Schief William R, Clark Rutilio H, Crevenna Alvaro H, Howard Jonathon
Department of Physiology and Biophysics, University of Washington, Seattle, WA 98195, USA.
Proc Natl Acad Sci U S A. 2004 Feb 3;101(5):1183-8. doi: 10.1073/pnas.0304369101. Epub 2004 Jan 20.
The motor protein kinesin couples a temporally periodic chemical cycle (the hydrolysis of ATP) to a spatially periodic mechanical cycle (movement along a microtubule). To distinguish between different models of such chemical-to-mechanical coupling, we measured the speed of movement of conventional kinesin along microtubules in in vitro motility assays over a wide range of substrate (ATP) and product (ADP and inorganic phosphate) concentrations. In the presence and absence of products, the dependence of speed on [ATP] was well described by the Michaelis-Menten equation. In the absence of products, the K(M) (the [ATP] required for half-maximal speed) was 28 +/- 1 microM, and the maximum speed was 904 nm/s. P(i) behaved as a competitive inhibitor with K(I) = 9 +/- 1 mM. ADP behaved approximately as a competitive inhibitor with K(I) = 35 +/- 2 microM. The data were compared to four-state kinetic models in which changes in nucleotide state are coupled to chemical and/or mechanical changes. We found that the deviation from competitive inhibition by ADP was inconsistent with models in which P(i) is released before ADP. This is surprising because all known ATPases (and GTPases) with high structural similarity to the motor domains of kinesin release P(i) before ADP (or GDP). Our result is therefore inconsistent with models, such as one-headed and inchworm mechanisms, in which the hydrolysis cycle takes place on one head only. However, it is simply explained by hand-over-hand models in which ADP release from one head precedes P(i) release from the other.
驱动蛋白这种马达蛋白将一个时间上周期性的化学循环（ATP水解）与一个空间上周期性的机械循环（沿微管移动）耦合起来。为了区分这种化学 - 机械耦合的不同模型，我们在体外运动分析中，在广泛的底物（ATP）和产物（ADP和无机磷酸）浓度范围内，测量了传统驱动蛋白沿微管的移动速度。在有和没有产物存在的情况下，速度对[ATP]的依赖性都能很好地用米氏方程来描述。在没有产物时，K(M)（达到最大速度一半时所需的[ATP]）为28±1微摩尔，最大速度为904纳米/秒。无机磷酸表现为竞争性抑制剂，K(I)=9±1毫摩尔。ADP表现得近似为竞争性抑制剂，K(I)=35±2微摩尔。我们将这些数据与四态动力学模型进行了比较，在这些模型中核苷酸状态的变化与化学和/或机械变化相耦合。我们发现，ADP对竞争性抑制的偏离与无机磷酸在ADP之前释放的模型不一致。这很令人惊讶，因为所有与驱动蛋白的马达结构域具有高度结构相似性的已知ATP酶（和GTP酶）都是在ADP（或GDP）之前释放无机磷酸。因此，我们的结果与诸如单头和尺蠖机制等模型不一致，在这些模型中水解循环仅在一个头部发生。然而，这可以通过双手交替模型简单地解释，在该模型中一个头部的ADP释放先于另一个头部的无机磷酸释放。
