Enzyme behavior at surfaces. Site-specific variants of subtilisin BPN' with enhanced surface stability

Enzyme adsorption and inactivation at the solid/liquid interface for subtilisin BPN' show a strong dependence on the nature of the solid surface. Adsorption of BPN' at the solid/liquid interface is considerably greater for a hydrophobic surface than for a hydrophilic one. Likewise, the rat...

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Bibliographic Details
Published in:The Journal of biological chemistry 1994-09, Vol.269 (38), p.23538-23543
Main Authors: P F Brode, 3rd, C R Erwin, D S Rauch, D S Lucas, D N Rubingh
Format: Article
Language:English
Subjects:
Adsorption
Autolysis
Bacillus - enzymology
Bacillus amyloliquefaciens
Enzymes, Immobilized - metabolism
Kinetics
Models, Molecular
Mutagenesis, Site-Directed
Solubility
Structure-Activity Relationship
Subtilisins - chemistry
Subtilisins - metabolism
Surface Properties
Surface-Active Agents - chemistry
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Summary:Enzyme adsorption and inactivation at the solid/liquid interface for subtilisin BPN' show a strong dependence on the nature of the solid surface. Adsorption of BPN' at the solid/liquid interface is considerably greater for a hydrophobic surface than for a hydrophilic one. Likewise, the rate of inactivation of the wild-type BPN' is over five times greater when equilibrated with a hydrophobic surface than with a hydrophilic surface. The rate data from these enzyme inactivation experiments performed at 50 degrees C are best fit by a second-order kinetic equation, suggesting a bimolecular pathway to inactivation. The role of increased surface adsorption on this bimolecular inactivation is discussed in terms of two different mechanisms. Several site-specific variants of subtilisin BPN' have been made in an attempt to alter the surface-inactivation of the wild-type enzyme. The extent of adsorption on the model surfaces is significantly lowered by certain lysine to phenylalanine changes in BPN'. Consequently, the surface autolytic stability shows a 4-fold improvement. The change in surface autolytic stability is achieved even though the basic kinetic parameters (kcat and KM) of the variant enzymes are not significantly different on a soluble substrate. The results provide insights into the use of mutagenesis to probe the mechanism of protein interactions with surfaces.
ISSN:0021-9258
1083-351X
DOI:10.1016/S0021-9258(17)31549-1