Towards the mechanism of trimeric purine nucleoside phosphorylases: stopped-flow studies of binding of multisubstrate analogue inhibitor - 2-amino-9-[2-(phosphonomethoxy)ethyl]-6-sulfanylpurine.

The binding of multisubstrate analogue inhibitor - 2-amino-9-[2-(phosphonomethoxy)ethyl]-6-sulfanylpurine (PME-6-thio-Gua) to purine nucleoside phosphorylase from Cellulomonas sp. at 20 degrees C, in 20 mM Hepes buffer with ionic strength adjusted to 50 mM using KCl, at several pH values between 6.5 and 8.2, was investigated using a stopped-flow spectrofluorimeter. The kinetic transients registered after mixing a protein solution with ligand solutions of different concentrations were simultaneously fitted by several association reaction models using nonlinear least-squares procedure based on numerical integration of the chemical kinetic equations appropriate for given model. It is concluded that binding of a PME-6-thio-Gua molecule by each of the binding sites is sufficiently well described by one-step process, with a model assuming interacting binding sites being more probable than a model assuming independent sites. The association rate constants derived from experimental data, assuming one step binding and independent sites, are decreasing with an increase in pH, changing from 30 to 6 microM(-1)s(-1) per binding site. The dissociation rate constants are in the range of 1-3 s(-1), and they are rather insensitive of changes in pH. Interestingly, for each pH value, the one-step binding model with interacting sites results in the association rate constant per site 1.5-4 times smaller for the binding of the first ligand molecule than that for the binding of the second one. Decrease of association constants with pH indicate that the enzyme does not prefer binding of the naturally occurring anionic form of the 6-thioguanine ring (pK(a) 8.7) resulting from a dissociation of N(1)-H. This finding supports the mechanism in which hydrogen bond interaction of N(1)-H with Glu204 (Glu 201 in mammalian PNPs) is crucial in the catalytic process. Results obtained also indicate that, in contrast to transition-state analogues, for which binding is followed by a conformational change, binding of multisubstrate analogue inhibitors to trimeric PNPs is a one-step process.

Stopped-flow studies of guanine binding by calf spleen purine nucleoside phosphorylase.

The binding of guanine to calf spleen purine nucleoside phosphorylase at 20 degrees C, in 20 mM Hepes-NaOH buffer, pH 7.0, at several ionic strength between 5 and 150 mM was investigated using a stopped-flow spectrofluorimeter. The kinetic transients registered after mixing a protein solution with ligand solutions of different concentrations were simultaneously fitted by several association reaction models using nonlinear least-squares procedure based on numerical integration of the chemical kinetic equations appropriate for given model. It is concluded that binding of a guanine molecule by each of the binding sites is a two-step process and that symmetrical trimeric calf spleen purine nucleoside phosphorylase represents a system of (identical) interacting binding sites. The interaction is visible through relations between the rate constants and non-additivity of changes in "molar" fluorescence for different forms of PNP-guanine complexes. It is also probable that electrostatic effects in guanine binding are weak, which indicates that it is the neutral form of the ligand which is bound and dissociated by PNP molecule.

Calf spleen purine nucleoside phosphorylase: structure of its ternary complex with an N(7)-acycloguanosine inhibitor and a phosphate anion.

The calf spleen purine nucleoside phosphorylase (PNP) ternary complex with an N(7)-acycloguanosine inhibitor and a phosphate ion has been crystallized in the cubic space group P2(1)3, with unit-cell parameter a = 94.11 A and one monomer per asymmetric unit. X-ray diffraction data were collected using synchrotron radiation (Station X31, EMBL Outstation, DESY, Hamburg). The crystal structure was refined to a resolution of 2.2 A and R and R(free) values of 17.5 and 24.5%, respectively. The acyclonucleoside inhibitor is bound in the active site in an inverted ('upside-down') orientation of the purine base compared with natural substrates. The side chain of Asp243 forms two hydrogen bonds with the base ring: N(delta) donates a hydrogen to N(3) and O(delta) accepts a hydrogen from the guanine N(2)-amino group. N(1)--H of the base is hydrogen bonded to O(epsilon) of Glu201, while N(9) accepts a hydrogen bond from Thr242 O(gamma). In addition, a water molecule (W417) bridges the N(2)-amino group of the base and O(epsilon) of Glu201. In the phosphate-binding site, a phosphate ion is bound to Ser33, His64, Arg84, His86, Ala116 and Ser220. The acyclic chain of the N(7)-acycloguanosine inhibitor is in a folded conformation and together with a water molecule (W388) occupies the pentose-binding site, with possible hydrogen bonds to Tyr88 O(eta) and His257 N(delta 1). This new binding mode fully accounts for the previously observed substrate properties of 7-beta-D-ribofuranosides of hypoxanthine and guanine. It also provides a new starting point for the design of inhibitors of PNP for therapeutic and other applications.

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.

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 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.

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.

Crystallization and preliminary X-ray studies of purine nucleoside phosphorylase from Cellulomonas sp.

The commercially available enzyme purine nucleoside phosphorylase (PNP) from Cellulomonas sp. was purified by ion--exchange chromatography, partially sequenced and crystallized in two different crystal forms using the hanging-drop vapour-diffusion technique. Crystal form A grows as polyeders and/or cubes in the cubic space group P4232 with unit-cell dimension a = 162.5 A. Crystal form B appears as thick plates in the space group P212121 with unit-cell dimensions a = 63.2, b = 108.3 and c = 117.4 A. Both crystal forms contain three monomers (one trimer) in the asymmetric unit.

Crystal structure of the ternary complex of E. coli purine nucleoside phosphorylase with formycin B, a structural analogue of the substrate inosine, and phosphate (Sulphate) at 2.1 A resolution.

The ternary complex of purine nucleoside phosphorylase from E. coli with formycin B and a sulphate or phosphate ion crystallized in the hexagonal space group P6122 with unit cell dimensions a=123.11, c=241.22 A and three monomers per asymmetric unit. The biologically active hexamer is formed through 2-fold crystallographic symmetry, constituting a trimer of dimers. High-resolution X-ray diffraction data were collected using synchrotron radiation (Daresbury, England). The crystal structure was determined by molecular replacement and refined at 2.1 A resolution to an R-value of 0.196. There is one active centre per monomer, composed of residues belonging to two subunits of one dimer. The phosphate binding site is strongly positively charged and consists of three arginine residues (Arg24, Arg87 and Arg43 from a neighbouring subunit), Ser90 and Gly20. It is occupied by a sulphate or phosphate anion, each oxygen atom of which accepts at least two hydrogen bonds or salt-bridges. The sulphate or phosphate anion is also in direct contact with the ribose moiety of formycin B. The ribose binding site is composed of Ser90, Met180, Glu181 and His4, the latter belonging to the neighbouring subunit. The base binding site is exposed to solvent, and the base is unspecifically bound through a chain of water molecules and aromatic-aromatic interactions. In all monomers the nucleosides are in the high syn conformation about the glycosidic bonds with chi in the range 100 to 130 degrees. The architecture of the active centre is in line with the known broad specificity and the kinetic properties of E. coli PNP.