Three-dimensional structural models of porcine aminoacylase 1 (pACY1) were constructed by homology modeling and aligning the structures of members of the M20 peptidase family. After energy minimization and quality evaluation, the best model from the homology modeling was chosen for docking with the best substrate (N-acetyl-L-methionine). The most reasonable binding mode was found among a large number of predicted complexes by using clustering analysis and screening with expert knowledge. Structural analysis revealed that the zinc ion is not likely to bind to the substrate, and that Arg348 and Glu146 play vital roles in binding and catalysis. In the site-directed mutagenesis experiments, mutation of His79, Asp112, Glu147, Arg348, and Glu146, resulted in significant reductions of specific activity, while the wild-type pACY1 overexpressed in Rosetta (DE3) had almost as high a specific activity as the native enzyme. On the basis of these observations, we proposed a revised catalytic mechanism for this metalloenzyme.