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 stoichiometries 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 for form the complex, Ca42+-calmodulin. This complex then interacts with the inactive catalytic subunit of the enzyme to form the active holoenzyme complex, Ca42+-calmodulin-enzyme. Formation of the holoenzyme follows simply hyperbolic kinetics, indicating 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 were found to have values of 10 microM 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.
D000818:Animals; D001923:Brain Chemistry; D002118:Calcium / Q000494:pharmacology*; D002135:Calcium-Binding Proteins / Q000494:pharmacology*; D002147:Calmodulin / Q000494:pharmacology*; D002417:Cattle; D004789:Enzyme Activation; D007700:Kinetics; D046911:Macromolecular Substances; D008433:Mathematics; D009132:Muscles / Q000201:enzymology*; D009219:Myosin-Light-Chain Kinase; D009218:Myosins / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism*; D011817:Rabbits
D002135:Calcium-Binding Proteins; D002147:Calmodulin; D046911:Macromolecular Substances; D011494:Protein Kinases; D009219:Myosin-Light-Chain Kinase; D009218:Myosins; D002118:Calcium