IMPORTANCE OF RESIDUES CARBOXYL-TERMINAL RELATIVE TO THE CLEAVAGE SITE IN SUBSTRATES OF MITOCHONDRIAL PROCESSING PEPTIDASE FOR THEIR SPECIFIC RECOGNITION AND CLEAVAGE

We previously identified distal and proximal arginine residues in the N-terminal portion and an aromatic amino acid at position 1 (P-1' site (3)) relative to the cleavage site as important recognition signals in substrates of mitochondrial processing peptidase [Niidome, T., Kitada , S., Shimokata, K., Ogishima, T., and Ito, A. (1994) J. Biol. Chem. 2 69, 24714-24722; Ogishima, T., Niidome, T., Shimokata,]K., Kitada, S., and Ito, A. (1995) ibid. 270, 30322-30326]. To further elucidate the elements required for the specific recognition and cleavage by the enz yme, we synthesized synthetic peptides that possessed only the distal and proximal arginine residues and phenylalanine at the P-1' site in a poly alanine sequence, and analyzed the processing reaction toward th em. They were not cleaved by the peptidase although they inhibited the peptidase activity. However, when serine was introduced into the C-te rminal portions of the sequence, processing was observed. The efficien cy of the resultant peptides improved as the number of serine residues was increased. A peptide with serine or histidine at P-2' and threoni ne at P-3' was processed most efficiently. These results indicate that the processing reaction catalyzed by the peptidase depends not only o n the N-terminal portion but also on the C-terminal portion from the c leavage site in the substrates.