title: Redox Switch for the Inhibited State of Yeast Glycogen Synthase Mimics Regulation by Phosphorylation.
abstract: Glycogen synthase (GS) is the rate limiting enzyme in the synthesis of glycogen. Eukaryotic GS is negatively regulated by covalent phosphorylation and allosterically activated by glucose-6-phosphate (G-6-P). To gain structural insights into the inhibited state of the enzyme, we solved the crystal structure of yGsy2-R589A/R592A to a resolution of 3.3 Å. The double mutant has an activity ratio similar to the phosphorylated enzyme and also retains the ability to be activated by G-6-P. When compared to the 2.88 Å structure of the wild-type G-6-P activated enzyme, the crystal structure of the low-activity mutant showed that the N-terminal domain of the inhibited state is tightly held against the dimer-related interface thereby hindering acceptor access to the catalytic cleft. On the basis of these two structural observations, we developed a reversible redox regulatory feature in yeast GS by substituting cysteine residues for two highly conserved arginine residues. When oxidized, the cysteine mutant enzyme exhibits activity levels similar to the phosphorylated enzyme but cannot be activated by G-6-P. Upon reduction, the cysteine mutant enzyme regains normal activity levels and regulatory response to G-6-P activation.
authors.affiliation: Department of Biochemistry and Molecular Biology, Indiana University School of Medicine , Indianapolis, Indiana 46202, United States.
authors.forename: Krishna K
authors.initials: KK
authors.lastname: Mahalingan
authors.affiliation: Department of Biochemistry and Molecular Biology, Indiana University School of Medicine , Indianapolis, Indiana 46202, United States.
authors.forename: Sulochanadevi
authors.initials: S
authors.lastname: Baskaran
authors.affiliation: Department of Biochemistry and Molecular Biology, Indiana University School of Medicine , Indianapolis, Indiana 46202, United States.
authors.forename: Anna A
authors.initials: AA
authors.lastname: DePaoli-Roach
authors.affiliation: Department of Biochemistry and Molecular Biology, Indiana University School of Medicine , Indianapolis, Indiana 46202, United States.
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Roach
authors.affiliation: Department of Biochemistry and Molecular Biology, Indiana University School of Medicine , Indianapolis, Indiana 46202, United States.
authors.forename: Thomas D
authors.initials: TD
authors.lastname: Hurley
mesh_terms: D003460:Crystallization; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects / Q000235:genetics; D019298:Glucose-6-Phosphate / Q000378:metabolism / Q000494:pharmacology; D006003:Glycogen / Q000378:metabolism; D006006:Glycogen Synthase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D010084:Oxidation-Reduction; D010766:Phosphorylation; D000072417:Protein Domains; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014532:Uridine Diphosphate Glucose / Q000378:metabolism
chemical_list: D029701:Saccharomyces cerevisiae Proteins; D019298:Glucose-6-Phosphate; D006003:Glycogen; D006006:Glycogen Synthase; D003545:Cysteine; D014532:Uridine Diphosphate Glucose