KINETIC ANALYSIS OF THE MICROTUBULE NCD ATPASE

这项建议的长期目标是建立结构性和 生物马达作用力的一般力学基础和 以微管为基础的ATPase特异性地激活了激动素和NCD。激动素是一种 微管依赖的运动负责细胞内的运动 轴突中的膜质细胞器。它是第一个具有特征性的蛋白质 一类与激动素相关的ATPase与多种 生物过程。Kinesin超家族的成员都有 运动蛋白结构域中显著的序列同源性，并且每个 动蛋白相关蛋白含有极高同源性的短片段 在ATP结合口袋内，微管结合结构域以及 以及t=尚未在功能上定义的其他区域。此外，每个 动蛋白相关蛋白包含一个独特的非运动结构域， 被认为赋予了马达生物专一性。NCD是一种激动素- 参与雌性减数分裂和胚胎有丝分裂的相关蛋白。这是一个 与激动素相比，运动研究很有趣，因为有40%-45% 运动区约350个氨基酸内的序列同源性 意味着这些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ATPase是截然不同的。 NCD促进微管向相反方向移位 动蛋白，易位明显比动蛋白慢得多，而且 迄今为止的实验表明，NCD并不是一种进行性的ATPase 激动素。这里提出的研究的目标是了解 这些差异的机制基础。具体目标是：1) 测量反应路径中每一步的动力学 微管-NCD-ATPase，2)确定NCD-ATPase是否 与微管相互作用呈进行性或分布性。至 实现这些目标将需要一个详细和全面的动力 以及使用稳态前动力学技术进行热力学研究 包括快速急冷和停流方法。只有这样才能做到 直接测量酶活性部位的事件可以 获得机械信息以了解能量的差异 NCD和Kinesin ATPase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.