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.