Iorgu, A.I., Baxter, N.J., Cliff, M.J. et al. (4 more authors) (2018) Nonequivalence of second sphere "noncatalytic" residues in pentaerythritol tetranitrate reductase in relation to local dynamics linked to H-transfer in reactions with NADH and NADPH coenzymes. ACS Catalysis, 8 (12). pp. 11589-11599. ISSN 2155-5435
Abstract
Many enzymes that catalyze hydride transfer reactions work via a mechanism dominated by quantum mechanical tunneling. The involvement of fast vibrational modes of the reactive complex is often inferred in these reactions, as in the case of the NAD(P)H-dependent pentaerythritol tetranitrate reductase (PETNR). Herein, we interrogated the H-transfer mechanism in PETNR by designing conservative (L25I and I107L) and side chain shortening (L25A and I107A) PETNR variants and using a combination of experimental approaches (stopped-flow rapid kinetics, X-ray crystallography, isotope/temperature dependence studies of H-transfer and NMR spectroscopy). X-ray data show subtle changes in the local environment of the targeted side chains but no major structural perturbation caused by mutagenesis of these two second sphere active site residues. However, temperature dependence studies of H-transfer revealed a coenzyme-specific and complex thermodynamic equilibrium between different reactive configurations in PETNR–coenzyme complexes. We find that mutagenesis of these second sphere “noncatalytic” residues affects differently the reactivity of PETNR with NADPH and NADH coenzymes. We attribute this to subtle, dynamic structural changes in the PETNR active site, the effects of which impact differently in the nonequivalent reactive geometries of PETNR−NADH and PETNR−NADPH complexes. This inference is confirmed through changes observed in the NMR chemical shift data for PETNR complexes with unreactive 1,4,5,6-tetrahydro-NAD(P) analogues. We show that H-transfer rates can (to some extent) be buffered through entropy–enthalpy compensation, but that use of integrated experimental tools reveals hidden complexities that implicate a role for dynamics in this relatively simple H-transfer reaction. Similar approaches are likely to be informative in other enzymes to understand the relative importance of (distal) hydrophobic side chains and dynamics in controlling the rates of enzymatic H-transfer.
Metadata
Item Type: | Article |
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Authors/Creators: |
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Copyright, Publisher and Additional Information: | © 2018 American Chemical Society. This is an open access article published under a Creative Commons Attribution (CC-BY) License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution and reproduction in any medium, provided the author and source are cited. |
Keywords: | Pentaerythritol tetranitrate reductase; kinetic isotope effects; hydride transfer; protein dynamics; protein NMR; protein crystallography |
Dates: |
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Institution: | The University of Sheffield |
Academic Units: | The University of Sheffield > Faculty of Science (Sheffield) > School of Biosciences (Sheffield) > Department of Molecular Biology and Biotechnology (Sheffield) |
Depositing User: | Symplectic Sheffield |
Date Deposited: | 18 Feb 2019 15:40 |
Last Modified: | 18 Feb 2019 15:40 |
Published Version: | https://doi.org/10.1021/acscatal.8b02810 |
Status: | Published |
Publisher: | American Chemical Society |
Refereed: | Yes |
Identification Number: | 10.1021/acscatal.8b02810 |
Related URLs: | |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:142170 |