Mammalian Pitrilysin: Substrate Specificity and Mitochondrial Targeting

The substrate specificity of the mitochondrial metallopeptidase proteinase 1 (MP1) was investigated and its mitochondrial targeting signal identified. the substrate specificity of MP1 was examined with physiological peptides as substrates. Although the enzyme exhibits broad substrate specificity, there is a trend for peptides containing 13 or more residues to exhibit K(m) values of 2 mu M or less. Three of four peptides containing 11 or fewer residues exhibited K(m) values above 10 mu M. Similarly, peptides containing 13 or more residues exhibited k(cat) values below 10 min(-1), while three of four peptides containing 11 or fewer residues exhibited k(cat) values above 30 min(-1). Many of the peptide cleavage sites of MP1 resemble that of the mitochondrial processing protease (MPP); however, MP1 does not process the precursor form of citrate synthase. the enzyme, however, does cleave the released prepeptide from precitrate synthase. A mitochondria localization was shown in MP1 transfected NT2 and HepG2 cells. Deletion of the N-terminal 15 amino acids caused MP1 to be mislocalized to the cytoplasm and nucleus. Furthermore, when fused to green flourescent protein, this 15-amino acid N-terminal sequence directed the fusion protein to the mitochondria.National Institute on Drug AbuseNational Center for Research Resources (NCRR)Univ Kentucky, Dept Mol & Cellular Biochem, Lexington, KY 40536 USAMayo Clin & Mayo Fdn, Dept Pediat & Adolescent Med & Biochem & Mol Biol, Rochester, MN 55905 USAEscola Paulista Med, Dept Biophys, BR-0404420 São Paulo, BrazilEscola Paulista Med, Dept Biophys, BR-0404420 São Paulo, BrazilNational Institute on Drug Abuse: DA 02243National Center for Research Resources (NCRR): P20RR02017Web of Scienc