Interaction of mant-adenosine nucleotides and magnesium with kinesin
Interaction of mant-adenosine nucleotides and magnesium with kinesin
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DOI：
10.1021/bi972742j
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发表时间：
1998-04-14
期刊：
影响因子：
2.9
通讯作者：
Hackney, DD
中科院分区：
文献类型：
--
作者：
Cheng, JQ;Jiang, W;Hackney, DD
过量 ATP 将荧光底物类似物甲基邻氨基甲酰基 ADP (mant-ADP) 从驱动蛋白中置换出来，从而产生双相荧光瞬变。速率和振幅的 pH 和微管依赖性表明，两相是通过释放结合的 mant-ADP 和过量的 3'-异构体产生的，随后游离的 2'-和 3'-异构体松弛至其平衡分布。 mant-ADP 释放的第一阶段由微管加速，并以与使用 [P-32]ADP 测量的 ADP 释放相同的速率发生。第二阶段受到碱催化，并且以与分离的 2'-或 3'-mant-ATP 异构化相同的速率发生，速率范围超过 100 倍。结合的 mant-ADP 异构体在 pH 8.2 下与驱动蛋白结合时迅速发生异构化，而与肌球蛋白结合时 mant-ADP 异构体仅缓慢相互转化。驱动蛋白颈域中的单个色氨酸和结合的 mant-ADP 之间不发生荧光共振能量转移，但蛋白质酪氨酸基团确实发生有效的能量转移。在没有微管的情况下，在 pH 7-8、2 mM Mg2+ 和 25 mM KCl 条件下，mant-ADP 的释放速率最小 (0.005 s(-1))，但在较低 pH 条件下释放速率加快 (pH 5.5 时为 0.04 s(-1))，且 [KCl] 较低或较高（0 和 800 mM 条件下为 0.01 和 0.06 s(-1)）分别为 KCl）。微管刺激的 ADP 释放速率在低 pH 条件下加速，并被高浓度的一价盐抑制。通过添加过量的 EDTA 减少游离 Mg2+，使 E.MgADP 中 mant-ADP 的释放增加至 0.03 s(-1)。低 Mg2+ 下的这种加速可能代表 0.03 s(-1) 时 Mg2+ 的连续释放，然后是 ADP 的快速释放，因为无 Mg E.ADP 的 ADP 释放速率很快（>0.5 s(-1)）。在高 Mg2+ 下，Mg2+ 与 E.ADP 重新结合，迫使 ADP 在 0.005 s(-1) 时从 E.MgADP 复合物中释放。
Displacement of the fluorescent substrate analogue methylanthraniloyl ADP (mant-ADP) from kinesin by excess ATP results in a biphasic fluorescent transient. The pH and microtubule dependence of the rates and amplitudes indicates that the two phases are produced by release of bound mant-ADP, with an excess of the 3'-isomer, followed by the subsequent relaxation of the free 2'- and 3'-isomers to their equilibrium distribution. The first phase for release of mant-ADP is accelerated by microtubules and occurs at the same rate as ADP release measured using [P-32]ADP. The second phase is subject to base catalysis and occurs at the same rate as the isomerization of isolated 2'- or 3'-mant-ATP over a 100-fold range of rates. The bound mant-ADP isomers undergo isomerization rapidly when bound to kinesin at pH 8.2, whereas mant-ADP isomers interconvert only slowly when bound to myosin. No fluorescence resonance energy transfer occurs between the single tryptophan in the kinesin neck domain and bound mant-ADP, but efficient energy transfer does occur from protein tyrosine groups. The rate of mant-ADP release in the absence of microtubules is minimal (0.005 s(-1)) at pH 7-8, 2 mM Mg2+, and 25 mM KCl but is accelerated at lower pH (0.04 s(-1) at pH 5.5) and either lower or higher [KCl] (0.01 and 0.06 s(-1) at 0 and 800 mM KCl, respectively). The microtubule-stimulated rate of ADP release is accelerated at low pH and inhibited by high concentrations of monovalent salts. Reduction of the free Mg2+ by addition of excess EDTA increases the release of mant-ADP from E.MgADP to 0.03 s(-1). This acceleration at low Mg2+ likely represents sequential release of Mg2+ at 0.03 s(-1) followed by rapid release of ADP, as the rate of ADP release from Mg-free E.ADP is fast (>0.5 s(-1)). At high Mg2+, rebinding of Mg2+ to E.ADP forces release of ADP from the E.MgADP complex at 0.005 s(-1).