Site-directed mutagenesis of a thermostable alpha-amylase from Bacillus stearothermophilus: putative role of three conserved residues.
The relationship between structure, activity, and stability of the thermostable Bacillus stearothermophilus alpha-amylase was studied by site-directed mutagenesis of the three most conserved residues. Mutation of His-238 to Asp involved in Ca2+ and substrate binding reduced the specific activity and thermal stability, but did not affect the pH and temperature optima. Replacement of Asp-331 by Glu in the active site caused almost total inactivation. Interestingly, in prolonged incubation this mutant enzyme showed an altered end-product profile by liberating only maltose and maltotriose. Conservative mutation of the conserved Arg-232 by Lys, for which no function has yet been proposed, resulted in lowered specific activity: around 12% of the parental enzyme. This mutant enzyme had a wider pH range but about the same temperature optimum and thermal stability as the wild-type enzyme. Results obtained with different mutants were interpreted by computer aided molecular modeling.
Department of Biochemistry, University of Turku, Finland.
M
M
Vihinen
P
P
Ollikka
J
J
Niskanen
P
P
Meyer
I
I
Suominen
M
M
Karp
L
L
Holm
J
J
Knowles
P
P
Mäntsälä
D000595:Amino Acid Sequence; D000681:Amylases / Q000235:genetics* / Q000378:metabolism; D001120:Arginine / Q000494:pharmacology / Q000502:physiology; D001224:Aspartic Acid / Q000502:physiology; D001426:Bacterial Proteins / Q000235:genetics; D004795:Enzyme Stability; D001411:Geobacillus stearothermophilus / Q000201:enzymology* / Q000235:genetics; D006639:Histidine / Q000502:physiology; D006358:Hot Temperature; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D013329:Structure-Activity Relationship
D001426:Bacterial Proteins; D001224:Aspartic Acid; D006639:Histidine; D001120:Arginine; D000681:Amylases