P450-catalyzed in-chain desaturation of valproic acid:: Isoform selectivity and mechanism of formation of Δ3-valproic acid generated by baculovirus-expressed CYP3A1
P450-catalyzed in-chain desaturation of valproic acid:: Isoform selectivity and mechanism of formation of Δ3-valproic acid generated by baculovirus-expressed CYP3A1
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DOI：
10.1006/abbi.1998.0742
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发表时间：
1998-08-01
影响因子：
3.9
通讯作者：
Rettie, AE
中科院分区：
文献类型：
--
作者：
Fisher, MB;Thompson, SJ;Rettie, AE
研究了大鼠肝微粒体形成链内不饱和脂肪酸代谢物 Delta(3)-丙戊酸 (Delta(3)-VPA) 的机制。在对照雌性大鼠肝微粒体催化的反应中，微粒体形成 Delta(3)-VPA 的速率低于可量化的限度，但在用三乙酰竹桃霉素和孕烯醇酮-16 α-腈预处理后，微粒体形成速率增加了 20 倍以上。用 3-羟基-VPA 或 [2-H-2,]VPA 进行的微粒体孵育表明，Delta(3)-VPA 不是由预制酒精脱水产生的，也不是可逆异构化为 Delta(2)-VPA。在杆状病毒表达载体系统中优化CYP3A1表达，添加P450还原酶的感染昆虫细胞膜催化形成3-OH-、4-OH-、5-OH-、Delta(3)-和Delta(4)-VPA，比例为160:35:6:3:1。使用 cDNA 表达的 CYP3A1 和 [3,3-H-2(2)]VPA 或 [4,4-H-2(2)]VPA 测定分子内氘同位素对代谢物形成的影响，得出 Delta(3)-VPA 的 k(H)/k(D) 值分别为 2.00 +/- 0.06 和 2.36 +/- 0.08。这些值显着低于在相同孵育中观察到的同位素效应，即从 3,3-D-2-VPA 形成 3-OH-VPA (k(H)/k(D) = 6.04 +/- 0,08)，或从 4,4-D-2-VPA 形成 4-OH-和 Delta(4)-VPA (k(H)/k(D) > 5)。总的来说，这些数据证明了微粒体 P450 依赖性链内脂肪酸去饱和酶系统的存在，该系统与有据可查的细胞色素 b(5) 连接的 CoA 去饱和酶不同，并进一步表明 Delta(3)-VPA 的 CYP3A1 依赖性形成是通过从 C-3 或 C-4 位非选择性的初始氢原子提取而产生的。 (C) 1998 年学术出版社。
The mechanism of formation of the in-chain, unsaturated fatty acid metabolite, Delta(3)-valproic acid (Delta(3)-VPA) by rat liver microsomes was examined. Microsomal rates of formation of Delta(3)-VPA were below quantifiable limits in reactions catalyzed by control female rat liver microsomes, but were induced more than 20-fold following pretreatment with triacetyloleandomycin and pregnenolone-16 alpha-carbonitrile. Microsomal incubations conducted with 3-hydroxy-VPA or [2-H-2,]VPA demonstrated that Delta(3)-VPA did not arise by dehydration of preformed alcohol nor was it reversibly isomerized to Delta(2)-VPA. CYP3A1 expression was optimized in the baculovirus expression vector system, and infected insect cell membranes which were supplemented with P450 reductase catalyzed formation of 3-OH-, 4-OH-, 5-OH-, Delta(3)-, and Delta(4)-VPA in ratios of 160:35: 6:3:1. Intramolecular deuterium isotope effects on metabolite formation, determined with cDNA-expressed CYP3A1 and either [3,3-H-2(2)]VPA or [4,4-H-2(2)]VPA, yielded k(H)/k(D) values for Delta(3)-VPA of 2.00 +/- 0.06 and 2.36 +/- 0.08, respectively. These values were significantly lower than the isotope effects observed in the same incubations for 3-OH-VPA formation from 3,3-D-2-VPA (k(H)/k(D) = 6.04 +/- 0,08), or for 4-OH- and Delta(4)-VPA formation from 4,4-D-2-VPA (k(H)/k(D) > 5). Collectively, these data demonstrate the existence of a microsomal P450-dependent in-chain fatty acid desaturase system distinct from the well-documented cytochrome b(5)-linked CoA desaturases and suggest further that CYP3A1-dependent formation of Delta(3)-VPA arises via nonselective, initial hydrogen atom abstraction from either the C-3 or the C-4 position. (C) 1998 Academic Press.