VITAMIN-K (MENAQUINONE) BIOSYNTHESIS IN BACTERIA - HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHIC ASSAY OF THE OVERALL SYNTHESIS OF O-SUCCINYLBENZOIC ACID AND OF 2-SUCCINYL-6-HYDROXY-2,4-CYCLOHEXADIENE-1-CARBOXYLIC ACID SYNTHASE
VITAMIN-K (MENAQUINONE) BIOSYNTHESIS IN BACTERIA - HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHIC ASSAY OF THE OVERALL SYNTHESIS OF O-SUCCINYLBENZOIC ACID AND OF 2-SUCCINYL-6-HYDROXY-2,4-CYCLOHEXADIENE-1-CARBOXYLIC ACID SYNTHASE
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DOI：
10.1016/0003-2697(89)90643-x
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发表时间：
1989-05-01
影响因子：
2.9
通讯作者：
BENTLEY, R
中科院分区：
文献类型：
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作者：
POPP, JL;BERLINER, C;BENTLEY, R
在菜籽酮(维生素K2)的生物合成中，最早的一种酶是由异甲叉酸合成2-succinyl-6-hydroxy-2，4-cyclohexadiene-1-carboxylic酸的合成酶(SHCHC)。然后，SHCHC被OSB合成酶芳构化为O-琥珀酸基苯甲酸(OSB)。建立了一种测定这两种酶的结合活性的方法(“OSB总合成”)，并用高效液相色谱法测定了OSB的含量。该分析方法仅测量0.1nmol的OSB，大大优于以前用来估计OSB合成总量的放射性气色谱方法。为了单独测定SHCHC合成酶的活性，将酶解形成的SHCHC非酶转化为OSB(加热到80℃，pH值10，10min)，然后用高效液相色谱法进行定量。还介绍了底物异甲酸的制备和制备高效液相色谱分离纯化方法。
An early enzyme in menaquinone (vitamin K2) biosynthesis is the synathase forming 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylic acid (SHCHC) from isochorismic acid. In turn, SHCHC is aromatized to o-succinylbenzoic acid (OSB) by OSB synthase. An assay for the combined activity of these two enzymnes ("overall OSB synthesis") has been developed using a high-performance liquid chromatographic method for the quantitation of OSB. The assay, which measures as little as 0.1 nmol of OSB, is vastly superior to the radiogas chromatographic method previously used to estimate overall OSB synthesis. To measure SHCHC synthase activity separately, the enzymatically formed SHCHC is converted nonenzymatically to OSB (heating to 80.degree.C, pH 10, 10 min), which is then quantitated by the HPLC assay. The preparation of the substrate, isochorismic acid, and its purification by preparative HPLC are also described.