Quantitation of energy coupling between Ca²⁺, calmodulin, skeletal muscle myosin light chain kinase, and kinase substrates

Interactions between Ca²⁺, calmodulin, rabbit skeletal muscle myosin light chain kinase, and kinase substrates were investigated by microequilibrium dialysis using ⁴⁵CaCl₂ and by fluorescence anisotropy using fluorescent labeled calmodulin. We have determined the free energy coupling for the interaction of Ca²⁺ and skeletal muscle myosin light chain kinase with calmodulin (ΔG(CM)), and the free energy coupling for the interaction of calmodulin and enzyme substrates with myosin light chain kinase (ΔG(SCaM)). The mean dissociation constants for Ca²⁺ interaction with calmodulin in the presence and absence of enzyme were 0.40 and 14 μM, respectively, yielding a minimal estimate for ΔG(CM) of -2.11 kcal/mol of Ca²⁺ (-8.44 kcal/4 mol of Ca²⁺). Dissociation constants for the interaction of fluorescent 5-iodo-amino-ethyl-amino-naphthalene-1-sulfonic acid-labeled calmodulin and native calmodulin with skeletal muscle myosin light chain kinase were 100 and 15 nM, respectively. In the presence of substrates (phosphorylatable skeletal muscle myosin light chain and App(NH)p) these dissociation constants were 30 and 3 nM, respectively. The free energy coupling for binding of calmodulin and substrates to myosin light chain kinase was -0.95 kcal/mol of calmodulin. These data confirm that interaction of Ca²⁺ with calmodulin is highly cooperative in the presence of myosin light chain kinase with the degree of cooperativity dependent upon the concentrations of Ca²⁺, enzyme, and substrates.