SITE-DIRECTED MUTAGENESIS OF ACTIVE-SITE RESIDUES IN BACILLUS-SUBTILIS ALPHA-AMYLASE

被引:58
作者
TAKASE, K [1 ]
MATSUMOTO, T [1 ]
MIZUNO, H [1 ]
YAMANE, K [1 ]
机构
[1] UNIV TSUKUBA,INST BIOL SCI,TSUKUBA,IBARAKI,JAPAN
关键词
AMYLASE; ACTIVE SITE; SITE-DIRECTED MUTAGENESIS; (BACILLUS-SUBTILIS);
D O I
10.1016/0167-4838(92)90249-D
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 k(cat) with a 5-fold increase in K(m) 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.
引用
收藏
页码:281 / 288
页数:8
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