The Roles of Surface Loop Insertions and Disulfide Bond in the Stabilization of Thermophilic Wf146 Protease.
From: College of Life Sciences, Wuhan University, Wuhan 430072, China.
FEBS letters
- Publish Date: Oct 2006
- ISSN: 0014-5793
- Volume: 580
- Issue: 25
- Pages: 6007-14
- Medium: Print
- Language: English
- Citation (JAMA): Bian Yan, Liang Xiaoliang, Fang Nan, et al. The Roles of Surface Loop Insertions and Disulfide Bond in the Stabilization of Thermophilic Wf146 Protease.. FEBS Lett. Oct 2006;580:6007-14
Abstract
Thermophilic WF146 protease possesses four surface loop insertions and a disulfide bond, resembling its psychrophilic (subtilisins S41 and S39) and mesophilic (subtilisins SSII and sphericase) homologs. Deletion of the insertion 3 (positions 193-197) or insertion 4 (positions 210-221) of WF146 protease resulted in a significant decrease of the enzyme stability. In addition, substitution of the residues Pro211 and Ala212 or residue Glu221 which localized in the vicinity of a Ca(2+) binding site of the enzyme by the corresponding residues in subtilisin S41 remarkably reduced the half-life of the enzyme at 70 degrees C, suggesting that the three residues contributed to the thermostability of the enzyme, probably by enhancing the affinity of enzyme to Ca(2+). In the presence of dithiothreitol, the WF146 protease suffered excessive autolysis, indicating that the Cys52-Cys65 disulfide bond played a critical role in stabilizing the WF146 protease against autolysis. The autolytic cleavage sites of the WF146 protease were identified to locate between residues Asn63-Gly64 and Cys65-Ala66 by N-terminal amino acid analysis of the autolytic product. It was noticed that the effect of the autolytic cleavage at Asn63-Gly64 could be compensated by the disulfide bond Cys52-Cys65 under non-reducing condition, and the disulfide bond cross-linked autolytic product remained active. The apparent stabilization effect of the disulfide bond Cys52-Cys65 in the WF146 protease might provide a rational basis for improving the stability of subtilase against autolysis by protein engineering.
Mesh Headings (Keywords): Amino Acid Sequence, Bacillus, Bacterial Proteins, Base Sequence, Binding Sites, Cysteine, DNA, Bacterial, Enzyme Stability, Heat, Kinetics, Molecular Sequence Data, Mutagenesis, Insertional, Mutagenesis, Site-Directed, Peptide Hydrolases, Recombinant Proteins, Sequence Homology, Amino Acid, Subtilisins
Check for Full Text / PubMed Unique Identifier (PMID): 17052711
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