通过定点诱变分析嗜热放线菌R-47α-淀粉酶II（TVA II）的催化残基。

Analysis of catalytic residues of Thermoactinomyces vulgaris R-47 alpha-amylase II (TVA II) by site-directed mutagenesis.

作者信息

Ichikawa K, Tonozuka T, Yokota T, Shimura Y, Sakano Y

机构信息

Department of Applied Biological Science, Faculty of Agriculture, Tokyo University of Agriculture and Technology, Fuchu, Japan.

出版信息

Biosci Biotechnol Biochem. 2000 Dec;64(12):2692-5. doi: 10.1271/bbb.64.2692.

DOI:10.1271/bbb.64.2692

PMID:11210138

Abstract

To confirm that the catalytic residues (Asp325, Glu354, and Asp421) are necessary for the hydrolysis of starch, pullulan, and cyclodextrins, we constructed TVA II mutated by site-directed mutagenesis. The mutated enzymes (D325N, E354Q, and D421N) had markedly reduced levels of activity, less than 0.006% of the wild type, indicating that these three residues are the catalytic sites for these substrates. Even E354D had reduced levels of activity, less than 0.05% of wild type. These four mutated enzymes retained a trace of activity. From the result of hydrolysis patterns for maltohexaose, in particular, D421N, unlike D325N and E354Q, catalyzed transglycosylation rather than hydrolysis. The results suggest that Asp421 could function to capture water molecules.

摘要

为了证实催化残基（Asp325、Glu354和Asp421）对于淀粉、支链淀粉和环糊精的水解是必需的，我们通过定点诱变构建了TVA II的突变体。突变酶（D325N、E354Q和D421N）的活性水平显著降低，不到野生型的0.006%，表明这三个残基是这些底物的催化位点。甚至E354D的活性水平也降低了，不到野生型的0.05%。这四种突变酶保留了微量活性。特别是从麦芽六糖的水解模式结果来看，与D325N和E354Q不同，D421N催化转糖基化而非水解。结果表明，Asp421可能起到捕获水分子的作用。