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Kinetic analysis of lauric acid hydroxylation by human cytochrome P450 4A11.

Kim D, Cha GS, Nagy LD, Yun CH, Guengerich FP
Biochemistry. 2014 53 (39): 6161-72

PMID: 25203493 · PMCID: PMC4188250 · 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-(2)H-substituted lauric acid. However, considerable "metabolic switching" to 11-hydroxylation was observed with [12-(2)H3]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-(3)H]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.

MeSH Terms (21)

Algorithms Binding, Competitive Biocatalysis Cytochrome P-450 CYP4A Cytochrome P-450 Enzyme System Cytochromes b5 Deuterium Electron Transport Ferric Compounds Ferrous Compounds Humans Hydroxylation Kinetics Lauric Acids Models, Chemical Models, Molecular Oxidation-Reduction Protein Binding Protein Structure, Tertiary Substrate Specificity Tritium

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