title: Cooperative formation of a substrate binding pocket by alpha- and beta-subunits of mitochondrial processing peptidase.
abstract: Mitochondrial processing peptidase (MPP) specifically recognizes a large variety of mitochondrial precursor proteins and cleaves off N-terminal extension peptides. The enzyme is a metalloprotease and forms a heterodimer consisting of structurally related alpha- and beta-subunits. To investigate the responsibility of MPP subunits for substrate recognition, we monitored interaction of the fluorescent-labeled peptide substrates with the MPP and its subunits. The specific binding of the peptide to the MPP was confirmed by findings of the direct participation of arginine residues in the binding, which are located at position -2 and the position distal to the cleavage site and are essential for the cleavage reaction. MPP bound the substrate peptides with high affinity only in the dimeric complex, and each subunit monomer had about a 30-fold less affinity than the complex. The individual subunit required arginines at different positions in the peptide for binding, although their affinities were much lower than that of MPP. Fluorescence quenching analysis showed that the peptide bound to MPP was buried in the enzyme. Thus, both subunits of MPP might be required for formation of a substrate binding pocket with multiple subsites lying across them.
authors.affiliation: Department of Chemistry, Faculty of Science, Kyushu University, Fukuoka 812-8581, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Kojima
authors.forename: S
authors.initials: S
authors.lastname: Kitada
authors.forename: K
authors.initials: K
authors.lastname: Shimokata
authors.forename: T
authors.initials: T
authors.lastname: Ogishima
authors.forename: A
authors.initials: A
authors.lastname: Ito
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D003374:Coumarins / Q000737:chemistry; D019281:Dimerization; D008666:Metalloendopeptidases / Q000378:metabolism*; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D051381:Rats; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D000096122:Mitochondrial Processing Peptidase
chemical_list: D003374:Coumarins; D008666:Metalloendopeptidases; D010455:Peptides; D000096122:Mitochondrial Processing Peptidase; C030123:coumarin