PROTEIN AND SOLVENT ENGINEERING OF SUBTILISIN BPN' IN NEARLY ANHYDROUS ORGANIC MEDIA

The combined effects of protein and solvent engineering have been stud ied using subtilisin BPN' as a model protease. The effects of site-spe cific mutations in the active site of subtilisin BPN' on the reactivit y and substrate specificity of the enzyme are strongly dependent on th e polarity of the substrate, active-site mutation, and solvent. In goi ng from a polar solvent such as acetone to a nonpolar solvent such as hexane, subtilisin BPN' catalysis is activated dramatically (up to 178 -fold) by employing a polar active-site mutation (Gly166 --> Asn). Thi s activation is proposed to be due to significant transition-state sta bilization afforded by the polar mutation on subtilisin catalysis. Ana lysis of the individual kinetic and binding constants for subtilisin i ndicates that the polar mutation in the S1 binding site of the enzyme results in improved catalysis over the wild-type solely because of inc reased enzyme-substrate interaction (decreased (K(m))true). Water also effects the kinetics of subtilisin catalysis. In dry tetrahydrofuran, acylation is rate limiting. Addition of small concentrations of water to the organic solvent (<2% v/v) results in both an increased rate co nstant for acylation and a decreased (K(m))true. At 2% (v/v) added wat er and above, subtilisin reverts to a deacylation rate-limiting reacti on on its ester substrates. These results suggest that water and polar mutations activate enzyme catalysis in nearly anhydrous solvents, alb eit by different mechanisms, and further increase our understanding of the nature of polarity on enzyme function. From a practical standpoin t, it is concluded that the effectiveness of protein engineering is st rongly dependent on the solvent conditions.