The glycogen debranching enzyme, in humans, is the protein encoded by the gene AGL. This enzyme is essential for the breakdown of glycogen, which serves as a store of glucose in the body. It has separate glucosyltransferase and glucosidase activities. Together with phosphorylases, the enzyme mobilize glucose reserves from glycogen deposits in the muscles and liver. This constitutes a major source of energy reserves in most organisms. Glycogen breakdown is highly regulated in the body, especially in the liver, by various hormones including insulin and glucagon, to maintain a homeostatic balance of blood-glucose levels. When glycogen breakdown is compromised by mutations in the glycogen debranching enzyme, metabolic diseases such as Glycogen storage disease type III can result. The two steps of glycogen breakdown, glucosyltransferase and glucosidase, are performed by a single enzyme in mammals, yeast, and some bacteria, but by two distinct enzymes in E. coli and other bacteria, complicating nomenclature. Proteins that catalyze both functions are referred to as glycogen debranching enzymes (GDEs). When glucosyltransferase and glucosidase are catalyzed by distinct enzymes, glycogen debranching enzyme usually refers to the glucosidase enzyme. In some literature, an enzyme capable only of glucosidase is referred to as a debranching enzyme.
Function Together with phosphorylase, glycogen debranching enzymes function in glycogen breakdown and glucose mobilization. When phosphorylase has digested a glycogen branch down to four glucose residues, it will not remove further residues. Glycogen debranching enzymes assist phosphorylase, the primary enzyme involved in glycogen breakdown, in the mobilization of glycogen stores. Phosphorylase can only cleave α-1,4-glycosidic bond between adjacent glucose molecules in glycogen but branches also exist as α-1,6 linkages. When phosphorylase reaches four residues from a branching point it stops cleaving; because 1 in 10 residues is branched, cleavage by phosphorylase alone would not be sufficient in mobilizing glycogen stores. Before phosphorylase can resume catabolism, debranching enzymes perform two functions:
4-α-D-glucanotransferase (EC 2.4.1.25), or glucosyltransferase, transfers three glucose residues from the four-residue glycogen branch to a nearby branch. This exposes a single glucose residue joined to the glucose chain through an α-1,6 glycosidic linkage Amylo-α-1,6-glucosidase (EC 3.2.1.33), or glucosidase, cleaves the remaining alpha-1,6 linkage, producing glucose and a linear chain of glycogen. The mechanism by which the glucosidase cleaves the α -1,6-linkage is not fully known because the amino acids in the active site have not yet been identified. It is thought to proceed through a two step acid base assistance type mechanism, with an oxocarbenium ion intermediate, and retention of configuration in glucose. This is a common method through which to cleave bonds, with an acid below the site of hydrolysis to lend a proton and a base above to deprotinate a water which can then act as a nucleophile. These acids and bases are amino acid side chains in the active site of the enzyme. A scheme for the mechanism is shown in the figure. Thus the debranching enzymes, transferase and α-1,6-glucosidase converts the branched glycogen structure into a linear one, paving the way for further cleavage by phosphorylase.
Structure and activity
Two enzymes In E. coli and other bacteria, glucosyltransferase and glucosidase functions are performed by two distinct proteins. In E. coli, Glucose transfer is performed by 4-alpha-glucanotransferase, a 78.5 kDa protein coded for by the gene malQ. A second protein, referred to as debranching enzyme, performs α-1,6-glucose cleavage. This enzyme has a molecular mass of 73.6 kDa, and is coded for by the gene glgX. Activity of the two enzymes is not always necessarily coupled. In E. coli glgX selectively catalyzes the cleavage of 4-subunit branches, without the action of glucanotransferase. The product of this cleavage, maltotetraose, is further degraded by maltodextrin phosphorylase. E. coli GlgX is structurally similar to the protein isoamylase. The monomeric protein contains a central domain in which eight parallel beta-strands are surrounded by eight parallel alpha strands. Notable within this structure is a groove 26 angstroms long and 9 angstroms wide, containing aromatic residues that are thought to stabilize a four-glucose branch before cleavage. The glycogen-degrading enzyme of the archaea Sulfolobus solfataricus, treX, provides an interesting example of using a single active site for two activities: amylosidase and glucanotransferase activities. TreX is structurally similar to glgX, and has a mass of 80kD and one active site. Unlike either glgX, however, treX exists as a dimer and tetramer in solution. TreX's oligomeric form seems to play a significant role in altering both enzyme shape and function. Dimerization is thought to stabilize a "flexible loop" located close to the active site. This may be key to explaining why treX (and not glgX) shows glucosyltransferase activity. As a tetramer, the catalytic efficiency of treX is increased fourfold over its dimeric form.
One enzyme with two catalytic sites In mammals and yeast, a single enzyme performs both debranching functions. The human glycogen debranching enzyme (gene: AGL) is a monomer with a molecular weight of 175 kDa. It has been shown that the two catalytic actions of AGL can function independently of each other, demonstrating that multiple active sites are present. This idea has been reinforced with inhibitors of the active site, such as polyhydroxyamine, which were found to inhibit glucosidase activity while transferase activity was not measurably changed. Glycogen debranching enzyme is the only known eukaryotic enzyme that contains multiple catalytic sites and is active as a monomer. Some studies have shown that the C-terminal half of yeast GDE is associated with glucosidase activity, while the N-terminal half is associated with glucosyltransferase activity. In addition to these two active sites, AGL appears to contain a third active site that allows it to bind to a glycogen polymer. It is thought to bind to six glucose molecules of the chain as well as the branched glucose, thus corresponding to 7 subunits within the active site, as shown in the figure below.
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