Hamberg M, Gerwick W H
Department of Physiological Chemistry, Karolinska Institutet, Stockholm, Sweden.
Arch Biochem Biophys. 1993 Aug 15;305(1):115-22. doi: 10.1006/abbi.1993.1400.
Biosynthesis of the vicinal diol fatty acid (12R,13S)-dihydroxy-(5Z,8Z,10E,14Z)-eicosatetrae noic acid from arachidonic acid was studied in preparations of the red alga Gracilariopsis lemaneiformis. The transformation consisted of initial 12-lipoxygenase-catalyzed oxygenation of arachidonic acid into (12S)-hydroperoxy-(5Z,8Z,10E,14Z)-eicosatetraeno ic acid followed by hydroperoxide isomerase-catalyzed conversion of the hydroperoxide into (12R,13S)-dihydroxyeicosatetraenoic acid. Short time incubations and trapping experiments with glutathione peroxidase revealed that (12S)-hydroperoxyeicosatetraenoic acid existed as a free intermediate in the overall conversion. The 12-lipoxygenase was mainly present in the soluble fraction of homogenate of G. lemaneiformis. Further, gel filtration experiments showed that the soluble 12-lipoxygenase was a protein having a molecular weight of 84,000-89,000. The enzymatic activity of 12-lipoxygenase isolated by gel filtration was weak; however, addition of 0.8-1 M sodium chloride to such desalted enzyme increased the activity 20-fold. Experiments with different salts revealed that sodium ion was specifically responsible for the stimulatory effect. Hydroperoxide isomerase was about equally distributed between the high speed supernatant and particulate fractions. Gel filtration of hydroperoxide isomerase present in the soluble fraction showed two peaks of activity corresponding to proteins having molecular weights of 220,000 or greater, and 40,000-45,000. The stereochemical course of the biosynthesis of vicinal diol fatty acids was determined using stereospecifically deuterated 6,9,12-octadecatrienoic acids. The 12-lipoxygenase-catalyzed reaction consisted of antarafacial hydrogen removal and oxygen insertion, whereas the hydroperoxide isomerase catalyzed an intramolecular oxygenation which occurred with retention of the configuration of the carbon atom hydroxylated.
在龙须菜的制备物中研究了从花生四烯酸生物合成邻二醇脂肪酸（12R，13S）-二羟基-（5Z，8Z，10E，14Z）-二十碳四烯酸的过程。该转化过程包括花生四烯酸首先由12-脂氧合酶催化氧化为（12S）-氢过氧-（5Z，8Z，10E，14Z）-二十碳四烯酸，随后由氢过氧化物异构酶催化将氢过氧化物转化为（12R，13S）-二羟基二十碳四烯酸。用谷胱甘肽过氧化物酶进行的短时间孵育和捕获实验表明，（12S）-氢过氧二十碳四烯酸在整个转化过程中作为游离中间体存在。12-脂氧合酶主要存在于龙须菜匀浆的可溶部分。此外，凝胶过滤实验表明，可溶性12-脂氧合酶是一种分子量为84,000 - 89,000的蛋白质。通过凝胶过滤分离的12-脂氧合酶的酶活性较弱；然而，向这种脱盐酶中加入0.8 - 1M氯化钠可使活性提高20倍。用不同盐进行的实验表明，钠离子对刺激作用具有特异性。氢过氧化物异构酶在高速上清液和颗粒部分中分布大致相等。对可溶部分中存在的氢过氧化物异构酶进行凝胶过滤显示出两个活性峰，分别对应分子量为220,000或更大以及40,000 - 45,000的蛋白质。使用立体特异性氘代的6,9,12 - 十八碳三烯酸确定了邻二醇脂肪酸生物合成的立体化学过程。12-脂氧合酶催化的反应包括反式面氢消除和氧插入，而氢过氧化物异构酶催化的是分子内氧合反应，该反应发生时羟基化碳原子的构型保持不变。
