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

• Published in: The Journal of biological chemistry Vol. 278; no. 3; pp. 1879 - 1885
• Main Authors: Kitada, Sakae, Yamasaki, Eiki, Kojima, Katsuhiko, Ito, Akio
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 17.01.2003; American Society for Biochemistry and Molecular Biology
• Subjects: Amino Acid Sequence; Animals; Binding Sites; Hydrolysis; Metalloendopeptidases - chemistry; Metalloendopeptidases - genetics; Metalloendopeptidases - isolation & purification; Metalloendopeptidases - metabolism; Mitochondrial Processing Peptidase; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Rats; Substrate Specificity
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M209263200

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.