title: Role of glutamine-169 in the substrate recognition of human aminopeptidase B.
abstract: BACKGROUND
  Aminopeptidase B (EC 3.4.11.6, APB) preferentially hydrolyzes N-terminal basic amino acids of synthetic and peptide substrates. APB is involved in the production and maturation of peptide hormones and neurotransmitters such as miniglucagon, cholecystokinin and enkephalin by cleaving N-terminal basic amino acids in extended precursor proteins. Therefore, the specificity for basic amino acids is crucial for the biological function of APB.
  
  
  METHODS
  Site-directed mutagenesis and molecular modeling of the S1 site were used to identify amino acid residues of the human APB responsible for the basic amino acid preference and enzymatic efficiency.
  
  
  RESULTS
  Substitution of Gln169 with Asn caused a significant decrease in hydrolytic activity toward the fluorescent substrate Lys-4-methylcoumaryl-7-amide (MCA). Substantial retardation of enzyme activity was observed toward Arg-MCA and substitution with Glu caused complete loss of enzymatic activity of APB. Substitution with Asn led to an increase in IC50 values of inhibitors that interact with the catalytic pocket of APB. The EC50 value of chloride ion binding was also found to increase with the Asn mutant. Gln169 was required for maximal cleavage of the peptide substrates. Molecular modeling suggested that interaction of Gln169 with the N-terminal Arg residue of the substrate could be bridged by a chloride anion.
  
  
  CONCLUSIONS
  Gln169 is crucial for obtaining optimal enzymatic activity and the unique basic amino acid preference of APB via maintaining the appropriate catalytic pocket structure and thus for its function as a processing enzyme of peptide hormones and neurotransmitters.
authors.affiliation: Faculty of Pharmaceutical Sciences, Teikyo Heisei University, Nakano, Tokyo 164-8530, Japan.
authors.forename: Yuko
authors.initials: Y
authors.lastname: Ogawa
authors.affiliation: Faculty of Pharmaceutical Sciences, Teikyo Heisei University, Nakano, Tokyo 164-8530, Japan.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Ohnishi
authors.affiliation: Faculty of Pharmaceutical Sciences, Teikyo Heisei University, Nakano, Tokyo 164-8530, Japan.
authors.forename: Yoshikuni
authors.initials: Y
authors.lastname: Goto
authors.affiliation: Faculty of Pharmaceutical Sciences, Teikyo Heisei University, Nakano, Tokyo 164-8530, Japan.
authors.forename: Yoshiki
authors.initials: Y
authors.lastname: Sakuma
authors.affiliation: Faculty of Pharmaceutical Sciences, Teikyo Heisei University, Nakano, Tokyo 164-8530, Japan.
authors.forename: Jobu
authors.initials: J
authors.lastname: Watanabe
authors.affiliation: Department of System Chemotherapy and Molecular Sciences, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto 606-8501, Japan.
authors.forename: Akira
authors.initials: A
authors.lastname: Hattori
authors.affiliation: Faculty of Pharmaceutical Sciences, Teikyo Heisei University, Nakano, Tokyo 164-8530, Japan. Electronic address: tsujimot@thu.ac.jp.
authors.forename: Masafumi
authors.initials: M
authors.lastname: Tsujimoto
mesh_terms: D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D020134:Catalytic Domain; D005973:Glutamine; D006801:Humans; D006868:Hydrolysis; D008958:Models, Molecular; D008969:Molecular Sequence Data; D012965:Sodium Chloride / Q000494:pharmacology; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D005973:Glutamine; D012965:Sodium Chloride; D000626:Aminopeptidases; C019721:aminopeptidase B
keywords: Aminopeptidase; Catalytic pocket; Proteolytic processing; Site-directed mutagenesis; Substrate specificity