Abstract
Glycogen synthase, a central enzyme in glucose metabolism, catalyzes the successive addition of α-1,4-linked glucose residues to the non-reducing end of a growing glycogen molecule. A non-catalytic glycogen-binding site, identified by x-ray crystallography on the surface of the glycogen synthase from the archaeon Pyrococcus abyssi, has been found to be functionally conserved in the eukaryotic enzymes. The disruption of this binding site in both the archaeal and the human muscle glycogen synthases has a large impact when glycogen is the acceptor substrate. Instead, the catalytic efficiency remains essentially unchanged when small oligosaccharides are used as substrates. Mutants of the human muscle enzyme with reduced affinity for glycogen also show an altered intracellular distribution and a marked decrease in their capacity to drive glycogen accumulation in vivo. The presence of a high affinity glycogen-binding site away from the active center explains not only the long-recognized strong binding of glycogen synthase to glycogen but also the processivity and the intracellular localization of the enzyme. These observations demonstrate that the glycogen-binding site is a critical regulatory element responsible for the in vivo catalytic efficiency of GS.
Highlights
Mobilization of glucose units when energy demand is high [1]
Polymerization is performed by glycogen synthase (GS),5 which catalyzes the formation of ␣-1,4-glycosidic bonds using UDP-Glc or ADP-Glc as the glucosyl donor, whereas the branching enzyme is responsible for introducing the ␣-1,6-linked branches every 11–14 glucose residues
Many of the enzymes that are involved in the glycogen metabolism possess, in addition to their catalytic sites, distinct non-catalytic carbohydrate binding modules (CBMs) or specific glycogen-binding sites [7], which provide these enzymes with high affinity for the polysaccharide
Summary
Expression Construct—The full-length PaGS cDNA [21] was cloned into the NdeI/SalI sites of pCold I (TaKaRa) [22], an expression vector that adds an N-terminal His tag to the expressed protein. Cell culture medium was removed, and 1 ml of cold lysis buffer (30 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.5% Nonidet P-40, 1 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride, 25 nM okadaic acid, 10 g/ml leupeptin, 10 g/ml aprotinin, and 10 g/ml pepstatin, 20% glycerol) was added per dish. The resin was transferred to an Eppendorf tube, where it was washed 5 times using 1 ml of cold wash buffer (30 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% Nonidet P-40, 20% glycerol) and transferred to a microspin column (GE Healthcare). The resin was incubated for 10 min with elution buffer (2.5 mM desthiobiotin, 30 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 20% glycerol), and protein was eluted. Data collection, phasing, and refinement statistics Values in parentheses are for the highest resolution shell
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