Structure of a low-population intermediate state in the release of an enzyme product

  1. Alfonso De Simone
  2. Francesco A Aprile
  3. Anne Dhulesia
  4. Christopher M Dobson
  5. Michele Vendruscolo  Is a corresponding author
  1. Imperial College London, United Kingdom
  2. University of Cambridge, United Kingdom

Abstract

Enzymes can increase the rate of biomolecular reactions by several orders of magnitude. Although the steps of substrate capture and product release are essential in the enzymatic process, complete atomic-level descriptions of these steps are difficult to obtain because of the transient nature of the intermediate conformations, which makes them largely inaccessible to standard structure determination methods. We describe here the determination of the structure of a low-population intermediate in the product release process by human lysozyme through a combination of NMR spectroscopy and molecular dynamics simulations. We validate this structure by rationally designing two mutations, the first engineered to destabilise the intermediate and the second to stabilize it, thus slowing down or speeding up, respectively, product release. These results illustrate how product release by an enzyme can be facilitated by the presence of a metastable intermediate with transient weak interactions between the enzyme and the product.

Article and author information

Author details

  1. Alfonso De Simone

    Division of Molecular Biosciences, Imperial College London, London, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  2. Francesco A Aprile

    Department of Chemistry, University of Cambridge, Cambridge, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  3. Anne Dhulesia

    Department of Chemistry, University of Cambridge, Cambridge, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  4. Christopher M Dobson

    Department of Chemistry, University of Cambridge, Cambridge, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  5. Michele Vendruscolo

    Department of Chemistry, University of Cambridge, Cambridge, United Kingdom
    For correspondence
    mv245@cam.ac.uk
    Competing interests
    The authors declare that no competing interests exist.

Reviewing Editor

  1. John Kuriyan, Howard Hughes Medical Institute, University of California, Berkeley, United States

Version history

  1. Received: March 13, 2014
  2. Accepted: December 16, 2014
  3. Accepted Manuscript published: January 9, 2015 (version 1)
  4. Version of Record published: January 30, 2015 (version 2)

Copyright

© 2015, De Simone et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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  1. Alfonso De Simone
  2. Francesco A Aprile
  3. Anne Dhulesia
  4. Christopher M Dobson
  5. Michele Vendruscolo
(2015)
Structure of a low-population intermediate state in the release of an enzyme product
eLife 4:e02777.
https://doi.org/10.7554/eLife.02777

Share this article

https://doi.org/10.7554/eLife.02777

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