title: Complete covalent structure of porcine liver acylamino acid-releasing enzyme and identification of its active site serine residue.
abstract: The complete covalent structure of porcine liver acylamino acid-releasing enzyme (AARE) [EC3.4.19.1], which catalyzes the hydrolysis of an N-terminally acylated peptide to release an N-acylamino acid, has been established. On basis of the amino acid sequence deduced from the cDNA sequence of porcine liver AARE [Mitta, M. et al. (1989) J. Biochem. 106, 548-555], sequence determination has been achieved by automated Edman degradation of peptides generated by chemical or enzymatic cleavages of the reduced and S-carboxymethylated protein. Ion-spray mass spectrometry was also successfully used to confirm the amino acid sequences of the peptides determined above and to elucidate both the N-terminal blocking group and the status of half-cystine residues of this protein. The protein consists of 732 amino acid residues, and the N-terminal methionine residue is blocked by an acetyl group. All of 18 half-cystine residues of this protein were proved to exist as cysteine residues. A serine residue reactive with diisopropyl fluorophosphate (DFP) was also identified as Ser587 by preparation of the AARE labeled with tritiated DFP followed by isolation and sequence analysis of a radioactive peptide obtained from its endoproteinase Asp-N digest.
authors.affiliation: Biotechnology Research Laboratories, Takara Shuzo Co., Ltd., Otsu.
authors.forename: M
authors.initials: M
authors.lastname: Miyagi
authors.forename: F
authors.initials: F
authors.lastname: Sakiyama
authors.forename: I
authors.initials: I
authors.lastname: Kato
authors.forename: S
authors.initials: S
authors.lastname: Tsunasawa
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D008099:Liver / Q000201:enzymology*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010447:Peptide Hydrolases / Q000737:chemistry*; D012694:Serine / Q000737:chemistry*; D013552:Swine
chemical_list: D012694:Serine; D010447:Peptide Hydrolases; C038220:acylaminoacyl-peptidase