Random mutagenesis to enhance the activity of subtilisin in organic solvents: Characterization of Q103R subtilisin E

Q103R subtilisin E was isolated following random mutagenesis and screening for improved activity in the presence of dimethylformamide (DMF). Our goal is to identify the mechanism(s) by which amino acid substitutions can enhance enzyme activity in polar organic solvents. A quantitative framework for comparing substrate binding and catalytic activities of mutant and wild-type enzymes in the presence and absence of DMF is outlined. Kinetic experiments performed at high salt concentration (1M KCl) reveal that the mechanism behind the Q103R variant's enhanced activity toward succinyl-Ala-Ala-Pro-Phe-p-nitroanilide is both electrostatic and nonelectrostatic in origin. Favorable electrostatic interactions between the negatively charged succinyl group of the substrate and the positive charge on Arg 103 are responsible for tighter substrate binding. This conclusion is supported by kinetic experiments performed on the related substrate Ala-Ala-Pro-Phe-p-nitroanilide and the hydrolysis kinetics of the Q103E, Q103K, and Q103S variants constructed by site-directed mutagenesis. These results highlight the importance of the choice of the substrate used to screen for improvements in catalytic activity.