Cooperative formation of a substrate binding pocket by α- and β- subunits of mitochondrial processing peptidase

Katsuhiko Kojima, Sakae Kitada, Kunitoshi Shimokata, Tadashi Ogishima, Akio Ito

Research output: Contribution to journalArticle

21 Citations (Scopus)

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 α- and β-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.

Original languageEnglish
Pages (from-to)32542-32546
Number of pages5
JournalJournal of Biological Chemistry
Volume273
Issue number49
DOIs
Publication statusPublished - Dec 4 1998

Fingerprint

Substrates
Peptides
Arginine
Protein Precursors
mitochondrial processing peptidase
Mitochondrial Proteins
Metalloproteases
Enzymes
Quenching
Monomers
Fluorescence

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Cooperative formation of a substrate binding pocket by α- and β- subunits of mitochondrial processing peptidase. / Kojima, Katsuhiko; Kitada, Sakae; Shimokata, Kunitoshi; Ogishima, Tadashi; Ito, Akio.

In: Journal of Biological Chemistry, Vol. 273, No. 49, 04.12.1998, p. 32542-32546.

Research output: Contribution to journalArticle

Kojima, Katsuhiko ; Kitada, Sakae ; Shimokata, Kunitoshi ; Ogishima, Tadashi ; Ito, Akio. / Cooperative formation of a substrate binding pocket by α- and β- subunits of mitochondrial processing peptidase. In: Journal of Biological Chemistry. 1998 ; Vol. 273, No. 49. pp. 32542-32546.
@article{553cb998c01e43db9c7a003077980514,
title = "Cooperative formation of a substrate binding pocket by α- and β- 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 α- and β-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.",
author = "Katsuhiko Kojima and Sakae Kitada and Kunitoshi Shimokata and Tadashi Ogishima and Akio Ito",
year = "1998",
month = "12",
day = "4",
doi = "10.1074/jbc.273.49.32542",
language = "English",
volume = "273",
pages = "32542--32546",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "49",

}

TY - JOUR

T1 - Cooperative formation of a substrate binding pocket by α- and β- subunits of mitochondrial processing peptidase

AU - Kojima, Katsuhiko

AU - Kitada, Sakae

AU - Shimokata, Kunitoshi

AU - Ogishima, Tadashi

AU - Ito, Akio

PY - 1998/12/4

Y1 - 1998/12/4

N2 - 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 α- and β-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.

AB - 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 α- and β-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.

UR - http://www.scopus.com/inward/record.url?scp=0032484020&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0032484020&partnerID=8YFLogxK

U2 - 10.1074/jbc.273.49.32542

DO - 10.1074/jbc.273.49.32542

M3 - Article

C2 - 9829989

AN - SCOPUS:0032484020

VL - 273

SP - 32542

EP - 32546

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 49

ER -