Manchester Interdisciplinary Biocentre, University of Manchester, 131 Princess Street, Manchester, M1 7DN, United Kingdom.
J Am Chem Soc. 2010 Aug 18;132(32):11329-35. doi: 10.1021/ja1048048.
The role of dynamical effects in enzyme catalysis is both complex and widely debated. Understanding how dynamics can influence the barrier to an enzyme catalyzed reaction requires the development of new methodologies and tools. In particular compressive dynamics-the focus of this study-may decrease both the height and width of a reaction barrier. By making targeted mutations in the active site of morphinone reductase we are able to alter the equilibrium of conformational states for the reactive complex in turn altering the donor-acceptor (D-A) distance for H-transfer. The sub-A changes which we induce are monitored using novel spectroscopic and kinetic "rulers". This new way of detecting variation in D-A distance allows us to analyze trends between D-A distance and the force constant of a compressive dynamical mode. We find that as the D-A distance decreases, the force constant for a compressive mode increases. Further, we demonstrate that-contrary to current dogma-compression may not always cause the magnitude of the primary kinetic isotope effect to decrease.
动态效应对酶催化的作用既复杂又备受争议。了解动力学如何影响酶催化反应的势垒需要开发新的方法和工具。特别是压缩动力学——本研究的重点——可能会降低反应势垒的高度和宽度。通过在吗啡酮还原酶的活性部位进行靶向突变，我们能够改变反应复合物的构象平衡状态，从而改变 H 转移的供体-受体 (D-A) 距离。我们使用新颖的光谱学和动力学“标尺”来监测所诱导的亚 A 变化。这种检测 D-A 距离变化的新方法使我们能够分析 D-A 距离与压缩动力学模式力常数之间的趋势。我们发现，随着 D-A 距离的减小，压缩模式的力常数增加。此外，我们证明与当前的教条相反，压缩不一定会导致主要动力学同位素效应的幅度减小。
