The noncatalytic β-propeller domain of prolyl oligopeptidase enhances the catalytic capability of the peptidase domain

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Abstract

Prolyl oligopeptidase, which is involved in memory disorders, is a member of a new family of serine peptidases. In addition to the peptidase domain, the enzyme contains a β-propeller, which excludes large peptides from the active site. The enzyme is inhibited with thiol reagents, possibly by reacting with Cys-255 located close to the substrate binding site. This assumption was tested with the Cys-255 → Thr, Cys-255 → Ala, and Cys-255 → Ser variants of prolyl oligopeptidase. In contrast to the wild type enzyme, the Cys-255 → Thr variant was not inhibited with N-ethylmaleimide, indicating that Cys-255, of the 16 free cysteine residues, exclusively accounts for the enzyme inhibition. Unlike the wild type enzyme that showed a doubly bell-shaped pH rate profile, the modified enzyme displayed a single bell-shaped pH dependence with benzyloxycarbonyl-Gly-Pro-naphthylamide. It was the high pH form of the enzyme that virtually disappeared with all three enzyme variants. A substantial reduction was also observed in k(cat)/K(m) for the aminobenzoyl-Ser-Pro-Phe(NO2)-Ala-OH substrate. The high pK(a) (9.77) of Cys-255 determined by titration with N-ethylmaleimide excluded the possibility that ionization of the thiol group was responsible for generation of the two active enzyme forms. The impaired activity of the enzyme variants could be rationalized in terms of weaker binding, which manifests itself in high K(m) for substrates and high K(i) for inhibitors, like benzyloxycarbonyl-Gly-Pro-OH and aminobenzoyl-Ser-D-Pro-Phe(NO2)-Ala-OH. It was concluded that, besides selecting substrates by size, the β-propeller domain containing Cys-255 remarkably contributed to catalysis of the peptidase domain.

Original languageEnglish
Pages (from-to)15000-15005
Number of pages6
JournalJournal of Biological Chemistry
Volume275
Issue number20
DOIs
Publication statusPublished - May 19 2000

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ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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