title: Probing the electrostatics of active site microenvironments along the catalytic cycle for Escherichia coli dihydrofolate reductase.
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.
authors: Department of Chemistry, Pennsylvania State University , University Park, Pennsylvania 16802, United States.
authors: C Tony
authors: CT
authors: Liu
authors: Joshua P
authors: JP
authors: Layfield
authors: Robert J
authors: RJ
authors: Stewart
authors: Jarrod B
authors: JB
authors: French
authors: Philip
authors: P
authors: Hanoian
authors: John B
authors: JB
authors: Asbury
authors: Sharon
authors: S
authors: Hammes-Schiffer
authors: Stephen J
authors: SJ
authors: Benkovic
mesh_terms: D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry*; D006860:Hydrogen Bonding; D007700:Kinetics; D056004:Molecular Dynamics Simulation; D015335:Molecular Probes / Q000737:chemistry; D019906:Nuclear Magnetic Resonance, Biomolecular; D011789:Quantum Theory; D017550:Spectroscopy, Fourier Transform Infrared; D055672:Static Electricity; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry*; D013861:Thiocyanates / Q000737:chemistry
chemical_list: D029968:Escherichia coli Proteins; D015335:Molecular Probes; D013861:Thiocyanates; D013762:Tetrahydrofolate Dehydrogenase; C031760:thiocyanate