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

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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.

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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.
Ariana Peck

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

Competing interests
The authors declare that no competing interests exist.
Julia Salzman

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

Competing interests
The authors declare that no competing interests exist.
Susanne Ressl

Molecular and Cellular Biochemistry Department, Indiana University Bloomington, Bloomington, United States

Competing interests
The authors declare that no competing interests exist.
Daniel Herschlag

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

Competing interests
The authors declare that no competing interests exist.

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