Site-directed mutagenesis of putative active site residues of 5-enolpyruvylshikimate-3-phosphate synthase.
Site-directed mutagenesis of putative active site residues of 5-enolpyruvylshikimate-3-phosphate synthase.
复制标题
5-烯醇丙酮莽草酸-3-磷酸合酶的假定活性位点残基的定点诱变。
DOI：
10.1021/bi9815142
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发表时间：
1999
期刊：
影响因子：
--
通讯作者：
Evans,JN
中科院分区：
文献类型：
--
作者：
Shuttleworth,WA;Pohl,ME;Helms,GL;Jakeman,DL;Evans,JN
报道了一些拟制的5-烯醇丙酮酰莽草酸-3-磷酸（EPSP）合成酶活性位点残基的定点突变。其中一些突变导致酶活性完全丧失，表明这些残基可能参与催化作用，特别是K22R、K411R、D384A、R27A、R100A和D242A。其中，K22R、R27A和D384A不结合底物莽草酸-3-磷酸（S3P）或草甘膦（GLP）。K411R和D242A突变体仅在GLP存在时结合S3P。报道了保持活性的突变体R100K、K340R和E418A的动力学特性。其中，R100K和K340R在平衡条件下不积累酶结合产物的酶中间体。这些残基虽然不是催化所必需的，但很可能对底物结合很重要。所有的突变体都被核磁共振光谱证明是正确折叠的。
The site-directed mutagenesis of a number of proposed active site residues of 5-enolpyruvyl shikimate-3-phosphate (EPSP) synthase is reported. Several of these mutations resulted in complete loss of enzyme activity indicating that these residues are probably involved with catalysis, notably K22R, K411R, D384A, R27A, R100A, and D242A. Of those, K22R, R27A, and D384A did not bind either the substrate shikimate-3-phosphate (S3P) or glyphosate (GLP). The K411R and D242A mutants bind S3P only in the presence of GLP. The kinetic characterization of mutants R100K, K340R, and E418A, which retain activity, is reported. Of those, R100K and K340R do not accumulate enzyme intermediate of enzyme-bound product under equilibrium conditions. These residues, while not essential for catalysis, are most likely important for substrate binding. All of the mutants are shown to be correctly folded by NMR spectroscopy.