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ATP结合一致序列（p -环）的重链以及中间链和轻链。我们从嗜热四膜虫的14S动力蛋白中纯化出一种单体动力蛋白（dynein-a），并对其进行了表征。在体外运动试验中，dynein-a围绕微管的纵轴旋转微管，并使微管的正端进行易位。在低水平ADP存在的情况下，1mm ATP的ATP酶活性增加了一倍。在ADP存在的情况下，atp酶活性和跨界速度都符合Michaelis- Menten方程。然而，在没有ADP的情况下，这两种活性都不遵循michaelis - menten型动力学，这可能是由于两个atp结合位点的影响。我们的研究结果还表明，dynein-a具有一个对ADP非常敏感的ATP结合位点，并影响催化位点的ATP水解。本研究表明，动力蛋白分子的单体形式通过ATP和ADP与自身的直接结合来调节其活性，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.