Truncated prolyl oligopeptidase from Pyrococcus furiosus

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The peptidase domain of prolyl oligopeptidase is covered by a propeller domain, which excludes large peptides and proteins from the catalytic triad. Previous studies indicated that some amino acids of the N-terminal region constitute a part of the substrate entrance to the active site. To investigate the catalytic role of the N-terminus, we removed the residues 1-32 from the enzyme and examined the kinetic, thermodynamic, and structural consequences of the deletion, using the thermophile Pyrococcus furiosus prolyl oligopeptidase. An about threefold decrease in the catalytic activity along with a 20°C reduction in the temperature optimum was observed. The pH-rate profile, the rate-limiting step, and the activation parameters did not change significantly. However, a substantial decrease was observed in the stability of the protein as demonstrated by circular dichroism and differential scanning calorimetry measurements, and by denaturation with guanidinium chloride. It was concluded that the N-terminal segment did not facilitate the substrate binding independent of the size of the substrate, but contributed principally to the protein stability required for the formation of the proper active site.

Original languageEnglish
Pages (from-to)633-643
Number of pages11
JournalProteins: Structure, Function and Genetics
Issue number3
Publication statusPublished - Nov 15 2007



  • Kinetic analysis
  • Protein stability
  • Refolding
  • Site specific mutagenesis
  • Thermophilic enzyme

ASJC Scopus subject areas

  • Structural Biology
  • Biochemistry
  • Molecular Biology

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