Mechanism of Processing of Mitochondrial Protein Precursor

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Mitochondrial processing peptodase (MPP) specifically recognaizes large variety of mitochondrial precursorproteins and cleaves off amino-terminal extension peptides. The enzymeforms a heterodimerconsisting of structurally related alpha-and beta-subunits. Structure common to all the extension peptides that is requiredfor specific recognition by MPP and role of each subunit in catalytic reaction and substrate recognition were studied.1. Two sets of basic amino acids in the peptide, the proximal arginine residue at position-2 and the distal basic residues at positions about-10 relative to the cleavagesite, and hydrophobicamino acids at position 1 are necessary for effective hydrolysis. Residues position 2 and 3 also contribute significantly to the efficiency of processing.2.To investigate the responsibility of MPP subunits for substrate recognition, we used the fluorescent-labeled peptide substrates with a coumarin derivative and found that MPP bound the substrate peptides with high affin … More ity (Kd ; 0.13muM) only in the dimericcomplex and each subunit had about a 30-foldless affinity than the somplex, indicating that the two subunits of MPP cooperatively form a substrate-binding pocket.3.Mutation experiments demonstrate that three glutamate residues, positions at 79,129, and 136, around the metal binding sitein beta-MPP,in addition to a putative active site glutamate at position 59, are essential for the function. Glu-79,129, and 136 appears to be involved in substrate binding, catalysis, and metal binding, respectively.4.Mutation of acidic amino acidresidues in the carboxy-terminal portion of the alpha-MPP causes a decreasein cleavage efficiency of precursors with a longer extension peptide.Taken together, we proposed a model for substrate recogmition of MPP,in which the two subunits of MPP cooperatively form the substrate binding pocket and recognize different structural elements in the extension peptide. The multiple-site recognition mechanism may make it possible to render strict spesificity and high affinity to MPP for precursors with structures little in common. Less