Duff Michael R, Borreguero Jose M, Cuneo Matthew J, Ramanathan Arvind, He Junhong, Kamath Ganesh, Chennubhotla S Chakra, Meilleur Flora, Howell Elizabeth E, Herwig Kenneth W, Myles Dean A A, Agarwal Pratul K
Biochemistry & Cellular and Molecular Biology Department , University of Tennessee , Knoxville , Tennessee , United States.
Neutron Data Analysis and Visualization Division , Oak Ridge National Laboratory , Oak Ridge , Tennessee , United States.
Biochemistry. 2018 Jul 24;57(29):4263-4275. doi: 10.1021/acs.biochem.8b00424. Epub 2018 Jul 6.
Optimal enzyme activity depends on a number of factors, including structure and dynamics. The role of enzyme structure is well recognized; however, the linkage between protein dynamics and enzyme activity has given rise to a contentious debate. We have developed an approach that uses an aqueous mixture of organic solvent to control the functionally relevant enzyme dynamics (without changing the structure), which in turn modulates the enzyme activity. Using this approach, we predicted that the hydride transfer reaction catalyzed by the enzyme dihydrofolate reductase (DHFR) from Escherichia coli in aqueous mixtures of isopropanol (IPA) with water will decrease by ∼3 fold at 20% (v/v) IPA concentration. Stopped-flow kinetic measurements find that the pH-independent k rate decreases by 2.2 fold. X-ray crystallographic enzyme structures show no noticeable differences, while computational studies indicate that the transition state and electrostatic effects were identical for water and mixed solvent conditions; quasi-elastic neutron scattering studies show that the dynamical enzyme motions are suppressed. Our approach provides a unique avenue to modulating enzyme activity through changes in enzyme dynamics. Further it provides vital insights that show the altered motions of DHFR cause significant changes in the enzyme's ability to access its functionally relevant conformational substates, explaining the decreased k rate. This approach has important implications for obtaining fundamental insights into the role of rate-limiting dynamics in catalysis and as well as for enzyme engineering.
最佳酶活性取决于多种因素，包括结构和动力学。酶结构的作用已得到充分认识；然而，蛋白质动力学与酶活性之间的联系引发了一场激烈的争论。我们开发了一种方法，利用有机溶剂的水性混合物来控制功能相关的酶动力学（不改变结构），进而调节酶活性。使用这种方法，我们预测，在异丙醇（IPA）与水的水性混合物中，大肠杆菌二氢叶酸还原酶（DHFR）催化的氢化物转移反应在IPA浓度为20%（v/v）时将降低约3倍。停流动力学测量发现，与pH无关的k速率降低了2.2倍。X射线晶体学酶结构没有明显差异，而计算研究表明，水和混合溶剂条件下的过渡态和静电效应是相同的；准弹性中子散射研究表明，酶的动态运动受到抑制。我们的方法为通过改变酶动力学来调节酶活性提供了一条独特的途径。此外，它提供了重要的见解，表明DHFR运动的改变导致酶获取其功能相关构象亚态的能力发生显著变化，解释了k速率的降低。这种方法对于深入了解限速动力学在催化中的作用以及酶工程具有重要意义。
