Probing the acyl‐binding pocket of aminoacylase‐1

In the metallopeptidase family M20, remodeling of a common scaffold has given rise to various amidohydrolases that feature a homodimerization domain nested within the Zn‐binding domain. Residues from both dimerization domains and one of the two Zn‐binding domains, respectively, contribute to the active sites. One such enzyme, the mammalian aminoacylase‐1 (Acy1), functions in the salvage of acetylated amino acids. The related Pseudomonas carboxypeptidase G2 (CPG2) is used in cancer therapy. CPG2 and Acy1 both deacylate amino acids, but with distinct selectivities toward the acyl portion: CPG2 prefers bulky acyl moieties whereas Acy1 selects for acetyl and small acyl groups. To map the apparently more restricted acyl‐binding pocket of Acy1, we designed and assayed a series of aliphatic acyl‐Met substrates for the human and porcine enzymes. 3D‐QSAR models were derived to relate measured substrate binding affinities (p K M ) to molecular topologies of the acyl fragments. The results indicate subtle differences between the two Acy1 enzymes. Docking of the 3D‐QSAR map for human Acy1 in the crystal structure of its Zn‐binding domain associates the I177, T347 and L372 side chains with detrimental contributions to the binding of larger acyl groups. We probed these predictions by site‐directed mutagenesis and enzymatic characterization. Lower K M values for T347S and L372V indicate improved binding affinities toward apposite acyl moieties. This strengthens our prediction that T347 and L372 specifically restrict the acyl‐binding pocket, and shows the potential for engineering of substrate specificity. Supported by the National Research Council Canada