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Thermal stability of pyrrolidone carboxyl peptidases from the hyperthermophilic Archaeon, Pyrococcus furiosus.
Eur J Biochem. 2001 Jun; 268(11):3233-42.EJ

Abstract

The temperature adaptation of pyrrolidone carboxyl peptidase (PCP) from a hyperthermophile, Pyrococcus furiosus (Pf PCP), was characterized in the context of an assembly form of the protein which is a homotetramer at neutral pH. The Pf PCP exhibited maximal catalytic activity at 90-95 degrees C and its activity was higher in the temperature range 30-100 degrees C than its counterpart from the mesophilic Bacillus amyloliquefaciens (BaPCP). Thermal stability was monitored by differential scanning calorimetry (DSC). Two clearly separated peaks appeared on the DSC curves for Pf PCP at alkaline and acidic pH. Using the oxidized Pf PCP and two mutant proteins (Pf C188S and Pf C142/188S), it was found that the peaks on the high and low temperature sides of the DSC curve of Pf PCP were produced by the forms with an intersubunit disulfide bridge between the two subunits and without the bridge, respectively, indicating the stabilization effect of intersubunit disulfide bridges. The denaturation temperature (Td) of Pf PCP with intersubunit disulfide bridges was higher by 53 degrees C at pH 9.0 than that of BaPCP. An analysis of the equilibrium ultracentrifugation patterns showed that the tetrameric Pf C142/188S dissociated into dimers with decreasing pH in the acidic region and became monomer subunits at pH 2.5. The heat denaturation of Pf PCP and its two Cys mutants was highly reversible in the dimeric forms, but completely irreversible in the tetrameric form. The Td of Pf C142/188S decreased as the enzyme became dissociated, but the monomeric form of the protein was still folded at pH 2.5, although BaPCP was completely denatured at acidic pH. These results indicate that subunit interaction plays an important role in stabilizing PCP from P. furiosus in addition to the intrinsic enhanced stability of its monomer.

Authors+Show Affiliations

Institute for Protein Research, Osaka University, Suita City, Osaka, Japan.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

11389725

Citation

Ogasahara, K, et al. "Thermal Stability of Pyrrolidone Carboxyl Peptidases From the Hyperthermophilic Archaeon, Pyrococcus Furiosus." European Journal of Biochemistry, vol. 268, no. 11, 2001, pp. 3233-42.
Ogasahara K, Khechinashvili NN, Nakamura M, et al. Thermal stability of pyrrolidone carboxyl peptidases from the hyperthermophilic Archaeon, Pyrococcus furiosus. Eur J Biochem. 2001;268(11):3233-42.
Ogasahara, K., Khechinashvili, N. N., Nakamura, M., Yoshimoto, T., & Yutani, K. (2001). Thermal stability of pyrrolidone carboxyl peptidases from the hyperthermophilic Archaeon, Pyrococcus furiosus. European Journal of Biochemistry, 268(11), 3233-42.
Ogasahara K, et al. Thermal Stability of Pyrrolidone Carboxyl Peptidases From the Hyperthermophilic Archaeon, Pyrococcus Furiosus. Eur J Biochem. 2001;268(11):3233-42. PubMed PMID: 11389725.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Thermal stability of pyrrolidone carboxyl peptidases from the hyperthermophilic Archaeon, Pyrococcus furiosus. AU - Ogasahara,K, AU - Khechinashvili,N N, AU - Nakamura,M, AU - Yoshimoto,T, AU - Yutani,K, PY - 2001/6/8/pubmed PY - 2001/7/28/medline PY - 2001/6/8/entrez SP - 3233 EP - 42 JF - European journal of biochemistry JO - Eur J Biochem VL - 268 IS - 11 N2 - The temperature adaptation of pyrrolidone carboxyl peptidase (PCP) from a hyperthermophile, Pyrococcus furiosus (Pf PCP), was characterized in the context of an assembly form of the protein which is a homotetramer at neutral pH. The Pf PCP exhibited maximal catalytic activity at 90-95 degrees C and its activity was higher in the temperature range 30-100 degrees C than its counterpart from the mesophilic Bacillus amyloliquefaciens (BaPCP). Thermal stability was monitored by differential scanning calorimetry (DSC). Two clearly separated peaks appeared on the DSC curves for Pf PCP at alkaline and acidic pH. Using the oxidized Pf PCP and two mutant proteins (Pf C188S and Pf C142/188S), it was found that the peaks on the high and low temperature sides of the DSC curve of Pf PCP were produced by the forms with an intersubunit disulfide bridge between the two subunits and without the bridge, respectively, indicating the stabilization effect of intersubunit disulfide bridges. The denaturation temperature (Td) of Pf PCP with intersubunit disulfide bridges was higher by 53 degrees C at pH 9.0 than that of BaPCP. An analysis of the equilibrium ultracentrifugation patterns showed that the tetrameric Pf C142/188S dissociated into dimers with decreasing pH in the acidic region and became monomer subunits at pH 2.5. The heat denaturation of Pf PCP and its two Cys mutants was highly reversible in the dimeric forms, but completely irreversible in the tetrameric form. The Td of Pf C142/188S decreased as the enzyme became dissociated, but the monomeric form of the protein was still folded at pH 2.5, although BaPCP was completely denatured at acidic pH. These results indicate that subunit interaction plays an important role in stabilizing PCP from P. furiosus in addition to the intrinsic enhanced stability of its monomer. SN - 0014-2956 UR - https://www.unboundmedicine.com/medline/citation/11389725/Thermal_stability_of_pyrrolidone_carboxyl_peptidases_from_the_hyperthermophilic_Archaeon_Pyrococcus_furiosus_ L2 - https://onlinelibrary.wiley.com/resolve/openurl?genre=article&sid=nlm:pubmed&issn=0014-2956&date=2001&volume=268&issue=11&spage=3233 DB - PRIME DP - Unbound Medicine ER -