Mechanism of calmodulin inhibition of cAMP-dependent protein kinase activation of phosphorylation kinase.
The activation of phosphorylase kinase (EC 2.7.1.38; ATP:phosphorylase b phosphotransferase) by the catalytic subunit of cAMP-dependent protein kinase (EC 2.7.1.37; ATP:protein phosphotransferase) is inhibited by calmodulin. The mechanism of that inhibition has been studied by kinetic measurements of the interactions of the three proteins. The binding constant for calmodulin with phosphorylase kinase was found to be 90 nM when measured by fluorescence polarization spectroscopy. Glycerol gradient centrifugation studies indicated that 1 mol of calmodulin was bound to each phosphorylase kinase. Phosphorylation of the phosphorylase kinase did not reduce the amount of calmodulin bound. Kinetic studies of the activity of the catalytic subunit of cAMP-dependent protein kinase on phosphorylase kinase as a function of phosphorylase kinase and calmodulin concentrations were performed. The results of those studies were compared with mathematical models of four different modes of inhibition: competitive, noncompetitive, substrate depletion, and inhibition by a complex between phosphorylase kinase and calmodulin. The data conform best to the model in which the inhibitory species is a complex of phosphorylase kinase and calmodulin. The complex apparently competes with the substrate, phosphorylase kinase, which does not have exogenous calmodulin bound to it. In contrast, the phosphorylation of the synthetic phosphate acceptor peptide, Kemptide, is not inhibited by calmodulin.
Department of Biochemistry, University of Minnesota, Medical School, Minneapolis 55455.
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D000255:Adenosine Triphosphate / Q000378:metabolism; D000465:Algorithms; D000818:Animals; D002147:Calmodulin / Q000494:pharmacology*; D002417:Cattle; D004789:Enzyme Activation; D007700:Kinetics; D009282:Naphthalenesulfonates / Q000378:metabolism; D010764:Phosphorylase Kinase / Q000378:metabolism*; D047428:Protein Kinase Inhibitors; D013816:Thermodynamics
C032162:AEDANS-calmodulin; D002147:Calmodulin; D009282:Naphthalenesulfonates; D047428:Protein Kinase Inhibitors; D000255:Adenosine Triphosphate; D010764:Phosphorylase Kinase