the molecular replacement method and refined at a 1.9A resolution to an r(free) value of 20.8%. The crystals of the binary complex with sulfate ion and ternary complexes with sulfate and adenosine or guanosine were also prepared and their crystal structures were refined at 2.1A, 2.4A and 2.4A, respectively. The overall structure of the T.thermophilus enzyme is similar to the structures of hexameric enzymes from Escherichia coli and Sulfolobus solfataricus, but significant differences are observed in the purine base recognition site. A base recognizing aspartic acid, which is conserved among the hexameric purine nucleoside phosphorylases, is Asn204 in the T.thermophilus enzyme, which is reminiscent of the base recognizing asparagine in trimeric purine nucleoside phosphorylases. Isothermal titration calorimetry measurements indicate that both adenosine and guanosine bind the enzyme with nearly similar affinity. However, the functional assays show that as in trimeric PNPs, only the guanosine is a true substrate of the T.thermophilus enzyme. In the case of adenosine recognition, the Asn204 forms hydrogen bonds with N6 and N7 of the base. While in the case of guanosine recognition, the Asn204 is slightly shifted together with the beta(9)alpha(7) loop and predisposed to hydrogen bond formation with O6 of the base in the transition state. The obtained experimental data suggest that the catalytic properties of the T.thermophilus enzyme are reminiscent of the trimeric rather than hexameric purine nucleoside phosphorylases.     

Probing the mechanism of purine nucleoside phosphorylase by steady-state kinetic studies and ligand binding characterization determined by fluorimetric titrations

Reversible reaction catalyzed by trimeric purine nucleoside phosphorylase (PNP) from Cellulomonas sp. with typical and non-typical substrates, including product inhibition patterns of both reaction directions, and interactions of the enzyme with bisubstrate analogue inhibitors, were investigated by the steady-state kinetic methods and fluorimetric titrations. The ligand chromophores exist most probably as neutral species, and not N(1)-H monoanions, in the complex with PNP, as shown by determination of inhibition constants vs. pH. This supports the mechanism in which hydrogen bond interaction of N(1)-H with Glu204 is crucial in the catalytic process. Stoichiometry of ligand binding, with possible exception of hypoxanthine, is three molecules per enzyme trimer. Kinetic experiments show that in principle the Michaelis-Menten model could not properly describe the reaction. However, this model seems to hold for certain experimental conditions. Data presented here are supported by earlier findings obtained by means of fluorimetric titrations and protective effects of ligands on thermal inactivation of the enzyme. All results are consistent with the following mechanism for trimeric PNPs: (i) random binding of substrates, (ii) potent binding and slow release of some reaction products leading to the circumstances that the chemical step is not the slowest one and that rapid-equilibrium assumptions do not hold, (iii) a dual role of phosphate--a substrate and also a reaction modifier.     

The purine nucleoside phosphorylase from Trichomonas vaginalis is a homologue of the bacterial enzyme

Trichomonas vaginalis is a parasitic protozoan and the causative agent of trichomoniasis. Its primary purine salvage system, consisting of a purine nucleoside phosphorylase (PNP) and a purine nucleoside kinase, presents potential targets for designing selective inhibitors as antitrichomonial drugs because of lack of de novo synthesis of purine nucleotides in this organism. cDNA encoding T. vaginalis PNP was isolated by complementation of an Escherichia coli strain deficient in PNP and expressed, and the recombinant enzyme was purified to apparent homogeneity. It bears only 28% sequence identity with that of human PNP but 57% identity with the E. coli enzyme. Gel filtration showed the enzyme in a hexameric form, similar to the bacterial PNPs. Steady-state kinetic analysis of T. vaginalis PNP-catalyzed reactions gave K(m)'s of 31.5, 59.7, and 6.1 microM for inosine, guanosine, and adenosine in the nucleosidase reaction and 45.6, 35.9, and 12.3 microM for hypoxanthine, guanine, and adenine in the direction of nucleoside synthesis. This substrate specificity appears to be similar to that of bacterial PNPs. The catalytic efficiency of this enzyme with adenine as substrate is 58-fold higher than that with either hypoxanthine or guanine, representing a distinct disparity with the mammalian PNPs, which have negligible activity with either adenine or adenosine. The kinetic mechanism of T. vaginalis PNP-catalyzed reactions, determined by product inhibition and equilibrium isotope exchange, was by random binding of substrates (purine base and ribose 1-phosphate) with ordered release of the purine nucleoside first, followed by inorganic phosphate. Formycin A, an analogue of adenosine known as an inhibitor of E. coli PNP without any effect on mammalian PNPs, was shown to inhibit T. vaginalis PNP with a K(is) of 2.3 microM by competing with adenosine. T. vaginalis PNP thus belongs to the family of bacterial PNPs and constitutes a target for antitrichomonial chemotherapy.     

Assignment of downfield proton resonances in purine nucleoside phosphorylase immucillin-H complex by saturation-transferred NOEs

Purine nucleoside phosphorylase (PNP) catalyzes N-ribosidic bond phosphorolysis in 6-oxypurine nucleosides and deoxynucleosides to form purine and alpha-D-phosphorylated ribosyl products. The transition state has oxacarbenium ion character with partial positive charge near C-1', ionic stabilization from the nearby phosphate anion, and protonation at N-7 of the purine. Immucillin-H (ImmH) has a protonated N-7 and resembles the transition-state charge distribution when N-4' is protonated to the cation. It binds tightly to the PNPs with a K(d) value 56 pM for human PNP. Previous NMR studies of PNP.ImmH.PO(4) have shown that the N-4' of bound ImmH is a cation and is postulated to have a significant contribution to its tight binding. Several unassigned downfield proton resonances (>11 ppm) are specific to the PNP.ImmH.PO(4) complex, suggesting the existence of strong hydrogen bonds. In this study, two of the proton resonances in this downfield region have been assigned. Using (15)N-7-labeled ImmH, a resonance at 12.5 ppm has been assigned to N-7H. The N-7H resonance is shifted downfield by only approximately 1 ppm from its position for ImmH free in aqueous solution, consistent with only a small change in the hydrogen bonding on N-7H upon binding of ImmH to PNP. In contrast, the downfield resonance at 14.9 ppm in the PNP.ImmH.PO(4) complex is assigned to N-1H of ImmH by using saturation-transferred NOE measurements on the PNP.ImmH complex. The approximately 4 ppm downfield shift of the N-1H resonance from its position for ImmH free in solution suggests that the hydrogen bonding to the N-1H in the complex has a significant contribution to the binding of ImmH to PNP. The crystal structure shows Glu201 is in a direct hydrogen bond with N-1H and to O-6 through a water bridge. In the complex with 6-thio-ImmH, the N-1H resonance is shifted further downfield by an additional 1.5 ppm to 16.4 ppm, but the relative shift from the value for 6-thio-ImmH free in solution is the same as in the ImmH complex. Since the binding affinity to hPNP for 6-thio-ImmH is decreased 440-fold relative to that for ImmH, the loss in binding energy is primarily due to the hydrogen bond energy loss at the 6-thiol.     

Inhibition and structure of Trichomonas vaginalis purine nucleoside phosphorylase with picomolar transition state analogues

Trichomonas vaginalis is a parasitic protozoan purine auxotroph possessing a unique purine salvage pathway consisting of a bacterial type purine nucleoside phosphorylase (PNP) and a purine nucleoside kinase. Thus, T. vaginalis PNP (TvPNP) functions in the reverse direction relative to the PNPs in other organisms. Immucillin-A (ImmA) and DADMe-Immucillin-A (DADMe-ImmA) are transition state mimics of adenosine with geometric and electrostatic features that resemble early and late transition states of adenosine at the transition state stabilized by TvPNP. ImmA demonstrates slow-onset tight-binding inhibition with TvPNP, to give an equilibrium dissociation constant of 87 pM, an inhibitor release half-time of 17.2 min, and a Km/Kd ratio of 70,100. DADMe-ImmA resembles a late ribooxacarbenium ion transition state for TvPNP to give a dissociation constant of 30 pM, an inhibitor release half-time of 64 min, and a Km/Kd ratio of 203,300. The tight binding of DADMe-ImmA supports a late SN1 transition state. Despite their tight binding to TvPNP, ImmA and DADMe-ImmA are weak inhibitors of human and P. falciparum PNPs. The crystal structures of the TvPNP x ImmA x PO4 and TvPNP x DADMe-ImmA x PO4 ternary complexes differ from previous structures with substrate analogues. The tight binding with DADMe-ImmA is in part due to a 2.7 A ionic interaction between a PO4 oxygen and the N1' cation of the hydroxypyrrolidine and is weaker in the TvPNP x ImmA x PO4 structure at 3.5 A. However, the TvPNP x ImmA x PO4 structure includes hydrogen bonds between the 2'-hydroxyl and the protein that are not present in TvPNP x DADMe-ImmA x PO4. These structures explain why DADMe-ImmA binds tighter than ImmA. Immucillin-H is a 12 nM inhibitor of TvPNP but a 56 pM inhibitor of human PNP. And this difference is explained by isotope-edited difference infrared spectroscopy with [6-18O]ImmH to establish that O6 is the keto tautomer in TvPNP x ImmH x PO4, causing an unfavorable leaving-group interaction.     

Plasmodium falciparum parasites are killed by a transition state analogue of purine nucleoside phosphorylase in a primate animal model

Plasmodium falciparum causes most of the one million annual deaths from malaria. Drug resistance is widespread and novel agents against new targets are needed to support combination-therapy approaches promoted by the World Health Organization. Plasmodium species are purine auxotrophs. Blocking purine nucleoside phosphorylase (PNP) kills cultured parasites by purine starvation. DADMe-Immucillin-G (BCX4945) is a transition state analogue of human and Plasmodium PNPs, binding with picomolar affinity. Here, we test BCX4945 in Aotus primates, an animal model for Plasmodium falciparum infections. Oral administration of BCX4945 for seven days results in parasite clearance and recrudescence in otherwise lethal infections of P. falciparum in Aotus monkeys. The molecular action of BCX4945 is demonstrated in crystal structures of human and P. falciparum PNPs. Metabolite analysis demonstrates that PNP blockade inhibits purine salvage and polyamine synthesis in the parasites. The efficacy, oral availability, chemical stability, unique mechanism of action and low toxicity of BCX4945 demonstrate potential for combination therapies with this novel antimalarial agent.     

Transition state structure of purine nucleoside phosphorylase and principles of atomic motion in enzymatic catalysis

Immucillin-H [ImmH; (1S)-1-(9-deazahypoxanthin-9-yl)-1,4-dideoxy-1,4-imino-D-ribitol] is a 23 pM inhibitor of bovine purine nucleoside phosphorylase (PNP) specifically designed as a transition state mimic [Miles, R. W., Tyler, P. C., Furneaux, R. H., Bagdassarian, C. K., and Schramm, V. L. (1998) Biochemistry 37, 8615-8621]. Cocrystals of PNP and the inhibitor are used to provide structural information for each step through the reaction coordinate of PNP. The X-ray crystal structure of free ImmH was solved at 0.9 A resolution, and a complex of PNP.ImmH.PO(4) was solved at 1.5 A resolution. These structures are compared to previously reported complexes of PNP with substrate and product analogues in the catalytic sites and with the experimentally determined transition state structure. Upon binding, ImmH is distorted to a conformation favoring ribosyl oxocarbenium ion formation. Ribosyl destabilization and transition state stabilization of the ribosyl oxocarbenium ion occur from neighboring group interactions with the phosphate anion and the 5'-hydroxyl of the ribosyl group. Leaving group activation of hypoxanthine involves hydrogen bonds to O6, N1, and N7 of the purine ring. Ordered water molecules provide a proton transfer bridge to O6 and N7 and permit reversible formation of these hydrogen bonds. Contacts between PNP and catalytic site ligands are shorter in the transition state analogue complex of PNP.ImmH.PO(4) than in the Michaelis complexes of PNP.inosine.SO(4) or PNP.hypoxanthine.ribose 1-PO(4). Reaction coordinate motion is dominated by translation of the carbon 1' of ribose between relatively fixed phosphate and purine groups. Purine and pyrimidine phosphoribosyltransferases and nucleoside N-ribosyl hydrolases appear to operate by a similar mechanism.     

Crystallographic structure of PNP from Mycobacterium tuberculosis at 1.9A resolution

Even being a bacterial purine nucleoside phosphorylase (PNP), which normally shows hexameric folding, the Mycobacterium tuberculosis PNP (MtPNP) resembles the mammalian trimeric structure. The crystal structure of the MtPNP apoenzyme was solved at 1.9 A resolution. The present work describes the first structure of MtPNP in complex with phosphate. In order to develop new insights into the rational drug design, conformational changes were profoundly analyzed and discussed. Comparisons over the binding sites were specially studied to improve the discussion about the selectivity of potential new drugs.     

Structures for the potential drug target purine nucleoside phosphorylase from Schistosoma mansoni causal agent of schistosomiasis

Despite the availability of effective chemotherapy, schistosomiasis continues to be one of the major parasitic infections to affect the human population worldwide. Currently, little is known of the structural biology of the parasites that are responsible for the disease and few attempts have been made to develop second generation drugs, which may become essential if resistance to those currently available becomes an issue. Here, we describe three crystal structures for the enzyme purine nucleoside phosphorylase (PNP) from Schistosoma mansoni, a component of the purine salvage pathway. PNP is known to be essential for the recovery of purine bases and nucleosides in schistosomes, due to an absence of the enzymes for de novo synthesis, making it a sensitive point in the parasite's metabolism. In all three structures reported here, acetate occupies part of the base-binding site and is directly bound to the conserved glutamic acid at position 203. One of the structures presents the crystallization additive sulfobetaine 195 (NDSB195) occupying simultaneously the ribose and phosphate binding sites, whilst a second presents only phosphate in the latter. The observation of sulfobetaine specifically bound to the protein active site was unexpected and is unique to this structure as far as we are aware. Considerable flexibility is observed in the active site, principally due to variable structural disorder in the regions centered on residues 64 and 260. This conformational plasticity extends to the way in which both NDSB195 and phosphate bind to the individual monomers of the trimeric structure reported here. Differences between the parasite and human enzymes are limited principally to the base-binding site, where the substitution of V245 in the mammalian enzymes by S247 introduces additional hydrogen bonding potential to the site. This is satisfied in the structures described here by a water molecule whose presence is normally observed only in complexes with 6-oxopurines. Residue Y202, which replaces F200 in human PNP, is able to reach over the ribose-binding site to interact with H259 and is predicted to form an additional hydrogen bond with the 5' hydroxyl of nucleoside substrates.     

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.