Determination of the Cleavage Site of the Presequence by Mitochondrial Processing Peptidase on the Substrate Binding Scaffold and the Multiple Subsites inside a Molecular Cavity*

Mitochondrial processing peptidase (MPP) recognizes a large variety of basic presequences of mitochondrial preproteins and cleaves the single site, often including arginine, at the −2 position (P₂). To elucidate the recognition and specific processing of the preproteins by MPP, we mutated to alanines at acidic residues conserved in a large internal cavity formed by the MPP subunits, α-MPP and β-MPP, and analyzed the processing efficiencies for various preproteins. We report here that alanine mutations at a subsite in rat β-MPP interacting with the P₂ arginine cause a shift in the processing site to the C-terminal side of the preprotein. Because of reduced interactions with the P₂ arginine, the mutated enzymes recognize not only the N-terminal authentic cleavage site with P₂ arginine but also the potential C-terminal cleavage site without a P₂arginine. In fact, it competitively cleaves the two sites of the preprotein. Moreover, the acidified site of α-MPP, which binds to the distal basic site in the long presequence, recognized the authentic P₂ arginine as the distal site in compensation for ionic interaction at the proximal site in the mutant MPP. Thus, MPP seems to scan the presequence from β- to α-MPP on the substrate binding scaffold inside the MPP cavity and finds the distal and P₂arginines on the multiple subsites on both MPP subunits. A possible mechanism for substrate recognition and cleavage is discussed here based on the notable character of a subsite-deficient mutant of MPP in which the substrate specificity is altered.