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

[Image: see text] 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 im...

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Autores principales: Kim, Donghak, Cha, Gun-Su, Nagy, Leslie D., Yun, Chul-Ho, Guengerich, F. Peter
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Chemical Society 2014
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4188250/
https://www.ncbi.nlm.nih.gov/pubmed/25203493
http://dx.doi.org/10.1021/bi500710e
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author Kim, Donghak
Cha, Gun-Su
Nagy, Leslie D.
Yun, Chul-Ho
Guengerich, F. Peter
author_facet Kim, Donghak
Cha, Gun-Su
Nagy, Leslie D.
Yun, Chul-Ho
Guengerich, F. Peter
author_sort Kim, Donghak
collection PubMed
description [Image: see text] 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)H(3)]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 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 O(2). Collectively, the results indicate that both the transfer of an electron to the ferrous·O(2) complex and C–H bond-breaking limit the rate of P450 4A11 ω-oxidation.
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spelling pubmed-41882502015-09-09 Kinetic Analysis of Lauric Acid Hydroxylation by Human Cytochrome P450 4A11 Kim, Donghak Cha, Gun-Su Nagy, Leslie D. Yun, Chul-Ho Guengerich, F. Peter Biochemistry [Image: see text] 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)H(3)]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 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 O(2). Collectively, the results indicate that both the transfer of an electron to the ferrous·O(2) complex and C–H bond-breaking limit the rate of P450 4A11 ω-oxidation. American Chemical Society 2014-09-09 2014-10-07 /pmc/articles/PMC4188250/ /pubmed/25203493 http://dx.doi.org/10.1021/bi500710e Text en Copyright © 2014 American Chemical Society Terms of Use (http://pubs.acs.org/page/policy/authorchoice_termsofuse.html)
spellingShingle Kim, Donghak
Cha, Gun-Su
Nagy, Leslie D.
Yun, Chul-Ho
Guengerich, F. Peter
Kinetic Analysis of Lauric Acid Hydroxylation by Human Cytochrome P450 4A11
title Kinetic Analysis of Lauric Acid Hydroxylation by Human Cytochrome P450 4A11
title_full Kinetic Analysis of Lauric Acid Hydroxylation by Human Cytochrome P450 4A11
title_fullStr Kinetic Analysis of Lauric Acid Hydroxylation by Human Cytochrome P450 4A11
title_full_unstemmed Kinetic Analysis of Lauric Acid Hydroxylation by Human Cytochrome P450 4A11
title_short Kinetic Analysis of Lauric Acid Hydroxylation by Human Cytochrome P450 4A11
title_sort kinetic analysis of lauric acid hydroxylation by human cytochrome p450 4a11
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4188250/
https://www.ncbi.nlm.nih.gov/pubmed/25203493
http://dx.doi.org/10.1021/bi500710e
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