Multiple Glycogen-binding Sites in Eukaryotic Glycogen Synthase Are Required for High Catalytic Efficiency toward Glycogen

Abstract

Glycogen synthase is a rate-limiting enzyme in the biosynthesis of glycogen and has an essential role in glucose homeostasis. The three-dimensional structures of yeast glycogen synthase (Gsy2p) complexed with maltooctaose identified four conserved maltodextrin-binding sites distributed across the surface of the enzyme. Site-1 is positioned on the N-terminal domain, site-2 and site-3 are present on the C-terminal domain, and site-4 is located in an interdomain cleft adjacent to the active site. Mutation of these surface sites decreased glycogen binding and catalytic efficiency toward glycogen. Mutations within site-1 and site-2 reduced the V max /S 0.5 for glycogen by 40-and 70-fold, respectively. Combined mutation of site-1 and site-2 decreased the V max /S 0.5 for glycogen by >3000-fold. Consistent with the in vitro data, glycogen accumulation in glycogen synthase-deficient yeast cells (⌬gsy1-gsy2) transformed with the site-1, site-2, combined site-1/site-2, or site-4 mutant form of Gsy2p was decreased by up to 40-fold. In contrast to the glycogen results, the ability to utilize maltooctaose as an in vitro substrate was unaffected in the site-2 mutant, moderately affected in the site-1 mutant, and almost completely abolished in the site-4 mutant. These data show that the ability to utilize maltooctaose as a substrate can be independent of the ability to utilize glycogen. Our data support the hypothesis that site-1 and site-2 provide a "toehold mechanism," keeping glycogen synthase tightly associated with the glycogen particle, whereas site-4 is more closely associated with positioning of the nonreducing end during catalysis.Glycogen synthase was the first reported intracellular target of insulin, and the enzyme catalyzes the linear polymerization of glucose residues from activated sugar donor molecules to the nonreducing end of the glycogen chain. Recent structural studies have shown that the enzyme folds into two Rossmann foldlike domains, with a deep cleft in between harboring the active site (1-3). Although the basic fold is conserved between the prokaryotic, archaeal, and eukaryotic enzymes, there are multiple sequence insertions in the eukaryotic enzymes. The largest of these insertions (a long coiled-coil insert in the C-terminal domain) gives rise to their unique tetrameric arrangement as well as the structural plasticity necessary for the complex regulation of glycogen synthase activity in eukaryotes (3). Furthermore, a conserved arginine cluster present in the C-terminal region of the eukaryotic enzymes mediates the sensitivity to inhibition by phosphorylation and activation by glucose 6-phosphate (4, 5). In our recent structural studies, we demonstrated that the middle two arginine residues 3 are necessary and sufficient to confer regulation by glucose 6-phosphate and that the first three arginine residues (Arg-580, Arg-581, and Arg-583) are required for full regulatory response to phosphorylation (3).The yeast Saccharomyces cerevisiae possesses two genes encoding glycogen synthase, GSY1 and GSY2, wh...

Selectivity of the surface binding site (SBS) on barley starch synthase I

“…Remarkably, as previously reported in the case of glycogen synthase (Baskaran et al 2011;Díaz et al 2011), the SBS in HvSSI was found to have much higher affinity for the substrates as compared to the active site.…”

“…A modest affinity may avoid that the product remains and blocks the active site, while the macromolecule undergoing ex-tension is still bound to the enzyme at the SBS, which moreover would facilitate a processive reaction on the growing chain (Baskaran et al 2011;Díaz et al 2011). The stoichiometry of the wild-type HvSSI with β-CD and M7 binding was 0.63 and 0.19, respectively, suggesting that the interaction takes place at only one site.…”

Glycogen and its metabolism: some new developments and old themes

“…When the first glycogen synthase structures were solved, the realization that it had the same protein fold as glycogen phosphorylase led to a thought that, like phosphorylase [131], perhaps glycogen synthase has its carbohydrate-binding sites integrated into its catalytic domain. Indeed, this has now been shown in the Escherichia coli , Pyrococcus abyssi and yeast Gsy2p enzymes [128,132,133]. These studies have revealed that glycogen phosphorylase and many forms of glycogen synthase retain a glycogen-association site on the surface of their respective N-terminal Rossmann-fold domains that is completely independent of the acceptor binding site within the catalytic cleft (Figures 9A–9C).…”

Analysis of surface binding sites (SBSs) in carbohydrate active enzymes with focus on glycoside hydrolase families 13 and 77 — a mini-review

“…In addition, α-glucan active enzymes containing an SBS include starch and glycogen synthases (Baskaran et al 2011;Diaz et al 2011;Cuesta-Seijo et al 2013), phosphatases (Gentry et al 2009;Meekins et al 2013) and glycogen phosphorylases (Fletterick et al 1976;Pinotsis et al 2003), which will not be addressed in the present review.…”