Abstract
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.
Highlights
Computer modeling suggested that a disulfide bond These engineered enzymes were expressed intracellularly could be built into Bacillus amyloliquefacienssubtili- in Escherichia coli and the disulfide bonds had to be produced sin betweenpositions 22 and87 (Ser) by chemical oxidation of the reduced enzymes i n uitro
DL-dithiohreitol, iodoacetamide, disulfide bonds are important in the conformational stability DTNB,4 and P-mercaptoethanol were from Sigma.Oxidized DTT
To addressthis issue more directly, disulfide bonds have been engineered into dihydrofolate reductase (Villafranca et al, 1983) and T4 lysozyme (Perry and Wetzel, 1984) by site-directed mutagenesis
Summary
We report the construction of two doubledisulfide cross-bridge loweretdhe autolytic stabilityof cysteine mutants in subtilisin Both enzymes were found to both double-cysteine mutants relative to thdeiisrulfide forms. This correlates witha lowered autolytic stability for thCeysZ2and Cyss7single-cysteine mutants,and the fact that an intramolechuyldarogen bond between the hydroxyl groupsof ThrZ2 andSers is likely to be be efficiently secreted and disulfide bonds were produced quantitatively i n uiuo. The stability of these disulfide bonds and the stability of these mutant enzymes against autolytic digestion are described.
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