Substrate specificity of phosphorylase kinase: effects of heparin and calcium.
Phosphorylase b and two peptides with sequences homologous to phosphorylation site 2 (syntide 2) and site 3 (syntide 3) of glycogen synthase were compared as substrates for purified muscle phosphorylase kinase. The substrate specificity of phosphorylase kinase varied according to whether heparin (at pH 6.5) or Ca2+ (at pH 8.2) was used as a stimulator of its activity. Phosphorylase b was preferentially phosphorylated in the presence of Ca2+; the rate of syntide 2 phosphorylation was the same for both stimulators; and the phosphorylation of syntide 3 was completely dependent on the presence of heparin. A kinetic analysis confirmed this stimulator-dependent substrate specificity since both the Vmax and Km for these substrates were affected diversely by heparin and Ca2+. Heparin stimulated phosphorylase kinase maximally at pH 6.5, whereas the effect of Ca2+ was optimal at a pH above 8. However, the stimulator-related substrate specificity could not be explained by the different pH values at which the effects of the stimulators were assessed. Nor did substrate-directed effects by heparin or Ca2+ apparently play a role. No indications were found for a stimulator-dependent specificity in the phosphorylation of sites in protein substrates of phosphorylase kinase (phosphorylase b, the alpha- and beta-subunits of phosphorylase kinase, or glycogen synthase). The diverse substrate specificity of the calcium- and heparin-dependent activities of phosphorylase kinase could be explained in two ways: either by the existence of separate calcium- and heparin-stimulated catalytic sites, or by just one catalytic site with two active conformations. The second possibility is favored by the observation that both calcium and heparin stimulated the isolated gamma-subunit (gamma X calmodulin complex) of phosphorylase kinase.
D000244:Adenosine Diphosphate / Q000494:pharmacology; D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology*; D002147:Calmodulin / Q000378:metabolism; D006006:Glycogen Synthase / Q000378:metabolism; D006493:Heparin / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010446:Peptide Fragments / Q000378:metabolism; D010764:Phosphorylase Kinase / Q000378:metabolism*; D010763:Phosphorylase b / Q000378:metabolism; D010766:Phosphorylation; D013379:Substrate Specificity; D014357:Trypsin / Q000378:metabolism
D002147:Calmodulin; D010446:Peptide Fragments; D000244:Adenosine Diphosphate; D006493:Heparin; D010763:Phosphorylase b; D006006:Glycogen Synthase; D010764:Phosphorylase Kinase; D014357:Trypsin; D002118:Calcium