Electrostatics in the active site of an alpha-amylase.
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 alpha-amylase by constructing site-directed mutants that were predicted to change the pKa values of the catalytic residues and thus change the pH-activity profile of the enzyme. To change the pKa 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 alpha-amylase.
Nielsen
J E
JE
EMBL, Heidelberg, Germany. Nielsen@EMBL-Heidelberg.DE
Beier
L
L
Otzen
D
D
Borchert
T V
TV
Frantzen
H B
HB
Andersen
K V
KV
Svendsen
A
A
D001407:Bacillus / Q000201:enzymology / Q000235:genetics; D020134:Catalytic Domain / Q000235:genetics; D004795:Enzyme Stability; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D015202:Protein Engineering; D055672:Static Electricity; D000516:alpha-Amylases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D000516:alpha-Amylases