Interaction of glycogen synthase from rabbit skeletal muscles with 1,5-gluconolactone

1.5-Gluconolactone was shown to inhibit in a competitive manner the activity of both I- and D-forms of rabbit skeletal muscle glycogen synthase. Unlike other known inhibitors (UDP and adenyl nucleotides) the affinity of the enzyme D-form for 1.5-gluconolactone is lower than that of the I-form. The joint inhibition of glycogen synthase by UDP and 1.5-gluconolactone is characterized by positive cooperativity. It was supposed that the binding of the nucleotide part of the substrate molecule is preceded by the UDPglucose glucosyl residue interaction with the enzyme and induces a closer resemblance to the transient state. The effect of the allosteric inhibitor, ADP, on the enzyme activity is conditioned by its effect on the conformational state of UDP-glucose glucosyl residue binding site. Phosphorylation of glycogen synthase results in conformational changes in the same active site region, although the pyrimidine base binding site also seems to be involved in this process.     

Escherichia coli K-12 glycogen synthase: ability to use UDPglucose and ADP glucose as glucosyl donors in the absence of added primer.

A mechanism of initiation of glycogen biosynthesis in Escherichia coli has been previously postulated: In a first step, the glucosyl groups would be transferred into an acceptor protein from UDPglucose or ADPglucose by two glucosyl transferases, distinct from the glycogen synthase. In this work, the activity of transfer from UDPglucose into a methanol-insoluble fraction could not be found in the crude extracts of six independently isolated glycogen synthase-deficient mutants of E. coli K-12. Purified E. coli K-12 glycogen synthase was able to catalyze the unprimed reaction from ADPglucose and UDPglucose but at a very low rate; the rate with UDPglucose is 6–7% the rate observed with ADPglucose. With these two substrates, the unprimed reaction was strongly stimulated by the simultaneous presence of salts and branching enzyme. However the activity with UDPglucose increased rapidly at low concentrations of branching enzyme and was inhibited at physiological concentrations whereas the activity with ADPglucose reached a maximum only at these concentrations. Consequently, the relative activities found with ADPglucose and UDPglucose varied with the branching enzyme concentration. Transfer from UDPglucose was inhibited by low concentrations of ADPglucose and high concentrations of glycogen. These results suggest that the same enzyme, namely the glycogen synthase, catalyzes the unprimed transfer from ADPglucose and UDPglucose and that ADPglucose is probably the most important physiological donor in glycogen biosynthesis in E. coli.     

Pyrophosphate may be involved in regulation of bacterial glycogen synthesis

Inorganic pyrophosphate is a potent inhibitor of the enzyme that catalyzes synthesis of the glucosyl donor for Escherichia coli glycogen synthesis, ADP-glucose pyrophosphorylase. The Ki is determined to be 40 microM and the substrate ATP, the activator, fructose 1,6-P2 or the allosteric inhibitor, AMP do not greatly affect the inhibition. PPi exhibits mixed type inhibition with the other substrate, glucose 1-P. The potential regulation of glycogen synthesis by PPi is discussed.     

Characterization of Escherichia coli B glycogen synthase enzymatic reactions and products.

Previous reports implicate UDPglucose as an active glucosyl donor for the unprimed reaction and “glucoprotein” formation in glycogen biosynthesis in Escherichia coli. Results presented here indicate that UDPglucose and GDPglucose are glucosyl donors in the primed and unprimed reactions catalyzed by purified E. coli B glycogen synthase at less than 5% the rate observed when ADPglucose is the donor. The unprimed reaction is stimulated by 0.25 m citrate and a high molecular weight product is formed similar to that produced when ADPglucose is the glucosyl donor. Physiological amounts of branching enzyme and high concentrations of glycogen inhibit transfer from UDPglucose and GDPglucose. In addition, transfer from UDPglucose is inhibited by ADPglucose. These results strongly suggest that ADPglucose is the physiological donor in both the primed and unprimed reactions. Furthermore, these and previously reported results suggest that one enzyme is involved in the catalysis of the primed, unprimed, and TCA-insoluble product formation reactions. Antiserum prepared against purified E. coli B glycogen synthase inactivates transfer of glucose from either ADPglucose or UDPglucose in the above reactions catalyzed by E. coli B crude extracts. Purified E. coli B glycogen synthase preparations contain significant amounts of α-glucan primer. Evidence shows that this glucan is not covalently attached to the enzyme. Results presented show that formation of material insoluble in TCA and previously considered to be due to “glucoprotein” formation, is in fact due to the generation of long chain length glucan molecules intrinsically acid insoluble. The data suggest that previous results purported to be de novo synthesis of glycogen are due to glucan associated with the glycogen synthase and not to formation of a “glucoprotein” intermediate which then acts as primer for further oligosaccharide synthesis.     

Biosynthesis of glycogen in Neurospora crassa. Existence of a glucoproteic intermediate in the initiation process.

A soluble enzyme preparation (20,000 X g supernatant fraction), prepared from the mycelia of wild-type Neurospora crassa, was capable of transferring [14C]glucose from UDP-[14C]glucose into both trichloroacetic acid (TCA)-soluble and TCA-insoluble macromolecule products in the absence of added primer. These reactions did not require either high concentrations of salts or any other chemical reagents. Two labeled products were formed; one was a glycogen-like polysaccharide and the other was a glycoprotein with glucosyl chains bound to protein through an acid-labile bond. After mild treatment of the glucoprotein with acid, the product liberated from the protein behaved as a mixture of malto-oligosaccharides and alpha-1,4-glucan with branches. The carbohydrate moiety of the glucoprotein seemed to be released upon prolonged incubation with the enzyme preparation. The glucan thus liberated from the glucoprotein may serve as a primer for the glycogen synthase. The results obtained are therefore suggestive of the existence of a glucoproteic intermediate in the initiation of glycogen biosynthesis.     

Crystal structure of an archaeal glycogen synthase: insights into oligomerization and substrate binding of eukaryotic glycogen synthases

Glycogen and starch synthases are retaining glycosyltransferases that catalyze the transfer of glucosyl residues to the non-reducing end of a growing alpha-1,4-glucan chain, a central process of the carbon/energy metabolism present in almost all living organisms. The crystal structure of the glycogen synthase from Pyrococcus abyssi, the smallest known member of this family of enzymes, revealed that its subunits possess a fold common to other glycosyltransferases, a pair of beta/alpha/beta Rossmann fold-type domains with the catalytic site at their interface. Nevertheless, the archaeal enzyme presents an unprecedented homotrimeric molecular arrangement both in solution, as determined by analytical ultracentrifugation, and in the crystal. The C-domains are not involved in intersubunit interactions of the trimeric molecule, thus allowing for movements, likely required for catalysis, across the narrow hinge that connects the N- and C-domains. The radial disposition of the subunits confers on the molecule a distinct triangular shape, clearly visible with negative staining electron microscopy, in which the upper and lower faces present a sharp asymmetry. Comparison of bacterial and eukaryotic glycogen synthases, which use, respectively, ADP or UDP glucose as donor substrates, with the archaeal enzyme, which can utilize both molecules, allowed us to propose the residues that determine glucosyl donor specificity.     

Dodecyl-{beta}-D-maltoside as a substrate for glucosyl and xylosyl transfer by glycogenin

Glycogenin is the core protein of glycogen proteoglycan andis, at the same time, a self-glucosylating enzyme which catalysesearly glucosyl transfer steps in the biosynthesis of glycogen.In the course of this process, glycogenin is glucosylated progressivelyuntil an oligosaccharide containing 8–11 glucose residueshas been formed. Although glycogenin, under physiological conditions,is both enzyme and acceptor in the glucosyl transfer reactions,it is also capable of utilizing p-nitrophenyl-linked malto-oligosaccharidesas exogenous acceptors. In view of the potential usefulnessof exogenous acceptors in the study of the mechanism of theglycogenin reaction, we have expanded the search for such compoundsand report here that several alkyl glucosides and alkyl maltosidesmay serve as acceptors in glucosyl transfer by beef kidney glycogenin.Dodecyl-ß-D-maltoside (K_m {small tilde}100 µM)was the most effective acceptor among the compounds tested andyielded 30 times as much product as p-nitrophenyl-     

Combined action of Escherichia coli glycogen synthase and branching enzyme in the so-called "unprimed" polyglucoside synthesis.

Mutants of Escherichia coli which are unable to synthesize glycogen were used to study the so-called “unprimed” synthesis of glycogen. The glycogen synthase has been partially purified from these mutants. During the purification, attempts were made to separate the activity which requires the addition of an exogenous primer (primed activity) from the activity which does not require a primer but is highly dependent on the presence of some salts such as citrate and EDTA (unprimed activity). No separation between these two activities could be achieved but the results obtained by chromatography on DEAE-Sephadex indicate that there is a single form of glycogen synthase which is responsible for both unprimed and primed activity. The evidence that a single protein was necessary to catalyze these two reactions was given by the findings that mutants defective in glycogen synthase activity were unable to catalyze glucosyl transfer without added primer. At low concentration, the glycogen synthase purified from a branching enzyme negative mutant catalyzed the unprimed reaction at a slow rate even in presence of salts. A protein activator of this reaction was found in mutants lacking glycogen synthase but not in mutants lacking branching enzyme. The hypothesis that this activator is the branching enzyme itself was supported by the observation that it co-purified with the branching enzyme from a E. coli strain defective in glycogen synthase activity. EDTA or Triton X-100 increased the stimulation of the unprimed synthesis by the branching enzyme. The apparent affinity of the glycogen synthase for glycogen was increased twofold in the presence of EDTA but the branching enzyme further increased the effect of EDTA. The combined action of the glycogen synthase and the branching enzyme on the endogenous glucan associated with the synthase may account for the unprimed activity observed in vitro.     

Amylopectin Synthase of Eimeria tenella: Identification and Kinetic Characterization

ABSTRACT. A soluble enzyme amylopectin synthase (UDP-glucose-α 1,4-glucan α-4-glucosyltransferase) which transfers glucose from uridine 5'-diphosphate glucose (UDP-glucose) to a primer to form α-I,4-glucosyl linkages has been identified in the extracts of unsporulated oocysts of Eimeria tenella . UDP-glucose and not ADP-glucose was the most active glucosyl donor. Corn amylopectin, rabbit liver glycogen, oyster glycogen and corn starch served as primers; the latter two were less efficient. The enzyme has an apparent pH optimum of 7.5 and exhibited typical Michaelis-Menten kinetics with dependence on both the primer and substrate concentrations. The Michaelis constants (Km). with respect to UDP-glucose, was 0.5 mM; and 0.25 mg/ml and 1.25 mg/ml with respect to amylopectin and rabbit liver glycogen. The product formed by the reaction was predominantly a glucan containing α-1,4 linkages. The specificity of the enzyme suggests that this enzyme is similar to glycogen synthase in eukaryotes and has been designated as amylopectin synthase (UDP-glucose-α-1,4-glucosetransferase EC 2.4.1.11).     

Inverse relationship of skeletal muscle glycogen from wild-type and genetically modified mice to their phosphorylase a activity.

Leg muscle was biopsied and frozen for storage at -70 degrees C. from 5 wild-type mice, two knocked out acid alpha-glucosidase (GAA) gene mice, and seven glycogen synthase plus glucose muscle transporter transgenic mice. All of the wild-type mice had very little muscle glycogen (3.58 +/- 1.67 micromols glucosyl subunits per g muscle), and 52% or more of its glycogen phosphorylase activity without AMP (69% +/- 17% glycogen phosphorylase a). In contrast the GAA knockout and transgenic mice had glycogen ranging from 63 to 297 micromols glucosyl subunits per g muscle, and very little or no glycogen phosphorylase activity without 1.00 mM AMP (4.8% and less glycogen phosphorylase a). This suggests that there is an inverse relationship between mouse muscle phosphorylase a and the muscle's glycogen content.     

Kinetic properties of ATP phosphoribosyltransferase of escherichia coli

Summary The kinetic constants for the series of glucosyl acceptors for homogeneous rabbit muscle glycogen synthaseI form free of glycogen were examined. The acceptors included glucose, maltose, G3, G4, G6, two hydrolyzed amyloses, amylodextrin and seven polysaccharides including amylopectin and glycogen. S_0.5 and relative V_max were estimated in each case. From these data a two site model of the enzyme is proposed, composed of a polysaccharide binding site and a separate catalytic site, the latter composed of several subsites.     

The initiation of glycogen biosynthesis in rat heart. Studies with a purified preparation

Two fractions of glycogen synthase were isolated from rat cardiac muscle on the basis of a different affinity for DEAE-cellulose and omega-aminobutyl-agarose. One of these fractions was able to transfer glucosyl residues from UDP-glucose not only to glycogen (GS-1 activity) but also to an endogenous acceptor. The latter reaction (GS-2 activity) occurred in the absence of added glycogen, and its reaction product was insoluble in trichloroacetic acid. This compound was degraded by amylolytic enzymes, thus showing that the product synthesized on the endogenous acceptor was an alpha 1,4-glucan. After incubation with alpha-amylase-free proteolytic enzyme, the compound was rendered trichloroacetic acid-soluble. Polyacrylamide gel electrophoresis, under both native and denaturing conditions, showed that GS-2 reaction products moved electrophoretically associated to protein. Our results give further evidence for the association between an alpha 1,4-glucan and protein, which we postulate is related to the initiation of glycogen biosynthesis.     

Elimination of glucose contamination from adipocyte glycogen extracts

Glycogen was quantified in rat adipocytes by isolation using conventional KOH digestion and ethanol precipitation, followed by hydrolysis and spectrophotometric assay of the glucose product. A concentration of 0.193+/-0.020 micromol glucosyl units/10(6)cells was recorded. When this procedure was modified by including a 4h incubation with glucose oxidase prior to glycogen hydrolysis, the glycogen concentration was found to be 0.055+/-0.008 micromol glucosyl units/10(6) cells. Therefore in adipocytes, conventional glycogen assays give substantial overestimates due to incomplete removal of glucose during glycogen isolation. Contaminant glucose can be scavenged in a simple manner by incubation with glucose oxidase prior to glycogen hydrolysis.     

Glucose formation in human skeletal muscle. Influence of glycogen content.

Eight men exercised at 66% of their maximal isometric force to fatigue after prior decrease in the glycogen store in one leg (low-glycogen, LG). The exercise was repeated with the contralateral leg (control) at the same relative intensity and for the same duration. Muscle (quadriceps femoris) glycogen content decreased in the LG leg from 199 +/- 17 (mean +/- S.E.M.) to 163 +/- 16 mmol of glucosyl units/kg dry wt. (P less than 0.05), and in the control leg from 311 +/- 23 to 270 +/- 18 mmol/kg (P less than 0.05). The decrease in glycogen corresponded to a similar accumulation of glycolytic intermediates. Muscle glucose increased in the LG leg during the contraction, from 1.8 +/- 0.1 to 4.3 +/- 0.6 mmol/kg dry wt. (P less than 0.01), whereas no significant increase occurred in the control leg (P greater than 0.05). It is concluded that during exercise glucose is formed from glycogen through the debranching enzyme when muscle glycogen is decreased to values below about 200 mmol/kg dry wt.     

The maltase, glucoamylase and transglucosylase activities of acid α-glucosidase from rabbit muscle

1. The maltase and glucoamylase activities of acid alpha-glucosidase purified from rabbit muscle exhibited marked differences in certain physicochemical properties. These included pH stability, inactivation by thiol-group reagents, inhibition by alphaalpha-trehalose, methyl alpha-d-glucoside, sucrose, turanose, polyols, glucono-delta-lactone and monosaccharides, pH optimum and the kinetics and pH-dependence of cation activation. 2. The results are interpreted in terms of the existence of at least two specific substrate-binding sites or sub-sites. One site is specific for the binding of maltose and probably other oligosaccharides. The second site binds polysaccharides such as glycogen. 3. The sites appear to be in close proximity, since glycogen and maltose are mutually inhibitory substrates and interact directly in transglucosylation reactions. 4. Acid alpha-glucosidase exhibited intrinsic transglucosylase activity. The enzyme catalysed glucosyl-transfer reactions from [(14)C]maltose (donor substrate) to polysaccharides (glycogen and pullulan) and to maltose itself (disproportionation). The pH optimum was 5.1, with a shoulder or secondary activity peak at pH5.4. The glucose transferred to glycogen was attached by alpha-1,4- and alpha-1,6-linkages. Three major oligosaccharide products of enzyme action on maltose (disproportionation) were detected. 5. The kinetics of enzyme action on [(14)C]maltose showed that the rate of transglucosylation increased in a sigmoidal fashion as a function of substrate concentration, approximately in parallel with a decrease in the rate of glucose release. 6. The results are interpreted to imply competitive interaction at a specific binding site between maltose and water as glucosyl acceptors. 7. The results are discussed in terms of the possible existence of multiple subgroups of glycogen-storage disease type II.     

Transfer of glucose from UDP-glucose in microsomal membranes of rat hepatocytes (author's transl)]

Microsomal preparations from rat liver mediate transfer of glucosyl units from UDP-glucose to three different kinds of acceptors: an endogenous glycoprotein, exogenous glycogen and collagen. Both glucosyl transferases work at acidic pH, 6.5 for transfer on endogeneous acceptor and glycogen and at pH 5.5 for transfer on collagen. None of these enzymes require divalent cations for activity. While transfers on endogenous acceptor and glycogen are inhibited by the presence of a non-ionic detergent, Triton X-100, the transfer on collagen is activated by the same detergent. Glycogen-synthase activity requires glucose 6-phosphate at an optimal concentration of 1 mM. The Km values for UDP-glucose are respectively: 0.5 mM, 0.33 mM, and 1 mM for transfer on endogenous acceptor, glycogen and collagen. Characterisation of the product indicates that a protein-bound alpha1-4 glucan is formed when no primer is added. Enzymatic and acidic hydrolyses of radioactive glycogen and collagen show only glucose as a radioactive sugar identified by thin layer chromatography on cellulose. Pre-treatment of microsomal membranes by alpha-amylase demonstrates that glucosyltransferases are not adsorbed on endogenous glycogen and seem to be really membranous enzymes.     

Asparagine-linked glycoprotein biosynthesis in rat brain: identification of glucosidase I, glucosidase II, and and endomannosidase (glucosyl mannosidase)

Previous studies from this laboratory provided evidence, largely based upon the presence of a novel alpha-D-mannosidase, suggesting that the biosynthesis of N-linked glycoproteins may be different in brain as compared to other tissues (Tulsiani, D. R. P., and Touster, O. (1985) J. Biol. Chem. 260, 13,081-13,087). In the present report we describe studies on the enzymes involved in early processing reactions. These studies indicate that the brain, like other tissues, contains glucosidases I and II. The two glucosidases were separated as distinct activities with some overlapping by chromatography on a DE-52 column. The differential inhibition studies and substrate specificity studies support our conclusion that, as in other tissues, rat brain glucosidase I cleaves alpha 1,2-linked terminal glucosyl residues, whereas glucosidase II prefers alpha 1,3-linked glucosyl residues. In addition to these two processing glucosidases, we have characterized an endo enzyme (glucosyl mannosidase) in rat brain. The endomannosidase cleaves a disaccharide (glucosyl alpha 1,3-mannose) from monoglucosylated oligosaccharides (GlcMan7-9GlcNAc). Little or no activity was observed when di- or triglucosylated oligosaccharide was used as a substrate. The pH optimum of the glucosyl mannosidase is 6.2-6.8. The enzyme appears to be an intrinsic microsomal membrane component, since washing of the microsomal membranes with salt solution did not release the enzyme in soluble form. A mixture of Triton X-100 and sodium deoxycholate is required for complete solubilization of the enzyme. The solubilized enzyme is eluted from a Bio-Gel A-1.5m column as a single peak with an apparent molecular weight of 380,000.     

Reassessment of the catalytic mechanism of glycogen debranching enzyme

The amylo-1,6-glucosidase catalytic activity of glycogen debranching enzyme allows it to hydrolyze alpha-D-glucosyl fluoride, in the absence or presence of glycogen or oligosaccharides, releasing equal amounts of fluoride and glucose at rates comparable to those seen with the natural substrates. 2-Deoxy-2-fluoro-alpha-D-glucosyl fluoride is found to be a poor substrate, rather than the covalent inhibitor that would be expected for a glucosidase which catalyzes hydrolysis of the glycosidic linkage with retention of anomeric configuration. In fact, analysis of the glucosidase reaction by NMR reveals that the debranching enzyme hydrolyzes the glycosidic linkage with inversion of configuration, releasing beta-D-glucose from both alpha-glucosyl fluoride and its natural substrate, the phosphorylase limit dextrin. In contrast, its transferase activity necessarily proceeds with retention of configuration. As has been seen with other "inverting" glycosidases, the debranching enzyme releases beta-D-glucose from beta-D-glucosyl fluoride in the presence of oligosaccharides such as maltohexaose and cyclomaltoheptaose but, unlike the others, not in their absence. An intermediate glucosyl-alpha-(1,6)-cyclomaltoheptaose has been detected by NMR analysis. In the presence of a water-soluble carbodiimide, a single mole of glycine ethyl ester is incorporated into each mole of the debranching enzyme, resulting in its inactivation when measured by the combined assay for both transferase and glucosidase activities. Measurement of the latter two activities independently indicates that it is the transferase activity which is inactivated, while the glucosidase activity, measured with alpha-D-glucosyl fluoride as substrate, is unaffected.(ABSTRACT TRUNCATED AT 250 WORDS)     

Manganese sulfate-dependent glycosylation of endogenous glycoproteins in human skeletal muscle is catalyzed by a nonglucose 6-P-dependent glycogen synthase and not glycogenin

Glycogenin, a Mn2+-dependent, self-glucosylating protein, is considered to catalyze the initial glucosyl transfer steps in glycogen biogenesis. To study the physiologic significance of this enzyme, measurements of glycogenin mediated glucose transfer to endogenous trichloroacetic acid precipitable material (protein-bound glycogen, i.e., glycoproteins) in human skeletal muscle were attempted. Although glycogenin protein was detected in muscle extracts, activity was not, even after exercise that resulted in marked glycogen depletion. Instead, a MnSO4-dependent glucose transfer to glycoproteins, inhibited by glycogen and UDP-pyridoxal (which do not affect glycogenin), and unaffected by CDP (a potent inhibitor of glycogenin), was consistently detected. MnSO4-dependent activity increased in concert with glycogen synthase fractional activity after prolonged exercise, and the MnSO4-dependent enzyme stimulated glucosylation of glycoproteins with molecular masses lower than those glucosylated by glucose 6-P-dependent glycogen synthase. Addition of purified glucose 6-P-dependent glycogen synthase to the muscle extract did not affect MnSO4-dependent glucose transfer, whereas glycogen synthase antibody completely abolished MnSO4-dependent activity. It is concluded that: (1) MnSO4-dependent glucose transfer to glycoproteins is catalyzed by a nonglucose 6-P-dependent form of glycogen synthase; (2) MnSO4-dependent glycogen synthase has a greater affinity for low molecular mass glycoproteins and may thus play a more important role than glucose 6-P-dependent glycogen synthase in the initial stages of glycogen biogenesis; and (3) glycogenin is generally inactive in human muscle in vivo.     

A transglucosidase necessary for starch degradation and maltose metabolism in leaves at night acts on cytosolic heteroglycans (SHG)

The recently characterized cytosolic transglucosidase DPE2 (EC 2.4.1.25) is essential for the cytosolic metabolism of maltose, an intermediate on the pathway by which starch is converted to sucrose at night. In in vitro assays, the enzyme utilizes glycogen as a glucosyl acceptor but the in vivo acceptor molecules remained unknown. In this communication we present evidence that DPE2 acts on the recently identified cytosolic water-soluble heteroglycans (SHG) as does the cytosolic phosphorylase (EC 2.4.1.1) isoform. By using in vitro two-step ¹⁴C labeling assays we demonstrate that the two transferases can utilize the same acceptor sites of the SHG. Cytosolic heteroglycans from a DPE2-deficient Arabidopsis mutant were characterized. Compared with the wild type the glucose content of the heteroglycans was increased. Most of the additional glucosyl residues were found in the outer chains of SHG that are released by an endo- α -arabinanase (EC 3.2.1.99). Additional starch-related mutants were characterized for further analysis of the increased glucosyl content. Based on these data, the cytosolic metabolism of starch-derived carbohydrates is discussed.