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 pK(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) + H3C - SO2-S-R --> Asn62Cys-S-R, where R may be infinitely variable, has demonstrated that pK(a) shifts of up to 0.72 unit are achievable and are accompanied by significant activity enhancements. The most dramatic pK(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 pK(a) demonstrates that the hydrophobicity in the vicinity of the catalytic triad modulates the pK(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.