Extensive site-directed mutagenesis reveals interconnected functional units in the Alkaline Phosphatase active site

  1. Fanny Sunden  Is a corresponding author
  2. Ariana Peck
  3. Julia Salzman
  4. Susanne Ressl
  5. Daniel Herschlag
  1. Stanford University, United States
  2. Indiana University Bloomington, United States

Abstract

Enzymes enable life by accelerating reaction rates to biological timescales. Conventional studies have focused on identifying the residues that have a direct involvement in an enzymatic reaction, but these so-called 'catalytic residues' are embedded in extensive interaction networks. Although fundamental to our understanding of enzyme function, evolution, and engineering, the properties of these networks have yet to be quantitatively and systematically explored. We dissected an interaction network of five residues in the active site of E. Coli Alkaline Phosphatase. Analysis of the complex catalytic interdependence of specific residues identified three energetically independent but structurally interconnected functional units with distinct modes of cooperativity. From an evolutionary perspective, this network is orders of magnitude more probable to arise than a fully cooperative network. From a functional perspective, new catalytic insights emerge. Further, such comprehensive energetic characterization will be necessary to benchmark the algorithms required to rationally engineer highly efficient enzymes.

Article and author information

Author details

  1. Fanny Sunden

    Department of Biochemistry, Beckman Center, Stanford University, Stanford, United States
    For correspondence
    fsunden@stanford.edu
    Competing interests
    The authors declare that no competing interests exist.
  2. Ariana Peck

    Department of Biochemistry, Beckman Center, Stanford University, Stanford, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Julia Salzman

    Department of Biochemistry, Beckman Center, Stanford University, Stanford, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Susanne Ressl

    Molecular and Cellular Biochemistry Department, Indiana University Bloomington, Bloomington, United States
    Competing interests
    The authors declare that no competing interests exist.
  5. Daniel Herschlag

    Department of Biochemistry, Beckman Center, Stanford University, Stanford, United States
    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

Publication history

  1. Received: December 20, 2014
  2. Accepted: April 22, 2015
  3. Accepted Manuscript published: April 22, 2015 (version 1)
  4. Version of Record published: May 20, 2015 (version 2)

Copyright

© 2015, Sunden 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. Fanny Sunden
  2. Ariana Peck
  3. Julia Salzman
  4. Susanne Ressl
  5. Daniel Herschlag
(2015)
Extensive site-directed mutagenesis reveals interconnected functional units in the Alkaline Phosphatase active site
eLife 4:e06181.
https://doi.org/10.7554/eLife.06181

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