Calmodulin-dependent protein kinase II. Kinetic studies on the interaction with substrates and calmodulin.
The kinetic reaction mechanism of calmodulin (CaM)-dependent protein kinase II (CaM-kinase II), including the regulatory mechanism by CaM, was studied by using microtubule-associated protein 2 (MAP2) as substrate under steady-state conditions. The detailed kinetic analyses of the phosphorylation of MAP2 and its inhibitions by the reaction products and by an ATP analogue, 5'-adenylylimidodiphosphate, revealed the rapid-equilibrium random mechanism. In the absence of Ca2+, CaM-kinase II was inactivated by incubation with ATP. The inactivation rate was dependent on the concentrations of ATP and MAP2, suggesting that these substrates can bind to the enzyme even in the absence of Ca2+/CaM. The activation of the enzyme by CaM reached the maximum when about 10 mol of CaM bound to 1 mol of CaM-kinase II, indicating the stoichiometry of the binding of one CaM to one subunit of the enzyme. The enzyme activity as a function of the concentration of CaM showed a sigmoidal curve. The concentration of CaM required for the half-maximal activation was dependent on the concentration of ATP at a fixed concentration of MAP2, although the Hill coefficient was unaffected by the concentration of ATP. A possible reaction mechanism of CaM-kinase II, including the phosphorylation of MAP2 by the enzyme and the binding of CaM to the enzyme, is discussed.
Katoh
T
T
Department of Biochemistry, Asahikawa Medical College, Japan.
Fujisawa
H
H
D000255:Adenosine Triphosphate / Q000378:metabolism; D000266:Adenylyl Imidodiphosphate / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002147:Calmodulin / Q000378:metabolism* / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D007700:Kinetics; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D008956:Models, Chemical; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000378:metabolism*; D013379:Substrate Specificity
D002147:Calmodulin; D008869:Microtubule-Associated Proteins; D047428:Protein Kinase Inhibitors; D000266:Adenylyl Imidodiphosphate; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002118:Calcium