Regulation of rat liver glycogen synthetase. Evidence for a lysyl residue essential for glucose 6-phosphate activation.

Glycogen synthetase D was inactivated by treatment with trlnitrobenzene sulfonate with the incorporation of 2.5 moles of trinitrophenyl group per mole of enzyme subunit. ‘I he sulfhydryl content of the enzyme was unchanged during trinitrophenylation indicating that derivatization was through the amino group. Glucose-6-P and other phosphate activators of glycogen synthetase D offered protection against modification. Partially active, trinitrophenylated derivatives of the enzyme were characterized by a decreased V,,, without any change in the K, for the glucose-6-P or the K,,, for the substrate, UDP-glucose. Pyridoxal-P was used to probe further these amino groups. As an inhibitor of glycogen synthetase D, pyridoxal-P showed competitive inhibition with respect to glucose-6-P. ‘Ihe unreactive analogues, pyridoxine-P and pyridoxamine-P, were weak activators of the enzyme by themselves, whereas pyridorine-P was a competitive inhibitor with respect to glucose6-P. The corresponding unphosphorylated forms of these analogues could not duplicate these effects, providing evidence that the site of action of pyridoxal-P was at or near the glucose-6-P-binding site involved in the activation of the enzyme. Reduction of the Schiff’s base complex between pyridoxalP and glycogen synthetase D by NaBH4 led to the irreversible inactivation of the enzyme. Inactivation was the result of the incorporation of 1.0 mole of phosphopyridoxal group per mole of enzyme subunit through the a-amino group of a lysyl residue. Glucose-6-P offered some protection against inactivation. Reduction of pyridoxine-Por pyridoxamineP-treated enzyme had no effect on enzyme activity. These results suggest that there is a lysyl residue located at or near the glucose-6-P binding site which is essential for the activation of glycogen synthetase D by glucose-6-P.