Moyer M L, Gilbert S P, Johnson K A
Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park 16802, USA.
Biochemistry. 1998 Jan 20;37(3):800-13. doi: 10.1021/bi9711184.
The ATPase mechanism for a monomeric Drosophila kinesin construct, K341, was determined by pre-steady-state kinetic methods and compared to dimeric kinesin, K401. We directly measured the kinetics of binding mantATP (a fluorescent ATP analog) to the microtubule K341 complex, the dissociation of K341 from the microtubule, and release of phosphate and ADP from K341. Measurements of phosphate release kinetics at low salt concentration show that K341 hydrolyzes 18 molecules of ATP per kinesin monomer prior to release from the microtubule. At a higher salt concentration the amplitude of the pre-steady-state burst of phosphate release was reduced to 8 molecules per kinesin monomer. The maximum rate of dissociation of K341 from the microtubule following the addition of ATP was 22 s-1. The rate of mantADP release from the M.K341.mantADP complex increased as a function of tubulin concentration with a second-order rate constant of 11 microM-1 s-1 for K341 binding to the microtubule and reached a maximum rate of mantADP release of 303 s-1. ADP release kinetics were also determined by monitoring the binding of mantATP to K341.ADP and K401.ADP after mixing with microtubules. We show that monomeric kinesin remains associated with the microtubule through multiple rounds of ATP hydrolysis. This apparent processivity implies that one of the functions of the cooperative interaction between the two kinesin heads in dimeric kinesin is for the reactions occurring on one kinesin head to facilitate the release of the adjacent head from the microtubule.
通过预稳态动力学方法确定了单体果蝇驱动蛋白构建体K341的ATP酶机制，并与二聚体驱动蛋白K401进行了比较。我们直接测量了mantATP（一种荧光ATP类似物）与微管K341复合物结合的动力学、K341从微管上的解离以及磷酸盐和ADP从K341上的释放。低盐浓度下磷酸盐释放动力学的测量表明，K341在从微管释放之前，每个驱动蛋白单体水解18个ATP分子。在较高盐浓度下，磷酸盐释放的预稳态爆发幅度降至每个驱动蛋白单体8个分子。添加ATP后，K341从微管上解离的最大速率为22 s⁻¹。mantADP从M.K341.mantADP复合物中释放的速率随着微管蛋白浓度的增加而增加，K341与微管结合的二级速率常数为11 μM⁻¹ s⁻¹，mantADP释放的最大速率达到303 s⁻¹。ADP释放动力学也通过监测mantATP与微管混合后与K341.ADP和K401.ADP的结合来确定。我们表明，单体驱动蛋白通过多轮ATP水解仍与微管结合。这种明显的持续性意味着二聚体驱动蛋白中两个驱动蛋白头部之间协同相互作用的功能之一是使一个驱动蛋白头部发生的反应促进相邻头部从微管上释放。
