Xu Fengyun, Falck John R, Ortiz de Montellano Paul R, Kroetz Deanna L
Department of Biopharmaceutical Sciences, University of California, San Francisco, CA 94143-0446, USA.
J Pharmacol Exp Ther. 2004 Mar;308(3):887-95. doi: 10.1124/jpet.103.059626. Epub 2003 Nov 21.
Arachidonic acid is omega-hydroxylated to 20-hydroxyeicosatetraenoic acid (20-HETE), which has effects on vasoactivity and renal tubular transport and has been implicated in the regulation of blood pressure. Cytochrome p450 (p450) 4A isoforms are generally considered the major arachidonic acid omega-hydroxylases; however, little is known about the role of rat CYP4F isoforms in 20-HETE formation. The rat CYP4F isoforms, CYP4F1, CYP4F4, CYP4F5, and CYP4F6, were heterologously expressed in Escherichia coli, and their substrate specificity in fatty acid metabolism was characterized. Substrate-binding assays indicated that leukotriene B(4) (LTB(4)) and arachidonic acid bound CYP4F1 and CYP4F4 in a type-I manner with a K(s) of 25 to 59 microM, and lauric acid bound CYP4F4 poorly. Reconstituted CYP4F1 and CYP4F4 catalyzed the omega-hydroxylation of LTB(4) with a K(m) of 24 and 31 microM, respectively, and CYP4F5 had minor activity in LTB(4) metabolism. Importantly, CYP4F1 and CYP4F4 catalyzed the omega-hydroxylation of arachidonic acid with an apparent k(cat) of 9 and 11 min(-1), respectively. Lauric acid was a poor substrate for all of the CYP4F isoforms, and CYP4F6 had no detectable fatty acid omega-hydroxylase activity. The p450 omega-hydroxylase inhibitors 17-octadecynoic acid, 10-undecynyl sulfate, and N-methylsulfonyl-12,12-dibromododec-11-enamide showed isoform-specific inhibition of CYP4F1- and CYP4F4-catalyzed omega-hydroxylation of arachidonic acid and potency differences between the CYP4A and CYP4F isoforms. These data support a significant role for CYP4F1 and CYP4F4 in the formation of 20-HETE and identify p450 inhibitors that can be used to understand the relative contribution of the CYP4A and CYP4F isoforms to renal 20-HETE formation.
花生四烯酸经ω-羟基化生成20-羟基二十碳四烯酸（20-HETE），其对血管活性和肾小管转运有影响，并与血压调节有关。细胞色素P450（P450）4A亚型通常被认为是主要的花生四烯酸ω-羟化酶；然而，关于大鼠CYP4F亚型在20-HETE形成中的作用知之甚少。大鼠CYP4F亚型CYP4F1、CYP4F4、CYP4F5和CYP4F6在大肠杆菌中进行了异源表达，并对它们在脂肪酸代谢中的底物特异性进行了表征。底物结合试验表明，白三烯B4（LTB4）和花生四烯酸以I型方式结合CYP4F1和CYP4F4，解离常数（K(s)）为25至59μM，月桂酸与CYP4F4结合较差。重组的CYP4F1和CYP4F4催化LTB4的ω-羟基化，米氏常数（K(m)）分别为24和31μM，CYP4F5在LTB4代谢中的活性较小。重要的是，CYP4F1和CYP4F4催化花生四烯酸的ω-羟基化，表观催化常数（k(cat)）分别为9和11分钟-1。月桂酸是所有CYP4F亚型的不良底物，CYP4F6没有可检测到的脂肪酸ω-羟化酶活性。P450ω-羟化酶抑制剂17-十八碳炔酸、10-十一炔基硫酸盐和N-甲基磺酰基-12,12-二溴十二碳-11-烯酰胺对CYP4F1和CYP4F4催化的花生四烯酸ω-羟基化表现出亚型特异性抑制，且CYP4A和CYP4F亚型之间存在效价差异。这些数据支持CYP4F1和CYP4F4在20-HETE形成中的重要作用，并鉴定出可用于了解CYP4A和CYP4F亚型对肾脏20-HETE形成相对贡献的P450抑制剂。
