SITE-DIRECTED MUTAGENESIS AT ASPARTATE AND GLUTAMATE RESIDUES OF XYLANASE FROM BACILLUS-PUMILUS
SITE-DIRECTED MUTAGENESIS AT ASPARTATE AND GLUTAMATE RESIDUES OF XYLANASE FROM BACILLUS-PUMILUS
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DOI：
10.1042/bj2880117
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发表时间：
1992-11-15
影响因子：
4.1
通讯作者：
OKADA, H
中科院分区：
文献类型：
--
作者：
KO, EP;AKATSUKA, H;OKADA, H
阐明木聚糖酶的反应机制。鉴定其催化所必需的氨基酸非常重要。研究表明，木聚糖酶的反应机制可能与鸡蛋溶菌酶相似，涉及酸性氨基酸残基。在此假设基础上，结合短小芽孢杆菌木聚糖酶的三维结构及其与其他不同来源木聚糖酶的氨基酸序列相似性，选择Asp-2 1、Glu-93和Glu-182这3种酸性氨基酸进行定点诱变。 Asp 残基改变为 Ser 或 Glu，Glu 残基改变为 Ser 或 Asp。纯化的突变木聚糖酶D21E、D21S、E93D、E93S、E182D和E182S在SDS/PAGE上显示出约26kDa的单一蛋白条带。光盘。这些突变酶的光谱显示对木聚糖酶的二级结构没有影响，但 D2] E 的二级结构除外，它显示出一点变化。此外，与Asp-21的突变相比，Glu-93和Glu-182的突变导致木聚糖酶的比活性急剧下降。根据这些结果，我们提出 Glu-93 和 Glu-182 是木聚糖酶必需催化残基的最佳候选者。
To elucidate the reaction mechanism of xylanase. the identification of amino acids essential for its catalysis is of importance. Studies have indicated the possibility that the reaction mechanism of xylanase is similar to that of hen's egg lysozyme, which involves acidic amino acid residues. On the basis of this assumption, together with the three-dimensional structure of Bacillus pumilus xylanase and its amino acid sequence similarity to other xylanases of different origins, three acidic amino acids, namely Asp-2 1, Glu-93 and Glu-182 were selected for site-directed mutagenesis. The Asp residue was altered to either Ser or Glu, and the Glu residues to Ser or Asp. The purified mutant xylanases D21E, D21S, E93D, E93S, E182D and E182S showed single protein bands of about 26 kDa on SDS/PAGE. C.d. spectra of these mutant enzymes show no effect on the secondary structure of xylanase, except that of D2] E, which shows a little variation. Furthermore, mutations of Glu-93 and Glu-182 resulted in a drastic decrease in the specific activity of xylanase as compared with mutation of Asp-21. On the basis of these results we propose that Glu-93 and Glu-182 are the best candidates for the essential catalytic residues of xylanase.