Importance of Tyr409 and Tyr414 in constructing the substrate pocket of human aminopeptidase B.

Abstract

Aminopeptidase B (APB, EC 3.4.11.6) preferentially hydrolyzes basic amino acids of synthetic substrates and requires a physiological concentration of chloride anions for optimal activity. Several amino acid residues of APB responsible for its enzymatic activity have been elucidated. In this study, we further searched for residues critical to its enzymatic activity, especially toward peptide substrates. APB residues Tyr409 (Y409) and Tyr414 (Y414), both of which were critical to its hydrolytic activity toward synthetic substrates, were predicted by molecular modeling to be involved in cleaving peptide substrates via its interaction with amino acids in the P1' cleavage site. Using site-directed mutagenesis, several mutant APBs were prepared. In contrast to synthetic substrates, wild-type and Y409F/Y414F double mutant enzymes showed P1'-dependent cleavage of peptide substrates, indicating that both tyrosine residues were not indispensable for hydrolytic activity toward peptide substrates. Moreover, the Y409F/Y414F double mutant enzyme cleaved peptides with a Pro residue at the P1' site, which is uncommon among the M1 family of aminopeptidases. These results suggested that Tyr409 and Tyr414 of APB play important roles in enzymatic function and characteristic properties of APB via proper formation of the S1' site.