Kinetic Analysis of Lauric Acid Hydroxylation by Human Cytochrome P450 4A11

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• 作者：Donghak Kim ; Gun-Su Cha ; Leslie D. Nagy ; Chul-Ho Yun ; F. Peter Guengerich
• 刊名：Biochemistry
• 出版年：2014
• 出版时间：October 7, 2014
• 年：2014
• 卷：53
• 期：39
• 页码：6161-6172
• 全文大小：518K
• ISSN：1520-4995

文摘

Cytochrome P450 (P450) 4A11 is the only functionally active subfamily 4A P450 in humans. P450 4A11 catalyzes mainly 蠅-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 蠅-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鈥?) for 12-hydroxylation with 12-²H-substituted lauric acid. However, considerable 鈥渕etabolic switching鈥?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鈥?078] and the use of tritium KIE analysis with [12-³H]lauric acid [Northrop, D. B. (1987) Methods Enzymol. 87, 607鈥?25] both indicated a high intrinsic KIE (>10). Cytochrome b₅ (b₅) stimulated steady-state lauric acid 蠅-hydroxylation 鈭?-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路O₂ complex and C鈥揌 bond-breaking limit the rate of P450 4A11 蠅-oxidation.