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The unusually slow unfolding rate causes the high stability of pyrrolidone carboxyl peptidase from a hyperthermophile, Pyrococcus furiosus: equilibrium and kinetic studies of guanidine hydrochloride-induced unfolding and refolding.
Biochemistry. 1998 Dec 15; 37(50):17537-44.B

Abstract

To elucidate the energetic features of the anomalously high-level stabilization of a hyperthermophile pyrrolidone carboxyl peptidase (PfPCP) from a hyperthermophilic archaeon, Pyrococcus furiosus, equilibrium and kinetic studies of the guanidine hydrochloride (GuHCl)-induced unfolding and refolding were carried out with CD measurements at 220 nm in comparison with those from the mesophile homologue (BaPCP) from Bacillus amyloliquefaciens. The mutant protein of PfPCP substituted with Ser at both Cys142 and Cys188 (PfC142/188S) was used. The GuHCl unfolding for PfC142/188S and BaPCP was reversible. It was difficult to obtain the equilibrated unfolding curve of the hyperthermophile proteins at temperatures below 50 degreesC and pH 7, because of the remarkably slow rate of the unfolding. The unfolding for PfC142/188S attained equilibrium after 7 days at 60 degreesC, resulting in the coincidence between the unfolding and refolding curves. The Gibbs energy change of unfolding, DeltaGH2O (56.6 kJ/mol), for PfC142/188S at 60 degreesC and pH 7 was dramatically higher than that (7.6 kJ/mol) for BaPCP at 40 degreesC and pH 7. The unfolding and refolding kinetics for PfC142/188S and BaPCP at both 25 and 60 degreesC at pH 7 were approximated as a single exponential. The rate constant in water (kuH2O) of the unfolding reaction for PfC142/188S (1.6 x 10(-)15 s-1) at 25 degreesC and pH 7 was drastically reduced by 7 orders of magnitude compared to that (1.5 x 10(-)8 s-1) for BaPCP, whereas the refolding rates (krH2O) in water for PfC142/188S (9.3 x 10(-)2 s-1) and BaPCP (3.6 x 10(-)1 s-1) at 25 degreesC and pH 7 were similar. These results indicate that the greater stability of the hyperthermophile PCP was characterized by the drastically slow unfolding rate.

Authors+Show Affiliations

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

9860869

Citation

Ogasahara, K, et al. "The Unusually Slow Unfolding Rate Causes the High Stability of Pyrrolidone Carboxyl Peptidase From a Hyperthermophile, Pyrococcus Furiosus: Equilibrium and Kinetic Studies of Guanidine Hydrochloride-induced Unfolding and Refolding." Biochemistry, vol. 37, no. 50, 1998, pp. 17537-44.
Ogasahara K, Nakamura M, Nakura S, et al. The unusually slow unfolding rate causes the high stability of pyrrolidone carboxyl peptidase from a hyperthermophile, Pyrococcus furiosus: equilibrium and kinetic studies of guanidine hydrochloride-induced unfolding and refolding. Biochemistry. 1998;37(50):17537-44.
Ogasahara, K., Nakamura, M., Nakura, S., Tsunasawa, S., Kato, I., Yoshimoto, T., & Yutani, K. (1998). The unusually slow unfolding rate causes the high stability of pyrrolidone carboxyl peptidase from a hyperthermophile, Pyrococcus furiosus: equilibrium and kinetic studies of guanidine hydrochloride-induced unfolding and refolding. Biochemistry, 37(50), 17537-44.
Ogasahara K, et al. The Unusually Slow Unfolding Rate Causes the High Stability of Pyrrolidone Carboxyl Peptidase From a Hyperthermophile, Pyrococcus Furiosus: Equilibrium and Kinetic Studies of Guanidine Hydrochloride-induced Unfolding and Refolding. Biochemistry. 1998 Dec 15;37(50):17537-44. PubMed PMID: 9860869.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - The unusually slow unfolding rate causes the high stability of pyrrolidone carboxyl peptidase from a hyperthermophile, Pyrococcus furiosus: equilibrium and kinetic studies of guanidine hydrochloride-induced unfolding and refolding. AU - Ogasahara,K, AU - Nakamura,M, AU - Nakura,S, AU - Tsunasawa,S, AU - Kato,I, AU - Yoshimoto,T, AU - Yutani,K, PY - 1998/12/23/pubmed PY - 1998/12/23/medline PY - 1998/12/23/entrez SP - 17537 EP - 44 JF - Biochemistry JO - Biochemistry VL - 37 IS - 50 N2 - To elucidate the energetic features of the anomalously high-level stabilization of a hyperthermophile pyrrolidone carboxyl peptidase (PfPCP) from a hyperthermophilic archaeon, Pyrococcus furiosus, equilibrium and kinetic studies of the guanidine hydrochloride (GuHCl)-induced unfolding and refolding were carried out with CD measurements at 220 nm in comparison with those from the mesophile homologue (BaPCP) from Bacillus amyloliquefaciens. The mutant protein of PfPCP substituted with Ser at both Cys142 and Cys188 (PfC142/188S) was used. The GuHCl unfolding for PfC142/188S and BaPCP was reversible. It was difficult to obtain the equilibrated unfolding curve of the hyperthermophile proteins at temperatures below 50 degreesC and pH 7, because of the remarkably slow rate of the unfolding. The unfolding for PfC142/188S attained equilibrium after 7 days at 60 degreesC, resulting in the coincidence between the unfolding and refolding curves. The Gibbs energy change of unfolding, DeltaGH2O (56.6 kJ/mol), for PfC142/188S at 60 degreesC and pH 7 was dramatically higher than that (7.6 kJ/mol) for BaPCP at 40 degreesC and pH 7. The unfolding and refolding kinetics for PfC142/188S and BaPCP at both 25 and 60 degreesC at pH 7 were approximated as a single exponential. The rate constant in water (kuH2O) of the unfolding reaction for PfC142/188S (1.6 x 10(-)15 s-1) at 25 degreesC and pH 7 was drastically reduced by 7 orders of magnitude compared to that (1.5 x 10(-)8 s-1) for BaPCP, whereas the refolding rates (krH2O) in water for PfC142/188S (9.3 x 10(-)2 s-1) and BaPCP (3.6 x 10(-)1 s-1) at 25 degreesC and pH 7 were similar. These results indicate that the greater stability of the hyperthermophile PCP was characterized by the drastically slow unfolding rate. SN - 0006-2960 UR - https://www.unboundmedicine.com/medline/citation/9860869/The_unusually_slow_unfolding_rate_causes_the_high_stability_of_pyrrolidone_carboxyl_peptidase_from_a_hyperthermophile_Pyrococcus_furiosus:_equilibrium_and_kinetic_studies_of_guanidine_hydrochloride_induced_unfolding_and_refolding_ L2 - https://doi.org/10.1021/bi9814585 DB - PRIME DP - Unbound Medicine ER -