Abstract
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 language | English |
|---|---|
| Pages (from-to) | 633-643 |
| Number of pages | 11 |
| Journal | Proteins: Structure, Function and Genetics |
| Volume | 69 |
| Issue number | 3 |
| DOIs | |
| Publication status | Published - Nov 15 2007 |
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Keywords
- Kinetic analysis
- Protein stability
- Refolding
- Site specific mutagenesis
- Thermophilic enzyme
ASJC Scopus subject areas
- Genetics
- Structural Biology
- Biochemistry
Cite this
Truncated prolyl oligopeptidase from Pyrococcus furiosus. / Juhász, T.; Szeltner, Z.; Polgár, L.
In: Proteins: Structure, Function and Genetics, Vol. 69, No. 3, 15.11.2007, p. 633-643.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Truncated prolyl oligopeptidase from Pyrococcus furiosus
AU - Juhász, T.
AU - Szeltner, Z.
AU - Polgár, L.
PY - 2007/11/15
Y1 - 2007/11/15
N2 - 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.
AB - 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.
KW - Kinetic analysis
KW - Protein stability
KW - Refolding
KW - Site specific mutagenesis
KW - Thermophilic enzyme
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UR - http://www.scopus.com/inward/citedby.url?scp=35449003305&partnerID=8YFLogxK
U2 - 10.1002/prot.21522
DO - 10.1002/prot.21522
M3 - Article
C2 - 17623862
AN - SCOPUS:35449003305
VL - 69
SP - 633
EP - 643
JO - Proteins: Structure, Function and Genetics
JF - Proteins: Structure, Function and Genetics
SN - 0887-3585
IS - 3
ER -