ACTIVE-SITE TITRATION OF SERINE PROTEASES IN ORGANIC-SOLVENTS

Calculation of kinetic constants of an enzymatic reaction in organic s olvents requires knowledge of the functional active-site concentration in organic solvents, and this can be significantly different than tha t in water. An experimental method for active-site titration of serine proteases in organic media has been developed based on the kinetics o f inhibition by phenylmethanesulfonyl fluoride (PMSF), a serine-specif ic inhibitor (or suicide substrate). This kinetic approach is fundamen tally different from other techniques that require complete titration of all accessible enzyme active sites. This active site titration meth od was applied to subtilisins BPN' and Carlsberg and alpha-chymotrypsi n 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 c oncentration of subtilisin BPN' and Carlsberg increased with increasin g 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 so lvent-induced inactivation caused by direct interaction of a hydrophil ic solvent with the enzyme. (C) 1996 John Wiley & Sons, Inc.