Free energy coupling in the interactions between Ca2+, calmodulin, and phosphorylase kinase.
Interactions between Ca2+, exogenous calmodulin, and white skeletal muscle phosphorylase kinase have been quantitatively studied by equilibrium gel filtrations and analyzed by means of the so-called "linked functions" theory (Weber, G. (1975) Adv. Protein Chem. 29, 1-83). Four moles of calmodulin, each saturated with at least 3 Ca2+ ions, bind to 1 mol of phosphorylase kinase with a Kdiss of 2.3 nM. The activation of the enzyme as a function of free [Ca2+] shows that the intrinsic Ca-binding properties of phosphorylase kinase do not change upon binding of exogenous calmodulin, and confirms that alpha beta gamma delta X Ca3 is the functional catalytic unit through which activation occurs. Direct binding studies reveal that the intrinsic Ca-binding properties of the enzyme remain the same in the presence of either 0.5 or 8 mM Mg2+, indicating that phosphorylase kinase is endowed with Ca-specific sites. Upon interaction with the enzyme, calmodulin acquires strong positive cooperativity in Ca2+-binding: whereas its first two stoichiometric Ca-binding constants are not significantly different from those of free calmodulin, the third Ca2+ ion binds with an affinity at least 10(5)-fold higher than the corresponding one in free calmodulin. Calmodulin liganded with 1 or 2 Ca2+ displays the same low affinity for the enzyme as calmodulin depleted of Ca2+ (approximate Kdiss = 10(-4)-10(-3) M). The alpha beta gamma delta X calmodulin X Ca3 complex is strengthened by a free energy coupling of -8 kcal/mol upon complexation. The quantitative analysis of our results predicts that in spite of this high free energy barrier the dissociation of the complex (i.e. the inactivation of phosphorylase kinase) occurs rapidly upon lowering free [Ca2+].
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D004789:Enzyme Activation; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008433:Mathematics; D010764:Phosphorylase Kinase / Q000378:metabolism*; D011817:Rabbits
D002147:Calmodulin; D010764:Phosphorylase Kinase; D008274:Magnesium; D002118:Calcium