Parallel Pathways and Free-Energy Landscapes for Enzymatic Hydride Transfer Probed by Hydrostatic Pressure
Parallel Pathways and Free-Energy Landscapes for Enzymatic Hydride Transfer Probed by Hydrostatic Pressure
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DOI：
10.1002/cbic.200900071
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发表时间：
2009-05-25
期刊：
影响因子：
3.2
通讯作者：
Scrutton, Nigel S.
中科院分区：
文献类型：
--
作者：
Pudney, Christopher R.;McGrory, Tom;Scrutton, Nigel S.
我们发现，在构象丰富的自由能环境中，由吗啡酮还原酶N189A突变体催化的氢化物从NADH到FMN的转移沿着平行的“化学”途径发生。我们已经开发了实验动力学和光谱工具，通过使用流体静压来探索这种自由能景观。N189A突变酶与非活性辅酶类似物NADH(4)形成的复合物的晶体结构表明，与野生型NADH(4)的相应结构相比，类似物的烟酰胺部分在构象上受到的限制较小。N189A酶构象自由度的增加引起了多反应构型(MRC)的概念，我们证明了这些状态在自由能版图上的相对数量可以作为压力的函数在实验上受到扰动。具体地说，在氢化物转移反应的停流研究中观察到的单个动力学相的幅度对压力很敏感；这表明压力推动了整个能量格局的改变分布。我们通过吸收光谱表明，酶-辅酶复合体电荷转移特性的丧失归因于景观上MRC数量的变化。在低压和高压下的分子动力学模拟支持N189A突变体中存在丰富的构象景观。这项工作为酶-辅酶复合体存在多个构象状态的平行途径提供了坚实的实验和计算支持。静水压力是多维能量图的一个强大而通用的探测器，可用于实验分析酶催化反应的平行路径。我们认为，这是特别是在蛋白质定向突变后的情况，这可能导致反应状态的增加，这在野生型酶的自由能景观中基本上是无法获得的。
We show that hydride transfer from NADH to FMN catalysed by the N189A mutant of morphinone reductase occurs along parallel "chemical" pathways in a conformationally rich free-energy landscape. We have developed experimental kinetic and spectroscopic tools by using hydrostatic pressure to explore this free-energy landscape. The crystal structure of the N189A mutant enzyme in complex with the unreactive coenzyme analogue NADH(4) indicates that the nicotinamide moiety of the analogue is conformationally less restrained than the corresponding structure of the wild-type NADH(4) complex. This increased degree of conformational freedom in the N189A enzyme gives rise to the concept of multiple reactive configurations (MRCs), and we show that the relative population of these states across the free-energy landscape can be perturbed experimentally as a function of pressure. Specifically, the amplitudes of individual kinetic phases that were observed in stopped-flow studies of the hydride transfer reaction are sensitive to pressure; this indicates that pressure drives an altered distribution across the energy landscape. We show by absorbance spectroscopy that the loss of charge-transfer character of the enzyme-coenzyme complex is attributed to the altered population of MRCs on the landscape. The existence of a conformationally rich landscape in the N189A mutant is supported by molecular dynamics simulations at low and high pressure. The work provides firm experimental and computational support for the existence of parallel pathways arising from multiple conformational states of the enzyme-coenzyme complex. Hydrostatic pressure is a powerful and general probe of multidimensional energy landscapes that can be used to analyse experimentally parallel pathways for enzyme-catalysed reactions. We suggest that this is especially the case following directed mutation of a protein, which can lead to increased population of reactant states that are essentially inaccessible in the free-energy landscape of wild-type enzyme.