Chemical Modifications at a Single Site Can Induce Significant Shifts in the pH Profiles of a Serine Protease†

Developing the ability to vary the pH−activity profile of an enzyme in a controlled manner has been a long sought-after goal. Such tailoring provides important insights into mechanism and permits optimization of enzyme performance in organic synthesis applications. The most successful approaches to date toward altering pH−activity profiles of enzymes have employed either site-directed mutagenesis or chemical modification to alter enzyme surface charge. We now report that, by combining these two methodologies, dramatic p K a changes can be induced in the serine protease subtilisin B. lentus . In particular, site specific incorporation of unnatural amino acid side chains by the following strategy, WT → Asn62Cys mutant + H 3 C−SO 2 −S−R → Asn62Cys-S−R, where R may be infinitely variable, has demonstrated that p K a shifts of up to 0.72 unit are achievable and are accompanied by significant activity enhancements. The most dramatic p K a shifts are caused by chemical modification with hydrophobic aliphatic moieties. A linear correlation between the hydrophobicity indicator log P for the side chain of the modification and the observed p K a demonstrates that the hydrophobicity in the vicinity of the catalytic triad modulates the p K a of the catalytic residue His64. Molecular modeling analysis reveals that the side chain R of Asn62Cys-S−R, where R is a hydrophobic modification such as decyl or cyclohexyl, positions itself over the imidazole moiety of His64 in the minimized structure.