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

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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 pKa 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 → Asn62Cysmutant + H3C−SO2−S−R → Asn62Cys-S−R, where R may be infinitely variable, has demonstrated that pKa shifts of up to 0.72 unit are achievable and are accompanied by significant activity enhancements. The most dramatic pKa shifts are caused by chemical modification with hydrophobic aliphatic moieties. A lin...