Tailoring the pH dependence of enzyme catalysis using protein engineering

One of the goals of protein engineering is to tailor the pH dependence of enzyme catalysis to optimize activity in industrial processes. Chemical modification studies have shown that the pH dependence of catalysis by serine proteases alters with changes in overall surface charge1, which suggests that a possible general method of modifying pH dependence is to alter the electrostatic environment of the active site by protein engineering and so change the pKa values of ionic catalytic groups. Electrostatic effects are of considerable importance in enzyme catalysis and are thought to play a major part in stabilizing charged transition states2–5. However, it is extremely difficult to calculate electrostatic effects in proteins because of the microheterogeneity of the dielectric constant. In the present study we show how the change of just one surface charge which is 14–15 Å from the active site of subtilisin has a significant effect on the pH dependence of the enzyme and that the magnitude of the effect changes with ionic strength in a manner qualitatively consistent with electrostatic theory. Such experiments should provide the basis for refining theoretical calculations of electrostatic effects in catalysis.