Modulation of kinesin half-site ADP release and kinetic processivity by a spacer between the head groups
Modulation of kinesin half-site ADP release and kinetic processivity by a spacer between the head groups
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DOI：
10.1021/bi0349118
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发表时间：
2003-10-21
期刊：
影响因子：
2.9
通讯作者：
Patterson, RA
中科院分区：
文献类型：
--
作者：
Hackney, DD;Stock, MF;Patterson, RA
构建了二聚体果蝇激酶颈连接子和颈线圈的一系列修饰，以确定头部取向和间距对atp酶动力学的影响。Ala（345）是颈部卷曲线圈中的第一个残基，用甘氨酸或脯氨酸替代它不会对微管（MT）激活ATP酶的k（cat）或k -0.5（MT）值产生显著变化，也不会对微管在与MT的过程中水解的ATP分子的平均数量的k（bi(ratio)）值产生显著变化。在连接处添加或删除单个氨基酸只会产生适度的变化，其动力学过程降低不到2倍。在颈连接器连接处插入6或12个额外氨基酸的间隔，使K-0.5（MT）值增加3-4倍，相应的动力学过程降低。在多运动条件下，所有突变体的滑动速度都在野生型的30%以内。所有具有单残基变化的构建体在与MT结合时都表现出半位点ADP释放。然而，具有长插入的构建体在与MT结合时迅速释放每个二聚体的ADP分子，这表明阻止ADP从野生型激酶栓系头部释放的空间限制已经被长插入缓解。具有长插入的结构体在ATP水解过程中降低了动力学进程，并且与MT分离的速度比野生型快3倍。
A series of modifications of the junction of the neck linker and neck coil of dimeric Drosophila kinesin were constructed to determine the influence of head orientation and spacing on the ATPase kinetics. Ala(345) is the first residue in the coiled-coil of the neck, and its replacement with glycine or proline produces no significant change in the k(cat) or K-0.5(MT) values for activation of their ATPase by microtubules (MTs) or in their k(bi(ratio)) value for the average number of ATP molecules hydrolyzed during a processive encounter with a MT. Addition or deletion of a single amino acid at the junction produces only modest changes with less than a 2-fold reduction in kinetic processivity. Insertion of a spacer of 6 or 12 additional amino acids at the neck linker junction increases the K-0.5(MT) value by 3-4-fold with a corresponding decrease in kinetic processivity. The sliding velocities of all the mutant constructs under multimotor conditions are within 30% of the wild-type value. All the constructs with single residue changes exhibit half-site ADP release on binding to MTs. The constructs with long insertion, however, rapidly release both ADP molecules per dimer on binding to a MT, indicating that the steric constraints that prevent release of ADP from the tethered head of wild-type kinesin have been relieved by the long insertions. The constructs with long inserts have decreased kinetic processivity and dissociate from the MT during ATP hydrolysis 3-fold faster than wild-type.