The σ-hole phenomenon of halogen atoms forms the structural basis of the strong inhibitory potency of C5 halogen substituted glucopyranosyl nucleosides towards glycogen phosphorylase b.

C5 halogen substituted glucopyranosyl nucleosides (1-(ß-D-glucopyranosyl)-5-X-uracil; X=Cl, Br, I) have been discovered as some of the most potent active site inhibitors of glycogen phosphorylase (GP), with respective K(i) values of 1.02, 3.27, and 1.94 µM. The ability of the halogen atom to form intermolecular electrostatic interactions through the σ-hole phenomenon rather than through steric effects alone forms the structural basis of their improved inhibitory potential relative to the unsubstituted 1-(ß-D-glucopyranosyl)uracil (K(i) =12.39 µM), as revealed by X-ray crystallography and modeling calculations exploiting quantum mechanics methods. Good agreement was obtained between kinetics results and relative binding affinities calculated by QM/MM-PBSA methodology for various substitutions at C5. Ex vivo experiments demonstrated that the most potent derivative (X=Cl) toward purified GP has no cytotoxicity and moderate inhibitory potency at the cellular level. In accordance, ADMET property predictions were performed, and suggest decreased polar surface areas as a potential means of improving activity in the cell.

Synthesis of variously coupled conjugates of D-glucose, 1,3,4-oxadiazole, and 1,2,3-triazole for inhibition of glycogen phosphorylase.

5-(O-Perbenzoylated-ß-D-glucopyranosyl)tetrazole was obtained from O-perbenzoylated-ß-D-glucopyranosyl cyanide by Bu(3)SnN(3) or Me(3)SiN(3)-Bu(2)SnO. This tetrazole was transformed into 5-ethynyl- as well as 5-chloromethyl-2-(O-perbenzoylated-ß-D-glucopyranosyl)-1,3,4-oxadiazoles by acylation with propiolic acid-DCC or chloroacetyl chloride, respectively. The chloromethyl oxadiazole gave the corresponding azidomethyl derivative on treatment with NaN(3). These compounds were reacted with several alkynes and azides under Cu(I) catalysed cycloaddition conditions to give, after removal of the protecting groups by the Zemplén protocol, ß-D-glucopyranosyl-1,3,4-oxadiazolyl-1,2,3-triazole, ß-D-glucopyranosyl-1,2,3-triazolyl-1,3,4-oxadiazole, and ß-D-glucopyranosyl-1,3,4-oxadiazolylmethyl-1,2,3-triazole type compounds. 5-Phenyltetrazole was also transformed under the above conditions into a series of aryl-1,3,4-oxadiazolyl-1,2,3-triazoles, aryl-1,2,3-triazolyl-1,3,4-oxadiazoles, and aryl-1,3,4-oxadiazolylmethyl-1,2,3-triazoles. The new compounds were assayed against rabbit muscle glycogen phosphorylase b and the best inhibitors had inhibition constants in the upper micromolar range (2-phenyl-5-[1-(ß-D-glucopyranosyl)-1,2,3-triazol-4-yl]-1,3,4-oxadiazole 36: K(i)=854µM, 2-(ß-D-glucopyranosyl)-5-[1-(naphthalen-2-yl)-1,2,3-triazol-4-yl]-1,3,4-oxadiazole 47: K(i)=745µM).

1-(3-Deoxy-3-fluoro-beta-d-glucopyranosyl) pyrimidine derivatives as inhibitors of glycogen phosphorylase b: Kinetic, crystallographic and modelling studies.

Design of inhibitors of glycogen phosphorylase (GP) with pharmaceutical applications in improving glycaemic control in type 2 diabetes is a promising therapeutic strategy. The catalytic site of muscle glycogen phosphorylase b (GPb) has been probed with five deoxy-fluro-glucose derivatives. These inhibitors had fluorine instead of hydroxyl at the 3' position of the glucose moiety and a variety of pyrimidine derivatives at the 1' position. The best of this carbohydrate-based family of five inhibitors displays a K(i) value of 46muM. To elucidate the mechanism of inhibition for these compounds, the crystal structures of GPb in complex with each ligand were determined and refined to high resolution. The structures demonstrated that the inhibitors bind preferentially at the catalytic site and promote the less active T state conformation of the enzyme by making several favorable contacts with residues of the 280s loop. Fluorine is engaged in hydrogen bond interactions but does not improve glucose potency. The pyrimidine groups are located between residues 284-286 of the 280s loop, Ala383 of the 380s loop, and His341 of the beta-pocket. These interactions appear important in stabilizing the inactive quaternary T state of the enzyme. As a follow up to recent computations performed on beta-d-glucose pyrimidine derivatives, tautomeric forms of ligands 1-5 were considered as potential binding states. Using Glide-XP docking and QM/MM calculations, the ligands 2 and 5 are predicted to bind in different tautomeric states in their respective GPb complexes. Also, using alpha-d-glucose as a benchmark model, a series of substitutions for glucose -OH at the 3' (equatorial) position were investigated for their potential to improve the binding affinity of glucose-based GPb catalytic site inhibitors. Glide-XP and quantum mechanics polarized ligand (QPLD-SP/XP) docking calculations revealed favorable binding at this position to be dominated by hydrogen bond contributions; none of the substitutions (including fluorine) out-performed the native -OH substituent which can act both as hydrogen bond donor and acceptor. The structural analyses of these compounds can be exploited towards the development of better inhibitors.

Endogenous effectors of human liver glycogen phosphorylase modulate effects of indole-site inhibitors.

Phosphorylase is regulated by a number of small-molecular-weight effectors that bind to three sites on the enzyme. Recently, a fourth site referred to as the indole-inhibitor site has been identified. Synthetic compounds bind to the site and inhibit activity. However, the effects of these compounds in the presence of other endogenous effectors are unknown. We have determined the effects of four indole derivative glycogen phosphorylase inhibitors (GPI) on recombinant human liver glycogen phosphorylase a activity. The GPIs tested were all potent inhibitors. However, the endogenous inhibitors (glucose, ADP, ATP, fructose 1-phosphate, glucose 6-phosphate, UDP-glucose) and the activator (AMP) markedly reduced the inhibitory effect of GPIs. Consistent with these in vitro findings, the IC50 for the inhibition of glycogenolysis in cells and the liver drug concentration associated with glucose-lowering activity in diabetic ob/ob mice in vivo were also significantly higher than those determined in in vitro enzyme assays. The inhibitory effect of indole-site effectors is modulated by endogenous small-molecular-weight effectors of phosphorylase a activity. However, at higher concentrations (10-30 microM), the GPI effect was dominant and resulted in inhibition of phosphorylase a activity irrespective of the presence or absence of the other modulators of the enzyme.

Characterization of a ligand-receptor binding event using receptor-dependent four-dimensional quantitative structure-activity relationship analysis.

Receptor-dependent four-dimensional quantitative structure-activity relationship (RD-4D-QSAR) analysis is used to map the ligand-receptor binding event characteristic of a set of 47 glucose analogue inhibitors of glycogen phosphorylase (GPb). Specifically, the geometric and energetic binding profiles are constructed, conformational changes are determined, and conformational couplings among structural units are identified for the composite set of ligand-receptor complexes. A pruned ligand-receptor model is used to estimate ligand-receptor thermodynamics. Rather than explicitly handling the large amount of structural data generated from each of the pruned ligand-receptor models, these complexes were divided into three subregions. The subregions consist of a "functional" region, the smallest region providing definitive information about inhibitor binding, and two "allosteric" regions that surround the "functional" region and are based on distances from the center of the catalytic site. Maximum information on inhibitor binding and/or inhibitor-receptor conformational changes is extracted from each of these subregions. The key sites for inhibitor binding and conformational changes in GPb are presented as grid cell occupancy descriptors (GCODs), which can be both numerically and graphically represented. An induced conformational change in both the inhibitor and the binding site of GPb occurs in a distinct manner for each complex. The inter-relationships (correlations) between GCODs from different regions are identified and probed. Such correlations validate the ligand-receptor interactions identified from the "functional" region. A long-range network of conformational associations involving ligands and the receptor is also found by exploring correlations among the GCODs for the set of inhibitors.

Fatty acid and amino acid modulation of glucose cycling in isolated rat hepatocytes.

We studied the influence of glucose/glucose 6-phosphate cycling on glycogen deposition from glucose in fasted-rat hepatocytes using S4048 and CP320626, specific inhibitors of glucose-6-phosphate translocase and glycogen phosphorylase respectively. The effect of amino acids and oleate was also examined. The following observations were made: (1) with glucose alone, net glycogen production was low. Inhibition of glucose-6-phosphate translocase increased intracellular glucose 6-phosphate (3-fold), glycogen accumulation (5-fold) without change in active (dephosphorylated) glycogen synthase (GSa) activity, and lactate production (4-fold). With both glucose 6-phosphate translocase and glycogen phosphorylase inhibited, glycogen deposition increased 8-fold and approached reported in vivo rates of glycogen deposition during the fasted-->fed transition. Addition of a physiological mixture of amino acids in the presence of glucose increased glycogen accumulation (4-fold) through activation of GS and inhibition of glucose-6-phosphatase flux. Addition of oleate with glucose present decreased glycolytic flux and increased the flux through glucose 6-phosphatase with no change in glycogen deposition. With glucose 6-phosphate translocase inhibited by S4048, oleate increased intracellular glucose 6-phosphate (3-fold) and net glycogen production (1.5-fold), without a major change in GSa activity. It is concluded that glucose cycling in hepatocytes prevents the net accumulation of glycogen from glucose. Amino acids activate GS and inhibit flux through glucose-6-phosphatase, while oleate inhibits glycolysis and stimulates glucose-6-phosphatase flux. Variation in glucose 6-phosphate does not always result in activity changes of GSa. Activation of glucose 6-phosphatase flux by fatty acids may contribute to the increased hepatic glucose production as seen in Type 2 diabetes.

Synthesis of and a comparative study on the inhibition of muscle and liver glycogen phosphorylases by epimeric pairs of d-gluco- and d-xylopyranosylidene-spiro-(thio)hydantoins and N-(d-glucopyranosyl) amides.

D-Gluco- and D-xylopyranosylidene-spiro-hydantoins and -thiohydantoins were prepared from the parent sugars in a six-step, highly chemo-, regio-, and stereoselective procedure. In the key step of the syntheses C-(1-bromo-1-deoxy-beta-D-glycopyranosyl)formamides were reacted with cyanate ion to give spiro-hydantoins with a retained configuration at the anomeric center as the major products. On the other hand, thiocyanate ions gave spiro-thiohydantoins with an inverted anomeric carbon as the only products. On the basis of radical inhibition studies, a mechanistic rationale was proposed to explain this unique stereoselectivity and the formation of C-(1-hydroxy-beta-D-glycopyranosyl)formamides as byproducts. Enzyme assays with a and b forms of muscle and liver glycogen phosphorylases showed spiro-hydantoin 12 and spiro-thiohydantoin 14 to be the best and equipotent inhibitors with K(i) values in the low micromolar range. The study of epimeric pairs of D-gluco and D-xylo configurated spiro-hydantoins and N-(D-glucopyranosyl)amides corroborated the role of specific hydrogen bridges in binding the inhibitors to the enzyme.

Hepatic glycogen synthesis is highly sensitive to phosphorylase activity: evidence from metabolic control analysis.

We used metabolic control analysis to determine the flux control coefficient of phosphorylase on glycogen synthesis in hepatocytes by titration with a specific phosphorylase inhibitor (CP-91149) or by expression of muscle phosphorylase using recombinant adenovirus. The muscle isoform was used because it is catalytically active in the b-state. CP-91149 inactivated phosphorylase with sequential activation of glycogen synthase. It increased glycogen synthesis by 7-fold at 5 mm glucose and by 2-fold at 20 mm glucose with a decrease in the concentration of glucose causing half-maximal rate (S(0.5)) from 26 to 19 mm. Muscle phosphorylase was expressed in hepatocytes mainly in the b-state. Low levels of phosphorylase expression inhibited glycogen synthesis by 50%, with little further inhibition at higher enzyme expression, and caused inactivation of glycogen synthase that was reversed by CP-91149. At endogenous activity, phosphorylase has a very high (greater than unity) negative control coefficient on glycogen synthesis, regardless of whether it is determined by enzyme inactivation or overexpression. This high control is attenuated by glucokinase overexpression, indicating dependence on other enzymes with high control. The high control coefficient of phosphorylase on glycogen synthesis affirms that phosphorylase is a strong candidate target for controlling hyperglycemia in type 2 diabetes in both the absorptive and postabsorptive states.

Dissociative mechanism of thermal denaturation of rabbit skeletal muscle glycogen phosphorylase b.

The thermal stability of rabbit skeletal muscle glycogen phosphorylase b was characterized using enzymological inactivation studies, differential scanning calorimetry, and analytical ultracentrifugation. The results suggest that denaturation proceeds by the dissociative mechanism, i.e., it includes the step of reversible dissociation of the active dimer into inactive monomers and the following step of irreversible denaturation of the monomer. It was shown that glucose 1-phosphate (substrate), glucose (competitive inhibitor), AMP (allosteric activator), FMN, and glucose 6-phosphate (allosteric inhibitors) had a protective effect. Calorimetric study demonstrates that the cofactor of glycogen phosphorylase-pyridoxal 5'-phosphate-stabilizes the enzyme molecule. Partial reactivation of glycogen phosphorylase b preheated at 53 degrees C occurs after cooling of the enzyme solution to 30 degrees C. The fact that the rate of reactivation decreases with dilution of the enzyme solution indicates association of inactive monomers into active dimers during renaturation. The allosteric inhibitor FMN enhances the rate of phosphorylase b reactivation.

A new allosteric site in glycogen phosphorylase b as a target for drug interactions.

BACKGROUND: In muscle and liver, glycogen concentrations are regulated by the coordinated activities of glycogen phosphorylase (GP) and glycogen synthase. GP exists in two forms: the dephosphorylated low-activity form GPb and the phosphorylated high-activity form GPa. In both forms, allosteric effectors can promote equilibrium between a less active T state and a more active R state. GP is a possible target for drugs that aim to prevent unwanted glycogen breakdown and to stimulate glycogen synthesis in non-insulin-dependent diabetes. As a result of a data bank search, 5-chloro-1H-indole-2-carboxylic acid (1-(4-fluorobenzyl)-2-(4-hydroxypiperidin-1-yl)-2-oxoethy l)amide, CP320626, was identified as a potent inhibitor of human liver GP. Structural studies have been carried out in order to establish the mechanism of this unusual inhibitor. RESULTS: The structure of the cocrystallised GPb-CP320626 complex has been determined to 2.3 A resolution. CP320626 binds at a site located at the subunit interface in the region of the central cavity of the dimeric structure. The site has not previously been observed to bind ligands and is some 15 A from the AMP allosteric site and 33 A from the catalytic site. The contacts between GPb and CP320626 comprise six hydrogen bonds and extensive van der Waals interactions that create a tight binding site in the T-state conformation of GPb. In the R-state conformation of GPa these interactions are significantly diminished. CONCLUSIONS: CP320626 inhibits GPb by binding at a new allosteric site. Although over 30 A from the catalytic site, the inhibitor exerts its effects by stabilising the T state at the expense of the R state and thereby shifting the allosteric equilibrium between the two states. The new allosteric binding site offers a further recognition site in the search for improved GP inhibitors.

Inhibitors of glycogen phosphorylase b: synthesis, biochemical screening, and molecular modeling studies of novel analogues of hydantocidin.

The synthesis and biochemical screening of four novel spironucleosides 1-4 against rabbit liver glycogen phosphorylase b (Gpb), along with molecular modeling studies on compound 2 and its 4-hydroxy analogue VII, have been presented. Gpb is a key enzyme of glycogen metabolism, and is known to be involved in the control of diabetes mellitus. The general strategy for synthesis involved base-catalyzed condensation of diethyl 2,4-dioxoimidazolidine-5-phosphonate (5) with either 2-deoxy-D-ribose or D-ribose, followed by sequential reactions involving ring-closure with phenylselenenyl chloride and reduction with tri-n-butyltin hydride catalyzed by azobisisobutyronitrile. Compounds 2 and 4 were found to be weak competitive inhibitors of Gpb, whereas 1 and 3 were inactive.

Alteration of the intramolecular dynamics of glycogen phosphorylase b by allosteric ligands.

Phosphorylase b (E.C. 2.4.1.1), prepared from rabbit skeletal muscle, was used to study whether the binding of allosteric ligands modifies the intramolecular dynamics of the protein matrix. Protein dynamics were monitored through the fluorescence and phosphorescence parameters of the 12 tryptophan (Trp) residues (one monomer) of the enzyme. The phosphorescence lifetime was measured at room temperature both in the absence and the presence of ligands. The addition of an allosteric inhibitor (ATP) decreased the lifetime, while the presence of activator (AMP) and/or substrate (G-1-P) had no detectable effect. The lifetime data allow us to conclude that the environment of the buried tryptophans becomes more flexible upon the binding of ATP, while the other ligands did not induce such change. The ATP-induced perturbation was also examined by the quenching of Trp fluorescence by acrylamide. The quenching parameters did not show any change, suggesting that the effect of ATP is localized to the vicinity of the phosphorescent Trp residues.

[Structure, regulation, and denaturation of muscle glycogen phosphorylase b]. / Struktura, reguliatsiia i denaturatsiia myshechnoi glikogenfosforilazy b.

The review summarizes data on structure, allosteric regulation, and denaturation of muscle glycogen phosphorylase b. Specific attention is paid to correlations between the structure and function of phosphorylase b and molecular mechanism of its allosteric regulation. Chemical and thermal denaturation of phosphorylase b is reviewed.

[Effect of specific ligands on heat inactivation of muscle glycogen phosphorylase b]. / Vliianie spetsificheskikh ligandov na teplovuiu inaktivatsiiu myshechnoi glikogenfosforilazy b.

It has been shown that the rate constant, k, for thermal inactivation of rabbit skeletal muscle glycogen phosphorylase b decreases as the enzyme concentration increases. This effect is interpreted within the framework of a kinetic model which includes two parallelly occurring processes, namely: phosphorylase b denaturation in solution and denaturation of the enzyme absorbed on test-tube walls. The contribution of the latter process increases with a decrease in the enzyme concentration. The protective effect of the allosteric activator (AMP), allosteric inhibitors (glucose 6-phosphate and FMN) and the competitive inhibitor (glucose) against heat denaturation of glycogen phosphorylase b has been demonstrated. Quantitative analysis of the dependence of the rate constant, k, on concentration of AMP, glucose 6-phosphate and FMN allows the calculation of microscopic constants for dissociation of phosphorylase b complexes with these ligands for the given experimental conditions as equal to 0.34, 0.50 and 0.30 mM, respectively. The S-shaped dependence of the rate constant of thermal inactivation on glucose concentration points to the existence of positive cooperative interactions between glucose-binding sites in the dimeric molecule of phosphorylase b (nH = 1.8).

[Reconstruction of muscle glycogen phosphorylase b from an apoenzyme and pyridoxal-5'-phosphate and its analogs. Interaction of apophosphorylase and the reconstructed enzyme with specific ligands]. / Rekonstruktsiia myshechnoi glikogenfosforilazy b iz apofermenta i piridoksal'-5'-fosfata i ego analogov. Vzaimodeistvie apofosforilazy i rekonstruirovannogo fermenta so spetsificheskimi ligandami.

Sedimentation methods were used to study the effects of modification of the pyridoxal-5'-phosphate (PLP) molecule at the 5th position on the affinity of reconstituted muscle glycogen phosphorylase b for the substrate (glycogen) and the allosteric inhibitor (FMN) as well as on the enzyme capacity to association induced by AMP. Reconstituted phosphorylase b was obtained with PLP analogs containing at the 5th position -CH2-CH2-COOH (analog I), trans-CH=CH-COOH (analog II) or -C identical to COOH (analog III) residues. Reconstitution of phosphorylase b is accompanied by the recovery of the enzyme quaternary structure. Phosphorylase b reconstituted with PLP or analogs I, II and III is not distinguished practically from the native enzyme in its affinity for glycogen. Substitution of the native coenzyme in the phosphorylase molecule with any tested PLP analog leads to lower enzyme affinity for FMN. Microscopic dissociation constants of the FMN-enzyme complexes increase in the following order: enzyme.I < enzyme.II < enzyme.III. Phosphorylase b reconstituted with analogs I, II and III differs substantially from the native enzyme in its capacity to association in the presence of 1 mM AMP: the reconstituted enzyme is represented practically by only the tetrameric form.

Caffeine inhibition of glycogen phosphorylase from Mytilus galloprovincialis mantle tissue.

A different caffeine inhibition of both phosphorylated and unphosphorylated forms of glycogen phosphorylase from Mytilus mantle has been demonstrated. Caffeine increases the allosteric constant of phosphorylase b 30-fold, acting as an allosteric inhibitor (nH = 2) of mixed type with respect to inorganic phosphate (Pi) and AMP, and of single competitive type with respect to glycogen. The Mytilus phosphorylated form is also caffeine inhibited through competitive inhibition in relation to Pi and glycogen. In this case, the inhibitor does not modify the allosteric constant (near 2), neither does it display allosteric effects (nH = 1). The results demonstrate the notable modification of the nucleotide site promoted by the phosphorylation process and the existence of a functional inhibitory nucleoside site in Mytilus phosphorylase.

Demonstration of a glycogen/glucose 1-phosphate cycle in hepatocytes from fasted rats. Selective inactivation of phosphorylase by 2-deoxy-2-fluoro-alpha-D-glucopyranosyl fluoride.

In search for a nonmetabolized, superior glucose analogue to study the mechanism of glucose-induced glycogen synthesis, we have tested 2-deoxy-2-fluoro-alpha-D-glucopyranosyl fluoride, which inhibits muscle phosphorylase beta 10-fold better than dose glucose (Street, I.P., Armstrong, C.R., and Withers, S.G. (1986) Biochemistry 25, 6021-6027). In a gel-filtered liver extract, 0.6 mM analogue and 10 mM glucose equally accelerated the inactivation of phosphorylase and shortened the latency before the activation of glycogen synthase. The analogue was not measurably defluorinated or phosphorylated by intact hepatocytes, as monitored by 19F NMR. When added to isolated hepatocytes, 10 mM analogue inactivated phosphorylase more extensively than did 50 mM glucose, but unlike glucose, it did not result in the activation of glycogen synthase. Therefore, the binding of glucose to phosphorylase alpha can account for the inactivation of phosphorylase, but the metabolism of glucose (probably to Glc-6-P) appears to be required to achieve activation of glycogen synthase. The livers of overnight-fasted, anesthetized mice contained appreciable amounts of both phosphorylase alpha and glycogen synthase alpha, without net glycogen accumulation. Likewise, hepatocytes isolated from fasted rats and incubated with 10 mM glucose contained 41% of phosphorylase and 32% of glycogen synthase in the alpha form, and these values remained stable for 1 h, while glycogen accumulated at only 22% of the rate expected from the glycogen synthase activity. The addition of 10 mM analogue decreased phosphorylase alpha to 10% without significant change in glycogen synthase alpha (38%), but with a 4-fold increased rate of glycogen accumulation. These findings imply that synthase alpha is fully active in the liver of the fasted animal and that the absence of net glycogen synthesis is due to continuous glycogenolysis by phosphorylase alpha.

Interaction between glycogen phosphorylase and sarcoplasmic reticulum membranes and its functional implications.

Skeletal muscle glycogen phosphorylase b binds to sarcoplasmic reticulum (SR) membranes with a dissociation constant of 1.7 +/- 0.6 mg of phosphorylase/ml at 25 degrees C at physiological pH and ionic strength. Raising the temperature to 37 degrees C produced a 2-3-fold decrease in the dissociation constant. The SR membranes could bind up to 1.1 +/- 0.1 mg of glycogen phosphorylase b/mg of SR protein, whereas liposomes prepared with endogenous SR lipids and reconstituted Ca(2+)-ATPase were unable to bind glycogen phosphorylase. Binding of glycogen phosphorylase b to SR membranes is accompanied by inhibition of its activity in the presence of AMP. The Vmax for glycogen phosphorylase b associated with SR membranes is 40 +/- 5% of that for purified glycogen phosphorylase and shows a decreased affinity for its allosteric activators, AMP and IMP. These kinetic effects are also observed with purified glycogen phosphorylase b when starch or alpha-amylose is used as substrate instead of glycogen. Treatment of SR membranes with alpha-amylase produced dissociation of glycogen phosphorylase b from the SR membranes. Thus, linear polysaccharide fragments of glycogen bound to the SR membranes are likely mediating the binding of glycogen phosphorylase b to these membranes.

[The effect of specific deimination of glycogen phosphorylase b by peptidylarginine deiminase on the allosteric properties of the enzyme and dimer-tetramer transition]. / Vliianie spetsificheskogo deziminirovanniia glikogenfosforilazy b peptidilarginindeziminazoi na allostericheskie svoistva fermenta i dimer-tetramernye perekhody.

The kinetics of the native glycogen phosphorylase b from rabbit skeletal muscle and of the enzyme specifically deiminated by peptidylarginine deiminase have been studied. According to the data on amino acid composition one arginine residue per phosphorylase b monomer is transformed into citrulline after 3 hours of incubation with peptidylarginine deiminase. The kinetics of the phosphorylase reaction were studied in the direction of glycogen synthesis. The native and the deiminated forms of phosphorylase b showed similar affinity to glucose 1-phosphate. The maximal velocity of the enzymatic reaction for the modified phosphorylase b is 8-20% higher than that for the native enzyme. Deiminated phosphorylase b like the native enzyme shows a positive kinetic cooperatively with respect to glucose 1-phosphate in the presence of the allosteric inhibitors (FMN, glucose), S-shaped dependences of the velocity of the enzymatic reaction on glucose 1-phosphate concentration (in the presence of FMN) pronouncing more distinctly for deiminated phosphorylase b than for the native enzyme (Hill coefficient is equal to 1.7 +/- 0.2 and 1.3 +/- 0.1, respectively). The affinity of the modified phosphorylase b to the allosteric activator AMP is one order of magnitude higher than that to the native enzyme. The cooperativity of AMP binding doesn't change significantly after deimination. The kinetics of inhibition of the native and modified phosphorylase b by FMN, glucose and glucose 6-phosphate are cooperative (the value of Hill coefficient is higher than unity). The more pronounced distinctions between two forms of the enzyme concern with the value of the "semisaturation" concentration [I]0.5. The deimination causes a pronounced reduction of the values of [I]0.5 for FMN and glucose, but the sensitivity of the deiminated enzyme to glucose 6-phosphate is much lower than that of the native phosphorylase b. Deiminated phosphorylase b unlike the native enzyme shows the positive cooperativity of the FMN binding (the value of the Hill coefficient is equal to 1.37 +/- 0.05). Deiminated phosphorylase b shows less capability to form tetramer in the presence of AMP as compared to the native enzyme.