Kinetic analysis of lauric acid hydroxylation by human cytochrome P450 4A11.
Kinetic analysis of lauric acid hydroxylation by human cytochrome P450 4A11.
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DOI：
10.1021/bi500710e
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发表时间：
2014-10-07
期刊：
影响因子：
2.9
通讯作者：
Guengerich, F. Peter
中科院分区：
文献类型：
--
作者：
Kim, Donghak;Cha, Gun-Su;Nagy, Leslie D.;Yun, Chul-Ho;Guengerich, F. Peter
细胞色素 P450 (P450) 4A11 是人类中唯一具有功能活性的 4A P450 亚家族。 P450 4A11 主要催化肝脏和肾脏中脂肪酸的 ω-羟基化；该过程不是主要的降解途径，但至少一种产物，20-羟基二十碳四烯酸，具有重要的信号传导特性。我们研究了 P450 4A11 的催化作用以及使用月桂酸 ω-羟基化（该酶的原型底物）的限速步骤问题。使用前稳态动力学方法研究了一些单独的反应步骤。底物和产物的结合和释放比总体催化速率快得多。三价铁 P450 4A11（还原为二价铁）的还原速度很快且不受速率限制。氘动力学同位素效应 (KIE) 实验对于 12-2H 取代的月桂酸的 12-羟基化产生较低但可重复的值 (1.2–2)。然而，[12-2H3]月桂酸观察到大量“代谢转换”为 11-羟基化。切换结果分析 [Jones, J. P., et al. (1986) J. Am。化学。 Soc.108, 7074–7078] 以及使用[12-3H]月桂酸进行氚 KIE 分析 [Northrop, D. B. (1987)Methods Enzymol.87, 607–625] 均表明具有较高的内在 KIE (>10)。细胞色素 b5 (b5) 刺激稳态月桂酸 ω-羟基化~2 倍；脱辅基蛋白无效，表明电子转移参与了 b5 增强。在亚铁 P450 与 O2 混合的情况下，b5 再氧化速率增加。总的来说，结果表明电子向亚铁·O2络合物的转移和C-H键断裂都限制了P450 4A11 ω-氧化的速率。
Cytochrome P450 (P450) 4A11 is the only functionally active subfamily 4A P450 in humans. P450 4A11 catalyzes mainly ω-hydroxylation of fatty acids in liver and kidney; this process is not a major degradative pathway, but at least one product, 20-hydroxyeicosatetraenoic acid, has important signaling properties. We studied catalysis by P450 4A11 and the issue of rate-limiting steps using lauric acid ω-hydroxylation, a prototypic substrate for this enzyme. Some individual reaction steps were studied using pre-steady-state kinetic approaches. Substrate and product binding and release were much faster than overall rates of catalysis. Reduction of ferric P450 4A11 (to ferrous) was rapid and not rate-limiting. Deuterium kinetic isotope effect (KIE) experiments yielded low but reproducible values (1.2–2) for 12-hydroxylation with 12-2H-substituted lauric acid. However, considerable “metabolic switching” to 11-hydroxylation was observed with [12-2H3]lauric acid. Analysis of switching results [Jones, J. P., et al. (1986) J. Am. Chem. Soc.108, 7074–7078] and the use of tritium KIE analysis with [12-3H]lauric acid [Northrop, D. B. (1987) Methods Enzymol.87, 607–625] both indicated a high intrinsic KIE (>10). Cytochrome b5 (b5) stimulated steady-state lauric acid ω-hydroxylation ∼2-fold; the apoprotein was ineffective, indicating that electron transfer is involved in the b5 enhancement. The rate of b5 reoxidation was increased in the presence of ferrous P450 mixed with O2. Collectively, the results indicate that both the transfer of an electron to the ferrous·O2 complex and C–H bond-breaking limit the rate of P450 4A11 ω-oxidation.
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影响因子：
1.8
作者：
Guengerich FP
通讯作者：
Guengerich FP
影响因子：
4.8
作者：
Cowart, LA;Wei, SZ;Capdevila, JH
通讯作者：
Capdevila, JH
影响因子：
4.8
作者：
Bell, LC;Guengerich, FP
通讯作者：
Guengerich, FP
DOI：
10.1111/j.1432-1033.1972.tb01845.x
发表时间：
1972-01-01
期刊：
EUROPEAN JOURNAL OF BIOCHEMISTRY
影响因子：
--
作者：
BJORKHEM, I
通讯作者：
BJORKHEM, I
影响因子：
3.9
作者：
Guengerich, FP
通讯作者：
Guengerich, FP