PROBING ENZYMATIC TRANSITION-STATE HYDROPHOBICITIES

Hydrophobic interactions are important in numerous biological processe s; however, the nature and extent of hydrophobic interactions in nonaq ueous enzymology remain poorly defined. We have estimated the free ene rgies of enzyme-substrate hydrophobic interactions for a model reactio n catalyzed by subtilisin BPN' (from Bacillus amyloliquefaciens) in va rious solvents. Transition state stabilization of subtilisin in water has contributions from both ground state destabilization of hydrophobi c substrates and intrinsic enzyme-substrate hydrophobic interactions. Both contributions are evident even in hydrophobic organic solvents an d can be modified by protein engineering of the enzyme's binding site, as well as by changing the hydrophobicity of the reaction medium. We have also developed a method to estimate the hydrophobicity of the enz ymic transition state involving systematic variation of the substrate and solvent hydrophobicities. The observed binding pocket hydrophobici ties were directly affected by replacing the Gly(166) residue, located at the back of the hydrophobic S-1 binding pocket of subtilisin BPN', with more hydrophobic amino acids such as alanine and valine. Thus, t he observed SI binding pocket hydrophobicities of the wild-type, G166A , and G166V mutants were measured to be 1.2, 1.8, and 2.6 log P units, respectively. Our method of calculating effective binding pocket hydr ophobicity was found to be applicable to other enzymes, including hors eradish peroxidase and alpha-chymotrypsin. Measurements of the binding pocket hydrophobicities have significant implications toward tailorin g enzyme function in aqueous as well as nonaqueous media.