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.


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. Study holds ProTherm entries: 9482, 9483, 9484 Extra Details: additive : EDTA(2 mM),DTE(0.1mM) was added in the experiment hyperthermophilic archaeon; mesophile homologue; Gibbs energy,change of unfolding

Submission Details

ID: ADxoLqTX4

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:38 p.m.

Version: 1

Publication Details
Ogasahara K;Nakamura M;Nakura S;Tsunasawa S;Kato I;Yoshimoto T;Yutani K,Biochemistry (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. PMID:9860869
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
1Z8X 2006-06-13 2.0 Structure of Mutant Pyrrolidone Carboxyl Peptidase (E192V) from a Hyperthermophile, Pyrococcus furiosus
1X10 2006-06-13 2.0 Structure of Mutant Pyrrolidone Carboxyl Peptidase (E192A) from a Hyperthermophile, Pyrococcus furiosus
1Z8W 2006-06-13 2.0 Structure of Mutant Pyrrolidone Carboxyl Peptidase (E192I) from a Hyperthermophile, Pyrococcus furiosus
1X12 2006-06-13 2.0 Structure of Mutant Pyrrolidone Carboxyl Peptidase (E192D) from a Hyperthermophile, Pyrococcus furiosus
1IOF 2001-03-21 2.2 X-RAY CRYSTALLINE STRUCTURES OF PYRROLIDONE CARBOXYL PEPTIDASE FROM A HYPERTHERMOPHILE, PYROCOCCUS FURIOSUS, AND ITS CYS-FREE MUTANT
2DF5 2007-03-06 2.3 Crystal Structure of Pf-PCP(1-204)-C
2EO8 2007-10-02 2.3 Crystal structure of a mutant pyrrolidone carboxyl peptidase (A199P) from P. furiosus
1Z8T 2006-06-13 2.5 Structure of Mutant Pyrrolidone Carboxyl Peptidase (E192Q) from a Hyperthermophile, Pyrococcus furiosus
1IOI 2001-03-21 2.7 x-ray crystalline structures of pyrrolidone carboxyl peptidase from a hyperthermophile, pyrococcus furiosus, and its cys-free mutant

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Pyrrolidone-carboxylate peptidase O73944 PCP_PYRFU