Site-directed mutagenesis and NMR studies of histidine-385 mutants of 5-enolpyruvylshikimate-3-phosphate synthase.
Site-directed mutagenesis and NMR studies of histidine-385 mutants of 5-enolpyruvylshikimate-3-phosphate synthase.
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5-烯醇丙酮莽草酸-3-磷酸合酶组氨酸-385 突变体的定点诱变和 NMR 研究。
DOI：
10.1021/bi00189a007
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发表时间：
1994
期刊：
影响因子：
2.9
通讯作者：
Evans,JN
中科院分区：
文献类型：
--
作者：
Shuttleworth,WA;Evans,JN
本文报道了5-烯醇式莽草酸-3-磷酸（EPSP）合成酶His-385的定点突变。将两个突变体H385 Q和H385 A的稳态动力学与野生型酶进行了比较。发现H385 Q EPSP合酶具有25%的野生型酶活性，而H385 A EPSP合酶保留1%的活性。P…的Am值和莽草酸3-磷酸不受影响，而磷酸烯醇丙酮酸（PEP）的Am对于H385 Q EPSP合酶增加了10倍。在H385 Q中EPSP的Am不受影响，但在H385 A EPSP合酶中升高了10倍。通过荧光光谱和31 P NMR光谱研究了草甘膦的结合。直接观察酶的中间复合物与[2，3- 13 C]磷酸烯醇丙酮酸的13 C NMR光谱进行了研究的突变酶，并与野生型相比。在平衡条件下，H385 A EPSP合酶不积累酶结合的EPSP。这些结果表明，虽然关键地位于PEP结合位点，但His-385不是负责通过PEP. 5-烯醇式莽草酸-3-磷酸（EPSP）合酶（EC 2.5. 1.19）催化莽草酸3-磷酸（S3 P，1）和磷酸烯醇丙酮酸（PEP，2）之间的反应以形成5-烯醇乙酰基莽草酸3-磷酸（EPSP，4），EPSP，4是导致分支酸并因此导致芳香族氨基酸的生物合成途径中的中间体（参见方案1）。EPSP合酶作为广谱芽后除草剂草甘膦（7V-（磷酸-N甲基）甘氨酸）的主要靶标是令人感兴趣的。EPSP合酶的作用机制和结构已被广泛研究（Barlow et
Revised Manuscript Received April 7, 1994* abstract: The site-directed mutagenesis of His-385 of 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase is reported. The steady-state kineticsfor two mutants, H385Q and H385A, are compared with that of the wild-type enzyme. H385Q EPSPsynthase was found to have 25% wild-type enzyme activity, whereas H385A EPSP synthase retained 1% activity. The Am values for P¡ and shikimate 3-phosphate were unaffected, whereas the Am for phosphoenolpyruvate (PEP) was increased 10 times for H385Q EPSP synthase. The Am for EPSP was unaffected in H385Q but raised by a factor of 10 in H385A EPSPsynthase. The binding of glyphosate was studied by fluorescence spectroscopy and by 31P NMR spectroscopy. Direct observation of the enzyme-intermediate complexes by 13C NMR spectroscopy with [2, 3-13C] phosphoenolpyruvate was studied for the mutant enzymes and compared with the wild type. Under equilibrium conditions, H385A EPSP synthase does not accumulate enzyme-bound EPSP. These results suggest that, while critically located in the PEP binding site, His-385 is not the residue responsible for initiating catalysis through the protonation of PEP.5-Enolpyruvylshikimate-3-phosphate (EPSP) synthase (EC 2.5. 1.19) catalyzes the reaction between shikimate 3-phosphate (S3P, 1) and phosphoenolpyruvate (PEP, 2) to form 5-enolpyruvylshikimate 3-phosphate (EPSP, 4) an intermedi-ate in the biosynthetic pathway leading to chorismate and, hence, to thearomatic amino acids (see Scheme 1). EPSP synthase is of interest as the primary target for the broad spectrum, postemergence herbicide, glyphosate (7V-(phospho-nomethyl) glycine). The mechanism of action and the structure of EPSP synthase have been studied extensively (Barlow et
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