Millisecond timescale fluctuations in dihydrofolate reductase are exquisitely sensitive to the bound ligands
Millisecond timescale fluctuations in dihydrofolate reductase are exquisitely sensitive to the bound ligands
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DOI：
10.1073/pnas.0914163107
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发表时间：
2010-01-26
影响因子：
11.1
通讯作者：
Wright, Peter E.
中科院分区：
文献类型：
--
作者：
Boehr, David D.;McElheny, Dan;Wright, Peter E.
酶催化可以描述为多维能量景观的进步，其中相互转化的构象亚状态的集合引导酶通过其催化循环。我们应用核磁共振弛豫分散来研究结合配体在调节大肠杆菌二氢叶酸还原酶的动力学和能量景观中的作用，以深入了解该酶在穿过其反应途径时有效采样功能构象的机制。尽管封闭底物二元复合物和产物三元复合物之间的结构差异非常小，但蛋白质动力学却存在显着差异。对于底物和产物二元复合物，辅因子结合裂隙中 mu s-ms 时间尺度上的主干波动是相似的，但仅在具有底物或底物类似物的复合物中观察到活性位点环中此时间尺度上的波动，而在二元产物复合物中未观察到。底物和产物二元复合物的动力学由完全不同的动力学和热力学参数控制。 E:THF:NADPH 和 E:THF:NADP(+) 产物三元复合物中的类似动态差异很难从基态结构合理化。对于这两种复合物，烟酰胺环位于基态活性位点袋的外部。然而，它们在结构、能量学和可接近的高能亚态动力学方面有所不同，其中烟酰胺环暂时占据活性位点。总体而言，我们的结果表明，二氢叶酸还原酶的动力学针对催化循环中的每个中间体进行了精确的“调整”。结构波动有效地引导酶通过功能相关的构象空间。
Enzyme catalysis can be described as progress over a multidimensional energy landscape where ensembles of interconverting conformational substates channel the enzyme through its catalytic cycle. We applied NMR relaxation dispersion to investigate the role of bound ligands in modulating the dynamics and energy landscape of Escherichia coli dihydrofolate reductase to obtain insights into the mechanism by which the enzyme efficiently samples functional conformations as it traverses its reaction pathway. Although the structural differences between the occluded substrate binary complexes and product ternary complexes are very small, there are substantial differences in protein dynamics. Backbone fluctuations on the mu s-ms timescale in the cofactor binding cleft are similar for the substrate and product binary complexes, but fluctuations on this timescale in the active site loops are observed only for complexes with substrate or substrate analog and are not observed for the binary product complex. The dynamics in the substrate and product binary complexes are governed by quite different kinetic and thermodynamic parameters. Analogous dynamic differences in the E:THF:NADPH and E:THF:NADP(+) product ternary complexes are difficult to rationalize from ground-state structures. For both of these complexes, the nicotinamide ring resides outside the active site pocket in the ground state. However, they differ in the structure, energetics, and dynamics of accessible higher energy substates where the nicotinamide ring transiently occupies the active site. Overall, our results suggest that dynamics in dihydrofolate reductase are exquisitely "tuned" for every intermediate in the catalytic cycle; structural fluctuations efficiently channel the enzyme through functionally relevant conformational space.