Abstract
Mitochondrial processing peptidase, a metalloendopeptidase consisting of α- and β-subunits, specifically recognizes a large variety of mitochondrial precursor proteins and cleaves off N-terminal extension peptides. The enzyme requires the basic amino acid residues in the extension peptides for effective and specific cleavage. To elucidate the mechanism involved in the molecular recognition of substrate by the enzyme, several glutamates around the active site of the rat β-subunit, which has a putative metal-binding motif, H56XXEH60, were mutated to alanines or aspartates, and effects on kinetic parameters, metal binding, and substrate binding of the enzyme were analyzed. None of mutant proteins analyzed was impaired in dimer formation with the α-subunit. Mutation of glutamates at positions 79, 129, and 136, in addition to an active-site glutamate at position 59, resulted in a marked decrease in cleavage efficiency. Together with sequence alignment data, glutamate 136 appears to be involved in metal binding. Glutamate 129 is mostly responsible for the catalysis, as there was a considerable decrease in k(cat) value by the mutation. Mutation of glutamate 79 led to decrease in k(cat) value and increase in K(m) values. Substrate binding experiments using an environmentally sensitive fluorescence probe attached to the peptide showed that the mutation caused a remarkable environmental change at the binding site to the N-terminal region of the substrate peptide and decreased binding of the peptide, thereby suggesting that glutamate 79 participates primarily in substrate binding. Thus, some glutamate residues required for substrate binding and cleavage activity have been identified.
| Original language | English |
|---|---|
| Pages (from-to) | 32547-32553 |
| Number of pages | 7 |
| Journal | Journal of Biological Chemistry |
| Volume | 273 |
| Issue number | 49 |
| DOIs | |
| Publication status | Published - Dec 4 1998 |
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All Science Journal Classification (ASJC) codes
- Biochemistry
- Molecular Biology
- Cell Biology
Cite this
Glutamate residues required for substrate binding and cleavage activity in mitochondrial processing peptidase. / Kitada, Sakae; Kojima, Katsuhiko; Shimokata, Kunitoshi; Ogishima, Tadashi; Ito, Akio.
In: Journal of Biological Chemistry, Vol. 273, No. 49, 04.12.1998, p. 32547-32553.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Glutamate residues required for substrate binding and cleavage activity in mitochondrial processing peptidase
AU - Kitada, Sakae
AU - Kojima, Katsuhiko
AU - Shimokata, Kunitoshi
AU - Ogishima, Tadashi
AU - Ito, Akio
PY - 1998/12/4
Y1 - 1998/12/4
N2 - Mitochondrial processing peptidase, a metalloendopeptidase consisting of α- and β-subunits, specifically recognizes a large variety of mitochondrial precursor proteins and cleaves off N-terminal extension peptides. The enzyme requires the basic amino acid residues in the extension peptides for effective and specific cleavage. To elucidate the mechanism involved in the molecular recognition of substrate by the enzyme, several glutamates around the active site of the rat β-subunit, which has a putative metal-binding motif, H56XXEH60, were mutated to alanines or aspartates, and effects on kinetic parameters, metal binding, and substrate binding of the enzyme were analyzed. None of mutant proteins analyzed was impaired in dimer formation with the α-subunit. Mutation of glutamates at positions 79, 129, and 136, in addition to an active-site glutamate at position 59, resulted in a marked decrease in cleavage efficiency. Together with sequence alignment data, glutamate 136 appears to be involved in metal binding. Glutamate 129 is mostly responsible for the catalysis, as there was a considerable decrease in k(cat) value by the mutation. Mutation of glutamate 79 led to decrease in k(cat) value and increase in K(m) values. Substrate binding experiments using an environmentally sensitive fluorescence probe attached to the peptide showed that the mutation caused a remarkable environmental change at the binding site to the N-terminal region of the substrate peptide and decreased binding of the peptide, thereby suggesting that glutamate 79 participates primarily in substrate binding. Thus, some glutamate residues required for substrate binding and cleavage activity have been identified.
AB - Mitochondrial processing peptidase, a metalloendopeptidase consisting of α- and β-subunits, specifically recognizes a large variety of mitochondrial precursor proteins and cleaves off N-terminal extension peptides. The enzyme requires the basic amino acid residues in the extension peptides for effective and specific cleavage. To elucidate the mechanism involved in the molecular recognition of substrate by the enzyme, several glutamates around the active site of the rat β-subunit, which has a putative metal-binding motif, H56XXEH60, were mutated to alanines or aspartates, and effects on kinetic parameters, metal binding, and substrate binding of the enzyme were analyzed. None of mutant proteins analyzed was impaired in dimer formation with the α-subunit. Mutation of glutamates at positions 79, 129, and 136, in addition to an active-site glutamate at position 59, resulted in a marked decrease in cleavage efficiency. Together with sequence alignment data, glutamate 136 appears to be involved in metal binding. Glutamate 129 is mostly responsible for the catalysis, as there was a considerable decrease in k(cat) value by the mutation. Mutation of glutamate 79 led to decrease in k(cat) value and increase in K(m) values. Substrate binding experiments using an environmentally sensitive fluorescence probe attached to the peptide showed that the mutation caused a remarkable environmental change at the binding site to the N-terminal region of the substrate peptide and decreased binding of the peptide, thereby suggesting that glutamate 79 participates primarily in substrate binding. Thus, some glutamate residues required for substrate binding and cleavage activity have been identified.
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UR - http://www.scopus.com/inward/citedby.url?scp=0032484217&partnerID=8YFLogxK
U2 - 10.1074/jbc.273.49.32547
DO - 10.1074/jbc.273.49.32547
M3 - Article
C2 - 9829990
AN - SCOPUS:0032484217
VL - 273
SP - 32547
EP - 32553
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 49
ER -