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Increased dynamic effects in a catalytically compromised variant of Escherichia coli dihydrofolate reductase.

ABSTRACT: Isotopic substitution ((15)N, (13)C, (2)H) of a catalytically compromised variant of Escherichia coli dihydrofolate reductase, EcDHFR-N23PP/S148A, has been used to investigate the effect of these mutations on catalysis. The reduction of the rate constant of the chemical step in the EcDHFR-N23PP/S148A catalyzed reaction is essentially a consequence of an increase of the quasi-classical free energy barrier and to a minor extent of an increased number of recrossing trajectories on the transition state dividing surface. Since the variant enzyme is less well set up to catalyze the reaction, a higher degree of active site reorganization is needed to reach the TS. Although millisecond active site motions are lost in the variant, there is greater flexibility on the femtosecond time scale. The "dynamic knockout" EcDHFR-N23PP/S148A is therefore a "dynamic knock-in" at the level of the chemical step, and the increased dynamic coupling to the chemical coordinate is in fact detrimental to catalysis. This finding is most likely applicable not just to hydrogen transfer in EcDHFR but also to other enzymatic systems.

SUBMITTER: Ruiz-Pernia JJ

PROVIDER: S-EPMC3949409 | biostudies-literature | 2013 Dec

REPOSITORIES: biostudies-literature

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Increased dynamic effects in a catalytically compromised variant of Escherichia coli dihydrofolate reductase.

Ruiz-Pernia J Javier JJ Luk Louis Y P LY García-Meseguer Rafael R Martí Sergio S Loveridge E Joel EJ Tuñón Iñaki I Moliner Vicent V Allemann Rudolf K RK

Journal of the American Chemical Society 20131126 49

Isotopic substitution ((15)N, (13)C, (2)H) of a catalytically compromised variant of Escherichia coli dihydrofolate reductase, EcDHFR-N23PP/S148A, has been used to investigate the effect of these mutations on catalysis. The reduction of the rate constant of the chemical step in the EcDHFR-N23PP/S148A catalyzed reaction is essentially a consequence of an increase of the quasi-classical free energy barrier and to a minor extent of an increased number of recrossing trajectories on the transition st ...[more]

PMID: 24252106

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Project description: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.

Dataset Information

Increased dynamic effects in a catalytically compromised variant of Escherichia coli dihydrofolate reductase.

Publications

Increased dynamic effects in a catalytically compromised variant of Escherichia coli dihydrofolate reductase.

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