EVIDENCE FOR ALTERNATING HEAD CATALYSIS BY KINESIN DURING MICROTUBULE-STIMULATED ATP HYDROLYSIS
EVIDENCE FOR ALTERNATING HEAD CATALYSIS BY KINESIN DURING MICROTUBULE-STIMULATED ATP HYDROLYSIS
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DOI：
10.1073/pnas.91.15.6865
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发表时间：
1994-07-19
影响因子：
11.1
通讯作者：
HACKNEY, DD
中科院分区：
文献类型：
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作者：
HACKNEY, DD
果蝇激动素α亚基的N端392个氨基酸(DKH392)在溶液中形成一个餐盘，与微管结合的两个紧密结合的ADP分子中只释放一个，而与微管结合的较短的单体结构(DKH340)释放大于或等于其一个结合的ADP的95%。二聚体DKH392的这种半中心活性在DKH392与微管的比例和稳态ATPase速率的很大范围内都可以观察到，这表明它是微管刺激的ATP水解机制的特征，而不是偶然的速率常数平衡的结果。当介质中含有[α-P-32]ATP时，P-32标记结合到与DKH392和微管结合的ADP池中的速度足够快，从而使结合的ADP成为ATP水解的主要途径的中间产物。这些和其他结果与二聚体DKH392通过一个头部结构域与微管捆绑的结果相一致，该结构域以僵硬的方式附着在没有结合核苷酸的情况下，而另一个头部没有附着在微管上，仍然含有紧密结合的ADP。这种性质的中间体以及DKH392在ATP存在下与微管的紧密结合表明了一种定向运动的机制，在这种机制中，二聚体DKH392的头域以顺序的方式交替。
The N-terminal 392 amino acids of the Drosophila kinesin alpha subunit (designated DKH392) form a diner in solution that releases only one of its two tightly bound ADP molecules on association with a microtubule, whereas a shorter monomeric construct (designated DKH340) releases greater than or equal to 95% of its one bound ADP on association with a microtubule. This half-site reactivity of dimeric DKH392 is observed over a wide range of ratios of DKH392 to microtubules and steady-state ATPase rates, indicating that it is characteristic of the mechanism of microtubule-stimulated ATP hydrolysis and not the result of a fortuitous balance of rate constants. When [alpha-P-32]ATP is included in the medium, incorporation of P-32 label into the pool of ADP that is bound to the complex of DKH392 and microtubules occurs rapidly enough for the bound ADP to be an intermediate on the main pathway of ATP hydrolysis. These and other results are consistent with the half-site reactivity being a consequence of the tethering of dimeric DKH392 to the microtubule through one head domain, which is attached in a rigor-like manner without bound nucleotide, whereas the other head is not attached to the microtubule and still contains a tightly bound ADP. An intermediate of this nature and the tight binding of DKH392 to microtubules in the presence of ATP suggest a mechanism for directed motility in which the head domains of dimeric DKH392 alternate in a sequential manner.