Abstract
Subtilisin and alpha-chymotrypsin vigorously act as catalysts in a variety of dry organic solvents. Enzymatic transesterifications in organic solvents follow Michaelis-Menten kinetics, and the values of V/Km roughly correlate with solvent's hydrophobicity. The amount of water required by chymotrypsin and subtilisin for catalysis in organic solvents is much less than needed to form a monolayer on its surface. The vastly different catalytic activities of chymotrypsin in various organic solvents are partly due to stripping of the essential water from the enzyme by more hydrophilic solvents and partly due to the solvent directly affecting the enzymatic process. The rate enhancements afforded by chymotrypsin and subtilisin in the transesterification reaction in octane are of the order of 100 billion-fold; covalent modification of the active center of the enzymes by a site-specific reagent renders them catalytically inactive in organic solvents. Upon replacement of water with octane as the reaction medium, the specificity of chymotrypsin toward competitive inhibitors reverses. Both thermal and storage stabilities of chymotrypsin are greatly enhanced in nonaqueous solvents compared to water. The phenomenon of enzymatic catalysis in organic solvents appears to be due to the structural rigidity of proteins in organic solvents resulting in high kinetic barriers that prevent the native-like conformation from unfolding.
Highlights
Subtilisin and a-chymotrypsin vigorously actas ca- those in aqueous solutions
It is easy to imagine how our understanding provide profound insights into such questions as protein foldof enzymatic catalysis could be enhanced if a new fundamental ing and dynamics, the role of water in enzyme catalysis and variable was introduced in the experimentation, namely the solvent (i.e.the reaction medium)
In the first approach (Luisi, 1985; Martinek et al, 1986), enzymes are dissolved in micropools of water which are emulsified in waterimmiscible solvents; the microemulsion is stabilized by surfactants that form “reverse micelles.’’ In thesecond approach (Klibanov, 1986), powdered enzymes are directly suspended in organic solvents
Summary
Bovine pancreatic achymotrypsin and Bacillus subtilis protease (subtilisin Carlsberg) were employed as model enzymes in organic solvents These two proteases are not associated with biological membranes in nature, and their physiological role is to hydrolyze water-soluble proteins; they represent enzymes whose natura environment and function involve aqueous solutions (in contrastto membrane-bound or lipolytic enzymes that act on interfaces and are accustomed to a nonaqueous milieu). In experiments involving immobilized chymotrypsin, the enzyme pancreatic lipase in organic solvents was greatly increased was covalently attached to CNBr-activated Sepharose4B following the procedure of h e n and Ernback (1971). This method afforded approximately 200 mg of chymotrypsin attached to 1 g of support.
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