The mechanism of the reverse recovery step, phosphate release, and actin activation of Dictyostelium myosin II
The mechanism of the reverse recovery step, phosphate release, and actin activation of Dictyostelium myosin II
复制标题
DOI：
10.1074/jbc.m708863200
复制
发表时间：
2008-03-28
影响因子：
4.8
通讯作者：
Malnasi-Csizmadia, Andras
中科院分区：
文献类型：
--
作者：
Gyimesi, Mate;Kintses, Balint;Malnasi-Csizmadia, Andras
假设肌球蛋白基础ATP酶的限速步骤（即，在不存在肌动蛋白的情况下）是杠杆臂的水解后摆动（反向恢复步骤），其限制了随后的快速产物释放步骤。然而，这一任务的直接实验证据是缺乏的。为了研究独立于杠杆臂运动的ADP和磷酸盐的结合和释放，使用了盘基网柄菌肌球蛋白II的两个单个含色氨酸的运动域。W129+和W501+构建体的单个双链体分别位于核苷酸结合口袋的入口处和杠杆臂附近。动力学实验表明，基础ATP酶循环中的限速步骤确实是反向恢复步骤，这是一个缓慢的平衡步骤（k（正向）= 0.05 s（-1），k（反向）= 0.15 s（-1）），在磷酸盐释放步骤之前。肌动蛋白直接激活反向恢复步骤，这在肌动蛋白结合形式中几乎变得不可逆，触发动力冲程。即使在低肌动蛋白浓度的动力中风发生在肌动蛋白附着状态，尽管低肌动蛋白亲和力的肌球蛋白在前动力中风的构象。
The rate-limiting step of the myosin basal ATPase (i.e. in absence of actin) is assumed to be a post-hydrolysis swinging of the lever arm ( reverse recovery step), that limits the subsequent rapid product release steps. However, direct experimental evidence for this assignment is lacking. To investigate the binding and the release of ADP and phosphate independently from the lever arm motion, two single tryptophan-containing motor domains of Dictyostelium myosin II were used. The single tryptophans of the W129+ and W501+ constructs are located at the entrance of the nucleotide binding pocket and near the lever arm, respectively. Kinetic experiments show that the rate-limiting step in the basal ATPase cycle is indeed the reverse recovery step, which is a slow equilibrium step (k(forward) = 0.05 s(-1), k(reverse) = 0.15 s(-1)) that precedes the phosphate release step. Actin directly activates the reverse recovery step, which becomes practically irreversible in the actin-bound form, triggering the power stroke. Even at low actin concentrations the power stroke occurs in the actin-attached states despite the low actin affinity of myosin in the pre-power stroke conformation.