Soliday C L, Kolattukudy P E
Department of Agricultural Chemistry and Program in Biochemistry and Biophysics, Washington State University, Pullman, Washington 99164.
Plant Physiol. 1977 Jun;59(6):1116-21. doi: 10.1104/pp.59.6.1116.
omega-Hydroxylation of fatty acids, which is a key reaction in the biosynthesis of cutin and suberin, has been demonstrated for the first time in a cell-free preparation from a higher plant. A crude microsomal fraction (105,000g pellet) from germinating embryonic shoots of Vicia faba catalyzed the conversion of palmitic acid to omega-hydroxypalmitic acid. As the crude cell-free preparation also catalyzes the formation of other hydroxy acids such as alpha- and beta-hydroxy acids, the omega-hydroxylation product was identified by gas chromatography on a polyester column and reverse phase, high performance liquid chromatography, two techniques which were shown to resolve the positional isomers. Gas chromatographic analysis of the dicarboxylic acid obtained by CrO(3) oxidation of the enzymic product also confirmed the identity of the enzymic omega-hydroxylation product. This enzymic hydroxylation required O(2) and NADPH, but substitution of NADH resulted in nearly half the reaction rate obtained with NADPH. Maximal rates of omega-hydroxylation occurred at pH 8 and the rate increased in a sigmoidal manner with increasing concentrations of palmitic acid. This omega-hydroxylation was inhibited by the classical mixed function oxidase inhibitors such as metal chelators (o-phenanthroline, 8-hydroxyquinoline, and alpha,alpha-dipyridyl), NaN(3) and thiol reagents (N-ethylmaleimide and p-chloromercuribenzoate). As expected of a hydroxylase, involving cytochrome P(450), the present omega-hydroxylase was inhibited by CO and this enzyme system showed unusually high sensitivity to this inhibition; 10% CO caused inhibition and 30% CO completely inhibited the reaction. Another unusual feature was that the inhibition caused by any level of CO could not be reversed by light (420-460 nm).
脂肪酸的ω-羟基化是角质和木栓质生物合成中的关键反应,首次在高等植物的无细胞制剂中得到证实。来自蚕豆发芽胚轴的粗微粒体部分(105,000g沉淀)催化了棕榈酸向ω-羟基棕榈酸的转化。由于这种粗无细胞制剂还催化其他羟基酸如α-和β-羟基酸的形成,因此通过在聚酯柱上的气相色谱法和反相高效液相色谱法鉴定了ω-羟基化产物,这两种技术被证明可分离位置异构体。通过CrO(3)氧化酶促产物获得的二羧酸的气相色谱分析也证实了酶促ω-羟基化产物的身份。这种酶促羟基化需要O(2)和NADPH,但用NADH替代导致反应速率接近用NADPH时获得的反应速率的一半。ω-羟基化的最大速率出现在pH 8时,并且随着棕榈酸浓度的增加,速率呈S形增加。这种ω-羟基化受到经典混合功能氧化酶抑制剂的抑制,如金属螯合剂(邻菲罗啉、8-羟基喹啉和α,α-联吡啶)、NaN(3)和硫醇试剂(N-乙基马来酰亚胺和对氯汞苯甲酸)。正如涉及细胞色素P(450)的羟化酶所预期的那样,目前的ω-羟化酶受到CO的抑制,并且该酶系统对这种抑制表现出异常高的敏感性;10%的CO会导致抑制,30%的CO会完全抑制反应。另一个不寻常的特征是,任何水平的CO引起的抑制都不能被光(420 - 460nm)逆转。