Mutations of muscle glycogen synthase that disable activation by glucose 6-phosphate.
Glycogen synthase, an enzyme of historical importance in the field of reversible protein modification, is inactivated by phosphorylation and allosterically activated by glucose 6-phosphate (glucose-6-P). Previous analysis of yeast glycogen synthase had identified a conserved and highly basic 13-amino-acid segment in which mutation of Arg residues resulted in loss of activation by glucose-6-P. The equivalent mutations R578R579R581A (all three of the indicated Arg residues mutated to Ala) and R585R587R590A were introduced into rabbit muscle glycogen synthase. Whether expressed transiently in COS-1 cells or produced in and purified from Escherichia coli, both mutant enzymes were insensitive to activation by glucose-6-P. The effect of phosphorylation was studied in two ways. Purified, recombinant glycogen synthase was directly phosphorylated by casein kinase 2 and glycogen synthase kinase 3, under conditions that inactivate the wild-type enzyme. In addition, phosphorylation sites were converted to Ala by mutagenesis in wild-type and in the glucose-6-P desensitized mutants expressed in COS-1 cells. Phosphorylation inactivated the R578R579R581A mutant but had little effect on the R585R587R590A. This result was surprising since phosphorylation had the opposite effects on the corresponding yeast enzyme mutants. The results confirm that the region of glycogen synthase, Arg-578-Arg-590, is required for activation by glucose-6-P and suggest that it is part of a sensitive and critical switch involved in transitions between different conformational states. However, the role must differ subtly between the mammalian and the yeast enzymes.
Hanashiro
Isao
I
Department of Biochemistry and Molecular Biology and Center for Diabetes Research, Indiana University School of Medicine, Indianapolis, Indiana 46202, USA.
Roach
Peter J
PJ
D000070:Acebutolol; D000818:Animals; D019556:COS Cells; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics; D019298:Glucose-6-Phosphate / Q000494:pharmacology*; D006006:Glycogen Synthase / Q000187:drug effects* / Q000235:genetics* / Q000378:metabolism; D009132:Muscles / Q000201:enzymology*; D010766:Phosphorylation; D011817:Rabbits; D011994:Recombinant Proteins / Q000378:metabolism; D015003:Yeasts / Q000201:enzymology
D011994:Recombinant Proteins; D019298:Glucose-6-Phosphate; D000070:Acebutolol; D006006:Glycogen Synthase