Sato Osamu, Li Xiang-Dong, Ikebe Mitsuo
Department of Physiology, University of Massachusetts Medical School, Worcester, Massachueetts 01655, USA.
J Biol Chem. 2007 May 4;282(18):13228-39. doi: 10.1074/jbc.M610766200. Epub 2007 Mar 14.
Vertebrate myosin Va is a typical processive motor with high duty ratio. Recent studies have revealed that the actin-activated ATPase activity of the full-length myosin Va (M5aFull) is inhibited at a low [Ca(2+)], which is due to the formation of a folded conformation of M5aFull. To clarify the underlying inhibitory mechanism, we analyzed the actin-activated ATP hydrolysis mechanism of the M5aFull at the inhibited and the activated states, respectively. Marked differences were found in the hydrolysis, P(i) release, and ADP release steps between the activated and the inhibited states. The kinetic constants of these steps of the activated state were similar to those of the unregulated S1 construct, in which the rate-limiting step was the ADP release step. On the other hand, the P(i) release rate from acto-M5aFull was decreased in EGTA by >1,000-fold, which makes this step the rate-limiting step for the actin-activated ATP hydrolysis cycle of M5aFull. The ADP off rate from acto-M5aFull was decreased by approximately 10-fold, and the equilibrium between the prehydrolysis state and the post hydrolysis state was shifted toward the former state in the inhibited state of M5aFull. Because of these changes, M5aFull spends a majority of the ATP hydrolysis cycling time in the weak actin binding state. The present results indicate that M5aFull molecules at a low [Ca(2+)] is inhibited as a cargo transporter not only due to the decrease in the cross-bridge cycling rate but also due to the decrease in the duty ratio thus being dissociated from actin.
脊椎动物肌球蛋白Va是一种典型的具有高占空比的持续性马达蛋白。最近的研究表明，全长肌球蛋白Va（M5aFull）的肌动蛋白激活的ATP酶活性在低[Ca(2+)]时受到抑制，这是由于M5aFull形成了折叠构象。为了阐明潜在的抑制机制，我们分别分析了M5aFull在抑制状态和激活状态下的肌动蛋白激活的ATP水解机制。在激活状态和抑制状态之间，水解、无机磷酸（P(i)）释放和ADP释放步骤存在显著差异。激活状态下这些步骤的动力学常数与未调节的S1构建体相似，其中限速步骤是ADP释放步骤。另一方面，在乙二醇双四乙酸（EGTA）存在下，肌动蛋白-M5aFull的P(i)释放速率降低了1000倍以上，这使得该步骤成为M5aFull肌动蛋白激活的ATP水解循环的限速步骤。肌动蛋白-M5aFull的ADP解离速率降低了约10倍，并且在M5aFull的抑制状态下，预水解状态和水解后状态之间的平衡向前者状态移动。由于这些变化，M5aFull在ATP水解循环的大部分时间处于弱肌动蛋白结合状态。目前的结果表明，低[Ca(2+)]时的M5aFull分子作为货物转运体受到抑制，不仅是由于横桥循环速率的降低，还由于占空比的降低，从而与肌动蛋白解离。
