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.