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 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.
Brode
P F
PF
Miami Valley Laboratories, Procter and Gamble Company, Cincinnati, Ohio 45239-8707.
Erwin
C R
CR
Rauch
D S
DS
Lucas
D S
DS
Rubingh
D N
DN
D000327:Adsorption; D001329:Autolysis; D001407:Bacillus / Q000201:enzymology; D004800:Enzymes, Immobilized / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D012995:Solubility; D013329:Structure-Activity Relationship; D013381:Subtilisins / Q000737:chemistry / Q000378:metabolism*; D013499:Surface Properties; D013501:Surface-Active Agents / Q000737:chemistry
D004800:Enzymes, Immobilized; D013501:Surface-Active Agents; D013381:Subtilisins