School of Chemistry and Cardiff Catalysis Institute, Cardiff University, Cardiff CF10 3AT, United Kingdom.
Proc Natl Acad Sci U S A. 2013 Oct 8;110(41):16344-9. doi: 10.1073/pnas.1312437110. Epub 2013 Sep 24.
Protein dynamics have controversially been proposed to be at the heart of enzyme catalysis, but identification and analysis of dynamical effects in enzyme-catalyzed reactions have proved very challenging. Here, we tackle this question by comparing an enzyme with its heavy ((15)N, (13)C, (2)H substituted) counterpart, providing a subtle probe of dynamics. The crucial hydride transfer step of the reaction (the chemical step) occurs more slowly in the heavy enzyme. A combination of experimental results, quantum mechanics/molecular mechanics simulations, and theoretical analyses identify the origins of the observed differences in reactivity. The generally slightly slower reaction in the heavy enzyme reflects differences in environmental coupling to the hydride transfer step. Importantly, the barrier and contribution of quantum tunneling are not affected, indicating no significant role for "promoting motions" in driving tunneling or modulating the barrier. The chemical step is slower in the heavy enzyme because protein motions coupled to the reaction coordinate are slower. The fact that the heavy enzyme is only slightly less active than its light counterpart shows that protein dynamics have a small, but measurable, effect on the chemical reaction rate.
蛋白质动力学一直被认为是酶催化的核心，但在酶催化反应中识别和分析动力学效应一直极具挑战性。在这里，我们通过比较酶与其重原子（(15)N、(13)C、(2)H 取代）对应物来解决这个问题，为重原子提供了一个微妙的动力学探针。反应的关键氢化物转移步骤（化学步骤）在重酶中进行得更慢。实验结果、量子力学/分子力学模拟和理论分析的结合确定了观察到的反应活性差异的起源。重酶中通常反应速度较慢反映了与氢化物转移步骤的环境耦合差异。重要的是，势垒和量子隧穿的贡献不受影响，这表明在驱动隧穿或调节势垒方面，“促进运动”没有重要作用。重酶中的化学步骤较慢是因为与反应坐标耦合的蛋白质运动较慢。重酶的活性仅略低于其轻酶对应物，这表明蛋白质动力学对化学反应速率有微小但可测量的影响。
