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Exhaustive mutagenesis of six secondary active-site residues in Escherichia coli chorismate mutase shows the importance of hydrophobic side chains and a helix N-capping position for stability and catalysis

Lassila, Jonathan Kyle and Keeffe, Jennifer R. and Kast, Peter and Mayo, Stephen L. (2007) Exhaustive mutagenesis of six secondary active-site residues in Escherichia coli chorismate mutase shows the importance of hydrophobic side chains and a helix N-capping position for stability and catalysis. Biochemistry, 46 (23). pp. 6883-6891. ISSN 0006-2960. doi:10.1021/bi700215x.

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Secondary active-site residues in enzymes, including hydrophobic amino acids, may contribute to catalysis through critical interactions that position the reacting molecule, organize hydrogen-bonding residues, and define the electrostatic environment of the active site. To ascertain the tolerance of an important model enzyme to mutation of active-site residues that do not directly hydrogen bond with the reacting molecule, all 19 possible amino acid substitutions were investigated in six positions of the engineered chorismate mutase domain of the Escherichia coli chorismate mutase-prephenate dehydratase. The six secondary active-site residues were selected to clarify results of a previous test of computational enzyme design procedures. Five of the positions encode hydrophobic side chains in the wild-type enzyme, and one forms a helix N-capping interaction as well as a salt bridge with a catalytically essential residue. Each mutant was evaluated for its ability to complement an auxotrophic chorismate mutase deletion strain. Kinetic parameters and thermal stabilities were measured for variants with in vivo activity. Altogether, we find that the enzyme tolerated 34% of the 114 possible substitutions, with a few mutations leading to increases in the catalytic efficiency of the enzyme. The results show the importance of secondary amino acid residues in determining enzymatic activity, and they point to strengths and weaknesses in current computational enzyme design procedures.

Item Type:Article
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URLURL TypeDescription DOIArticle
Keeffe, Jennifer R.0000-0002-5317-6398
Kast, Peter0000-0002-0209-8975
Mayo, Stephen L.0000-0002-9785-5018
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Additional Information:© 2007 American Chemical Society. Received January 31, 2007; Revised Manuscript Received April 12, 2007, Published on Web 05/17/2007. This work was supported by the Howard Hughes Medical Institute, the Defense Advanced Research Projects Agency, the Institute for Collaborative Biotechnologies (ARO), the Ralph M. Parsons foundation, and an IBM Shared University Research Grant.
Funding AgencyGrant Number
Howard Hughes Medical Institute (HHMI)UNSPECIFIED
Defense Advanced Research Projects Agency (DARPA)UNSPECIFIED
Army Research Office (ARO)UNSPECIFIED
Ralph M. Parsons FoundationUNSPECIFIED
Subject Keywords:Amino Acid Sequence; Bacterial Proteins; Protein Conformation; Amino Acid Substitution; Models: Molecular; Catalysis; Mutagenesis: Site-Directed; Escherichia coli Proteins; Binding Sites; Chorismate Mutase; Enzyme Stability; Sequence Alignment; Escherichia coli; Recombinant Proteins
Issue or Number:23
Record Number:CaltechAUTHORS:20110913-160001644
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Official Citation:Exhaustive Mutagenesis of Six Secondary Active-Site Residues in Escherichia coli Chorismate Mutase Shows the Importance of Hydrophobic Side Chains and a Helix N-Capping Position for Stability and Catalysis
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:25325
Deposited By: Marie Ary
Deposited On:16 Sep 2011 18:13
Last Modified:09 Nov 2021 16:32

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