Effect of phosphorylation in the motor domain of human myosin IIIA on its ATP hydrolysis cycle.
Effect of phosphorylation in the motor domain of human myosin IIIA on its ATP hydrolysis cycle.
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DOI：
10.1021/bi902211w
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发表时间：
2010-05-04
期刊：
影响因子：
2.9
通讯作者：
Ikebe M
中科院分区：
文献类型：
--
作者：
Komaba S;Watanabe S;Umeki N;Sato O;Ikebe M
先前的研究表明，人类肌球蛋白IIIA （HM3A）的运动活性受到磷酸化的影响[Kambara， T., et al.(2006)]。化学学报，28 (1),37291-37301；然而，磷酸化如何控制HM3A的运动活性尚不清楚。在本研究中，我们阐明了磷酸化对HM3A （huM3AMD）马达结构域ATP水解周期影响的动力学基础。在ATP存在的情况下，人肌球蛋白IIIA对丝状肌动蛋白的亲和力因磷酸化而降低了100倍以上，而ATP的最大周转率几乎没有变化。从动磷酸化的huM3AMD释放ADP的速率是整个循环速率的6倍，因此不是速率决定步骤。肌动蛋白解离形式的ATP水解步骤的速率常数因磷酸化而显著增加了30倍。磷酸化使atp结合形式的huM3AMD与肌动蛋白解离的解离常数大大增加，这一结果与稳态atp酶反应中Kactin值的显著增加非常吻合。Pi off步骤的速率常数大于60 s−1，表明该步骤不限制ATP水解循环的总速率。我们的动力学模型表明，在ATP水解周期中，磷酸化诱导了huM3AMD与肌动蛋白的解离，这是由于磷酸化依赖性的huM3AMD·ATP对肌动蛋白的亲和力显著降低，而在肌动蛋白解离状态下，huM3AMDin的ATP水解速率增加。这些结果表明，肌凝蛋白IIIA的磷酸化显著降低了占空比，这可能会影响天然形式的肌凝蛋白IIIA的货物运输能力，后者在尾部含有不依赖atp的肌动蛋白结合位点。
Previous findings suggested that the motor activity of human myosin IIIA (HM3A) is influenced by phosphorylation [Kambara, T., et al. (2006) J. Biol. Chem. 281, 37291–37301]; however, how phosphorylation controls the motor activity of HM3A is obscure. In this study, we clarify the kinetic basis of the effect of phosphorylation on the ATP hydrolysis cycle of the motor domain of HM3A (huM3AMD). The affinity of human myosin IIIA for filamentous actin in the presence of ATP is more than 100-fold decreased by phosphorylation, while the maximum rate of ATP turnover is virtually unchanged. The rate of release of ADP from acto-phosphorylated huM3AMD is 6-fold greater than the overall cycle rate, and thus not a rate-determining step. The rate constant of the ATP hydrolysis step of the actin-dissociated form is markedly increased by phosphorylation by 30-fold. The dissociation constant for dissociation of the ATP-bound form of huM3AMD from actin is greatly increased by phosphorylation, and this result agrees well with the significant increase in the Kactin value of the steady-state ATPase reaction. The rate constant of the Pi off step is greater than 60 s−1, suggesting that this step does not limit the overall ATP hydrolysis cycle rate. Our kinetic model indicates that phosphorylation induces the dissociation of huM3AMD from actin during the ATP hydrolysis cycle, and this is due to the phosphorylation-dependent marked decrease in the affinity of huM3AMD· ATP for actin and the increase in the ATP hydrolysis rate of huM3AMDin the actin-dissociated state. These results suggest that the phosphorylation of myosin IIIA significantly lowers the duty ratio, which may influence the cargo transporting ability of the native form of myosin IIIA that contains the ATP-independent actin binding site in the tail.
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