Department of Chemistry, University of Iowa , Iowa City, Iowa 52242-1727, United States.
Division of Chemical Biology and Medicinal Chemistry, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill , Chapel Hill, North Carolina 27599, United States.
J Am Chem Soc. 2017 Dec 6;139(48):17405-17413. doi: 10.1021/jacs.7b08359. Epub 2017 Nov 27.
Isotopically labeled enzymes (denoted as "heavy" or "Born-Oppenheimer" enzymes) have been used to test the role of protein dynamics in catalysis. The original idea was that the protein's higher mass would reduce the frequency of its normal-modes without altering its electrostatics. Heavy enzymes have been used to test if the vibrations in the native enzyme are coupled to the chemistry it catalyzes, and different studies have resulted in ambiguous findings. Here the temperature-dependence of intrinsic kinetic isotope effects of the enzyme formate dehydrogenase is used to examine the distribution of H-donor to H-acceptor distance as a function of the protein's mass. The protein dynamics are altered in the heavy enzyme to diminish motions that determine the transition state sampling in the native enzyme, in accordance with a Born-Oppenheimer-like effect on bond activation. Findings of this work suggest components related to fast frequencies that can be explained by Born-Oppenheimer enzyme hypothesis (vibrational) and also slower time scale events that are non-Born-Oppenheimer in nature (electrostatic), based on evaluations of protein mass dependence of donor-acceptor distance and forward commitment to catalysis along with steady state and single turnover measurements. Together, the findings suggest that the mass modulation affected both local, fast, protein vibrations associated with the catalyzed chemistry and the protein's macromolecular electrostatics at slower time scales; that is, both Born-Oppenheimer and non-Born-Oppenheimer effects are observed. Comparison to previous studies leads to the conclusion that isotopic labeling of the protein may have different effects on different systems, however, making heavy enzyme studies a very exciting technique for exploring the dynamics link to catalysis in proteins.
同位素标记的酶（表示为“重”或“Born-Oppenheimer”酶）已被用于测试蛋白质动力学在催化中的作用。最初的想法是，蛋白质的质量更高会降低其正常模式的频率，而不会改变其静电。重酶已被用于测试天然酶中的振动是否与它催化的化学反应耦合，不同的研究得出了模棱两可的结果。在这里，使用甲酸脱氢酶的酶形式的固有动力学同位素效应的温度依赖性来检查蛋白质质量作为函数的 H-供体到 H-受体距离的分布。重酶中的蛋白质动力学发生改变，以减少决定天然酶中过渡态采样的运动，这符合对键激活的 Born-Oppenheimer 样效应。这项工作的结果表明，与 Born-Oppenheimer 酶假设（振动）相关的快速频率成分以及与静电相关的较慢时间尺度事件（非 Born-Oppenheimer），这是基于对供体-受体距离的蛋白质质量依赖性的评估以及与稳态和单轮测量一起对催化的前进承诺。总的来说，这些发现表明，质量调制既影响与催化化学相关的局部、快速的蛋白质振动，也影响蛋白质在较慢时间尺度上的大分子静电；也就是说，既观察到 Born-Oppenheimer 效应，也观察到非 Born-Oppenheimer 效应。与以前的研究进行比较得出的结论是，蛋白质的同位素标记可能对不同的系统有不同的影响，然而，重酶研究是探索蛋白质催化动力学联系的一种非常令人兴奋的技术。
