4P68

Electrostatics of Active Site Microenvironments for E. coli DHFR

4P68 の概要

• エントリーDOI: 10.2210/pdb4p68/pdb
• 関連するPDBエントリー: 4P66
• 分子名称: Dihydrofolate reductase, METHOTREXATE, NADP NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE, ... (6 entities in total)
• 機能のキーワード: electrostatics, catalysis, oxidoreductase, dhfr
• 由来する生物種: Escherichia coli
• タンパク質・核酸の鎖数: 1
• 化学式量合計: 19382.29

構造登録者

Liu, C.T.,Layfield, J.P.,Stewart III, R.J.,French, J.B.,Hanoian, P.,Asbury, J.B.,Hammes-Schiffer, S.,Benkovic, S.J. (登録日: 2014-03-22, 公開日: 2014-07-16, 最終更新日: 2024-11-13)

主引用文献

Liu, C.T.,Layfield, J.P.,Stewart, R.J.,French, J.B.,Hanoian, P.,Asbury, J.B.,Hammes-Schiffer, S.,Benkovic, S.J.
Probing the electrostatics of active site microenvironments along the catalytic cycle for Escherichia coli dihydrofolate reductase.
J.Am.Chem.Soc., 136:10349-10360, 2014

PubMed Abstract: Electrostatic interactions play an important role in enzyme catalysis by guiding ligand binding and facilitating chemical reactions. These electrostatic interactions are modulated by conformational changes occurring over the catalytic cycle. Herein, the changes in active site electrostatic microenvironments are examined for all enzyme complexes along the catalytic cycle of Escherichia coli dihydrofolate reductase (ecDHFR) by incorporation of thiocyanate probes at two site-specific locations in the active site. The electrostatics and degree of hydration of the microenvironments surrounding the probes are investigated with spectroscopic techniques and mixed quantum mechanical/molecular mechanical (QM/MM) calculations. Changes in the electrostatic microenvironments along the catalytic environment lead to different nitrile (CN) vibrational stretching frequencies and (13)C NMR chemical shifts. These environmental changes arise from protein conformational rearrangements during catalysis. The QM/MM calculations reproduce the experimentally measured vibrational frequency shifts of the thiocyanate probes across the catalyzed hydride transfer step, which spans the closed and occluded conformations of the enzyme. Analysis of the molecular dynamics trajectories provides insight into the conformational changes occurring between these two states and the resulting changes in classical electrostatics and specific hydrogen-bonding interactions. The electric fields along the CN axes of the probes are decomposed into contributions from specific residues, ligands, and solvent molecules that make up the microenvironments around the probes. Moreover, calculation of the electric field along the hydride donor-acceptor axis, along with decomposition of this field into specific contributions, indicates that the cofactor and substrate, as well as the enzyme, impose a substantial electric field that facilitates hydride transfer. Overall, experimental and theoretical data provide evidence for significant electrostatic changes in the active site microenvironments due to conformational motion occurring over the catalytic cycle of ecDHFR.

PubMed: 24977791
DOI: 10.1021/ja5038947

実験手法

X-RAY DIFFRACTION (2.26 Å)