Expression and characterization of active site mutants of hevamine, a chitinase from the rubber tree Hevea brasiliensis
Expression and characterization of active site mutants of hevamine, a chitinase from the rubber tree Hevea brasiliensis
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DOI：
10.1046/j.0014-2956.2001.02721.x
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发表时间：
2002-02-01
期刊：
影响因子：
--
通讯作者：
Beintema, JJ
中科院分区：
文献类型：
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作者：
Bokma, E;Rozeboom, HJ;Beintema, JJ
Hevamine是来自橡胶树Hevea brasiliensis的几丁质酶。其活性部位含有Asp 125、Glu 127。Tyr 183其与底物的-1糖残基相互作用。为了研究它们在催化中的作用，我们已经成功地表达了野生型酶和这些残基的突变体在大肠杆菌中的包涵体。经过复性和纯化，其特征在于结构和酶动力学研究。Tyr 183突变为苯丙氨酸产生的酶具有比野生型酶低的k（cat）和略高的K-m。将Asp 125和Glu 127突变为丙氨酸，得到具有约2%残余活性的突变体。相比之下，Asp 125 Asn突变体保留了大量的活性，与野生型酶相比，k（cat）约低两倍，K-m约高两倍。更有趣的是，它显示出比其他变体更高的pH值的活性。Asp 125 Ala/Glu 127 Ala双突变体经壳聚糖浸泡后的X射线结构表明。与野生型hevamine相比。-1糖残基的N-乙酰基的羰基氧原子已从该残基的Cl原子旋转离开。结合的结构和动力学数据表明，Asp 125和Tyr 183通过将N-乙酰基的羰基氧定位在Cl原子附近而有助于催化。这使得稳定的一个带正电荷的瞬态中间体，在协议与以前的建议，酶利用底物辅助催化。
Hevamine is a chitinase from the rubber tree Hevea brasiliensis. Its active site contains Asp125, Glu127. and Tyr183. which interact with the -1 sugar residue of the substrate. To investigate their role in catalysis, we have successfully expressed wild-type enzyme and mutants of these residues as inclusion bodies in Escherichia coli. After refolding and purification they were characterized by both structural and enzyme kinetic studies. Mutation of Tyr183 to phenylalanine produced an enzyme with a lower k(cat) and a slightly higher K-m than the wild-type enzyme. Mutating Asp125 and Glu127 to alanine gave mutants with; approximate to2% residual activity. In contrast, the Asp125Asn mutant retained substantial activity, with an approximately twofold lower k(cat) and an approximately twofold higher K-m than the wild-type enzyme. More interestingly, it showed activity to higher pH values than the other variants. The X-ray structure of the Asp125Ala/Glu127Ala double mutant soaked with chitotetraose shows that. compared with wild-type hevamine. the carbonyl oxygen atom of the N-acetyl group of the -1 sugar residue has rotated away from the Cl atom of that residue. The combined structural and kinetic data show that Asp125 and Tyr183 contribute to catalysis by positioning the carbonyl oxygen of the N-acetyl group near to the Cl atom. This allows the stabilization of a positively charged transient intermediate, in agreement with a previous proposal that the enzyme makes use of substrate-assisted catalysis.