KINETIC ANALYSIS OF THE MICROTUBULE NCD ATPASE

这项建议的长期目标是建立结构和 一般生物马达产生力的机械基础， 基于微管的ATP酶特别是驱动蛋白和ncd。 驱动蛋白是一种 微管依赖性马达负责细胞内运动， 轴突中的膜性细胞器。 它是第一个被鉴定的蛋白质， 一类与驱动蛋白相关的ATP酶， 生物过程。 驱动蛋白超家族的成员 在驱动蛋白马达结构域内具有显著的序列同源性，并且每个 驱动蛋白相关蛋白含有高度同源的短片段 在ATP结合口袋内，微管结合结构域以及 以及t=功能上尚未定义的其他区域。 另外每个 驱动蛋白相关蛋白含有一个独特的非运动域， 被认为赋予了马达生物特异性。 Ncd是一种驱动蛋白- 参与雌性减数分裂和胚胎有丝分裂的相关蛋白质。 这是一 与驱动蛋白相比，研究有趣的运动，因为有40-45% 在马达结构域的约350个氨基酸内具有序列同一性， 这意味着蛋白质将具有非常相似的三维结构， 结构. 然而，作为发动机，这两种ATP酶是截然不同的。 Ncd促进微管易位的相反方向 在驱动蛋白中，易位明显比驱动蛋白慢， 迄今为止的实验表明，NCD不是一种进行性ATP酶， 驱动蛋白 本文提出的研究目标是了解 这些差异的机制基础。 具体目标是：（1） 测量反应途径中每个步骤的动力学， 微管-ncd ATP酶，2）以确定ncd ATP酶是否 在与微管的相互作用中是进行性的或分布性的。 到 要实现这些目标，需要一个详细而全面的动力学 用预稳态动力学技术进行热力学研究 包括快速骤冷和停流方法。 只有这样 直接测量酶活性位点上的事件可以 获得机械信息以了解能量的差异 通过ncd和驱动蛋白ATP酶的转导。

The long term aims of this proposal are to establish the structural and mechanistic basis for force production by biological motors in general and the microtubule-based ATPases kinesin and ncd specifically. Kinesin is a microtubule dependent motor responsible for the intracellular movements of membranous organelles in axons. It is the first characterized protein of a class of kinesin-related ATPases implicated ina wide variety of biological processes. The members of the kinesin superfamily all share significant sequence homology within the kinesin motor domain, and each kinesin-related protein contains short segments of extremely high homology within the ATP binding pocket, the microtubule binding domain as well as well as t=other areas not yet defined functionally. In addition, each kinesin-related protein contains a nonmotor domain that is unique and is believed to confer biological specificity to the motor. Ncd is a kinesin- related protein involved in female meiosis and embryonic mitosis. It is an interesting motor to study in comparison to kinesin because there is 40-45% sequence identity within about 350 amino acids of the motor domain which implies that the proteins will have very similar three-dimensional structures. Yet as motors, the two ATPases are distinctively different. Ncd promotes microtubule translocations in the opposite direction of kinesin, translocations are significantly slower than kinesin's, and the experiments to date suggest that ncd is not a processive ATPase as is kinesin. The goal of the research proposed here is to understand the mechanistic basis of these differences. The specific aims are 1) to measure the kinetics of each step in the reaction pathway of the microtubule-ncd ATPase, 2) to determine whether the ncd ATPase is processive or distributive in its interactions with the microtubule. To accomplish these goals will require a detailed and comprehensive kinetic and thermodynamic investigation using presteady state kinetic techniques including rapid quench and stopped-flow approaches. It is only by this direct measurement of event at the active site of the enzyme can the mechanistic information be obtained to understand the differences in energy transduction by the ncd and kinesin ATPases.