Activation of Skeletal Muscle Myosin Light Chain Kinase by Calcium(2+) and Calmodulin

Many biological processes are now known to be regulated by Ca²⁺ via calmodulin (CM). Although a general mechanistic model by which Ca²⁺ and calmodulin modulate many of these activities has been proposed, an accurate quantitative model is not available. A detailed analysis of skeletal muscle myosin light chain kinase activation was undertaken in order to determine the stoichiometrics and equilibrium constants of Ca²⁺, calmodulin, and enzyme catalytic subunit in the activation process. The analysis indicates that activation is a sequential, fully reversible process requiring both Ca²⁺ and calmodulin. The first step of the activation process appears to require binding of Ca²⁺ to all four divalent metal binding sites on calmodulin to form the complex, Ca₄ ²⁺•calmodulin. This complex then interacts with the inactive catalytic subunit of the enzyme to form the active holoenzyme complex, Ca₄ ²⁺•calmodulin-enzyme. Formation of the holoenzyme follows simple hyperbolic kinetics, indicating a 1 : 1 stoichiometry of Ca₄ ²⁺•calmodulin to catalytic subunit. The rate equation derived from the mechanistic model was used to determine the values of K_Ca ²⁺ and K_CM, the intrinsic activation constants for each step of the activation process. K_Ca ²⁺ and K_CM were found to have values of 10 µM and 0.86 nM, respectively, at 10 mM Mg²⁺. The rate equation using these equilibrium constants accurately predicts the extent of enzyme activation over a wide range of Ca²⁺ and calmodulin concentrations. The kinetic model and analytical techniques employed herein may be generally applicable to other enzymes with similar regulatory schemes.