Katoh T, Fujisawa H
Department of Biochemistry, Asahikawa Medical College, Japan.
Biochim Biophys Acta. 1991 Jan 31;1091(2):205-12. doi: 10.1016/0167-4889(91)90063-4.
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.
在稳态条件下，以微管相关蛋白2（MAP2）为底物，研究了钙调蛋白（CaM）依赖性蛋白激酶II（CaM激酶II）的动力学反应机制，包括CaM的调节机制。对MAP2磷酸化及其受反应产物和ATP类似物5'-腺苷酰亚胺二磷酸抑制的详细动力学分析揭示了快速平衡随机机制。在没有Ca2+的情况下，CaM激酶II与ATP孵育会失活。失活速率取决于ATP和MAP2的浓度，这表明即使在没有Ca2+/CaM的情况下，这些底物也能与酶结合。当约10摩尔的CaM与1摩尔的CaM激酶II结合时，CaM对酶的激活达到最大值，表明一个CaM与酶的一个亚基结合的化学计量关系。酶活性作为CaM浓度的函数呈S形曲线。在MAP2固定浓度下，半最大激活所需的CaM浓度取决于ATP浓度，尽管希尔系数不受ATP浓度影响。讨论了CaM激酶II可能的反应机制，包括该酶对MAP2的磷酸化以及CaM与该酶的结合。
