Isotopic Labeling of Formate Dehydrogenase Perturbs the Protein Dynamics
Isotopic Labeling of Formate Dehydrogenase Perturbs the Protein Dynamics
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DOI：
10.1021/acs.jpcb.9b08426
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发表时间：
2019-12-12
影响因子：
3.3
通讯作者：
Cheatum, Christopher M.
中科院分区：
文献类型：
--
作者：
Ranasinghe, Chethya;Pagano, Philip;Cheatum, Christopher M.
Isotope substitution of enzymes has become a means of addressing the participation of protein motions in enzyme-catalyzed reactions. The idea is that only the enzyme mass will be altered and not the electrostatics, so that the protein dynamics are essentially the same but at lower frequencies because of the mass change. In this study, we variably label all carbon atoms in formate dehydrogenase (FDH) with C-13, all nitrogen atoms with N-15, and all nonexchangeable hydrogen atoms with deuterium and investigate the impact that isotopic substitution has on the dynamics at the active site by two-dimensional infrared spectroscopy and compare with the measurements of the temperature dependence of the intrinsic kinetic isotope effects (KIEs). We show that N-15 labeling of FDH has the largest effect and makes the active site more heterogeneous, whereas the addition of nonexchangeable deuterium appears to have the opposite effect of N-15 on active-site dynamics, resulting in a behavior similar to that of native FDH. Nevertheless, the temperature dependence of the KIEs shows a monotonic trend with protein mass that does not correspond with the changes in dynamics. These results suggest that isotope labeling has more than just a mass effect on enzyme dynamics and may influence electrostatics in ways that complicate the interpretation of the protein isotope effect.
