Pechatnikova E, Taylor E W
Department of Molecular Genetics and Cell Biology, The University of Chicago, Chicago, Illinois 60637, USA.
Biophys J. 1999 Aug;77(2):1003-16. doi: 10.1016/S0006-3495(99)76951-1.
The kinetic mechanism of the nonclaret disjunctional protein (Ncd) motor was investigated using the dimer termed MC1 (residues 209-700), which has been shown to exhibit negative-end directed motility (Chandra et al., 1993). The kinetic properties are similar to those of the monomeric Ncd motor domain (Pechatnikova and Taylor, 1997). The maximum steady-state ATPase activity of 1.5 s(-1) is half as large as for the monomeric motor. Dissociation constants in the presence of nucleotides showed the same trend but with approximately a two-fold decrease in the values: K(d) values are 1.0 microM for ADP-AlF(4), 1.1 microM for ATPgammaS, 1.5 microM for ATP, 3 microM for ADP, and 10 microM for ADP-vanadate (in 25 mM NaCl, 22 degrees C). The apparent second-order rate constants for the binding of ATP and ADP to the microtubule-motor complex (MtMC1) are 2 microM(-1) s(-1). Based on measurements at high microtubule concentrations the kinetic steps were fitted to the scheme,[see text] where N refers to one head of the dimer and T, D, and P stand for ATP, ADP, and inorganic phosphate. k(1) and k(-4) are the first-order rate constants of the transition induced by the binding of mant ATP and mant ADP respectively. ADP release is the main rate-limiting step in the MtMC1 mechanism. The binding of the MC1-mant ADP complex to microtubules released less than half of the mant ADP (alternating site reactivity). The second mant ADP is only released by the binding of nucleotides that dissociate the MtMC1 complex (ATP and ADP but not AMPPNP). The apparent rate constant for dissociation of the second mant ADP is four times smaller than the first and much smaller than the rate of dissociation of MtMC1 by ATP or ADP. These results are explained by a model in which MC1.ADP is first dissociated from the microtubule by ATP, followed by rebinding to the microtubule by the ADP-containing head. Ncd may follow a different reaction pathway than does kinesin, but the differences in rate constants do not explain the opposite direction of motion. The kinetic evidence and the high ratio of motile velocity to ATPase support a nonprocessive, low duty cycle mechanism for the Ncd motor.
使用称为MC1（残基209 - 700）的二聚体研究了非红化分离蛋白（Ncd）马达的动力学机制，该二聚体已被证明表现出负端定向运动性（钱德拉等人，1993年）。其动力学特性与单体Ncd马达结构域的相似（佩查特尼科娃和泰勒，1997年）。最大稳态ATP酶活性为1.5 s⁻¹，仅为单体马达的一半。核苷酸存在时的解离常数呈现相同趋势，但数值大约降低了两倍：ADP - AlF₄的Kd值为1.0 μM，ATPγS为1.1 μM，ATP为1.5 μM，ADP为3 μM，ADP - 钒酸盐为10 μM（在25 mM NaCl，22℃条件下）。ATP和ADP与微管 - 马达复合物（MtMC1）结合的表观二级速率常数为2 μM⁻¹ s⁻¹。基于在高微管浓度下的测量，动力学步骤拟合到如下反应式，[见原文]其中N指二聚体的一个头部，T、D和P分别代表ATP、ADP和无机磷酸。k₁和k⁻₄分别是由mant ATP和mant ADP结合诱导的转变的一级速率常数。ADP释放是MtMC1机制中的主要限速步骤。MC1 - mant ADP复合物与微管的结合释放的mant ADP不到一半（交替位点反应性）。第二个mant ADP仅通过使MtMC1复合物解离的核苷酸（ATP和ADP而非AMPPNP）的结合而释放。第二个mant ADP解离的表观速率常数比第一个小四倍，且远小于ATP或ADP使MtMC1解离的速率。这些结果由一个模型解释，即MC1·ADP首先通过ATP从微管上解离，然后由含ADP的头部重新结合到微管上。Ncd可能遵循与驱动蛋白不同的反应途径，但速率常数上的差异并不能解释相反的运动方向。动力学证据以及运动速度与ATP酶的高比值支持Ncd马达的非连续、低占空比机制。
