Abstract
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+].
Highlights
It is generallyaccepted that CaM-Ca” activation of enzymes proceedsvia thesequentialformation of two fully reversible complexes as originally proposed for Ca2+-dependent cyclic nucleotide phosphodiesterase (10-12): the presence of either 0.5 or 8 mM Mg2+,indicating that phosphorylase kinase is endowed with Ca-specific
Whereas its first two stoichiometric Ca-binding constants arenot significantly different from those of free According to this scheme, at saturationof a critical number calmodulin, the thirdCa2+ion binds with an affinityat of sites, CaMundergoes aconformational change anbdecomes least 106-fold higher than the corresponding one in a biologically active species, designated as CaM*
- displays the same low affinity for the enzyme as cal- (13)studying the activationof myosin light chain kinase, and modulin depleted of Ca2+(approximate Kdiss =
Summary
It is generallyaccepted that CaM-Ca” activation of enzymes proceedsvia thesequentialformation of two fully reversible complexes as originally proposed for Ca2+-dependent cyclic nucleotide phosphodiesterase (10-12): the presence of either 0.5 or 8 mM Mg2+,indicating that phosphorylase kinase is endowed with Ca-specific. Whereas its first two stoichiometric Ca-binding constants arenot significantly different from those of free According to this scheme, at saturationof a critical number calmodulin, the thirdCa2+ion binds with an affinityat of sites, CaMundergoes aconformational change anbdecomes least 106-fold higher than the corresponding one in a biologically active species, designated as CaM*. The aflyh.calmodulin.Ca, complex is strength- equilibrium reactions of the above scheme to onesingle equaened by a free energy coupling of -8 kcal/mol upon tion, which was linearized assuming thatonly the fully satucomplexation. According to their experimental data, these authorcsoncluded that CaM. In thcaese of activation of bovine brain phosphodiesterase(17),adenylate cyclase (18), and human
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