title: 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 (Δgsy1-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.
authors.affiliation: Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana 46202, USA.
authors.forename: Sulochanadevi
authors.initials: S
authors.lastname: Baskaran
authors.forename: Vimbai M
authors.initials: VM
authors.lastname: Chikwana
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Contreras
authors.forename: Keri D
authors.initials: KD
authors.lastname: Davis
authors.forename: Wayne A
authors.initials: WA
authors.lastname: Wilson
authors.forename: Anna A
authors.initials: AA
authors.lastname: DePaoli-Roach
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Roach
authors.forename: Thomas D
authors.initials: TD
authors.lastname: Hurley
mesh_terms: D001665:Binding Sites; D006003:Glycogen / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006006:Glycogen Synthase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D009844:Oligosaccharides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D009844:Oligosaccharides; D029701:Saccharomyces cerevisiae Proteins; C101183:maltooctaose; D006003:Glycogen; D006006:Glycogen Synthase