Mogami Toshifumi, Kon Takahide, Ito Kohji, Sutoh Kazuo
Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, Komaba 3-8-1, Tokyo, Japan.
J Biol Chem. 2007 Jul 27;282(30):21639-44. doi: 10.1074/jbc.M701914200. Epub 2007 Jun 4.
According to the power stroke model of dynein deduced from electron microscopic and fluorescence resonance energy transfer studies, the power stroke and the recovery stroke are expected to take place at the two isomerization steps of the ATPase cycle at the primary ATPase site. Here, we have conducted presteady-state kinetic analyses of these two isomerization steps with the single-headed motor domain of Dictyostelium cytoplasmic dynein by employing fluorescence resonance energy transfer to probe ATPase steps at the primary site and tail positions. Our results show that the recovery stroke at the first isomerization step proceeds quickly ( approximately 180 s(-1)), whereas the power stroke at the second isomerization step is very slow ( approximately 0.2 s(-1)) in the absence of microtubules, and that the presence of microtubules accelerates the second but not the first step. Moreover, a comparison of the microtubule-induced acceleration of the power stroke step and that of steady-state ATP hydrolysis implies the intriguing possibility that microtubules simultaneously accelerate the ATPase activity not only at the primary site but also at other site(s) in the motor domain.
根据从电子显微镜和荧光共振能量转移研究中推导出来的动力蛋白的动力冲程模型，动力冲程和恢复冲程预计在初级ATP酶位点的ATP酶循环的两个异构化步骤发生。在这里，我们通过利用荧光共振能量转移来探测初级位点和尾部位置的ATP酶步骤，对盘基网柄菌细胞质动力蛋白的单头运动结构域的这两个异构化步骤进行了预稳态动力学分析。我们的结果表明，在没有微管的情况下，第一个异构化步骤的恢复冲程进行得很快（约180 s⁻¹），而第二个异构化步骤的动力冲程非常缓慢（约0.2 s⁻¹），并且微管的存在加速了第二个步骤而非第一个步骤。此外，对微管诱导的动力冲程步骤加速和稳态ATP水解加速的比较暗示了一种有趣的可能性，即微管不仅同时加速初级位点的ATP酶活性，还加速运动结构域中其他位点的ATP酶活性。
