Role of basic amino acids in the cleavage of synthetic peptide substrates by mitochondrial processing peptidase

Our recent experiments using model peptides of rat malate dehydrogenase (MDH) indicated that a proximal arginine and a distal basic amino acid are important for processing by mitochondrial processing peptidase (MPP). [Niidome, T., Kitada, S., Shimokata, K., Ogishima, T., and Ito, A. (1994) J. Biol. Chem. 269, 24719-24722]. To elucidate if the recognition elements apply to other precursor proteins, we analyzed cleavage of model peptides of human ornithine aminotransferase (OAT). Purified peptidase cleaved peptides that corresponded to N-terminal 1-25 and 3-25 at the correct site (Gly17-Val18) at nearly equal rates. Replacement of Arg16 (-2 position) with lysine or alanine reduced the processing efficiency by 95- and 380-fold, respectively. Either deletion from Met1 to Arg10 or replacement of the basic amino acids between them decreased the processing efficiency considerably. A peptide containing Arg7 in addition to Lys4 and Arg10 was more effective than the control peptide. However, a peptide with one and two consecutive basic amino acids in the distal region had a processing efficiency close to the control peptide. These results indicated that processing of OAT was enhanced by an increase in the number of basic amino acids with a suitable distance between them. In other respects, the processing signal of OAT was essentially the same as that of MDH.