Department of Physiology, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, Massachusetts 01655-0127, USA.
Biochemistry. 2010 May 4;49(17):3695-702. doi: 10.1021/bi902211w.
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 K(actin) value of the steady-state ATPase reaction. The rate constant of the P(i) 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 huM3AMD in 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.
先前的研究结果表明，人肌球蛋白 IIIA（HM3A）的运动活性受到磷酸化的影响[Kambara，T.等人。（2006）J. Biol. Chem. 281，37291-37301]；然而，磷酸化如何控制 HM3A 的运动活性尚不清楚。在这项研究中，我们阐明了磷酸化对 HM3A 运动结构域（huM3AMD）的 ATP 水解循环的影响的动力学基础。在存在 ATP 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 多倍，而 ATP 周转率的最大速率几乎不变。与总循环速率相比，磷酸化的 huM3AMD 从肌动蛋白释放 ADP 的速率增加了 6 倍，因此不是限速步骤。磷酸化使与肌动蛋白解离的形式的 ATP 水解步骤的速率常数显著增加了 30 倍。磷酸化大大增加了 huM3AMD 从肌动蛋白上结合的 ATP 形式的解离常数，这一结果与稳态 ATPase 反应中 K（肌动蛋白）值的显著增加一致。P(i) 脱离步骤的速率常数大于 60 s(-1)，表明该步骤不会限制整体 ATP 水解循环速率。我们的动力学模型表明，磷酸化诱导 huM3AMD 在 ATP 水解循环过程中与肌动蛋白解离，这是由于磷酸化依赖性 huM3AMD 对肌动蛋白的亲和力显著降低以及在与肌动蛋白解离的状态下 huM3AMD 的 ATP 水解速率增加所致。这些结果表明，肌球蛋白 IIIA 的磷酸化显著降低了 duty ratio，这可能会影响含有尾部非依赖于 ATP 的肌动蛋白结合位点的天然形式的肌球蛋白 IIIA 的货物运输能力。
