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 recomb inant 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 phosphorylati on by the cyclin-dependent protein kinase Pcl10p/Pho85p decreased V-max/K-m by similar to 30-fold (state I). In the presence of glucose-6-P, state III is achieved regardless of phosphorylation state. The enzyme forms complexe s 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 i nactive. Five mutant proteins were altered in their activation by glucose-6 -P, and two were completely unaffected by the hexose phosphate, One of thes e, R586A/R588A/R591A (all three of the indicated Arg residues mutated to Al a), had wild-type activity and was normally inactivated by phosphorylation. A second mutant, R579A/R580A/R582A, had somewhat reduced V-max, but its ac tivity was not greatly reduced by phosphorylation. The Arg residues in thes e two mutants are restricted to a highly conserved, 13-residue segment of G sy2p that me propose to be important for glucose-6-P binding and/or the abi lity of the enzyme to undergo transitions between activity states.