Document Detail

In vivo formation and stability of engineered disulfide bonds in subtilisin.
MedLine Citation:
PMID:  3516996     Owner:  NLM     Status:  MEDLINE    
Computer modeling suggested that a disulfide bond could be built into Bacillus amyloliquefaciens subtilisin between positions 22 (wild-type, Thr) and 87 (Ser) or between positions 24 (Ser) and 87 (Ser). Single cysteines were introduced into this cysteine-free protease at positions 22, 24, or 87 by site-directed mutagenesis of the cloned subtilisin gene. The corresponding double-cysteine mutants were constructed, and recombinant plasmids were expressed in Bacillus subtilis. Double-cysteine mutant enzymes were secreted as efficiently as wild-type, and disulfide bonds were formed quantitatively in vivo. These disulfide bonds were introduced approximately 24 A away from the catalytic site and had no detectable effect on either the specific activities or the pH optima of the mutant enzymes. The equilibrium constants for the reduction of the mutant disulfide bonds by dithiothreitol were determined to be 82 +/- 22 and 20 +/- 5 for Cys22/Cys87 and Cys24/Cys87, respectively. Studies of autoproteolytic inactivation of wild-type subtilisin support a relationship between autolytic stability and conformational stability of the protein. The stabilities of Cys24/Cys87 and wild-type enzymes to autolysis were essentially the same; however, Cys22/Cys87 was actually less stable to autolysis. Reduction of the disulfide cross-bridge lowered the autolytic stability of both double-cysteine mutants relative to their disulfide forms. This correlates with a lowered autolytic stability for the Cys22 and Cys87 single-cysteine mutants, and the fact that an intramolecular hydrogen bond between the hydroxyl groups of Thr22 and Ser87 is likely to be disrupted in the Cys22 and Cys87 single-cysteine mutant proteins.
J A Wells; D B Powers
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Publication Detail:
Type:  Journal Article    
Journal Detail:
Title:  The Journal of biological chemistry     Volume:  261     ISSN:  0021-9258     ISO Abbreviation:  J. Biol. Chem.     Publication Date:  1986 May 
Date Detail:
Created Date:  1986-06-16     Completed Date:  1986-06-16     Revised Date:  2000-12-18    
Medline Journal Info:
Nlm Unique ID:  2985121R     Medline TA:  J Biol Chem     Country:  UNITED STATES    
Other Details:
Languages:  eng     Pagination:  6564-70     Citation Subset:  IM    
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MeSH Terms
Bacillus / genetics
Disulfides / analysis*
Dithiothreitol / pharmacology
Models, Molecular
Subtilisins / analysis*,  genetics
Reg. No./Substance:
0/Disulfides; 3483-12-3/Dithiothreitol; 52-90-4/Cysteine; 57-13-6/Urea; EC 3.4.21.-/Subtilisins

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine

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