Control of phosphorylase b conformation by a modified cofactor: crystallographic studies on R-state glycogen phosphorylase reconstituted with pyridoxal 5''-diphosphate.

Previous crystallographic studies on glycogen phosphorylase have described the different conformational states of the protein (T and R) that represent the allosteric transition and have shown how the properties of the 5'-phosphate group of the cofactor pyridoxal phosphate are influenced by these conformational states. The present work reports a study on glycogen phosphorylase b (GPb) complexed with a modified cofactor, pyridoxal 5'-diphosphate (PLPP), in place of the natural cofactor. Solution studies (Withers, S.G., Madsen, N.B., & Sykes, B.D., 1982, Biochemistry 21, 6716-6722) have shown that PLPP promotes R-state properties of the enzyme indicating that the cofactor can influence the conformational state of the protein. GPb complexed with pyridoxal 5'-diphosphate (PLPP) has been crystallized in the presence of IMP and ammonium sulfate in the monoclinic R-state crystal form and the structure refined from X-ray data to 2.8 A resolution to a crystallographic R value of 0.21. The global tertiary and quaternary structure in the vicinity of the Ser 14 and the IMP sites are nearly identical to those observed for the R-state GPb-AMP complex. At the catalytic site the second phosphate of PLPP is accommodated with essentially no change in structure from the R-state structure and is involved in interactions with the side chains of two lysine residues (Lys 568 and Lys 574) and the main chain nitrogen of Arg 569. Superposition of the T-state structure shows that were the PLPP to be incorporated into the T-state structure there would be a close contact with the 280s loop (residues 282-285) that would encourage the T to R allosteric transition. The second phosphate of the PLPP occupies a site that is distinct from other dianionic binding sites that have been observed for glucose-1-phosphate and sulfate (in the R state) and for heptulose-2-phosphate (in the T state). The results indicate mobility in the dianion recognition site, and the precise position is dependent on other linkages to the dianion. In the modified cofactor the second phosphate site is constrained by the covalent link to the first phosphate of PLPP. The observed position in the crystal suggests that it is too far from the substrate site to represent a site for catalysis.     

Multiple phosphate positions in the catalytic site of glycogen phosphorylase: structure of the pyridoxal-5''-pyrophosphate coenzyme-substrate analog.

The three-dimensional structure of an R-state conformer of glycogen phosphorylase containing the coenzyme-substrate analog pyridoxal-5'-diphosphate at the catalytic site (PLPP-GPb) has been refined by X-ray crystallography to a resolution of 2.87 A. The molecule comprises four subunits of phosphorylase related by approximate 222 symmetry. Whereas the quaternary structure of R-state PLPP-GPb is similar to that of phosphorylase crystallized in the presence of ammonium sulfate (Barford, D. & Johnson, L.N., 1989, Nature 340, 609-616), the tertiary structures differ in that the two domains of the PLPP-GPb subunits are rotated apart by 5 degrees relative to the T-state conformation. Global differences among the four subunits suggest that the major domains of the phosphorylase subunit are connected by a flexible hinge. The two different positions observed for the terminal phosphate of the PLPP are interpreted as distinct phosphate subsites that may be occupied at different points along the reaction pathway. The structural basis for the unique ability of R-state dimers to form tetramers results from the orientation of subunits with respect to the dyad axis of the dimer. Residues in opposing dimers are in proper registration to form tetramers only in the R-state.     

Functional attributes of the phosphate group binding cup of pyridoxal phosphate-dependent enzymes

Twenty-four structures of pyridoxal-5'-phosphate (PLP)-dependent enzymes that represent five different folds are shown to share a common recognition pattern for the phosphate group of their PLP-ligands. All atoms that interact with the phosphate group of PLP in these proteins are organized within a two-layer structure so that the first interacting layer contains from five to seven atoms and parallel with this is a second layer containing from three to seven interacting atoms. In order to identify features of the phosphate-binding site common to PLP-dependent enzymes, a simple procedure is described that assigns relative positions to all interacting atoms unambiguously, such that the networks of interactions for different proteins can be compared. On the basis of these diagrams for 24 enzyme-cofactor complexes, a detailed comparison of the two-layer structures of PLP-dependent enzymes, with both similar and different folds, was made. A majority of the structurally defined PLP-dependent proteins use the same atom types in analogous "key" positions to bind their PLP-ligands. In some instances, proteins use water molecules when a key position is unoccupied. A similar two-layer recognition pattern extends to protein recognition of at least one other, non-PLP ligand, glucosamine 6-phosphate. We refer to this three-dimensional recognition pattern as the phosphate-binding cup. In general, the phosphate-binding cup provides a very stable anchoring point for PLP. When numerous water molecules occur within the cup, however, then the phosphate group of PLP participates directly in the enzymatic reactions with inorganic phosphate replacing the water molecules of the cup. With glucosamine-6-phosphate synthase, the water molecules of the phosphate-binding cup facilitate the entry of substrate and the exit of product.     

The turnover of skeletal muscle glycogen phosphorylase studied using the cofactor, pyridoxal phosphate, as a specific label

The turnover of glycogen phosphorylase has been measured using the cofactor, pyridoxal phosphate, as a label specific for this enzyme in skeletal muscle. Radiolabelled pyridoxine administered in vivo is incorporated into a protein-bound fraction in skeletal muscle, shown by several criteria to be equivalent to glycogen phosphorylase. This pool of radiolabel disapears slowly with a half-life of 11.9 days, taken to be a good estimate of the intracellular half-life of the enzyme. The use of the cofactor in this fashion minimises overestimation of half-life that results from reincorporation of the label. Further, premature dissociation of the cofactor from native enzyme, which would lead to underestimation of half-life, is unlikely. At the level of sensitivity given by this method there was little evidence for the appearance of pyridoxal phosphate-labelled degradation intermediates of the enzyme.     

Similarity between pyridoxal/pyridoxamine phosphate-dependent enzymes involved in dideoxy and deoxyaminosugar biosynthesis and other pyridoxal phosphate enzymes.

A multiple sequence alignment among aspartate aminotransferase, dialkylglycine decarboxylase, and serine hydroxymethyltransferase (DAS) was used for profile databank search. The DAS profile could detect similarities to other pyridoxal or pyridoxamine phosphate-dependent enzymes, like several gene products involved in dideoxysugar and deoxyaminosugar synthesis. The alignment among DAS and such gene products shows the conservation of aspartate 222 and lysine 258, which, in aspartate aminotransferase, interacts with the N1 of the coenzyme pyridine ring and forms the internal Schiff base, respectively. The lysine is replaced by histidine in the pyridoxamine phosphate-dependent gene products. The alignment indicates also that the region encompassing the coenzyme binding site is the most conserved.     

Localization of pyrodoxal phosphate binding site on the mero-receptor domain of the glucocorticoid receptor

Previous studies have demonstrated that the vitamin pyridoxal phosphate can alter the physicochemical properties of glucocorticoid receptors. We now report the localization of a pyridoxal phosphate binding site within the mero-receptor domain of this glucocorticoid receptor. Mero-glucocorticoid receptors that are generated by trypsin (10 μg/ml) or chymotrypsin (100 μg/ml) digestion of intact receptors sediment as 2.6 S species on 5–20% sucrose gradients in the presence or absence of pyridoxal phosphate. Mero-glucocoritcoid receptors prepared by exogenous proteinases are hydrophobic and show no affinity for DEAE Bio-Gel A. Treating either trypsin-generated or chymotrypsin-generated mero-receptors with pyridoxal phosphate rapidly converts the proteins (60 and 35%, respectively) into forms that bind to DEAE Bio-Gel A. Induction of DEAE binding is specific to pyridoxal phosphate, for treating mero-receptors with pyridoxal, pyridoxamine or pyridoxine phosphate is ineffective. Furthermore, DEAE binding cannot be induced by adding other pyridoxal phosphate-treated cytosols to untreated mero-receptors. High-resolution polyacrylamide gel isoelectric focussing studies indicated that treating mero-receptor generated by either proteinase with pyridoxal phosphate shifted the isoelectric points of lower pH values. The conversion of the mero-receptor to a more acidic form also occurred when the intact glucocorticoid receptor was treated with the vitamin prior to proteolysis. These studies localize at least one pyridoxal phosphate binding site on the mero-receptor domain of the rat thymocyte glucocorticoid receptor.     

Aminolevulinate synthase: lysine 313 is not essential for binding the pyridoxal phosphate cofactor but is essential for catalysis.

5-Aminolevulinate synthase is the first enzyme of the heme biosynthetic pathway in animals and some bacteria. Lysine-313 of the mouse erythroid aminolevulinate synthase was recently identified to be linked covalently to the pyridoxal 5'-phosphate cofactor (Ferreira GC, Neame PJ, Dailey HA, 1993, Protein Sci 2:1959-1965). Here we report on the effect of replacement of aminolevulinate synthase lysine-313 by alanine, histidine, and glycine, using site-directed mutagenesis. Mutant enzymes were purified to homogeneity, and the purification yields were similar to those of the wild-type enzyme. Although their absorption spectra indicate that the mutant enzymes bind pyridoxal 5'-phosphate, they bind noncovalently. However, addition of glycine to the mutant enzymes led to the formation of external aldimines. The formation of an external aldimine between the pyridoxal 5'-phosphate cofactor and the glycine substrate is the first step in the mechanism of the aminolevulinate synthase-catalyzed reaction. In contrast, lysine-313 is an essential catalytic residue, because the K313-directed mutant enzymes have no measurable activity. In summary, site-directed mutagenesis of the aminolevulinate synthase active-site lysine-313, to alanine (K313A), histidine (K313H), or glycine (K313G) yields enzymes that bind the pyridoxal 5'-phosphate cofactor and the glycine substrate to produce external aldimines, but which are inactive. This suggests that lysine-313 has a functional role in catalysis.     

The external anion binding site of the human erythrocyte anion transporter: DNDS binding and competition with chloride

Summary The interaction between chloride and the anion transport inhibitor DNDS (4,4-dinitro stilbene-2,2-disulfonate) at the external anion binding site of the human erythrocyte anion transporter was examined by two techniques: a) chloride tracer flux experiments in the presence of varying concentrations of DNDS, and b) DNDS equilibrium binding experiments in the presence of varying concentrations of intracellular and extracellular chloride, Cl _i and Cl _o . DNDS inhibited competitively the Cl _o -stimulated chloride efflux from intact red cells at 0°C and pH 7.8 with an inhibitor constant of 90nm. Under the same conditions DNDS bound reversibly to one class of binding sites on intact cells with a capacity of 8.5×10⁵ molecules/cell. Cl _o competitively inhibited DNDS binding with an inhibitor constant of 6mm. In the absence of Cl _o the DNDS binding constant was 84mm. The competition between chloride and DNDS was also tested in nystatintreated cells in which Cl _o always equaled Cl _i . Under these conditions the values of the DNDS binding constant and the chloride inhibitor constant were significantly larger. All these data were in quantitative agreement with a single-site, alternating access kinetic scheme with ping-pong-type kinetics that we have previously developed for modeling chloride exchange transport. The data also served to rule out special cases of an alternative two-sited sequential-type kinetic scheme. DNDS binding experiments were also performed at 10 and 20°C. We found that neither the DNDS binding constant nor the Cl _o inhibitor constant were significantly changed compared to 0°C.     

The binding of beta- and gamma-cyclodextrins to glycogen phosphorylase b: kinetic and crystallographic studies

A number of regulatory binding sites of glycogen phosphorylase (GP), such as the catalytic, the inhibitor, and the new allosteric sites are currently under investigation as targets for inhibition of hepatic glycogenolysis under high glucose concentrations; in some cases specific inhibitors are under evaluation in human clinical trials for therapeutic intervention in type 2 diabetes. In an attempt to investigate whether the storage site can be exploited as target for modulating hepatic glucose production, alpha-, beta-, and gamma-cyclodextrins were identified as moderate mixed-type competitive inhibitors of GPb (with respect to glycogen) with K(i) values of 47.1, 14.1, and 7.4 mM, respectively. To elucidate the structural basis of inhibition, we determined the structure of GPb complexed with beta- and gamma-cyclodextrins at 1.94 A and 2.3 A resolution, respectively. The structures of the two complexes reveal that the inhibitors can be accommodated in the glycogen storage site of T-state GPb with very little change of the tertiary structure and provide a basis for understanding their potency and subsite specificity. Structural comparisons of the two complexes with GPb in complex with either maltopentaose (G5) or maltoheptaose (G7) show that beta- and gamma-cyclodextrins bind in a mode analogous to the G5 and G7 binding with only some differences imposed by their cyclic conformations. It appears that the binding energy for stabilization of enzyme complexes derives from hydrogen bonding and van der Waals contacts to protein residues. The binding of alpha-cyclodextrin and octakis (2,3,6-tri-O-methyl)-gamma-cyclodextrin was also investigated, but none of them was bound in the crystal; moreover, the latter did not inhibit the phosphorylase reaction.     

Biosynthesis of bacterial glycogen. Incorporation of pyridoxal phosphate into the allosteric activator site and an ADP-glucose-protected pyridoxal phosphate binding site of Escherichia coli B ADP-glucose synthase.

[3H]Pyridoxal-P can be covalently incorporated into Escherichia coli B mutant strain AC70R1 ADP-glucose synthase by reduction with NaBH4. Two distinct lysine residues can be modified by the allosteric activator pyridoxal-P. Incorporation of [3H]pyridoxal-P in the presence of substrate ADP-glucose + MgCl2 prevents pyridoxylation of an ADP-glucose-protected site and allows modification of the allosteric activator site. Incorporation of [3H]pyridoxal-P in the presence of allosteric effectors fructose-P2, 5'-AMP, or hexanediol-1,6-P2, protects against pyridoxylation of the allosteric activator site, and allows modification of the ADP-glucose-protected site. Incorporation of pyridoxal-P into the allosteric activator site results in modified enzyme of high activity form, even in the absence of fructose-P2. This modified enzyme, when assayed in the absence of fructose-P2, exhibits activation kinetics similar to nonpyridoxylated enzyme assayed in the presence of fructose-P2 and is still inhibited by 5'-AMP. These data suggest that the allosteric activator site of pyridoxylation is the fructose-P2 binding site, and is distinct from the inhibitor 5'-AMP binding site. Incorporation of pyridoxal-P into the ADP-glucose-protected site results in a decrease in enzyme activity. This pyridoxylated lysine could be involved with the binding of thesubstrates ADP-glucose, alpha-glucose-1-P, or PPi, or participate in the catalytic mechanism of the enzyme.     

Metal binding to pyridoxal derivatives. An NMR study of the interaction of Eu(III) with pyridoxal phosphate and pyridoxamine phosphate

The solution conformations of pyridoxal-5′ -phosphate and pyridoxamine-5′-phosphate have been investigated using Eu(III) as a nuclear magnetic resonance shift probe. Binding of Eu(III) to pyridoxal phosphate results in the formation of two complexes, at the phosphate group and theo-hydroxy-aldehyde moiety, which are in slow exchange on the nuclear magnetic resonance time-scale. The lanthanide-induced pseudo contact shifts calculated using the McConnell-Robertson equation (J. Chem. Soc. (1950), 22, 1561) are in good agreement with the experimentally observed values for both pyridoxal phosphate and pyridoxamine phosphate and lead to a family of closely related conformations. Contribution No. 130 from the Molecular Biophysics Unit.     

N-acetyl-β-D-glucopyranosylamine: A potent T-state inhibitor of glycogen phosphorylase. A comparison with α-D-glucose

Structure-based drug design has led to the discovery of a number of glucose analogue inhibitors of glycogen phosphorylase that have an increased affinity compared to α-D-glucose (K_i = 1.7 mM). The best inhibitor in the class of N-acyl derivatives of β-D-glucopyranosylamine, N-acetyl-β-D-glucopyranosylamine (1-GlcNAc), has been characterized by kinetic, ultracentrifugation, and crystallographic studies. 1-GlcNAc acts as a competitive inhibitor for both the b (K_i = 32 μM) and the α (K_i = 35 μM) forms of the enzyme with respect to glucose 1-phosphate and in synergism with caffeine, mimicking the binding of glucose. Sedimentation velocity experiments demonstrated that 1-GlcNAc was able to induce dissociation of tetrameric phosphorylase α and stabilization of the dimeric T-state conformation. Co-crystals of the phosphorylase b-1-GlcNAc-IMP complex were grown in space group P4₃2₁2, with native-like unit cell dimensions, and the complex structure has been refined to give a crystallographic R factor of 18.1%, for data between 8 and 2.3 Å resolution. 1-GlcNAc binds tightly at the catalytic site of T-state phosphorylase b at approximately the same position as that of α-D-glucose. The ligand can be accommodated in the catalytic site with very little change in the protein structure and stabilizes the T-state conformation of the 280s loop by making several favorable contacts to Asn 284 of this loop. Structural comparisons show that the T-state phosphorylase b-1-GlcNAc-IMP complex structure is overall similar to the T-state phosphorylase b-α-D-glucose complex structure. The structure of the 1-GlcNAc complex provides a rational for the biochemical properties of the inhibitor.     

The nature of the binding site for aromatic compounds in glycogen phosphorylase b.

The inhibition of rabbit muscle glycogen phosphorylase b (1,4-alpha-D-glucan--orthophosphate alpha-glucosyltransferase, EC 2.4.1.1) by aromatic compounds was examined with 15 compounds. The relative effectiveness of the inhibitors correlated well with increasing substituent constant, pi, indicating the hydrophobic nature of the binding site. The inhibition was not affected by the ionic-strength variation of the assay mixtures. The results predict that the course of chemical modification of this enzyme and the properties of the derivatives depend on the nature of the reagent and on the incorporated groups. Many of the dissimilar and sometimes contradictory results reported for chemical-modification studies and for chemically modified phosphorylase b are explained by the findings presented in the paper.     

A novel dominant selectable system for the selection of transgenic plants under in vitro and greenhouse conditions based on phosphite metabolism

Antibiotic and herbicide resistance genes are currently the most frequently used selectable marker genes for plant research and crop development. However, the use of antibiotics and herbicides must be carefully controlled because the degree of susceptibility to these compounds varies widely among plant species and because they can also affect plant regeneration. Therefore, new selectable marker systems that are effective for a broad range of plant species are still needed. Here, we report a simple and inexpensive system based on providing transgenic plant cells the capacity to convert a nonmetabolizable compound (phosphite, Phi) into an essential nutrient for cell growth (phosphate) trough the expression of a bacterial gene encoding a phosphite oxidoreductase (PTXD). This system is effective for the selection of Arabidopsis transgenic plants by germinating T0 seeds directly on media supplemented with Phi and to select transgenic tobacco shoots from cocultivated leaf disc explants using nutrient media supplemented with Phi as both a source of phosphorus and selective agent. Because the ptxD/Phi system also allows the establishment of large‐scale screening systems under greenhouse conditions completely eliminating false transformation events, it should facilitate the development of novel plant transformation methods.     

Interaction of pyridoxal phosphate with the amino groups at the active site of 5- aminolevulinic acid dehydratase in maize

Pyridoxal 5′-phosphate strongly and reversibly inhibited maize leaf 5-amino levulinic acid dehydratase. The inhibition was linearly competitive with respect to the substrate 5-aminolevulinic acid at pH values between 7 to 9.0. Pyridoxal was also effective as an inhibitor of the enzyme but pyridoxamine phosphate was not inhibitory. The results suggest that pyridoxal 5′-phosphate may be interacting with the enzyme either close to or at the 5-aminolevulinic acid binding site. This conclusion was further corroborated by the detection of a Schiff base between the enzyme and the substrate, 5-aminolevulinic acid and by reduction of pyridoxal phosphate and substrate complexes with sodium borohydride     

Kinetic properties of tetrameric glycogen phosphorylase b in solution and in the crystalline state.

R-state monoclinic P2(1) crystals of phosphorylase have been shown to be catalytically active in the presence of an oligosaccharide primer and glucose-1-phosphate in 0.9 M ammonium sulfate, 10 mM beta-glycerophosphate, 0.5 mM EDTA, and 1 mM dithiothreitol, the medium in which the crystals are grown or equilibrated for crystallographic studies (Barford, D. & Johnson, L.N., 1989, Nature 360, 609-616; Barford, D., Hu, S.-H., & Johnson, L.N., 1991, J. Mol. Biol. 218, 233-260). Kinetic data suggest that the activity of crystalline tetrameric phosphorylase is similar to that determined in solution for the enzyme tetramer. However, large differences were found in the maximal velocities for both oligosaccharide or glucose-1-phosphate substrates between the soluble dimeric and crystalline tetrameric enzyme.     

The structure of a glycogen phosphorylase glucopyranose spirohydantoin complex at 1.8 A resolution and 100 K: the role of the water structure and its contribution to binding.

A glucopyranose spirohydantoin (a pyranose analogue of the potent herbicide, hydantocidin) has been identified as the highest affinity glucose analogue inhibitor of glycogen phosphorylase b (GPb). In order to elucidate the structural features that contribute to the binding, the structures of GPb in the native T state conformation and in complex with glucopyranose spirohydantoin have been determined at 100 K to 2.0 A and 1.8 A resolution, respectively, and refined to crystallographic R values of 0.197 (R[free] 0.248) and 0.182 (R[free] 0.229), respectively. The low temperature structure of GPb is almost identical to that of the previously determined room temperature structure, apart from a decrease in overall atomic temperature factors ((B) room temperature GPb = 34.9 A2; (B) 100 K GPb = 23.4 A2). The glucopyranose spirohydantoin inhibitor (Ki = 3.0 microM) binds at the catalytic site and induces small changes in two key regions of the protein: the 280s loop (residues 281-286) that results in a decrease in mobility of this region, and the 380s loop (residues 377-385) that undergoes more significant shifts in order to optimize contact to the ligand. The hydantoin group, that is responsible for increasing the affinity of the glucose compound by a factor of 10(3), makes only one hydrogen bond to the protein, from one of its NH groups to the main chain oxygen of His377. The other polar groups of the hydantoin group form hydrogen bonds to five water molecules. These waters are involved in extensive networks of hydrogen bonds and appear to be an integral part of the protein structure. Analysis of the water structure at the catalytic site of the native enzyme, shows that five waters are displaced by ligand binding and that there is a significant decrease in mobility of the remaining waters on formation of the GPb-hydantoin complex. The ability of the inhibitor to exploit existing waters, to displace waters and to recruit new waters appears to be important for the high affinity of the inhibitor.