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Enhanced active-site electric field accelerates enzyme catalysis

Nature Chemistry volume 15, pages 1715–1721 (2023)Cite this article

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Abstract

The design and improvement of enzymes based on physical principles remain challenging. Here we demonstrate that the principle of electrostatic catalysis can be leveraged to substantially improve a natural enzyme’s activity. We enhanced the active-site electric field in horse liver alcohol dehydrogenase by replacing the serine hydrogen-bond donor with threonine and replacing the catalytic Zn2+ with Co2+. Based on the electric field enhancement, we make a quantitative prediction of rate acceleration—50-fold faster than the wild-type enzyme—which was in close agreement with experimental measurements. The effects of the hydrogen bonding and metal coordination, two distinct chemical forces, are described by a unified physical quantity—electric field, which is quantitative, and shown here to be additive and predictive. These results suggest a new design paradigm for both biological and non-biological catalysts.

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Fig. 1: Electrostatic catalysis of LADH.
Fig. 2: Metal exchange in the active site of LADH.
Fig. 3: Hydrogen-bond perturbation in the active site of LADH.
Fig. 4: A unifying electrostatic basis for enzyme catalysis and design.

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Data availability

The X-ray coordinates and structural factors of LADH variants complexed with NADH and CXF have been deposited in the Protein Data Bank (PDB) as entries 7UQ9 (LADHS48T), 8EIW (LADHCo), 7UTW (LADHCd), 7U9N (LADHS48A), 8EIY (LADHCo,S48T) and 8EIX (LADHCo,S48A). The structure 2OHX was also used in this study and is accessible in the PDB. All the data that support the finding of this study are available within this article, Supplementary Information and provided source data.

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Acknowledgements

We thank B. V. Plapp at the University of Iowa for providing detailed guidance on the expression and purification of LADH and much other valuable advice. We thank I. Andersson at Uppsala University and W. Maret at King’s College London for their advice on the metal substitution of LADH. We thank A. Braun, A. Heyer and M. Brueggemeyer for the data analysis and discussion of LADHCo; G. Li at the Stanford Environmental Measurements Facility (EMF) for the inductively coupled plasma atomic emission spectroscopy data collection; T. McLaughlin from Stanford University Mass Spectrometry (SUMS) for the measurements of native mass spectrometry; T. Carver at the Stanford Nano Shared Facilities (SNSF) for nickel coating Stark windows. C.Z. is grateful for a Stanford Center for Molecular Analysis and Design (CMAD) Fellowship. This work was supported by NIH grant GM118044 (to S.G.B.). Use of the Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, is supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences under contract number DE-AC02-76SF00515. The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research, and by the National Institutes of Health, National Institute of General Medical Sciences (P30GM133894). The contents of this publication are solely the responsibility of the authors and do not necessarily represent the official views of NIGMS or NIH. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the paper.

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Author notes

  1. These authors contributed equally: Chu Zheng, Zhe Ji.

Authors and Affiliations

  1. Department of Chemistry, Stanford University, Stanford, CA, USA

    Chu Zheng, Zhe Ji & Steven G. Boxer

  2. Stanford Synchrotron Radiation Lightsource, Menlo Park, CA, USA

    Irimpan I. Mathews

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  1. Chu Zheng
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Contributions

C.Z. and S.G.B. designed the research. C.Z. and Z.J. performed most of the experiments and data analysis, including expression and purification of LADH variants, metal substitution of LADH, infrared spectroscopy and enzyme kinetic studies. C.Z. and I.I.M. performed X-ray crystallography and solved the crystal structures of LADH variants. C.Z., Z.J. and S.G.B discussed the results and wrote the paper.

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Correspondence to Steven G. Boxer.

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Source data

Source Data Fig. 2

Numerical data for optical spectra and enzyme kinetics.

Source Data Fig. 2

X-ray crystallographic data.

Source Data Fig. 3

Numerical data for optical spectra and enzyme kinetics.

Source Data Fig. 3

X-ray crystallographic data.

Source Data Fig. 4

Numerical data for optical spectra and scatter plots.

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Zheng, C., Ji, Z., Mathews, I.I. et al. Enhanced active-site electric field accelerates enzyme catalysis. Nat. Chem. 15, 1715–1721 (2023). https://doi.org/10.1038/s41557-023-01287-x

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