Gilbert S P, Moyer M L, Johnson K A
Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park 16802, USA.
Biochemistry. 1998 Jan 20;37(3):792-9. doi: 10.1021/bi971117b.
The processivity of the microtubule-kinesin ATPase has been investigated using stopped-flow kinetic methods to measure the binding of each motor domain of the dimeric kinesin (K401) to the microtubule and the release of the fluorescent ADP analog, 2'(3')-O-(N-methylanthraniloyl)adenosine 5'-diphosphate (mantADP) from the active site of the motor domain. The results show that the release of two molecules of ADP from dimeric kinesin (K401) after the binding of kinesin ADP to the microtubule is a sequential process leading to biphasic kinetics. The maximum rate of release of mantADP from the first motor domain of K401 or monomeric K341 is fast (300 s-1) and independent of added nucleotide. The rate of mantADP release from the second motor domain of K401 is slow in the absence of added nucleotide (0.4 s-1) and reaches a maximum rate of 300 s-1 at saturating concentrations of ATP. High concentrations of ADP stimulate mantADP release from the second head to a maximum rate of 3.8 s-1. The nonhydrolyzable analog AMP-PNP and ATP-gamma S also stimulate ADP release from the second head (maximum rate of 30 s-1), suggesting that ATP hydrolysis is not necessary to stimulate the ADP release. These experiments establish an alternating site mechanism for dimeric kinesin whereby ATP binding to one kinesin active site stimulates the release of ADP from the second site such that the reactions occurring at the active sites of the two monomer units are kept out of phase from each other by interactions between the heads. These results define the steps of the ATPase pathway that lead to the efficient coupling of ATP hydrolysis to force production in a processive reaction whereby force production in forming a tight microtubule complex by one head is coupled to the rate-limiting release of the other head from the microtubule.
利用停流动力学方法研究了微管驱动蛋白ATP酶的持续合成能力，以测量二聚体驱动蛋白（K401）的每个运动结构域与微管的结合以及荧光ADP类似物2'(3')-O-(N-甲基邻氨基苯甲酰基)腺苷5'-二磷酸（mantADP）从运动结构域活性位点的释放。结果表明，驱动蛋白ADP与微管结合后，二聚体驱动蛋白（K401）释放两分子ADP是一个导致双相动力学的连续过程。从K401的第一个运动结构域或单体K341释放mantADP的最大速率很快（300 s-1），且与添加的核苷酸无关。在没有添加核苷酸的情况下，从K401的第二个运动结构域释放mantADP的速率很慢（0.4 s-1），在ATP饱和浓度下达到最大速率300 s-1。高浓度的ADP刺激mantADP从第二个头部释放，最大速率为3.8 s-1。不可水解类似物AMP-PNP和ATP-γS也刺激ADP从第二个头部释放（最大速率为30 s-1），这表明ATP水解对于刺激ADP释放不是必需的。这些实验建立了二聚体驱动蛋白的交替位点机制，即ATP与一个驱动蛋白活性位点的结合刺激ADP从第二个位点释放，使得两个单体单元活性位点发生的反应通过头部之间的相互作用而彼此不同步。这些结果定义了ATP酶途径的步骤，这些步骤导致ATP水解与在一个连续反应中产生力的有效偶联，在该连续反应中，一个头部形成紧密微管复合物时产生的力与另一个头部从微管的限速释放相偶联。
