Glutamate residues required for substrate binding and cleavage activity in mitochondrial processing peptidase

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

研究成果: Contribution to journalArticle査読

31 被引用数 (Scopus)

抄録

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.

本文言語英語
ページ(範囲)32547-32553
ページ数7
ジャーナルJournal of Biological Chemistry
273
49
DOI
出版ステータス出版済み - 12 4 1998

All Science Journal Classification (ASJC) codes

  • 生化学
  • 分子生物学
  • 細胞生物学

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