Electrostatics in the active site of an alpha-amylase

The importance of electrostatics in catalysis has been emphasized in the li terature for a large number of enzymes. I;Ve examined this hypothesis for t he Bacillus licheniformis alpha-amylase by constructing site-directed mutan ts that were predicted to change the pK(a) values of the catalytic residues and thus change the pH-activity profile of the enzyme. To change the pK(a) of the catalytic residues in the active site, we constructed mutations tha t altered the hydrogen bonding network, mutations that changed the solvent accessibility, and mutations that altered the net charge of the molecule. T he results show that changing the hydrogen bonding network near an active s ite residue or changing the solvent accessibility of an active site residue will very likely result in an enzyme with drastically reduced activity. Th e differences in the pH-activity profiles for these mutants were modest. pH -activity profiles of mutants which change the net charge on the molecule w ere significantly different from the wild-type pi-I-activity profile. The d ifferences were, however, difficult to correlate with the electrostatic fie ld changes calculated. In several cases we observed that pr-I-activity prof iles shifted in the opposite direction compared to the shift predicted from electrostatic calculations. This strongly suggests that electrostatic effe cts cannot be solely responsible for the pi-I-activity profile of the B. li cheniformis a-amylase.