The rate-limiting step in microtubule-stimulated ATP hydrolysis by dimeric kinesin head domains occurs while bound to the microtubule.
The rate-limiting step in microtubule-stimulated ATP hydrolysis by dimeric kinesin head domains occurs while bound to the microtubule.
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发表时间：
1994-06
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通讯作者：
D. Hackney
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作者：
D. Hackney
DKH 392是含有果蝇驱动蛋白α-亚基的前392个氨基酸并且在溶液中是二聚体的构建体（Huang，T. G.，Suhan，J.，和哈克尼，D. D.（1994）J.Biol.Chem.269，16502-16507）。DKH 392的ATP酶速率为0.005 s-1。一个ADP紧密结合到每个亚基和释放的ADP是ATP水解的限速步骤。微管加速ADP释放速率，并使稳态ATP水解速率增加近10，000倍（kcat =约45 s-1）。在8 nM DKH 392下，微管对ATP酶反应的刺激饱和的KMT 0.5，ATP酶值为50 nM，但在较低浓度的DKH 392下降低。在1 mM MgATP存在下，DKH 392与微管的物理结合导致微管对ATP酶活性的刺激饱和，这表明当DKH 392与微管结合时，微管刺激的ATP水解发生限速步骤。这些结果表明，微管刺激的ATP水解DKH 392可能是进行性的多个ATP分子的水解过程中，DKH 392与微管的每个扩散遇到。
DKH392 is a construct which contains the first 392 amino acids of the alpha-subunit of Drosophila kinesin and is dimeric in solution (Huang, T.-G., Suhan, J., and Hackney, D. D. (1994) J. Biol. Chem. 269, 16502-16507). The ATPase rate of DKH392 was 0.005 s-1 in the absence of MTs. One ADP bound tightly to each subunit and the release of this ADP was the rate-limiting step in ATP hydrolysis. Microtubules accelerated the rate of ADP release and increased the rate of steady state ATP hydrolysis by almost 10,000-fold (kcat = approximately 45 s-1). The KMT0.5,ATPase value for saturation of the stimulation of the ATPase reaction by microtubules was 50 nM at 8 nM DKH392, but decreased at lower concentrations of DKH392. Physical binding of DKH392 to microtubules in the presence of 1 mM MgATP paralleled saturation of the stimulation of the ATPase activity by microtubules indicating that the rate-limiting step in microtubule-stimulated ATP hydrolysis occurs while DKH392 is bound to the microtubule. These results suggest that microtubule-stimulated ATP hydrolysis by DKH392 may be processive with the hydrolysis of multiple ATP molecules during each diffusional encounter of DKH392 with a microtubule.