Site-directed mutagenesis of active site residues in Bacillus subtilis alpha-amylase.
Site-directed mutagenesis of Bacillus subtilis N7 alpha-amylase has been performed to evaluate the roles of the active site residues in catalysis and to prepare an inactive catalytic-site mutant that can form a stable complex with natural substrates. Mutation of Asp-176, Glu-208, and Asp-269 to their amide forms resulted in over a 15,000-fold reduction of its specific activity, but all the mutants retained considerable substrate-binding abilities as estimated by gel electrophoresis in the presence of soluble starch. Conversion of His-180 to Asn resulted in a 20-fold reduction of kcat with a 5-fold increase in Km for a maltopentaose derivative. The relative affinities for acarbose vs. maltopentaose were also compared between the mutants and wild-type enzyme. The results are consistent with the roles previously proposed in Taka-amylase A and porcine pancreatic alpha-amylase based on their X-ray crystallographic analyses, although different pairs had been assigned as catalytic residues for each enzyme. Analysis of the residual activity of the catalytic-site mutants by gel electrophoresis has suggested that it derived from the wild-type enzyme contaminating the mutant preparations, which could be removed by use of an acarbose affinity column; thus, these mutants are completely devoid of activity. The affinity-purified mutant proteins should be useful for elucidating the complete picture of the interaction of this enzyme with starch.
Takase
K
K
Department of Molecular Biology, National Institute of Agrobiological Resources, Ibaraki, Japan.
Matsumoto
T
T
Mizuno
H
H
Yamane
K
K
D020909:Acarbose; D001412:Bacillus subtilis / Q000201:enzymology / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000096:biosynthesis; D013213:Starch / Q000737:chemistry; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014312:Trisaccharides / Q000737:chemistry / Q000494:pharmacology; D000516:alpha-Amylases / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics*
D011994:Recombinant Proteins; D014312:Trisaccharides; D013213:Starch; D000516:alpha-Amylases; D020909:Acarbose