Activation of skeletal muscle myosin light chain kinase by calcium(2+) and calmodulin

Publisher Website
Google Scholar
Abstract
Many biological processes are regulated by Ca2+ via calmodulin (CM). Although a general mechanistic model by which Ca2+ and calmodulin modulate many of these activities was proposed, an accurate quantitative model is not available. A detailed analysis of skeletal muscle myosin light chain kinase activation was undertaken to determine the stoichiometries and equilibrium constants of Ca2+, calmodulin and enzyme catalytic subunit in the activation process. Activation apparently is a sequential, fully reversible process requiring Ca2+ and calmodulin. The 1st step of the activation process appears to require binding of Ca2+ to all 4 divalent metal binding sites on calmodulin to form the complex, Ca42+-calmodulin. This complex interacts with the inactive catalytic subunit of the enzyme to form the active holoenzyme complex, Ca42+-calmodulin-enzyme. Formation of the holoenzyme follows simple hyperbolic kinetics, indicating a 1:1 stoichiometry of Ca42+-calmodulin to catalytic subunit. The rate equation derived from the mechanistic model was used to determine the values of KCa2+ and KCM, the intrinsic activation constants for each step of the activation process. KCa2+ and KCM had values of 10 .mu.M 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. These kinetic model and analytical techniques employed may be generally applicable to other enzymes with similar regulatory schemes.