Probing the mechanism of ATP hydrolysis on F-actin using vanadate and the structural analogs of phosphate BeF-3 and A1F-4.
Probing the mechanism of ATP hydrolysis on F-actin using vanadate and the structural analogs of phosphate BeF-3 and A1F-4.
复制标题
DOI：
10.1016/s0021-9258(19)77854-5
复制
发表时间：
1988-11
期刊：
影响因子：
--
通讯作者：
Ckile Combeau;M F Carlier
中科院分区：
文献类型：
--
作者：
Ckile Combeau;M F Carlier
研究了磷酸类似物与f - adp -肌动蛋白丝的结合及其对聚合物动力学的影响。正钒酸盐以与磷酸盐相同的亲和力与f -肌动蛋白结合，在低饱和水平下，以pi样的方式稳定丝结构；在较高的浓度下，它促进了长丝的不稳定。BeF-3和A1F-4与F-ADP-actin的结合与Pi竞争，其化学计量值为1 mol/mol的F-actin亚基，亲和力比Pi高3个数量级（BeF-3和A1F-4的KD分别为2和25微米）。BeF-3和A1F-4在稳定f -肌动蛋白方面与Pi相似，并将肌动蛋白从纤维中分离的速度降低了150倍。只有1%的F-ADP-BeF3亚基提供灯丝的广泛稳定。提出了用BeF-3进行稳定化的定量分析。当Pi与F-ADP-actin快速平衡时，BeF-3以非常慢的速度与F-ADP-actin结合并解离（k+ = 4 M-1 S-1; k = 8.10(-6) S-1）。此外，虽然功能上与重组的F-ADP-Pi相似，但F-ADP-BeF3具有不同的构象，与BeF-3结合相连的芘基荧光猝灭17%。我们认为BeF-3可能是过渡态F-ADP-P*的良好模拟物，并且F-actin上ATP裂解后的Pi释放可能受到F-ADP-P*异构化的速率限制。
The binding of phosphate analogs to F-ADP-actin filaments and their effect on the dynamics of the polymer have been investigated. Orthovanadate binds to F-actin with the same affinity as phosphate and, at low saturation levels, stabilizes the filament structure in a Pi-like fashion; at higher concentration, it promotes destabilization of the filament. BeF-3 and A1F-4 bind to F-ADP-actin in competition with Pi, with a stoichiometry of 1 mol/mol of F-actin subunit and an affinity 3 orders of magnitude higher than Pi (KD = 2 and 25 microM for BeF-3 and A1F-4, respectively). BeF-3 and A1F-4 mimic Pi in stabilizing F-actin and slow down the rate of actin dissociation from filaments 150-fold. Only 1% of F-ADP-BeF3 subunits provide extensive stabilization of the filament. A quantitative analysis of the stabilization by BeF-3 is proposed. While Pi appears in rapid equilibrium with F-ADP-actin, BeF-3 binds to and dissociates from F-ADP-actin at very slow rates (k+ = 4 M-1 S-1; k = 8.10(-6) S-1). In addition, although functionally similar to the reconstituted F-ADP-Pi species, F-ADP-BeF3 has a different conformation as indicated by the 17% quenching of pyrenyl fluorescence linked to BeF-3 binding. We suggest that BeF-3 may be a good analog of the transition state F-ADP-P* and that Pi release following cleavage of ATP on F-actin might be rate-limited by the isomerization of F-ADP-P*.