Involvement of Phenylalanine 297 in the Construction of the Substrate Pocket of Human Aminopeptidase B.
Aminopeptidase B (APB, EC 3.4.11.6) preferentially hydrolyzes the N-terminal basic amino acids of synthetic and peptide substrates and requires a physiological concentration of NaCl for optimal activity. In this study, we used site-directed mutagenesis and molecular modeling to search for an amino acid residue that is critical for the enzymatic properties of human APB. Substitution of Phe297 with Tyr caused a significant decrease in hydrolytic activity toward synthetic and peptide substrates as well as chloride anion sensitivity. Molecular modeling suggests that Phe297 contributes to the construction of the substrate pocket of APB, which is wide enough to hold a chloride anion and allow the interaction of Gln169 with the N-terminal Arg residue of the substrate through bridging with the chloride anion. These results indicate that Phe297 is crucial for the optimal enzymatic activity and chloride anion sensitivity of APB via formation of the optimal structure of the catalytic pocket.
Faculty of Pharmaceutical Sciences, Teikyo Heisei University , Nakano, Tokyo 164-8530, Japan.
Atsushi
A
Ohnishi
Faculty of Pharmaceutical Sciences, Teikyo Heisei University , Nakano, Tokyo 164-8530, Japan.
Jobu
J
Watanabe
Faculty of Pharmaceutical Sciences, Teikyo Heisei University , Nakano, Tokyo 164-8530, Japan.
Yuko
Y
Ogawa
Faculty of Pharmaceutical Sciences, Teikyo Heisei University , Nakano, Tokyo 164-8530, Japan.
Yoshikuni
Y
Goto
Department of System Chemotherapy and Molecular Sciences, Graduate School of Pharmaceutical Sciences, Kyoto University , Kyoto 606-8501, Japan.
Akira
A
Hattori
Faculty of Pharmaceutical Sciences, Teikyo Heisei University , Nakano, Tokyo 164-8530, Japan.
Masafumi
M
Tsujimoto
D019943:Amino Acid Substitution; D000626:Aminopeptidases / Q000737:chemistry* / Q000235:genetics; D020134:Catalytic Domain; D006801:Humans; D008958:Models, Molecular; D010649:Phenylalanine / Q000737:chemistry* / Q000235:genetics
D010649:Phenylalanine; D000626:Aminopeptidases; C019721:aminopeptidase B