title: Aspartic acid 405 contributes to the substrate specificity of aminopeptidase B.
abstract: Aminopeptidase B (EC 3.4.11.6, ApB) specifically cleaves in vitro the N-terminal Arg or Lys residue from peptides and synthetic derivatives. Ap B was shown to have a consensus sequence found in the metallopeptidase family. We determined the putative zinc binding residues (His324, His328, and Glu347) and the essential Glu325 residue for the enzyme using site-directed mutagenesis (Fukasawa, K. M., et al. (1999) Biochem. J. 339, 497-502). To identify the residues binding to the amino-terminal basic amino acid of the substrate, rat cDNA encoding ApB was cloned into pGEX-4T-3 so that recombinant protein was expressed as a GST fusion protein. Twelve acidic amino acid residues (Glu or Asp) in ApB were replaced with a Gln or Asn using site-directed mutagenesis. These mutants were isolated to characterize the kinetic parameters of enzyme activity toward Arg-NA and compare them to those of the wild-type ApB. The catalytic efficiency (kcat/Km) of the mutant D405N was 1.7 x 10(4) M(-1) s(-1), markedly decreased compared with that of the wild-type ApB (6.2 x 10(5) M(-1) s(-1)). The replacement of Asp405 with an Asn residue resulted in the change of substrate specificity such that the specific activity of the mutant D405N toward Lys-NA was twice that toward Arg-NA (in the case of wild-type ApB; 0.4). Moreover, when Asp405 was replaced with an Ala residue, the kcat/Km ratio was 1000-fold lower than that of the wild-type ApB for hydrolysis of Arg-NA; in contrast, in the hydrolysis of Tyr-NA, the kcat/Km ratios of the wild-type (1.1 x 10(4) M(-1) s(-1)) and the mutated (8.2 x 10(3) M(-1) s(-1)) enzymes were similar. Furthermore, the replacement of Asp-405 with a Glu residue led to the reduction of the kcat/Km ratio for the hydrolysis of Arg-NA by a factor of 6 and an increase of that for the hydrolysis of Lys-NA. Then the kcat/Km ratio of the D405E mutant for the hydrolysis of Lys-NA was higher than that for the hydrolysis of Arg-NA as opposed to that of wild-type ApB. These data strongly suggest that the Asp 405 residue is involved in substrate binding via an interaction with the P1 amino group of the substrate's side chain.
authors.affiliation: Department of Hard Tissue Research, Graduate School of Oral Medicine, Matsumoto Dental University, School of Dentistry, Shiojiri, Nagano 399-0781, Japan. kmf@po.mdu.ac.jp
authors.forename: Kayoko M
authors.initials: KM
authors.lastname: Fukasawa
authors.forename: Junzo
authors.initials: J
authors.lastname: Hirose
authors.forename: Toshiyuki
authors.initials: T
authors.lastname: Hata
authors.forename: Yukio
authors.initials: Y
authors.lastname: Ono
mesh_terms: D000409:Alanine / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000626:Aminopeptidases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D001120:Arginine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D001224:Aspartic Acid / Q000378:metabolism*; D004851:Epoxide Hydrolases / Q000737:chemistry / Q000378:metabolism; D018698:Glutamic Acid / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D008954:Models, Biological; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D016415:Sequence Alignment; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D050505:Mutant Proteins; D011994:Recombinant Proteins; D001224:Aspartic Acid; D018698:Glutamic Acid; C029772:arginine beta-naphthylamide; D001120:Arginine; D004851:Epoxide Hydrolases; D000626:Aminopeptidases; C019721:aminopeptidase B; D000409:Alanine; C043134:leukotriene A4 hydrolase