Kinetic resolution of a conformational transition and the ATP hydrolysis step using relaxation methods with a Dictyostelium myosin II mutant containing a single tryptophan residue
Kinetic resolution of a conformational transition and the ATP hydrolysis step using relaxation methods with a Dictyostelium myosin II mutant containing a single tryptophan residue
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DOI：
10.1021/bi010963q
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发表时间：
2001-10-23
期刊：
影响因子：
2.9
通讯作者：
Bagshaw, CR
中科院分区：
文献类型：
--
作者：
Málnási-Csizmadia, A;Pearson, DS;Bagshaw, CR
来自位于盘状二胞囊藻肌球蛋白II马达结构域的中继环（F466至L516）中的保守色氨酸残基（W501）的荧光发射强度对ATP结合和水解敏感。最初的结合过程伴随着荧光的小淬灭，然后是与水解步骤一致出现的大增强。使用温度和压力跳跃的方法，我们表明，增强过程是动力学不同的，但耦合到水解步骤。荧光增强对应于开-闭跃迁（k（obs）在20 ℃下约为1000 s（-1））。从整体稳态荧光信号和弛豫瞬态的存在或不存在，我们得出结论，ADP状态主要是在开放状态，而ADP.AlF4状态主要是关闭的。在20摄氏度时，AMP.PNP和ADP.BeFx复合物的开闭平衡接近于1，并且很容易受到温度和压力的干扰。在ATP的情况下，该步骤的平衡略微有利于开放状态，但与随后的水解步骤的偶联在稳态中产生主要的封闭状态。稳态ATP周转期间的压力跃变揭示了快速开-闭转变和较慢水解步骤的明显瞬变。
The fluorescence emission intensity from a conserved tryptophan residue (W501) located in the relay loop (F466 to L516) of the Dicytostelium discoideum myosin Il motor domain is sensitive to ATP binding and hydrolysis. The initial binding process is accompanied by a small quench in fluorescence, and this is followed by a large enhancement that appears coincident with the hydrolysis step. Using temperature and pressure jump methods, we show that the enhancement process is kinetically distinct from but coupled to the hydrolysis step. The fluorescence enhancement corresponds to the open-closed transition (k(obs) approximate to 1000 s(-1) at 20 degreesC). From the overall steady-state fluorescence signal and the presence or absence of a relaxation transient, we conclude that the ADP state is largely in the open state, while the ADP.AlF4 state is largely closed. At 20 degreesC the open-closed equilibria for the AMP.PNP and ADP.BeFx complexes are close to unity and are readily perturbed by temperature and pressure. In the case of ATP, the equilibrium of this step slightly favors the open state, but coupling to the subsequent hydrolysis step gives rise to a predominantly closed state in the steady state. Pressure jump during steady-state ATP turnover reveals the distinct transients for the rapid open-closed transition and the slower hydrolysis step.