Replacing the catalytic C188 by serine or glycine caused a considerable loss of activity, which could be recovered by mutagenesis of the hydrophobic pocket. Using a combination of empirical valence bond, conventional molecular dynamics, and well-tempered metadynamics simulations, we showed that the amino acid substitutions during the iterative and simultaneous saturation mutagenesis substantially increased the hydrophobicity of the active-site pocket of AMDase and formed a second hydrophobic pocket. This enhancement potentially promotes unfavorable interactions with the leaving carboxylate that leads to its cleavage.