The mechanism of the amidases: Mutating the glutamate adjacent to the catalytic triad inactivates the enzyme due to substrate mispositioning

Background:A cysteine, a glutamic acid, and a lysine are the well known amidase catalytic residues. Results:Mutating the neighboring, structurally conserved Glu-142 inactivates the enzyme, but the active site cysteine still reacts with acrylamide via its double bond. Conclusion:Glu-142 positions the amide for productive nucleophilic attack by the cysteine. Significance:An intact catalytic tetrad is required for amidase activity. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc.