The role of large-scale motions in catalysis by dihydrofolate reductase.
The role of large-scale motions in catalysis by dihydrofolate reductase.
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DOI：
10.1021/ja208844j
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发表时间：
2011-12-21
影响因子：
15
通讯作者：
Allemann, Rudolf K.
中科院分区：
文献类型：
--
作者：
Loveridge, E. Joel;Tey, Lai-Hock;Behiry, Enas M.;Dawson, William M.;Evans, Rhiannon M.;Whittaker, Sara B. -M.;Guenther, Ulrich L.;Williams, Christopher;Crump, Matthew P.;Allemann, Rudolf K.
二氢叶酸还原酶长期以来被用作研究蛋白质运动与酶促氢化物转移耦合的模型系统。通过研究环境对来自冷适应细菌深莫里特菌（MpDHFR）的二氢叶酸还原酶（DHFR）中氢化物转移的影响，并将该酶的灵活性与来自大肠杆菌（EcDHFR）的DHFR进行比较，我们证明了影响大规模（即，长距离但不一定是大幅度）蛋白质运动对氢化物转移的动力学同位素效应或其温度依赖性没有影响，尽管催化反应的速率受到影响。通过NMR光谱法的氢/氘交换研究表明，MpDHFR是比EcDHFR更灵活的酶。NMR实验与EcDHFR在共溶剂的存在下，表明在构象合奏的酶的差异。来自不同环境小生境和具有不同灵活性的酶对氢化物转移表现出相同的动力学同位素效应的事实强烈地表明，虽然蛋白质运动对于产生反应就绪构象是重要的，具有正确的静电和几何形状的最佳构象用于反应发生，但它们不影响化学步骤本身的性质;大尺度运动不直接耦合到DHFR中的氢化物转移。
Dihydrofolate reductase has long been used as a model system to study the coupling of protein motions to enzymatic hydride transfer. By studying environmental effects on hydride transfer in dihydrofolate reductase (DHFR) from the cold-adapted bacterium Moritella profunda (MpDHFR) and comparing the flexibility of this enzyme to that of DHFR from Escherichia coli (EcDHFR), we demonstrate that factors that affect large-scale (i.e., long-range, but not necessarily large amplitude) protein motions have no effect on the kinetic isotope effect on hydride transfer or its temperature dependence, although the rates of the catalyzed reaction are affected. Hydrogen/deuterium exchange studies by NMR-spectroscopy show that MpDHFR is a more flexible enzyme than EcDHFR. NMR experiments with EcDHFR in the presence of cosolvents suggest differences in the conformational ensemble of the enzyme. The fact that enzymes from different environmental niches and with different flexibilities display the same behavior of the kinetic isotope effect on hydride transfer strongly suggests that, while protein motions are important to generate the reaction ready conformation, an optimal conformation with the correct electrostatics and geometry for the reaction to occur, they do not influence the nature of the chemical step itself; large-scale motions do not couple directly to hydride transfer proper in DHFR.
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DOI：
10.1126/science.1198542
发表时间：
2011-04-08
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Bhabha G;Lee J;Ekiert DC;Gam J;Wilson IA;Dyson HJ;Benkovic SJ;Wright PE
通讯作者：
Wright PE
影响因子：
3.9
作者：
Allemann, Rudolf K.;Evans, Rhiannon M.;Loveridge, E. Joel
通讯作者：
Loveridge, E. Joel
影响因子：
2.9
作者：
Goldberg, JM;Kirsch, JF
通讯作者：
Kirsch, JF
DOI：
10.1073/pnas.1111252108
发表时间：
2011-08-23
影响因子：
11.1
作者：
Adamczyk, Andrew J.;Cao, Jie;Warshel, Arieh
通讯作者：
Warshel, Arieh
影响因子：
2.9
作者：
BROUWER, AC;KIRSCH, JF
通讯作者：
KIRSCH, JF