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The roles of surface loop insertions and disulfide bond in the stabilization of thermophilic WF146 protease
- Source :
- FEBS Letters; Oct2006, Vol. 580 Issue 25, p6007-6014, 8p
- Publication Year :
- 2006
-
Abstract
- 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<superscript>2+</superscript> binding site of the enzyme by the corresponding residues in subtilisin S41 remarkably reduced the half-life of the enzyme at 70°C, suggesting that the three residues contributed to the thermostability of the enzyme, probably by enhancing the affinity of enzyme to Ca<superscript>2+</superscript>. 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. [Copyright &y& Elsevier]
Details
- Language :
- English
- ISSN :
- 00145793
- Volume :
- 580
- Issue :
- 25
- Database :
- Complementary Index
- Journal :
- FEBS Letters
- Publication Type :
- Academic Journal
- Accession number :
- 22796772
- Full Text :
- https://doi.org/10.1016/j.febslet.2006.09.068