Kinetic analysis of the fastest motor protein, Chara myosin.

Chara corallina XI类肌球蛋白是目前最快的分子马达。为了研究这种快速运动的分子机制，我们对Chara肌球蛋白的重组运动结构域进行了动力学分析。我们通过测量ADP从肌动蛋白-马达结构域复合物中解离的速率常数和atp诱导的马达结构域从肌动蛋白中解离的速率常数来估计与肌动蛋白在强结合状态下花费的时间。在生理ATP浓度下，ADP与肌动蛋白分离的速率常数为2,200 s^<-1>。根据这些数据，与肌动蛋白处于强结合状态的时间估计为<0.82 ms。该值是各种肌凝蛋白已知值中最短的，其占空比<0.3，肌动蛋白激活的ATPase活性Vmax值为390 s 1。肌球蛋白Va的长颈结构域加入到Chara运动结构域，在不增加ATP水解循环速率的情况下，极大地提高了运动速度，与摆动杠杆模型一致。此外，本研究还揭示了Chara肌球蛋白适合快速运动的一些显著动力学特征：肌动蛋白释放ADP的速度急剧加快（1000倍），ATP结合速度极快。

Chara corallina class XI myosin is by far the fastest molecular motor. To investigate the molecular mechanism of this fast movement, we performed a kinetic analysis of a recombinant motor domain of Chara myosin. We estimated the time spent in the strongly bound state with actin by measuring rate constants of ADP dissociation from actin-motor domain complex and ATP-induced dissociation of the motor domain from actin. The rate constant of ADP dissociation from acto-motor domain was >2,800 s^<-1> and the rate constant of ATP-induced dissociation of the motor domain from actin at physiological ATP concentration was 2,200 s^<-1>. From these data, the time spent in the strongly bound state with actin was estimated to be <0.82 ms. This value is the shortest among known values for various myosins, and yields the duty ratio of <0.3 with the Vmax value of the actin-activated ATPase activity of 390 s 1. The addition of the long neck domain of myosin Va to the Chara motor domain largely increased the velocity of the motility without increasing the ATP hydrolysis cycle rate, consistent with the swinging lever model. In addition, this study reveals some striking kinetic features of Chara myosin that are suited for the fast movement: a dramatic acceleration of ADP release by actin (1,000-fold) and extremely fast ATP binding rate.