IDENTIFICATION OF THE CATALYTIC TRIAD RESIDUES OF PORCINE LIVER ACYLAMINO ACID-RELEASING ENZYME

Acylamino acid-releasing enzyme (AARE) [EC 3.4.19.1] is a tetrameric s erine protease, which belongs to the oligopeptidase family and specifi cally removes acetyl amino acids from N-terminally acetylated peptides , By using diisopropyl fluorophosphate, we previously identified one o f the residues comprising the catalytic triad of this enzyme as Ser(58 7) [Miyagi, M. et al, (1995) J, Biochem. 118, 771-779], To elucidate t he other two residues forming the catalytic triad of this new serine-t ype protease, wild-type and four mutant AAREs, in which each candidate residue of the catalytic triad deduced from sequence alignment with o ther oligopeptidases was substituted by site-directed mutagenesis, wer e expressed in Escherichia coli as fusion proteins with short peptide chains at both N- and C-termini of a subunit of porcine liver enzyme. All of the recombinant AAREs were estimated to have similar conformati onal and quaternary structures to the native porcine liver enzyme horn their CD spectra and behavior on gel-filtration, but the mutants in w hich Ala(587), Asn(675) or Tyr(707) was substituted for Ser(587), Asp( 675), Or His(707) respectively, did not show detectable hydrolytic act ivity toward acetyl-L-methionyl L-alanine, These facts suggest that Se r(587), Asp(675), and His(707) are essential residues for the AARE act ivity and comprise the catalytic triad of the enzyme in this order. Th us, AARE has been shown to have a protease-like domain in its C-termin al region, as do other proteins classified as members of the oligopept idase family.