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

• Published in: Biochemistry (Easton) 2014-10, Vol.53 (39), p.6161-6172
• Main Authors: Kim, Donghak, Cha, Gun-Su, Nagy, Leslie D, Yun, Chul-Ho, Guengerich, F. Peter
• Format: Article
• Language: English
• Subjects: Algorithms; Binding, Competitive; Biocatalysis; Cytochrome P-450 CYP4A; Cytochrome P-450 Enzyme System - chemistry; Cytochrome P-450 Enzyme System - metabolism; Cytochromes b5 - chemistry; Cytochromes b5 - metabolism; Deuterium; Electron Transport; Ferric Compounds - chemistry; Ferric Compounds - metabolism; Ferrous Compounds - chemistry; Ferrous Compounds - metabolism; Humans; Hydroxylation; Kinetics; Lauric Acids - chemistry; Lauric Acids - metabolism; Models, Chemical; Models, Molecular; Oxidation-Reduction; Protein Binding; Protein Structure, Tertiary; Substrate Specificity; Tritium
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi500710e

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 b 5 (b 5) stimulated steady-state lauric acid ω-hydroxylation ∼2-fold; the apoprotein was ineffective, indicating that electron transfer is involved in the b 5 enhancement. The rate of b 5 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.