Pyridoxal(5')diphospho(1)-alpha-D-glucose. A potent R-state inhibitor of glycogen phosphorylase

Pyridoxal(5')diphospho(1)-alpha-D-glucose has been tested as an inhibitor of native glycogen phosphorylases a and b. Its inhibition patterns with respect to substrate, glucose 1-phosphate, and activator, adenosine monophosphate, show it to be a potent (Ki = 40 microM) R-state inhibitor of phosphorylase b, mimicking the binding of glucose-1-phosphate, and, as predicted for an R-state inhibitor, its binding to AMP-activated phosphorylase a is even tighter (Ki = 10 microM). Moreover, it is demonstrated that its binding does not involve covalent imine formation from the pyridoxal aldehyde to an active-site lysine residue. It thus represents the tightest binding R-state inhibitor reported to date, and a 31P NMR study of the effects of binding of this inhibitor upon 31P resonances for the coenzyme phosphate and that of the nucleotide activator is presented. Results obtained are essentially identical to those obtained previously using glucose cyclic 1,2-phosphate, corroborating the previous conclusions. A rationale for the tightness of the binding is presented, as are other possible uses of this compound in studies on glycogen phosphorylase and other similar enzymes.     

Flexibility in the phosphorylase catalytic reaction. Glucosyltransfer from pyridoxal (5')-triphospho(1)-alpha-D-glucose to glycogen catalyzed by phosphorylase

When rabbit muscle phosphorylase reconstituted with pyridoxal (5')-diphospho(1)-alpha-D-glucose is incubated with glycogen, its glucosyl moiety is transferred to the nonreducing end of glycogen with the formation of a new alpha-1,4-glucosidic linkage. This finding provided the first evidence for the direct phosphate-phosphate interaction between the coenzyme pyridoxal 5'-phosphate and the substrate alpha-D-glucose 1-phosphate in the phosphorylase catalytic reaction (Takagi, M., Fukui, T., and Shimomura, S. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 3716-3719). We have examined whether pyridoxal(5')triphospho(1)-alpha-D-glucose can act in a similar manner to the diphospho compound or not. In the absence of glucan the enzyme-bound triphospho compound was stable for 1 day at pH 6-9. In the presence of glucan, however, its glucosidic linkage was cleaved, and the glucosyl moiety liberated was transferred to glycogen with the formation of a new alpha-1,4-glucosidic linkage. Allosteric activator AMP accelerated the reaction and allosteric inhibitor glucose 6-phosphate showed the reverse effect. The pH optimum of the reaction was pH 8.1-8.4. Mg2+ slightly but significantly accelerated the reaction, whereas Mn2+ and Ca2+ inhibited the reaction. These results indicate that the glucosyltransfer from the triphospho compound occurs in an identical manner to that from the diphospho compound. Based on the present and previous data, we discuss the catalytic mechanism of phosphorylase, especially in comparison with that of phosphoryltransferases.     

Nucleotide binding to glycogen phosphorylase b in the crystal.

The binding of the allosteric activator, AMP, and the inhibitor, ATP, to glycogen phosphorylase b has been studied in the crystal at 3 Å resolution. The nucleotides bind to two sites on the enzyme which are identified as site N, the allosteric effector site which is close to the subunit-subunit interface, and site I, a nucleoside inhibitor site which blocks the entrance to the active site crevasse. AMP when bound at the allosteric effector site makes several defined interactions with the enzyme in agreement with the results of solution studies. The contacts involve the N-10 position of the base, the 2′ hydroxyl of the ribose and the phosphate. IMP, analysed at 4 Å resolution, appears to bind in an identical conformation to AMP. At 3 Å resolution no well defined conformational changes are observed on binding AMP, although there are indications of a disturbance of the crystal lattice. It is concluded that the forces which stabilise the crystal lattice prevent the allosteric response of the enzyme in the crystal.     

The binding of beta-glycerophosphate to glycogen phosphorylase b in the crystal

The binding of beta-glycerophosphate (glycerol-2-P) to glycogen phosphorylase b in the crystal has been studied by X-ray diffraction at 3 A resolution. Glycerol-2-P binds to the allosteric effector site in a position close to that of AMP, glucose-6-P, UDP-Glc, and phosphate. In this position, glycerol-2-P is stabilized through interactions of its phosphate moiety with the guanidinium groups of Arg 309 and Arg 310 which undergo conformational changes, and the hydroxyl group of Tyr 75, while the same residues and solvent are involved in van der Waals interactions with the remaining part of the molecule. Kinetic experiments indicate that glycerol-2-P partially competes with both the activator (AMP) and the inhibitor (glucose 6-phosphate) of phosphorylase b. A comparison of the positions of glycerol-2-P, AMP, glucose 6-phosphate, UDP-Glc, and Pi at the allosteric site is presented.     

AMP and IMP binding to glycogen phosphorylase b. A calorimetric and equilibrium dialysis study

Reaction microcalorimetry and equilibrium dialysis have been used to study the binding of AMP and IMP to glycogen phosphorylase b (EC 2.4.1.1) at 25 degrees C and pH 6.9. The combination of both techniques has enabled us to obtain some of the thermodynamic parameters for these binding processes. Four binding sites were found to be present in the dimeric active enzyme for both AMP and IMP. The binding to two high-affinity sites, which, in our opinion, correspond to the activator sites, seems to be cooperative. The two low-affinity sites, which would then correspond to the inhibitor sites, appear to be independent when the nucleotides bind to the enzyme. The negative delta G0 of binding/site at 25 degrees C is the result in all cases of a balance between negative enthalpy and entropy changes. The large differences in delta H and delta S0 for the binding of AMP to the activator sites (-27 and -70 kJ mol-1; -22 and -150 J X K-1 mol-1) suggest the existence of rather extensive conformational changes taking place in phosphorylase b on binding with the allosteric activator. Whereas the affinity of AMP for the activator sites is about 1 order of magnitude higher than that of IMP, the affinity of both nucleotides, including their delta H and delta S0 values, seems to be the same for the inhibitor sites.     

Allosteric interactions of glycogen phosphorylase b. A crystallographic study of glucose 6-phosphate and inorganic phosphate binding to di-imidate-cross-linked phosphorylase b.

The binding to glycogen phosphorylase b of glucose 6-phosphate and inorganic phosphate (respectively allosteric inhibitor and substrate/activator of the enzyme) were studied in the crystal at 0.3 nm (3A) resolution. Glucose 6-phosphate binds in the alpha-configuration at a site that is close to the AMP allosteric effector site at the subunit-subunit interface and promotes several conformational changes. The phosphate-binding site of the enzyme for glucose 6-phosphate involves contacts to two cationic residues, Arg-309 and Lys-247. This site is also occupied in the inorganic-phosphate-binding studies and is therefore identified as a high-affinity phosphate-binding site. It is distinct from the weaker phosphate-binding site of the enzyme for AMP, which is 0.27 nm (2.7A) away. The glucose moiety of glucose 6-phosphate and the adenosine moiety of AMP do not overlap. The results provide a structural explanation for the kinetic observations that glucose 6-phosphate inhibition of AMP activation of phosphorylase b is partially competitive and highly co-operative. The results suggest that the transmission of allosteric conformational changes involves an increase in affinity at phosphate-binding sites and relative movements of alpha-helices. In order to study glucose 6-phosphate and phosphate binding it was necessary to cross-link the crystals. The use of dimethyl malondi-imidate as a new cross-linking reagent in protein crystallography is discussed.     

Substrate-cofactor interactions for glycogen phosphorylase b: a binding study in the crystal with heptenitol and heptulose 2-phosphate

The structural relationships between substrate and pyridoxal phosphate in glycogen phosphorylase b (EC 2.4.1.1) have been studied by X-ray diffraction experiments at 3-A resolution. Recent work [Klein, H. W., Im, M. J., & Helmreich, E. J. M. (1984) in Chemical and Biological Aspects of Vitamin B6 Catalysis (Evangelopoulos, A. E., Ed.) pp 147-160, Liss, New York] has shown that phosphorylase in the presence of inorganic phosphate catalyzes the conversion of heptenitol to heptulose 2-phosphate. The latter compound is a dead-end product and a most potent inhibitor (Ki = 14 microM). The X-ray diffraction studies show that heptenitol binds at the catalytic site of phosphorylase in a position essentially identical with that observed for the glucopyranose moiety of glucose 1-phosphate. Incubation of a phosphorylase b crystal for 50 h in a solution containing the substrates heptenitol and inorganic phosphate and the activators AMP and maltohetaose resulted in the formation of a phosphorylated product bound at the active site. The structure of this product, as analyzed by a difference Fourier synthesis at 3 A, is consistent with that of heptulose 2-phosphate. Analysis of the surrounding soak solution by thin-layer chromatography showed that heptulose 2-phosphate was produced under these conditions. Heptulose 2-phosphate binds with its glucopyranose moiety in the same position as that for glucose 1-phosphate, but there is a marked difference in phosphate positions. The presence of the methyl group in the beta-configuration in heptulose 2-phosphate forces a change in the torsion angle O5-C1-O1-P from 117 degrees as observe in glucose 1-phosphate to -136 degrees in heptulose 2-phosphate. The "down" position of the phosphate (with respect to the crystallographic z axis) results in a change in the distance between the 5'-phosphorus atom of the pyridoxal phosphate and the phosphorus atom of the substrate from 6.8 (with glucose 1-phosphate) to 4.5 A (with heptulose 2-phosphate). The closest distance between the phosphate oxygen of the cofactor and a phosphate oxygen of heptulose 2-phosphate is 2.7 A, and it is assumed that there must be a hydrogen bond between them. These observations are consistent with the NMR experiments reported in the preceding paper in which sharing of a proton between heptulose 2-phosphate and pyridoxal 5'-phosphate is observed [Klein, H.W., Im, M. J., Palm, D., & Helmreich, E. J. M. (1984) Biochemistry (preceding paper in this issue)].(ABSTRACT TRUNCATED AT 400 WORDS)     

Comparison of AMP and NADH binding to glycogen phosphorylase b

The binding sites for the allosteric activator, AMP, to glycogen phosphorylase b are described in detail utilizing the more precise knowledge of the native structure obtained from crystallographic restrained least-squares refinement than has hitherto been available. Localized conformational changes are seen at the allosteric effector site that include shifts of between 1 and 2 A for residues Tyr75 and Arg309 and very small shifts for the region of residues 42 to 44 from the symmetry-related subunit. Kinetic studies demonstrate that NADH inhibits the AMP activation of glycogen phosphorylase b. Crystallographic binding studies at 3.5 A resolution show that NADH binds to the same sites on the enzyme as AMP, i.e. the allosteric effector site N, which is close to the subunit-subunit interface, and the nucleoside inhibitor site I, which is some 12 A from the catalytic site. The conformations of NADH at the two sites are different but both conformations are "folded" so that the nicotinamide ring is close (approx. 6 A) to the adenine ring. These conformations are compared with those suggested from solution studies and with the extended conformations observed in the single crystal structure of NAD+ and for NAD bound to dehydrogenases. Possible mechanisms for NADH inhibition of phosphorylase activation are discussed.     

Catalysis in the crystal: synchrotron radiation studies with glycogen phosphorylase b.

Direct observation of the progress of a catalysed reaction in crystals of glycogen phosphorylase b has been made possible through fast crystallographic data collection achieved at the Synchrotron Radiation source at Daresbury, UK. In the best experiments, data to 2.7 A resolution (some 108,300 measurements; 21,200 unique reflections) were measured in 25 min. In a series of time-resolved studies in which the control properties of the enzyme were exploited in order to slow down the reaction, the conversion of heptenitol to heptulose-2-phosphate, the phosphorylysis of maltoheptaose to yield glucose-1-phosphate and the oligosaccharide synthesis reaction involving maltotriose and glucose-1-phosphate have been monitored in the crystal. Changes in electron density in the difference Fourier maps are observed as the reaction proceeds not only at the catalytic site but also the allosteric and glycogen storage sites. Phosphorylase b is present in the crystals in the T state and under these conditions exhibits low affinity for both phosphate and oligosaccharide substrates. There are pronounced conformational changes associated with the formation and binding of the high-affinity dead-end product, heptulose-2-phosphate, which show that movement of an arginine residue, Arg 569, is critical for formation of the substrate-phosphate recognition site. The results are discussed with reference to proposals for the enzymic mechanism of phosphorylase. The feasibility for time-resolved studies on other systems and recent advances in this area utilizing Laue diffraction are also discussed.     

A spectrophotometric study of vitamin B2 and its coenzyme forms binding to muscle glycogen phosphorylase b

The vitamin B2 and its coenzyme forms binding to glycogen phosphorylase b from rabbit skeletal muscle has been studied by the spectrophotometric method. The spectral properties of riboflavin, FMN and FAD bound to muscle glycogen phosphorylase b were found to be identical at the wavelengths of 300 to 500 nm. According to data on spectrophotometric titration of muscle glycogen phosphorylase b by FMN, each subunit of the enzyme contains one flavin-binding site.     

Comparison of the binding of glucose and glucose 1-phosphate derivatives to T-state glycogen phosphorylase b

The binding of T-state- and R-state-stabilizing ligands to the catalytic C site of T-state glycogen phosphorylase b has been investigated by crystallographic methods to study the interactions made and the conformational changes that occur at the C site. The compounds studied were alpha-D-glucose, 1, a T-state-stabilizing inhibitor of the enzyme, and the R-state-stabilizing phosphorylated ligands alpha-D-glucose 1-phosphate (2), 2-deoxy-2-fluoro-alpha-D-glucose 1-phosphate (3), and alpha-D-glucose 1-methylenephosphonate (4). The complexes have been refined, giving crystallographic R factors of less than 19%, for data between 8 and 2.3 A. Analysis of the refined structures shows that the glucosyl portions of the phosphorylated ligands bind in the same orientation as glucose and retain most of the interactions formed between glucose and the enzyme. However, the phosphates of the phosphorylated ligands adopt different conformations in each case; the stability of these conformations have been studied by using computational methods to rationalize the different binding modes. Binding of the phosphorylated ligands is accompanied by movement of C-site residues, most notably a shift of a loop out of the C site and toward the exterior of the protein. The C-site alterations do not include movement of Arg569, which has been observed in both the refined complex with 1-deoxy-D-gluco-heptulose 2-phosphate (5) [Johnson, L. N., et al (1990) J. Mol. Biol. 211, 645-661] and in the R-state enzyme [Barford, D. & Johnson, L. N. (1989) Nature 340, 609-616]. Refinement of the ligand complexes has also led to the observation of additional electron density for residues 10-19 at the N-terminus which had not previously been localized in the native structure. The conformation of this stretch of residues is different from that observed in glycogen phosphorylase a.     

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.     

AMP interaction sites in glycogen phosphorylase b. A thermodynamic analysis

The binding of AMP to activator site N and to inhibitor site I in glycogen phosphorylase b has been characterized by calorimetry, potentiometry and ultracentrifugation in the pH range 6.5-7.5 at 25 degrees C (mu = 0.1). Calorimetric titration data of phosphorylase b with adenosine 5'-phosphoramidate are also reported at pH 6.9 (T = 25 degrees C, mu = 0.1). Calorimetric curves have been analyzed on the basis of potentiometric and sedimentation velocity results to determine thermodynamic quantities for AMP binding to the enzyme. The comparison of calorimetric titration data of AMP and adenosine 5'-phosphoramidate at pH 6.9 supports the hypothesis previously suggested that the dianionic phosphate form of the nucleotide preferentially binds to the allosteric activator site. The thermodynamic parameters for AMP binding to site N are as follows: delta G0 = -22 kJ mol-1, delta H0 = -34 kJ mol-1 and delta S0 = -40 J mol-1 K-1. The binding of the nucleotide to site I was found to be strongly dependent on the pH. This behaviour may be explained in terms of coupled protonations of three groups having pKa values of 6.0, 6.0 and 6.1 in the unbound form and 7.0, 7.5 and 7.2 in the enzyme-nucleotide complex. The thermodynamic parameters for nucleotide binding to site I for the enzymatic form in which all the modified groups are completely deprotonated or protonated have been calculated to be: delta G0 = -7.7 kJ mol-1, delta H0 = -28 kJ mol-1 and delta S0 = -68 J mol-1 K-1 and delta G0 = -28 kJ mol-1, delta H0H = -10 kJ mol-1 and delta S0H = 61 J mol-1 K-1, respectively. These results suggest that attractive dispersion forces are of primary significance for AMP binding to activator site N, although electrostatic interactions act as a stabilizing factor in the nucleotide binding. The protonation states of those residues of which the pKa values are modified by AMP binding to site I highly influence the thermodynamic parameters for the nucleotide binding to this site.     

The pyridoxal 5' -phosphate site in rabbit skeletal muscle glycogen phosphorylase b: an ultraviolet and 1H and 31P nuclear magnetic resonance spectroscopic study.

1 H NMR spectra of the 3-0-methylpyridoxal 5'-phosphate-n-butylamine reaction product indicated that this analogue forms a Schiff base in aprotic solvent. The uv spectral properties of 3-0-methylpyridoxal-5'-phosphate phosphorylase b correspond to those of the n-butylamine Schiff base derivative in dimethyl sulfoxide. On the basis of that and auxiliary uv and 1H NMR spectra of pyridoxal and pyridoxal 5'-phosphate and the corresponding Schiff base derivatives we have verified that pyridoxal 5' -phosphate is also bound as a Schiff base to phosphorylase and not as an aldamine. Since 3-0-methylpyridoxal-5'-phosphate phosphorylase is active, a proton shuttle between the 3-hydroxyl group and the pyridine nitrogen is excluded. This directs attention to the 5' -phosphate group of the cofactor as a candidate for a catalytic function. 31P NMR spectra of pyridoxal 5' -phosphate in phosphorylase b indicated that deprotonation of the 5' -phosphate group was unresponsive to external pH. Interaction of phosphorylase b with adenosine 5' -monophosphate, the allosteric effector required activity, and arsenate, which substitutes for phosphate as substrate, triggered a conformational change which resulted in deprotonation of the 5' -phosphate group of pyridoxal 5' at pH 7.6. It now behaved like in the pyridoxal-phosphate-epsilon-aminocaproate Schiff base in aqueous buffer, where the diionized form is dominant at this pH. Differences of line widths of the adenosine 5' -monophosphate signal point to different life times of the allosteric effector- enzyme complexes in the presence and absence of substrate (arsenate).