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Cooperative formation of a substrate binding pocket by alpha- and beta-subunits of mitochondrial processing peptidase
K Kojima1, S Kitada, K Shimokata
1Department of Chemistry, Faculty of Science, Kyushu University, Fukuoka 812-8581, Japan.
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.
Insights
Mitochondrial processing peptidase (MPP) requires both alpha and beta subunits for high-affinity substrate binding. This enzyme complex, crucial for protein processing, utilizes specific arginine residues for efficient substrate recognition and cleavage.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Mitochondrial processing peptidase (MPP) is a metalloprotease responsible for cleaving N-terminal extension peptides from mitochondrial precursor proteins.
- MPP functions as a heterodimer composed of structurally related alpha- and beta-subunits.
Purpose of the Study:
- To investigate the role of individual MPP subunits in substrate recognition and binding affinity.
- To elucidate the specific interactions between MPP and its peptide substrates.
Main Methods:
- Fluorescently labeled peptide substrates were used to monitor interactions with MPP and its isolated subunits.
- Fluorescence quenching analysis was employed to study substrate burial within the enzyme complex.
Main Results:
- MPP exhibited high-affinity binding to substrate peptides only in its dimeric form; individual subunits showed significantly lower affinity (approximately 30-fold less).
- Specific arginine residues at positions -2 and distal to the cleavage site were identified as crucial for peptide binding and cleavage.
- Fluorescence quenching confirmed that bound peptides are largely buried within the MPP enzyme complex.
Conclusions:
- Both alpha and beta subunits of MPP are essential for forming a functional substrate binding pocket with multiple subsites.
- Subunit cooperation is critical for high-affinity substrate recognition and efficient processing of mitochondrial proteins.