Aarhus University Seal / Aarhus Universitets segl

Electrostatics in the active site of an α-amylase

Research output: Contribution to journal/Conference contribution in journal/Contribution to newspaperJournal articleResearchpeer-review

  • Jens E. Nielsen, European Molecular Biology Laboratory Heidelberg
  • ,
  • Lars Beier, Novo Nordisk AS
  • ,
  • Daniel Otzen
  • Torben V. Borchert, Novo Nordisk AS
  • ,
  • Henrik B. Frantzen, Novo Nordisk AS
  • ,
  • Kim V. Andersen, Novo Nordisk AS
  • ,
  • Allan Svendsen, Novo Nordisk AS

The importance of electrostatics in catalysis has been emphasized in the literature for a large number of enzymes. We examined this hypothesis for the Bacillus licheniformis α-amylase by constructing site-directed mutants 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 that altered the hydrogen bonding network, mutations that changed the solvent accessibility, and mutations that altered the net charge of the molecule. The results show that changing the hydrogen bonding network near an active site residue or changing the solvent accessibility of an active site residue will very likely result in an enzyme with drastically reduced activity. The differences in the pH-activity profiles for these mutants were modest, pH- activity profiles of mutants which change the net charge on the molecule were significantly different from the wild-type pH-activity profile. The differences were, however, difficult to correlate with the electrostatic field changes calculated. In several cases we observed that pH-activity profiles shifted in the opposite direction compared to the shift predicted from electrostatic calculations. This strongly suggests that electrostatic effects cannot be solely responsible for the pH-activity profile of the B. licheniformis α-amylase.

Original languageEnglish
JournalEuropean Journal of Biochemistry
Volume264
Issue3
Pages (from-to)816-824
Number of pages9
ISSN0014-2956
DOIs
Publication statusPublished - 15 Sep 1999

    Research areas

  • α-Amylase, Active site, Electrostatics, pH-activity profile, Protein engineering

See relations at Aarhus University Citationformats

ID: 139497192