Wise M L, Rossi J, Gerwick W H
Department of Biochemistry and Biophysics, College of Pharmacy, Oregon State University, Corvallis 97331, USA.
Biochemistry. 1997 Mar 11;36(10):2985-92. doi: 10.1021/bi962158v.
The substrate binding site of polyenoic fatty acid isomerase (PFI) has been investigated using a series of alternate substrates and by examination of the pH dependence on the kinetic parameters of PFI with selected substrates. The pH dependence profile of PFI with EPA [(5Z,8Z,11Z,14Z,17Z)-eicosapentaenoic acid] shows the enzyme to be catalytically active over a wide pH range, with activity being optimal below pH 6.0. Analysis of the kinetic parameters of DHA [(4Z,7Z,10Z,13Z,16Z,19Z)-docosahexen oic acid]; adrenic acid [(7Z,10Z,13Z,16Z)-docosatetraenoic acid]; EPA; arachidonic acid [(5Z,8Z,11Z,14Z)-eicosatetraenoic acid]; anandamide (arachidonyl-N-ethanolamide); and eicosatrienoic acid [(5Z,8Z,11Z)-eicosatrienoic acid] demonstrates that substrates possessing omega-3 olefins (DHA and EPA) have the lowest K(m) values (1.9 and 9.6 microM, respectively). EPA and arachidonic acid showed the highest V(max) values (6.0 and 2.8 micromol min(-1) mg(-1), respectively). The twenty carbon omega-9 fatty acid eicosatrienoic acid showed a relatively large K(m) and had a V(max) approximately 20-fold less than EPA. Anandamide, a substrate analog lacking an ionizable carboxylate, showed a K(m) similar to the other omega-6 fatty acids (arachidonic acid and adrenic acid); however, the V(max) was approximately 5-fold lower than arachidonic acid and 8-fold lower than EPA. Moreover, anandamide demonstrated no pH dependency on its kinetic parameters over a range where EPA showed a 27-fold decrease in V/K(m). NMR spectroscopy was used to determine the structure of the product from reaction of PFI with DHA. These data showed the compound to be (4Z,7Z,9E,11E,16Z,19Z)-docosahexenoi c acid. Reaction of PFI with dihomo-gamma-linolenic acid resulted in the development of two products, one with the characteristic chromophore of a conjugated triene, the other with a chromophore characteristic of a conjugated diene. Analysis of the products from these reactions of PFI, in conjunction with the kinetic parameters from the alternate substrates, provides compelling evidence that the enzyme preferentially orients the substrate in the catalytic site with respect to the methyl terminus.
已使用一系列替代底物并通过研究pH对多烯脂肪酸异构酶（PFI）与选定底物的动力学参数的依赖性，对多烯脂肪酸异构酶（PFI）的底物结合位点进行了研究。PFI与EPA [（5Z，8Z，11Z，14Z，17Z）-二十碳五烯酸]的pH依赖性曲线表明，该酶在较宽的pH范围内具有催化活性，在pH 6.0以下活性最佳。对DHA [（4Z，7Z，10Z，13Z，16Z，19Z）-二十二碳六烯酸]、肾上腺酸[（7Z，10Z，13Z，16Z）-二十二碳四烯酸]、EPA、花生四烯酸[（5Z，8Z，11Z，14Z）-二十碳四烯酸]、花生四烯乙醇胺（花生四烯酰基-N-乙醇胺）和二十碳三烯酸[（5Z，8Z，11Z）-二十碳三烯酸]的动力学参数分析表明，具有ω-3烯烃的底物（DHA和EPA）的K（m）值最低（分别为1.9和9.6 microM）。EPA和花生四烯酸的V（max）值最高（分别为6.0和2.8微摩尔·分钟-1·毫克-1）。二十碳ω-9脂肪酸二十碳三烯酸的K（m）相对较大，其V（max）比EPA低约20倍。花生四烯乙醇胺是一种缺乏可电离羧酸盐的底物类似物，其K（m）与其他ω-6脂肪酸（花生四烯酸和肾上腺酸）相似；然而，其V（max）比花生四烯酸低约5倍，比EPA低8倍。此外，在EPA的V/K（m）下降27倍的范围内，花生四烯乙醇胺的动力学参数没有pH依赖性。利用核磁共振光谱法确定了PFI与DHA反应产物的结构。这些数据表明该化合物为（4Z，7Z，9E，11E，16Z，19Z）-二十二碳六烯酸。PFI与二高-γ-亚麻酸反应产生了两种产物，一种具有共轭三烯的特征发色团，另一种具有共轭二烯的特征发色团。对PFI这些反应产物的分析，结合替代底物的动力学参数，提供了令人信服的证据，表明该酶在催化位点相对于甲基末端优先使底物定向。
