Mutational and crystallographic analyses of the active site residues of the bacillus circulans xylanase
Mutational and crystallographic analyses of the active site residues of the bacillus circulans xylanase
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DOI：
10.1002/pro.5560030312
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发表时间：
1994-03
期刊：
影响因子：
8
通讯作者：
W. Wakarchuk;R. Campbell;W. Sung;J. Davoodi;M. Yaguchi
中科院分区：
文献类型：
--
作者：
W. Wakarchuk;R. Campbell;W. Sung;J. Davoodi;M. Yaguchi
使用定点诱变，我们已经研究了来自环状芽孢杆菌的木聚糖酶中的催化残基。对突变体E78 D和E172 D的分析表明，这些保守残基中的突变不会严重改变酶的结构，并且这些残基参与催化机制。我们现在已经确定了酶-底物复合物的晶体结构，使用无催化能力的突变体（E172 C）的分辨率为1.8 nm。除了催化残基Glu 78和Glu 172外，我们还鉴定了2个酪氨酸残基Tyr 69和Tyr 80，它们可能在底物结合中起作用，以及精氨酸残基Arg 112，其在该酶的活性位点中起重要作用。基于我们的工作，我们提出Glu 78是亲核试剂，Glu 172是反应中的酸碱催化剂。
Using site‐directed mutagenesis we have investigated the catalytic residues in a xylanase from Bacillus circulans. Analysis of the mutants E78D and E172D indicated that mutations in these conserved residues do not grossly alter the structure of the enzyme and that these residues participate in the catalytic mechanism. We have now determined the crystal structure of an enzyme‐substrate complex to 1.8 Å resolution using a catalytically incompetent mutant (E172C). In addition to the catalytic residues, Glu 78 and Glu 172, we have identified 2 tyrosine residues, Tyr 69 and Tyr 80, which likely function in substrate binding, and an arginine residue, Arg 112, which plays an important role in the active site of this enzyme. On the basis of our work we would propose that Glu 78 is the nucleophile and that Glu 172 is the acid‐base catalyst in the reaction.