Direction Of Glycogen Synthesis Research Articles

Discovering benzamide derivatives as glycogen phosphorylase inhibitors and their binding site at the enzyme

A series of novel benzamide derivatives was designed, synthesized, and their inhibitory activities against glycogen phosphorylase (GP) in the direction of glycogen synthesis by the release of phosphate from glucose-1-phosphate were evaluated. The structure-activity relationships (SAR) of these compounds are also presented. Within this series of compounds, 4m is the most potent GPa inhibitor (IC 50 = 2.68 μM), which is nearly 100 times more potent than the initial compound 1. Analysis of mapping between pharmacophores of different binding sites and each compound demonstrated that these benzamide derivatives bind at the dimer interface of the rabbit muscle enzyme, and possible docking modes of compound 4m were explored by molecular docking simulation.

Crystallographic studies on N-azidoacetyl-β- d-glucopyranosylamine, an inhibitor of glycogen phosphorylase: Comparison with N-acetyl-β- d-glucopyranosylamine

N-Acetyl-β- d-glucopyranosylamine (NAG) is a potent inhibitor ( K i = 32 μM) of glycogen phosphorylase b (GPb), and has been employed as a lead compound for the structure-based design of new analogues, in an effort to utilize its potential as a hypoglycaemic agent. Replacement of the acetamido group by azidoacetamido group resulted in an inhibitor, N-azidoacetyl-β- d-glucopyranosylamine (azido-NAG), with a K i value of 48.7 μM, in the direction of glycogen synthesis. In order to elucidate the mechanism of inhibition, we determined the ligand structure in complex with GPb at 2.03 Å resolution, and the structure of the fully acetylated derivative in the free form. The molecular packing of the latter is stabilized by a number of bifurcated hydrogen bonds of which the one involving a bifurcated C–H⋯N⋯H–C type hydrogen bonding is rather unique in organic azides. Azido-NAG can be accommodated in the catalytic site of T-state GPb at approximately the same position as that of NAG and stabilizes the T-state conformation of the 280s loop by making several favourable contacts to residues of this loop. The difference observed in the K i values of the two analogues can be interpreted in terms of desolvation effects, subtle structural changes of protein residues and changes in water structure.

Kinetic mechanism of the glycogen-phosphorylase-catalysed reaction in the direction of glycogen synthesis: co-operative interactions of AMP and glucose 1-phosphate during catalysis.

We employed our newly developed, continuous, spectrophotometric method [Sergienko and Srivastava (1994) Anal. Biochem. 221, 348-355] for measuring the glycogen-phosphorylase-catalysed reaction in the direction of glycogen synthesis, utilizing varied concentrations of AMP (2-400 microM) and glucose 1-phosphate (G1P; 4 microM to 41 mM). The experimental data revealed that the enzyme catalysis exhibits sigmoidal dependence on both AMP and G1P concentrations, with Hill coefficient and EC50 values (mutually) affected by the concentrations of the above substrates. A detailed kinetic analysis of the substrate-dependent activation, as well as glucose-inhibition data, lead us to propose the following mechanistic features of the glycogen-phosphorylase-catalysed reaction. (1) The enzyme exhibits catalytic activity when two molecules of AMP and two molecules of G1P are bound to the dimeric unit. (2) The binding of one molecule of glucose (the competitive inhibitor of G1P) per dimeric unit results into a complete loss of the enzyme activity. (3) There is no restriction of binding of AMP or G1P when one of the dimeric subunits is already bound with the other ligand. For example, one or two G1P molecules can bind to the enzyme dimer when zero, one or two molecules of AMP are already bound. The magnitudes of rate and equilibrium constants for the glycogen-phosphorylase-catalysed reaction, derived from analyses of the experimental data in the light of a few selected minimal models, are presented.

Sulphate-activated phosphorylase b: the pH-dependence of catalytic activity

The pH-dependence of sulphate-activated phosphorylase b has been studied in the direction of glycogen synthesis. The bell-shaped curve of the pH-dependence of the catalytic constant for the AMP-activated enzyme showed pK values of 6.1 and 7.3, but the curve for the enzyme activated by 0.9 M ammonium sulphate showed a drop of activity on the acid side at much higher pH values. Its bell was centred at pH 7.8 but it was too narrow to be characterized by only two pK values. The narrowness of the curve could be explained by positive co-operativity, but not its unusually steep acid side. We suggest that the fall on the acid side is due to more than one hydronation (addition of H+). The points can be fitted by a curve with two de-activating hydronations and a de-activating dehydronation having identical titration pK values of 7.5, and hence molecular values of 7.0, 7.5 and 8.0. If both 0.9 M ammonium sulphate and 5 mM AMP are added, the bell is as broad as with AMP alone, but is somewhat raised in pH optimum. The results are discussed in the light of new structural data from crystallographic studies on binary complexes of the enzyme.

Photometric Microtiter Assay of Inorganic Phosphate in the Presence of Acid-Labile Organic Phosphates

A simple and rapid colorimetric microassay for inorganic phosphate in the mild acid pH range has enabled us to perform extensive enzyme kinetic studies even in the presence of high concentrations of acid-labile substrates. The assay is performed in a 96-well microtiter plate using 30-μl samples containing between 0.5 and 100 nmol Pi. Compared to existing microassays, color formation is linear over a much wider range of phosphate concentrations and reaction conditions. Using a computer-assisted microtiter plate reader, the data can be directly transferred to kinetic analysis software. The above assay was used to determine simultaneously up to 12 rates of the Escherichia coli maltodextrin phosphorylase-catalyzed reaction in the direction of glycogen synthesis under conditions which include screening of substrates and inhibitors at variable substrate concentrations and pH on a single 96-well microtiter plate.

A Continuous Spectrophotometric Method for the Determination of Glycogen Phosphorylase-Catalyzed Reaction in the Direction of Glycogen Synthesis

We offer a "continuous" spectrophotometric method for the determination of the glycogen phosphorylase-catalyzed reaction in the direction of glycogen synthesis. This method relies on a coupled enzyme procedure, involving purine nucleoside phosphorylase and its chromophoric substrate, 2-amino-6-mercapto-7-methyl ribonucleoside (7-methyl-6-thioguanosine (MTGuo)), for the estimation of inorganic phosphate (M. R. Webb, Proc. Natl. Acad. Sci. USA 89, 4884-4887, 1992). We have examined the effects of the reaction components on the catalytic activities of both "primary" and "coupling" enzymes. While MTGuo exhibits no effect on the glycogen phosphorylase-catalyzed reaction, glucose 1-phosphate and AMP are partially inhibitory to nucleoside phosphorylase. However, the latter effects pose no problem as long as the coupling enzyme is maintained at a relatively higher concentration in the assay system. The coupled enzyme assay system, standardized for the measurement of glycogen phosphorlase activity, has enabled us to demonstrate explicitly that the rate of the enzyme-catalyzed reaction exhibits sigmoidal dependence on both AMP and glucose 1-phosphate concentrations. We argue that these sigmoidal profiles have been observed due to the sensitivity and precision of the present assay system.

Kinetic Mechanism of Activation of Muscle Glycogen Phosphorylase b by Adenosine 5′-Monophosphate

The kinetic analysis of the enzymatic reaction catalyzed by glycogen phosphorylase b from the rabbit skeletal muscle has been carried out in the direction of glycogen synthesis under the conditions of the saturation of the enzyme by glycogen. The applicability of 12 different variants of the Monod-Wyman-Changeux model and 6 kinetic schemes involving the interaction of AMP- and glucose-1-phosphate-binding sites in the dimeric enzyme molecule is considered. The kinetic model has been proposed, which assumes (i) the independent binding of AMP and one molecule of glucose 1-phosphate with the enzyme saturated by glycogen, (ii) the exclusive binding of the second molecule of glucose 1-phosphate by the enzyme containing two molecules of AMP and one molecule of glucose 1-phosphate, and (iii) the exclusive ability of the complex of the enzyme with glycogen, two molecules of AMP, and two molecules of glucose 1-phosphate to undergo the catalytic transformation. The parameters of the equation of the initial steady-state rate of the enzymatic reaction are calculated by the nonlinear regression method. The proposed kinetic scheme is shown to satisfy to the following criteria: (i) the convergence under the regressional analysis, (ii) the reliability of the values of the parameters of the model, (iii) the minimality of the sum of the weighted squares of the differences between the experimental and calculated values of the reaction rate.

A kinetic study of glycogen phosphorylase b from the mantle tissue of Mytilus galloprovincialis, Lmk

1. 1. In order to assign a meaningful role to the phosphorolytic pathway in Mytilus glycogen metabolism the kinetic mechanism of phosphorylase b, and its allosteric control, were studied. 2. 2. The kinetic parameters of phosphorylase b from the mussel Mytilus galloprovincialis were determined. Michaelis constants ( K m or S 0.5) were in the range of 0.32–2.49 mg/ml for glycogen, 7–16 mM for P i and 114–423 μM for AMP. In the direction of glycogen synthesis, the K m value for glucose-1-P was approximately 180 mM. 3. 3. The enzyme displayed homotropic co-operativity towards the binding of co-substrate and AMP (Hill coefficients of 2 and 1.4, respectively) and heterotropic co-operativity between substrates and AMP. 4. 4. The concentration of glycogen in the Mytilus mantle is between 38- and 125-fold higher than the apparent K m of phosphorylase b; the concentration of AMP varies throughout the year from 10 to 175 μM, up to a value close to the apparent K m for the effector. 5. 5. The apparent K m for P i is close to the concentration found in the mantle. This ligand showed more important regulatory effects than the effector AMP.

The binding of d-gluconohydroximo-1,5-lactone to glycogen phosphorylase. Kinetic, ultracentrifugation and crystallographic studies

Combined kinetic, ultracentrifugation and X-ray-crystallographic studies have characterized the effect of the beta-glucosidase inhibitor gluconohydroximo-1,5-lactone on the catalytic and structural properties of glycogen phosphorylase. In the direction of glycogen synthesis, gluconohydroximo-1,5-lactone was found to competitively inhibit both the b (Ki 0.92 mM) and the alpha form of the enzyme (Ki 0.76 mM) with respect to glucose 1-phosphate in synergism with caffeine. In the direction of glycogen breakdown, gluconohydroximo-1,5-lactone was found to inhibit phosphorylase b in a non-competitive mode with respect to phosphate, and no synergism with caffeine could be demonstrated. Ultracentrifugation and crystallization experiments demonstrated that gluconohydroximo-1,5-lactone was able to induce dissociation of tetrameric phosphorylase alpha and stabilization of the dimeric T-state conformation. A crystallographic binding study with 100 mM-gluconohydroximo-1,5-lactone at 0.24 nm (2.4 A) resolution showed a major peak at the catalytic site, and no significant conformational changes were observed. Analysis of the electron-density map indicated that the ligand adopts a chair conformation. The results are discussed with reference to the ability of the catalytic site of the enzyme to distinguish between two or more conformations of the glucopyranose ring.

The enzymic synthesis and N.M.R. characterisation of specifically deoxygenated and fluorinated glycogens

The incorporation of several deoxy- and deoxyfluoro- d-glucose analogues into glycogen has been achieved through the action of rabbit muscle glycogen phosphorylase on a number of deoxy- and deoxyfluoro- analogues of α- d-glucopyranosyl phosphate. Time courses for the incorporation of these analogues into glycogen and maltopentaose have been determined, and the introduction of 4-deoxy- or 4-deoxy-4-fluoro- d-glucose units has been demonstrated to terminate after the introduction of one sugar unit per non-reducing terminus. Glycogen analogues containing sugars modified at the 3- and 4-positions have been isolated and characterised by 1H-n.m.r. and 19F-n.m.r. spectroscopy, and the extent of incorporation has been confirmed by integration of the new resonances associated with the incorporated residues. Longitudinal (T 1) relaxation times have been determined for the two 19F-n.m.r. resonances observed for 3-deoxy-3-fluoro-glycogen, and through comparison with the T 1 measured for 4-deoxy-4-fluoro-glycogen, the identity (terminal or internal) of each of these two resonances was determined. Kinetic studies indicate that neither 4-deoxy- nor 4-deoxy-4-fluoro-glycogen can serve as a substrate for glycogen phosphorylase in the direction of glycogen synthesis, proving that these glycogen analogues have been fully substituted. Both of these 4-substituted glycogens are good inhibitors of glycogen phosphorylase.

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 Å 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 Å resolution is 0.201, and the overall root-mean-square difference between the native and complexed structure is 0.58 Å 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.

In vitro regulation of glycogen phosphorylase of the larval fat body of Tenebrio molitor

1. 1. Glycogen phosphorylase activity in last larval instar Tenebrio fat body was assayed in the direction of glycogen synthesis in the presence or absence of 5′-AMP. The active form of the enzyme was found to represent 45% of total phosphorylase. 2. 2. Cold exposure of the in vitro fat body strongly activates phosphorylase: after 2 hr of incubation at 4°C, the active form is found to represent 75% of total phosphorylase, ie. 65% increase. When intestinal insulin-like peptide (I.L.P.) is applied after cold activation, this induces a decrease of phosphorylase activity, while I.L.P. fails to modify phosphorylase activity at 25°C. 3. 3. A dose-dependent activation of phosphorylase is induced in vitro by either corpora cardiaca (CC) extracts, or synthetic locust AKH I. The increase of activity of phosphorylase reaches 50% with one pair of CC, and 59% with 10 pmol AKH I. 4. 4. We conclude that larval Tenebrio fat body glycogen phosphorylase may be under hormonal control in vitro.

Purification and characterization of glycogen phosphorylase from Drosophila melanogaster

Glycogen phosphorylase b was purified from Drosophila melanogaster with a yield of 15% by low speed centrifugation, DEAE-Sepharose CL-6B chromatography and affinity chromatography on 5′-AMP-Sepharose 4B. The preparation proved to be homogeneous by SDS-polyacrylamide gel electrophoresis and showed a subunit molecular weight of 95,000. Gel filtration of the native enzyme on Sephacryl S-300 revealed a molecular weight of 176,000 suggesting that phosphorylase b is a dimer composed of two identical subunits. The fruit fly phosphorylase b has a maximal specific activity of 36 U/mg when assayed in the direction of glycogen synthesis. It requires AMP for activity ( A 0.5 = 0.4 mM, Hill coefficient: 1.8). The K M for glycogen is 0.4% and the S 0.5 for glucose-1-phosphate is 20 mM (Hill coefficient: 1.3). The enzyme is inhibited by glucose, UDPG, caffeine, glucose-6-phosphate, ATP and IMP.

Pyridoxal phosphate is not the acid catalyst in the glycogen phosphorylase catalytic mechanism

Pyridoxal-reconstituted phosphorylase has been shown previously (Parrish, R.F., Uhing, R.J. and Graves, D.J. (1977), Biochemistry 16 , 4824) to be activated to a very similar extent by phosphite (pK a=6.6) and fluorophosphate (pK a=4.8). This paper shows that the pH dependence of the enzyme activity is also similar for these two activators. This is interpreted to mean that the coenzyme phosphate is not involved as an essential acid catalyst in the actual reaction mechanism and is probably not involved in any form of proton transfer process. The pH/activity profile for native phosphorylase b in the direction of glycogen synthesis is also presented and a comparison of the two sets of profiles is discussed.

The catalytic activity of phosphorylase b in the liver. With a note on the assay in the glycogenolytic direction.

1. The activity and the kinetic properties of purified hepatic phosphorylases a and b from rabbit and rat have been investigated in the glycogenolytic direction with a radiochemical assay. 2. In contrast with the a form, phosphorylase b has an absolute requirement for both AMP and a lyotropic salt. When the latter effectors are included, the b/a-form activity ratio remains low (0.03-0.15) at the hepatic concentration of Pi, because the b form has an exceedingly low affinity for this substrate. 3. Only phosphorylase b is significantly inhibited by glucose, glucose 6-phosphate and MgATP2-. Assays in the presence of substrastes, stimulators and inhibitors in the physiological concentration range indicate that glycogenolysis in the liver depends strictly on the conversion of phosphorylase b into a. Even at 1 mM-AMP the b/a-form activity ratio does not exceed 0.01. 4. Current spectrophotometric procedures for the glycogenolytic assay of phosphorylase in crude liver preparations are highly specific for the a form; the measurement of total phosphorylase (a + b) would require impractical modifications, and is better performed in the direction of glycogen synthesis.

A method for continuous monitoring of phosphorylase b activity during glycogen degradation and synthesis

Phosphorylase b (1,4-α-glucaniorthophosphate glucosyltransferase, EC. 2.4.1.1) catalyzes the glycogen synthesis and degradation reaction. It is the latter which is the physiological function of the enzyme. Its action may be described by a random order bi-bi kinetic mechanism. The rate-limiting step is an interconversion between ternary complexes (1). Activity determination can be carried out in the direction of glycogen synthesis by measuring the amount of inorganic phosphate (P j) released during the reaction (2). This method requires termination of the enzyme action after an appropriate time interval and only yields information about the total amount of the product without giving details of the time course of the process. In the reverse direction, the glycogen degradation can be followed in a more complicated and indirect way by coupled reactions of additional enzymes (3). The present communication describes a convenient method for continuous monitoring of the reaction in both directions with a fluorescence spectrometer.

Kinetic properties of α-glucan phosphorylase from Indocibium guttattam

The kinetic properties of α-glucan phosphorylase b (α- d-1,4-glucan : orthophosphate α- d-glucosyltransferase, EC 2.4.1.1) purified from a deep-sea fish Indocibium guttattam were studied in the direction of glycogen synthesis. There was no homotropic site-site interaction between glucose 1-phosphate sites and between glycogen sites. However, heterotropic interaction was observed between substrate and activator sites. The kinetic data obtained for the fish enzyme were consistent with the rapid equilibrium random mechanism reported for α-glucan phosphorylase from other sources. All the dissociation constants were 2–3 times higher for the fish enzyme than for the rabbit enzyme. Although the fish enzyme exhibited a greater affinity for AMP at 30°C as Compared to the rabbit enzyme, these sites were characterized by a lack of homotropic cooperativity. Heterotropic cooperativity was observed between AMP and glucose 1-phosphate sites.

Corticosteroid-induced changes in glucose metabolism of chondrocytes.

Immature mice were treated for up to 12 weeks with daily doses of triamcinolone diacetate. Using quantitative histochemical methods, the proximal epiphyseal plate of the humerus was studied at regular intervals. By the tenth injection significant decrease was noted in the acid glycosaminoglycans content (25%) and in the neutral mucopolysaccharides (30%). Concomitantly, a marked increase was noted in the intracellular depots of glycogen (70%). It is suggested that the hormone's antiglycolytic effect in cartilage caused the glucose metabolic pathway to divert in the direction of glycogen synthesis, and thereby interfered with the normal synthetic pathway of the chondrocytes. The hormone's primary effect on the cells' hexose metabolism could be responsible, at least in part, for its inhibitory influence on bone growth.

Human liver glycogen phosphorylase. Kinetic properties and assay in biopsy specimens.

1. The two forms of glycogen phosphorylase were purified from human liver, and some kinetic properties were examined in the direction of glycogen synthesis. The b form has a limited catalytic capacity, resembling that of the rabbit liver enzyme. It is characterized by a low affinity for glucose 1-phosphate, which is unaffected by AMP, and a low V, which becomes equal to that of the a form in the presence of the nucleotide. Lyotropic anions stimulate phosphorylase b and inhibit phosphorylase a by modifying the affinity for glucose 1-phosphate. Both enzyme forms are easily saturated with glycogen. 2. These kinetic properties have allowed us to design a simple assay method for total (a + b) phosphorylase in human liver. It requires only 0.5 mg of tissue, and its average efficiency is 90% when the enzyme is predominantly in the b form. 3. The assay of total phosphorylase allows the unequivocal diagnosis of hepatic glycogen-storage disease caused by phosphorylase deficiency. One patient with a complete deficiency is reported. 4. The assay of human liver phosphorylase a is based on the preferential inhibition of the b form by caffeine. The a form displays the same activity when measured by either of the two assays.

Kinetics of glycogen phosphorylase a from the muscle of the blue crab, Callinectes danae

The kinetics of purified glycogen phosphorylase a from the muscle of the blue crab ( Callinectes danae) were studied in the direction of glycogen synthesis, and in the direction of glycogen degradation with P i or arsenate as substrates. The effects of AMP, UDPG, G-6- P, glucose, and arsenate on the appropriate systems were studied. AMP is an activator of the enzyme. Inhibition by UDPG with respect to P i changes from noncompetitive to competitive when AMP is added; it changes from noncompetitive to mixed with respect to glycogen when AMP is added. G-6- P is a competitive inhibitor of G-1- P and arsenate. Inhibition by glucose with respect to glycogen changes from noncompetitive to competitive when AMP is added in the direction of glycogen breakdown; it is noncompetitive with respect to P i. Arsenate is a competitive inhibitor with respect to P i. The K m for AMP increases in the presence of UDPG, and decreases with increasing concentrations of P i or glycogen. We propose a model in which the enzyme bears three interacting sites: an active site, an activator (AMP) site, and an inhibitor (glucose) site. The active site has three subsites: one for P i, one for glycogen, and one for a glucose moiety which may be part of the substrates or inhibitors.