Citation

Wangikar PP, Carmichael D, Clark DS, Dordick JS. Biotechnol. Bioeng. 1996; 50(3): 329-335.

Copyright

(Copyright © 1996, John Wiley and Sons)

DOI

10.1002/(SICI)1097-0290(19960505)50:3<329::AID-BIT11>3.0.CO;2-I

PMID

unavailable

Abstract

Calculation of kinetic constants of an enzymatic reaction in organic solvents requires knowledge of the functional active-site concentration in organic solvents, and this can be significantly different than that in water. An experimental method for active-site titration of serine proteases in organic media has been developed based on the kinetics of inhibition by phenylmethanesulfonyl fluoride (PMSF), a serine-specific inhibitor (or suicide substrate). This kinetic approach is fundamentally different from other techniques that require complete titration of all accessible enzyme active sites. This active site titration method was applied to subtilisins BPN' and Carlsberg and α-chymotrypsin and resulted in fractions of active sites that ranged from 8 to 62% (of the fraction active in water) depending on the enzyme, the method of enzyme preparation, and the organic solvent used. The active-site concentration of subtilisin BPN' and Carlsberg increased with increasing hydrophobicity of the solvent and with increasing solvent hydration in tetrahydrofuran. The dependence of the fraction of active sites on the nature of the organic solvent appears to be governed largely by solvent-induced inactivation caused by direct interaction of a hydrophilic solvent with the enzyme.; Calculation of kinetic constants of an enzymatic reaction in organic solvents requires knowledge of the functional active-site concentration in organic solvents, and this can be significantly different than that in water. An experimental method for active-site titration of serine proteases in organic media has been developed based on the kinetics of inhibition by phenylmethanesulfonyl fluoride (PMSF), a serine-specific inhibitor (or suicide substrate). This kinetic approach is fundamentally different from other techniques that require complete titration of all accessible enzyme active sites. This active site titration method was applied to subtilisins BPN′ and Carlsberg and α-chymotrypsin and resulted in fractions of active sites that ranged from 8 to 62% (of the fraction active in water) depending on the enzyme, the method of enzyme preparation, and the organic solvent used. The active-site concentration of subtilisin BPN′ and Carlsberg increased with increasing hydrophobicity of the solvent and with increasing solvent hydration in tetrahydrofuran. The dependence of the fraction of active sites on the nature of the organic solvent appears to be governed largely by solvent-induced inactivation caused by direct interaction of a hydrophilic solvent with the enzyme.

Language: en

Keywords

article; nonhuman; enzyme inhibition; Reaction kinetics; Catalysis; Enzymes; enzyme active site; enzyme mechanism; serine proteinase; hydrophobicity; Titration; Hydrophobicity; Organic solvents; organic solvent; Chymotrypsin; enzyme kinetics; Hydration; Calculations; Active site titration; active-site titration; Aromatic compounds; deacylation; enzyme conformation; Enzyme inhibition; gel permeation chromatography; organic solvents; Phenyl methanesulfonyl fluoride; serine proteases; Serine proteases; stoichiometry; Subtilisin; Tetrahydrofuran