The role of multiple ATP-binding sites in dynein motor domain

动力蛋白是一种巨大的复杂分子，包括具有多个（4个）ATP结合共有序列（P环）的重链以及中间链和轻链。我们从嗜热四膜虫的14 S动力蛋白中分离纯化了一种动力蛋白单体（dynein-a），并对其进行了表征。ATP酶活性在1 mM ATP的存在下，在低水平的ADP的两倍。ATP酶活性和跨膜速度均符合米氏方程.而在ADP不存在时，两种酶的活性均不符合米氏动力学，这可能是由于两个ATP结合位点的作用。我们的研究结果还表明，动力蛋白-a有一个ATP结合位点，这是非常敏感的ADP和影响ATP水解的催化位点。这项研究表明，单体形式的动力蛋白分子通过直接与ATP和ADP结合来调节其活性，因此动力蛋白分子具有分子内调节系统。

Dynein is a huge complex molecule including heavy chain(s) which have multiple (4) ATP binding consensus sequences (P-loops) as well as intermediate and light chains. We purified a monomeric form of dynein (dynein-a) from 14S dyneins of Tetrahymena thermophila and characterized it. In in vitro motility assays, dynein-a rotated microtubules around their longitudinal axis as well as translocated them with their plus-ends leading. ATPase activity at 1 mM ATP was doubled in the presence of a low level of ADP. Both ATPase activity and transnational velocities in the presence of ADP fit the Michaelis- Menten equation well. However in the absence of ADP, neither of the activities followed the Michaelis-Menten-type kinetics probably due to the effect of two-ATP binding sites. Our results also indicate that dynein-a has an ATP-binding site that is very sensitive to ADP and affects ATP hydrolysis at the catalytic site. This study shows that a monomeric form of dynein molecule regulates its activity by direct binding of ATP and ADP to itself, and thus the dynein molecule has an intramolecular regulating system.