Rabbit muscle phosphorylase derivatives with oligosaccharides covalently bound to the glycogen storage site

Linear maltooligosaccharides, e.g., maltoheptaose or terminal 4-O-methylmaltoheptaose, activated by cyanogen bromide, react covalently with rabbit muscle phosphorylases b and a (EC 2.4.1.1). Site-specific modification prevents further binding to glycogen and shifts the phosphorylase a tetramer-dimer equilibrium in favor of the dimer. Use was made of these properties to separate by affinity chromatography and gel filtration phosphorylase a dimers with specifically bound oligosaccharide from unspecifically modified products. The phosphorylase a-maltoheptaose derivative carries one oligosaccharide residue per monomer and can be distinguished from the native enzyme by its electrophoretic mobility in polyacrylamide gels or by affinity electrophoresis. Phosphorylase a preparations with covalently bound maltooligosaccharides are enzymatically active in the presence of a primer and alpha-D-glucopyranose 1-phosphate (glucose-1-P). Methylation of the nonreducing chain terminus of the bound oligosaccharide has no effect on glycogen synthesis. These findings exclude the participation of bound oligosaccharides in chain elongation. Purified covalent phosphorylase a-maltoheptaose complexes are stable dimers. They are no longer activated by glycogen. The properties of covalently modified phosphorylase-oligosaccharides are consistent with and provide direct evidence for the existence of a glycogen storage site in rabbit muscle phosphorylases. Covalent occupation of the storage site renders the affinity of glucose-1-P to phosphorylase a independent of modulation by glycogen, supporting the assumption that the glycogen storage site is involved in interactions with the catalytic site.     

Phosphorylase: control and activity

Recent results from the crystallographic studies on glycogen phosphorylase b at 2 A resolution are reviewed with special reference to other themes of the meeting. The structural similarity of the fold of 150 residues in phosphorylase to the observed in lactate dehydrogenase is discussed and the binding sites for NADH in phosphorylase are described. The binding of the potent inhibitor glucose-1,2-cyclic phosphate to phosphorylase b in the crystal has been studied at 3 A resolution. The results are compared with those previously obtained for glucose-1-phosphate and discussed with reference to proposals for a mechanism of catalysis that involves the essential cofactor pyridoxal phosphate.     

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.     

Effect of polymyxins on glycogen phosphorylase

AMP-dependent activity of glycogen phosphorylase b is stimulated by the polymyxins A, B, D, and E. Kinetic studies indicate that these cyclic peptide antibiotics at low concentrations greatly enhance AMP-activation of the enzyme. The presence of polymyxins in the assay system leads to (a) partial desensitization of allosteric interactions toward AMP, (b) lowering of K_m for the substrates glucose-1-phosphate and glycogen, and (c) reversal of the glucose-6-phosphate inhibition. in contrast to phosphorylase b, neither AMP-phosphorylase b′ system nor phosphorylase a (with or without AMP) is considerably activated by polymyxins.     

Laue and monochromatic diffraction studies on catalysis in phosphorylase b crystals.

The conversion of substrate, heptenitol, to product, beta-1-C-methyl, alpha-D-glucose-1-phosphate (heptulose-2-P), in crystals of glycogen phosphorylase b has been studied by Laue and monochromatic diffraction methods. The phosphorolysis reaction in the crystal was started following liberation of phosphate from a caged phosphate compound, 3,5-dinitrophenyl phosphate (DNPP). The photolysis of DNPP, stimulated by flashes from a xenon flash lamp, was monitored in the crystal with a diode array spectrophotometer. In the Laue diffraction experiments, data to 2.8 A resolution were collected and the first time shot was obtained at 3 min from the start of reaction, and data collection comprised three 800-ms exposures. Careful data processing of Laue photographs for the large enzyme resulted in electron density maps of almost comparable quality to those produced by monochromatic methods. The difference maps obtained from the Laue measurements showed that very little catalysis had occurred 3 min and 1 h after release of phosphate, and a distinct peak consistent with the position expected for phosphate, in the attacking position was observed. Data collection times with monochromatic crystallographic methods on a home source took 16 h for data to 2.3 A resolution. Sufficient phosphate was released from the caged phosphate in the crystal from 5 flashes with a xenon flashlamp within 1 min for the reaction to go to completion within the time scale of the monochromatic data collection procedures. The heptulose-2-P product complex has been refined and the model agrees with that obtained previously with the major difference that the interchange of an aspartic acid (Asp 283) by an arginine (Arg 569) was not observed at the catalytic site. This change is part of the activation process of glycogen phosphorylase and may not have taken place in the current experiments because the caged compound binds weakly at the inhibitor site, restricting conformational change, and because activators of the enzymic reaction were not present in the crystal. In experiments with monochromatic radiation in which low phosphate concentrations were generated either by fewer photons or by diffusion of known phosphate concentrations, mixtures of substrate and product were observed. It was not possible through crystallographic refinement at 2.3 A resolution to establish the fractional occupancies of the enzyme-substrate and enzyme-product complexes, but the results did indicate that the reaction was proceeding slowly, consistent with approximate calculations for the likely rate of the reaction in the crystal.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of commonly used cryoprotectants on glycogen phosphorylase activity and structure

The effects of a number of cryoprotectants on the kinetic and structural properties of glycogen phosphorylase b have been investigated. Kinetic studies showed that glycerol, one of the most commonly used cryoprotectants in X-ray crystallographic studies, is a competitive inhibitor with respect to substrate glucose-1-P with an apparent Ki value of 3.8% (v/v). Cryogenic experiments, with the enzyme, have shown that glycerol binds at the catalytic site and competes with glucose analogues that bind at the catalytic site, thus preventing the formation of complexes. This necessitated a change in the conditions for cryoprotection in crystallographic binding experiments with glycogen phosphorylase. It was found that 2-methyl-2,4-pentanediol (MPD), polyethylene glycols (PEGs) of various molecular weights, and dimethyl sulfoxide (DMSO) activated glycogen phosphorylase b to different extents, by stabilizing its most active conformation, while sucrose acted as a noncompetitive inhibitor and ethylene glycol as an uncompetitive inhibitor with respect to glucose-1-P. A parallel experimental investigation by X-ray crystallography showed that, at 100 K, both MPD and DMSO do not bind at the catalytic site, do not induce any significant conformational change on the enzyme molecule, and hence, are more suitable cryoprotectants than glycerol for binding studies with glycogen phosphorylase.     

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.     

Kinetic study on the dimer-tetramer interconversion of glycogen phosphorylase a.

Kinetic theory of dissociating enzyme systems has been applied to a study of the dimer-tetramer interconversion of glycogen phosphorylase a. All kinetic constants for the dissociating-associating reaction of phosphorylase a have been determined. The results indicate that (a) the presence of glucose-1-phosphate has no influence on either the rate of dissociation or the rate of association, and hence does not shift the dimer-tetramer equilibrium of phosphorylase a; (b) the binding og glycogen to the enzyme decreases the association rate of the dimer to form the tetramer, but has no effect on the dissociation rate of the tetramer; (c) both the dimeric and tetrameric form of phosphorylase a can bind glycogen, but the tetrameric form has a lower affinity for glycogen and is catalytically inactive.     

Inhibition of rabbit muscle glycogen phosphorylase by D-gluconohydroximo-1,5-lactone-N-phenylurethane

The effect of the beta-glycosidase inhibitor D-gluconohydroximo-1,5-lactone-N-phenylurethane (PUG) on the kinetic and ultracentrifugation properties of glycogen phosphorylase has been studied. Recent crystallographic work at 2.4 A resolution [D. Barford et al. (1988) Biochemistry 27, 6733-6741] has shown that PUG binds in the catalytic site of phosphorylase b crystals with its gluconohydroximolactone moiety occupying a position similar to that observed for other glucosyl compounds and the N-phenylurethane side chain fitting into an adjacent cavity with little conformational change in the enzyme. In solution, PUG was shown to be a potent inhibitor of phosphorylase b, directly competitive with alpha-D-glucopyranose 1-phosphate (glucose-1-P) (Ki = 0.40 mM) and noncompetitive with respect to glycogen and AMP. When PUG was tested for synergistic inhibition in the presence of caffeine, the Dixon plots of reciprocal velocity versus PUG concentration at different fixed caffeine concentrations provided intersecting lines with interaction constant (alpha) values of 0.95-1.38, indicating that the binding of one inhibitor is not significantly affected by the binding of the other. For glycogen phosphorolysis, PUG was noncompetitive with respect to phosphate, suggesting that it can bind to the central enzyme-AMP-glycogen-phosphate complex. PUG was shown to inhibit phosphorylase alpha (without AMP) activity (Ki = 0.43 mM) in a manner similar to that of the b form. However, in the presence of AMP, PUG exhibited complex kinetics, acting as a noncompetitive inhibitor with respect to glucose-1-P, while a twofold decrease of PUG binding to the enzyme-AMP-glycogen complex was observed. Ultracentrifugation experiments demonstrated that PUG does not cause any significant dissociation of phosphorylase alpha tetramer. Furthermore the dimerization of phosphorylase alpha by glucose is completely prevented in the presence of PUG. These observations are consistent with PUG binding to both the R and the T conformations of phosphorylase.     

Purification and characterization of a glycogen phosphorylase analog missing the amino-terminal segment

A glycogen phosphorylase analog missing only the amino-terminal 16 to 18 residues, which include the phosphorylation site, was produced by subtilisin Carlsberg cleavage of phosphorylase b in the presence of caffeine. The analog, named phosphorylase b's, was purified, and its enzymatic properties were compared with those of phosphorylase b. The KM's for glucose 1-phosphate are similar, but phosphorylase b's has a VM 43% higher than that of phosphorylase b. Also, phosphorylase b's is less sensitive to inhibition by glucose 6-phosphate and stimulation by sodium fluoride than is phosphorylase b. The subunit interactions in the two enzyme forms were also compared. The monomer-monomer interactions in phosphorylase b's are weaker than in phosphorylase b, as evidenced by a faster rate of resolution of the coenzyme, pyridoxal phosphate, from phosphorylase b's. The dimer-dimer interactions are also weaker in phosphorylase b's than in phosphorylase b, because phosphorylase b's does not form tetramers or crystals as readily as does phosphorylase b. Because removal of the amino-terminal segment changes the properties of the enzyme, this segment must be interacting with other parts of the protein. This statement conflicts with previous interpretation of X-ray crystallographic data that suggest that the amino-terminal region of phosphorylase b is freely mobile. Possible explanations for this contradiction are discussed.     

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.     

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.     

Inhibition by 2-Deoxyglucose and 1,5-Gluconolactone of Glycogen Mobilization in Astroglia-Rich Primary Cultures

Abstract: The presence of glycogen in astroglia-rich primary cultures derived from the brains of newborn rats depends on the availability of glucose in the culture medium. On glucose deprivation, glycogen vanishes from the astroglial cultures. This decrease of glycogen content is completely prevented if 2-deoxyglucose in a concentration of > 1 m M or 1,5-gluconolactone (20 m M ) is present in the culture medium. 2-Deoxyglucose itself or 3- O -methylglucose, a glucose derivative that is not phosphorylated by hexokinase, does not reduce the activity of glycogen phosphorylase purified from bovine brain or in the homogenate of astroglia-rich rat primary cultures. In contrast, deoxyglucose-6-phosphate strongly inhibits the glycogen phosphorylase activities of the preparations. Half-maximal effects were obtained at deoxyglucose-6-phosphate concentrations of 0.75 (phosphorylase a, astroglial culture), 5 (phosphorylase b, astroglial culture), 2 (phosphorylase a, bovine brain), or 9 m M (phosphorylase b, bovine brain). Thus, the block of glycogen degradation in these cells appears to be due to inhibition of glycogen phosphorylase by deoxyglucose-6-phosphate rather than deoxyglucose itself. These results suggest that glucose-6-phosphate, rather than glucose, acts as a physiological negative feedback regulator of the brain isoenzyme of phosphorylase and thus of glycogen degradation in astrocytes.     

Phosphorescent analysis of the intramolecular dynamics of the muscle glycogen phosphorylase b

Large-scale functionally significant changes in the intramolecular dynamics of muscle glycogen phosphorylase b (EC 2.4.1.1) in solution upon ligand binding, transition from dimeric to tetrameric form, temperature denaturation and aggregation were registered at room temperature using the tryptophan phosphorescence technique. It was shown that binding of glucose-1-phosphate (substrate, 0.25-4 mM) and glucose (competitive inhibitor, 0.5-8 mM) to the active site and temperature-induced protein aggregation decrease large-scale structural fluctuations of the protein matrix at the level of domains and subunits; whereas the transition of glycogen phosphorylase b to tetrameric form (R-conformation) leads to a dramatic increase in the structural flexibility of the peripheral parts of the protein globule.     

The interaction of ligands with chemically modified phosphorylase b.

Phosphorylase b which had been inactivated with 5-diazo1H-tetrazole was specifically labelled with 4-iodoacetamidosalicylic acid (a fluorescent probe) or with N-(1-oxyl-2,2,6,6,-tetramethyl-4-piperidinyl)iodoacetamide (a spin label probe) so that the binding of ligands and accompanying conformational changes could be determined by fluorescence or electron spin resonance changes, respectively. The allosteric effector, AMP, causes conformational changes similar to those caused in the native enzyme. The affinity of binding of phosphate or AMP to the inhibited protein is the same as for the unmodified protein. The heterotropic interactions between glucose-1-phosphate or glycogen and AMP are much less in the inactivated enzyme than in unmodified phosphorylase. Using a light scattering assay, it is shown that the modified enzyme binds to glycogen less strongly than the native protein. Phosphorylase b which had been inactivated by carbodimide in the presence of glycine ethyl ester, resulting in the modification of one or more carboxyl groups, was labelled with the spin label probe described above. The modified enzyme has an affinity for AMP similar to that of the native enzyme. AMP binding to the modified enzyme is tightened by glycogen, weakened by glucose-6-phosphate and is unaffected by glucose-1-phosphate. The actions of 5-diazo-1H-tetrazole and carbodimide on phosphorylase are discussed in the light of the above observation.     

A tentative mechanism of the ternary complex formation between phosphorylase kinase, glycogen phosphorylase b and glycogen

The kinetics of rabbit skeletal muscle phosphorylase kinase interaction with glycogen has been studied. At pH 6.8 the binding of phosphorylase kinase to glycogen proceeds only in the presence of Mg2+, whereas at pH 8.2 formation of the complex occurs even in the absence of Mg2+. On the other hand, the interaction of phosphorylase kinase with glycogen requires Ca2+ at both pH values. The initial rate of the complex formation is proportional to the enzyme and glycogen concentrations, suggesting the formation of the complex with stoichiometry 1:1 at the initial step of phosphorylase kinase binding by glycogen. According to the kinetic and sedimentation data, the substrate of the phosphorylase kinase reaction, glycogen phosphorylase b, favors the binding of phosphorylase kinase with glycogen. We suggest a model for the ordered binding of phosphorylase b and phosphorylase kinase to the glycogen particle that explains the increase in the tightness of phosphorylase kinase binding with glycogen in the presence of phosphorylase b.     

Enzymatic catalysis in crystals of Escherichia coli maltodextrin phosphorylase

The bacterial enzyme maltodextrin phosphorylase (MalP) catalyses the phosphorolysis of an alpha-1,4-glycosidic bond in maltodextrins, removing the non-reducing glucosyl residues of linear oligosaccharides as glucose-1-phosphate (Glc1P). In contrast to the well-studied muscle glycogen phosphorylase (GP), MalP exhibits no allosteric properties and has a higher affinity for linear oligosaccharides than GP. We have used MalP as a model system to study catalysis in the crystal in the direction of maltodextrin synthesis. The 2.0A crystal structure of the MalP/Glc1P binary complex shows that the Glc1P substrate adopts a conformation seen previously with both inactive and active forms of mammalian GP, with the phosphate group not in close contact with the 5'-phosphate group of the essential pyridoxal phosphate (PLP) cofactor. In the active MalP enzyme, the residue Arg569 stabilizes the negative-charged Glc1P, whereas in the inactive form of GP this key residue is held away from the catalytic site by loop 280s and an allosteric transition of the mammalian enzyme is required for activation. The comparison between MalP structures shows that His377, through a hydrogen bond with the 6-hydroxyl group of Glc1P substrate, triggers a conformational change of the 380s loop. This mobile region folds over the catalytic site and contributes to the specific recognition of the oligosaccharide and to the synergism between substrates in promoting the formation of the MalP ternary complex. The structures solved after the diffusion of oligosaccharides (either maltotetraose, G4 or maltopentaose, G5) into MalP/Glc1P crystals show the formation of phosphate and elongation of the oligosaccharide chain. These structures, refined at 1.8A and at 2.2A, confirm that only when an oligosaccharide is bound to the catalytic site will Glc1P bend its phosphate group down so it can contact the PLP 5' phosphate group and promote catalysis. The relatively large oligosaccharide substrates can diffuse quickly into the MalP/Glc1P crystals and the enzymatic reaction can occur without significant crystal damage. These structures obtained before and after catalysis have been used as frames of a molecular movie. This movie reveals the relative positions of substrates in the catalytic channel and shows a minimal movement of the protein, involving mainly Arg569, which tracks the substrate phosphate group.     

Refined crystal structure of the phosphorylase-heptulose 2-phosphate-oligosaccharide-AMP complex

The crystal structure of phosphorylase b-heptulose 2-phosphate complex with oligosaccharide and AMP bound has been refined by molecular dynamics and crystallographic least-squares with the program XPLOR. Shifts in atomic positions of up to 4 A from the native enzyme structure were correctly determined by the program without manual intervention. The final crystallographic R value for data between 8 and 2.86 A resolution is 0.201, and the overall root-mean-square difference between the native and complexed structure is 0.58 A for all protein atoms. The results confirm the previous observation that there is a direct hydrogen bond between the phosphate of heptulose 2-phosphate and the pyridoxal phosphate 5'-phosphate group. The close proximity of the two phosphates is stabilized by an arginine residue, Arg569, which shifts from a site buried in the protein to a position where it can make contact with the product phosphate. There is a mutual interchange in position between the arginine and an acidic group, Asp283. These movements represent the first stage of the allosteric response which converts the catalytic site from a low to a high-affinity binding site. Communication of these changes to other sites is prevented in the crystal by the lattice forces, which also form the subunit interface. The constellation of groups in the phosphorylase transition state analogue complex provides a structural basis for understanding the catalytic mechanism in which the cofactor pyridoxal phosphate 5'-phosphate group functions as a general acid to promote attack by the substrate phosphate on the glycosidic bond when the reaction proceeds in the direction of glycogen degradation. In the direction of glycogen synthesis, stereoelectronic effects contribute to the cleavage of the C-1-O-1 bond. In both reactions the substrate phosphate plays a key role in transition state stabilization. The details of the oligosaccharide, maltoheptaose, interactions with the enzyme at the glycogen storage site are also described.     

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.     

The interplay between covalent and non-covalent regulation of glycogen phosphorylase. The role of different effectors of phosphorylase b on the phosphorylase b to a conversion rate.

Glycogen phosphorylase b is converted to glycogen phosphorylase a, the covalently activated form of the enzyme, by phosphorylase kinase. Glc-6-P, which is an allosteric inhibitor of phosphorylase b, and glycogen, which is a substrate of this enzyme, are already known to have respectively an inhibiting and activating effect upon the rate of conversion from phosphorylase b to phosphorylase a by phosphorylase kinase. In the former case, this effect is due to the binding of glucose-6-phosphate to glycogen phosphorylase b. In order to investigate whether or not the rate of conversion of glycogen phosphorylase b to phosphorylase a depends on the conformational state of the b substrate, we have tested the action of the most specific effectors of glycogen phosphorylase b activity upon the rate of conversion from phosphorylase b to phosphorylase a at 0 degrees C and 22 degrees C : AMP and other strong activators, IMP and weak activators, Glc-6-P, glycogen. Glc-1-P and phosphate. AMP and strong activators have a very important inhibitory effect at low temperature, but not at room temperature, whereas the weak activators have always a very weak, if even existing, inhibitory effect at both temperatures. We confirmed the very strong inhibiting effect of Glc-6-P at both temperatures, and the strong activating effect of glycogen. We have shown that phosphate has a very strong inhibitory effect, whereas Glc-1-P has an activating effect only at room temperature and at non-physiological concentrations. The concomitant effects of substrates and nucleotides have also been studied. The observed effects of all these ligands may be either direct ones on phosphorylase kinase, or indirect ones, the ligand modifying the conformation of phosphorylase b and its interaction with phosphorylase kinase. Since we have no control experiments with a peptidic fragment of phosphorylase b, the interpretation of our results remains putative. However, the differential effects observed with different nucleotides are in agreement with the simple conformational scheme proposed earlier. Therefore, it is suggested that phosphorylase kinase recognizes differently the different conformations of glycogen phosphorylase b. In agreement with such an explanation, it is shown that the inhibiting effect of AMP is mediated by a slow isomerisation which has been previously ascribed to a quaternary conformational change of glycogen phosphorylase b. The results presented here (in particular, the important effect of glycogen and phosphate) are also discussed in correlation with the physiological role of the different ligands as regulatory signals in the in vivo situation where phosphorylase is inserted into the glycogen particle.