Gyimesi Máté, Kintses Bálint, Bodor Andrea, Perczel András, Fischer Stefan, Bagshaw Clive R, Málnási-Csizmadia András
Department of Biochemistry, Institute of Biology, Eötvös University, Pázmány Péter Sétány 1/A, Budapest, Hungary.
J Biol Chem. 2008 Mar 28;283(13):8153-63. doi: 10.1074/jbc.M708863200. Epub 2008 Jan 21.
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.
肌球蛋白基础ATP酶（即不存在肌动蛋白时）的限速步骤被认为是杠杆臂水解后的摆动（反向恢复步骤），这限制了随后的快速产物释放步骤。然而，缺乏关于这一归属的直接实验证据。为了独立于杠杆臂运动研究ADP和磷酸的结合与释放，使用了盘基网柄菌肌球蛋白II的两个含单个色氨酸的运动结构域。W129 +和W501 +构建体的单个色氨酸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（正向速率常数k(forward) = 0.05 s(-1)，反向速率常数k(reverse) = 0.15 s(-1)），在磷酸释放步骤之前。肌动蛋白直接激活反向恢复步骤，在肌动蛋白结合形式下该步骤实际上变得不可逆，从而引发动力冲程。即使在低肌动蛋白浓度下，尽管在动力冲程前构象中肌球蛋白对肌动蛋白的亲和力较低，但动力冲程仍在肌动蛋白附着状态下发生。
