Protein engineering of disulfide bonds in subtilisin BPN'.
Five single-disulfide mutants were studied in subtilisin BPN', a cysteine-free, secreted serine protease from Bacillus amyloliquefaciens. The disulfides were engineered between residues 26-232, 29-119, 36-210, 41-80, and 148-243. These bonds connected a variety of secondary structural elements, located in buried or exposed positions at least 10 A from the catalytic Ser-221, and linked residues that were separated by 39 up to 206 amino acids. All disulfide bonds formed in the enzyme when the expressed protein was secreted from Bacillus subtilis, and the disulfides had only minor effects on the enzyme kinetics. Although these disulfide bonds varied by over 50-fold in their equilibrium constants for reduction with dithiothreitol, there was no correlation between the strength of the disulfide bond and the stability it imparted to the enzyme to irreversible inactivation. In some cases, the disulfide-bonded protein was stabilized greatly relative to its reduced counterpart. However, no disulfide mutant was substantially more stable than wild-type subtilisin BPN'. Some of these results can be rationalized by destabilizing effects of the cysteine mutations that disrupt interactions present in the folded enzyme structure. It is also possible that the rate of irreversible inactivation depends upon the kinetics and not the thermodynamics of unfolding and so the entropically stabilizing effect expected from a disulfide bond may not apply.
Mitchinson
C
C
Department of Biomolecular Chemistry, Genentech, Inc., South San Francisco, California 94080.
Wells
J A
JA
D001407:Bacillus / Q000235:genetics; D001412:Bacillus subtilis / Q000235:genetics; D004220:Disulfides / Q000096:biosynthesis*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D006358:Hot Temperature; D009154:Mutation; D010084:Oxidation-Reduction; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis*; D013381:Subtilisins / Q000096:biosynthesis* / Q000235:genetics; D013816:Thermodynamics
D004220:Disulfides; D011994:Recombinant Proteins; D013381:Subtilisins