PARTIAL ACTIVATION OF MUSCLE PHOSPHORYLASE BY REPLACEMENT OF SERINE-14 WITH ACIDIC RESIDUES AT THE SITE OF REGULATORY PHOSPHORYLATION

Phosphorylation of glycogen phosphorylase at residue Ser14 triggers a conformational transition that activates the enzyme. The N-terminus of the protein, in response to phosphorylation, folds into a 3(10) helix and moves from its location near a cluster of acidic residues on the protein surface to a site at the dimer interface where a pair of argin ine residues form charged hydrogen bonds with the phosphoserine. Site- directed mutagenesis was used to replace Ser14 with Asp and Glu residu es, analogs of the phosphoserine, that might be expected to participat e in ionic interactions with the arginine side chains at the dimer int erface. Kinetic analysis of the mutants indicates that substitution of an acidic residue in place of Ser14 at the site of regulatory phospho rylation partially activates the enzyme. The S14D mutant shows a 1.6-f old increase in V-max, a 10-fold decrease in the apparent dissociation constant for AMP, and a 3-fold decrease in the S-0.5 for glucose 1-ph osphate. The S14E mutant behaves similarly, showing a 2.2-fold increas e in V-max, a 6-fold decrease in the apparent dissociation constant fo r AMP, and a 2-fold decrease in the S-0.5 for glucose l-phosphate. The ability of the mutations to enhance binding of AMP and glucose l-phos phate and to raise catalytic activity suggests that the introduction o f a carboxylate side chain at position 14 promotes docking of the N-te rminus at the subunit interface and concomitant stabilization of the a ctivated conformation of the enzyme. Like the native enzyme, both muta nts show significant activity only in the presence of the activator, A MP. Full activation, analogous to that provided by covalent phosphoryl ation of the enzyme, likely is not achieved because of differences in the charge and the geometry of ionic interactions at the phosphorylati on site.