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 μM or less. Three of four peptides containing 11 or fewer residues exhibited K m values above 10 μM. 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 local zation 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.
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