Regulation of glycogen synthase. Identification of residues involved in regulation by the allosteric ligand glucose-6-P and by phosphorylation.
The major yeast glycogen synthase, Gsy2p, is inactivated by phosphorylation and activated by the allosteric ligand glucose-6-P. From studies of recombinant proteins, the control can be accommodated by a three-state model, in which unphosphorylated enzyme has intermediate activity (state II). Glucose-6-P increased V(max)/K(m) by about 2-fold (state III), whereas phosphorylation by the cyclin-dependent protein kinase Pcl10p/Pho85p decreased V(max)/K(m) by approximately 30-fold (state I). In the presence of glucose-6-P, state III is achieved regardless of phosphorylation state. The enzyme forms complexes in solution with the yeast glycogenin Glg2p, but this interaction appears not to affect control either by glucose-6-P binding or by phosphorylation. Scanning mutagenesis was applied to identify residues potentially involved in ligand binding. Of 22 mutant enzymes analyzed, seven were essentially inactive. Five mutant proteins were altered in their activation by glucose-6-P, and two were completely unaffected by the hexose phosphate. One of these, R586A/R588A/R591A (all three of the indicated Arg residues mutated to Ala), had wild-type activity and was normally inactivated by phosphorylation. A second mutant, R579A/R580A/R582A, had somewhat reduced V(max), but its activity was not greatly reduced by phosphorylation. The Arg residues in these two mutants are restricted to a highly conserved, 13-residue segment of Gsy2p that we propose to be important for glucose-6-P binding and/or the ability of the enzyme to undergo transitions between activity states.
Pederson
B A
BA
Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, and the Indiana University Center for Diabetes Research, Indianapolis, Indiana 46202-5122, USA.
Cheng
C
C
Wilson
W A
WA
Roach
P J
PJ
D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D016384:Consensus Sequence; D019298:Glucose-6-Phosphate / Q000378:metabolism*; D006006:Glycogen Synthase / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008099:Liver / Q000201:enzymology; D008954:Models, Biological; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000201:enzymology; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D008024:Ligands; D011994:Recombinant Proteins; D019298:Glucose-6-Phosphate; D006006:Glycogen Synthase