Multiple glycogen-binding sites in eukaryotic glycogen synthase are required for high catalytic efficiency toward glycogen

Sulochanadevi Baskaran, Vimbai M. Chikwana, Christopher J. Contreras, Keri D. Davis, Wayne A. Wilson, Anna De Paoli-Roach, Peter Roach, Thomas Hurley

Research output: Contribution to journalArticle

17 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)33999-34006
Number of pages8
JournalJournal of Biological Chemistry
Volume286
Issue number39
DOIs
StatePublished - Oct 30 2011

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Glycogen Synthase
Glycogen
Binding Sites
Yeast
Mutation
Yeasts
Biosynthesis
Substrates
Enzymes
Catalysis
Catalytic Domain
Homeostasis
Cells
Glucose

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology

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Multiple glycogen-binding sites in eukaryotic glycogen synthase are required for high catalytic efficiency toward glycogen. / Baskaran, Sulochanadevi; Chikwana, Vimbai M.; Contreras, Christopher J.; Davis, Keri D.; Wilson, Wayne A.; De Paoli-Roach, Anna; Roach, Peter; Hurley, Thomas.

In: Journal of Biological Chemistry, Vol. 286, No. 39, 30.10.2011, p. 33999-34006.

Research output: Contribution to journalArticle

Baskaran, Sulochanadevi ; Chikwana, Vimbai M. ; Contreras, Christopher J. ; Davis, Keri D. ; Wilson, Wayne A. ; De Paoli-Roach, Anna ; Roach, Peter ; Hurley, Thomas. / Multiple glycogen-binding sites in eukaryotic glycogen synthase are required for high catalytic efficiency toward glycogen. In: Journal of Biological Chemistry. 2011 ; Vol. 286, No. 39. pp. 33999-34006.
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AB - 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.

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