Catalytic Activity and Isoform-Specific Inhibition of Rat Cytochrome P450 4F Enzymes

Fengyun Xu, J R Falck, Paul R. Ortiz De Montellano, Deanna L. Kroetz

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Abstract

Arachidonic acid is ω-hydroxylated to 20-hydroxyeicosatetraenoic acid (20-HETE), which has effects on vasoactivity and renal tubular transport and has been implicated in the regulation of blood pressure. Cytochrome P450 (P450) 4A isoforms are generally considered the major arachidonic acid ω-hydroxylases; however, little is known about the role of rat CYP4F isoforms in 20-HETE formation. The rat CYP4F isoforms, CYP4F1, CYP4F4, CYP4F5, and CYP4F6, were heterologously expressed in Escherichia coli, and their substrate specificity in fatty acid metabolism was characterized. Substrate-binding assays indicated that leukotriene B4 (LTB 4) and arachidonic acid bound CYP4F1 and CYP4F4 in a type-I manner with a Ks of 25 to 59 μM, and lauric acid bound CYP4F4 poorly. Reconstituted CYP4F1 and CYP4F4 catalyzed the ω-hydroxylation of LTB 4 with a Km of 24 and 31 μM, respectively, and CYP4F5 had minor activity in LTB4, metabolism. Importantly, CYP4F1 and CYP4F4 catalyzed the ω-hydroxylation of arachidonic acid with an apparent kcat of 9 and 11 min-1, respectively. Lauric acid was a poor substrate for all of the CYP4F isoforms, and CYP4F6 had no detectable fatty acid ω-hydroxylase activity. The P450 ω-hydroxylase inhibitors 17-octadecynoic acid, 10-undecynyl sulfate, and N-methylsulfonyl-12,12-dibromododec-11-enamide showed isoform-specific inhibition of CYP4F1- and CYP4F4-catalyzed ω-hydroxylation of arachidonic acid and potency differences between the CYP4A and CYP4F isoforms. These data support a significant role for CYP4F1 and CYP4F4 in the formation of 20-HETE and identify P450 inhibitors that can be used to understand the relative contribution of the CYP4A and CYP4F isoforms to renal 20-HETE formation.

Original languageEnglish (US)
Pages (from-to)887-895
Number of pages9
JournalJournal of Pharmacology and Experimental Therapeutics
Volume308
Issue number3
DOIs
StatePublished - Mar 2004

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Cytochrome P-450 Enzyme System
Protein Isoforms
Arachidonic Acid
lauric acid
Leukotriene B4
Hydroxylation
Cytochrome P-450 CYP4A
Mixed Function Oxygenases
Fatty Acids
Kidney
Substrate Specificity
Sulfates
Escherichia coli
Blood Pressure
20-hydroxy-5,8,11,14-eicosatetraenoic acid

ASJC Scopus subject areas

  • Pharmacology

Cite this

Catalytic Activity and Isoform-Specific Inhibition of Rat Cytochrome P450 4F Enzymes. / Xu, Fengyun; Falck, J R; Ortiz De Montellano, Paul R.; Kroetz, Deanna L.

In: Journal of Pharmacology and Experimental Therapeutics, Vol. 308, No. 3, 03.2004, p. 887-895.

Research output: Contribution to journalArticle

Xu, Fengyun ; Falck, J R ; Ortiz De Montellano, Paul R. ; Kroetz, Deanna L. / Catalytic Activity and Isoform-Specific Inhibition of Rat Cytochrome P450 4F Enzymes. In: Journal of Pharmacology and Experimental Therapeutics. 2004 ; Vol. 308, No. 3. pp. 887-895.
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abstract = "Arachidonic acid is ω-hydroxylated to 20-hydroxyeicosatetraenoic acid (20-HETE), which has effects on vasoactivity and renal tubular transport and has been implicated in the regulation of blood pressure. Cytochrome P450 (P450) 4A isoforms are generally considered the major arachidonic acid ω-hydroxylases; however, little is known about the role of rat CYP4F isoforms in 20-HETE formation. The rat CYP4F isoforms, CYP4F1, CYP4F4, CYP4F5, and CYP4F6, were heterologously expressed in Escherichia coli, and their substrate specificity in fatty acid metabolism was characterized. Substrate-binding assays indicated that leukotriene B4 (LTB 4) and arachidonic acid bound CYP4F1 and CYP4F4 in a type-I manner with a Ks of 25 to 59 μM, and lauric acid bound CYP4F4 poorly. Reconstituted CYP4F1 and CYP4F4 catalyzed the ω-hydroxylation of LTB 4 with a Km of 24 and 31 μM, respectively, and CYP4F5 had minor activity in LTB4, metabolism. Importantly, CYP4F1 and CYP4F4 catalyzed the ω-hydroxylation of arachidonic acid with an apparent kcat of 9 and 11 min-1, respectively. Lauric acid was a poor substrate for all of the CYP4F isoforms, and CYP4F6 had no detectable fatty acid ω-hydroxylase activity. The P450 ω-hydroxylase inhibitors 17-octadecynoic acid, 10-undecynyl sulfate, and N-methylsulfonyl-12,12-dibromododec-11-enamide showed isoform-specific inhibition of CYP4F1- and CYP4F4-catalyzed ω-hydroxylation of arachidonic acid and potency differences between the CYP4A and CYP4F isoforms. These data support a significant role for CYP4F1 and CYP4F4 in the formation of 20-HETE and identify P450 inhibitors that can be used to understand the relative contribution of the CYP4A and CYP4F isoforms to renal 20-HETE formation.",
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T1 - Catalytic Activity and Isoform-Specific Inhibition of Rat Cytochrome P450 4F Enzymes

AU - Xu, Fengyun

AU - Falck, J R

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AU - Kroetz, Deanna L.

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N2 - Arachidonic acid is ω-hydroxylated to 20-hydroxyeicosatetraenoic acid (20-HETE), which has effects on vasoactivity and renal tubular transport and has been implicated in the regulation of blood pressure. Cytochrome P450 (P450) 4A isoforms are generally considered the major arachidonic acid ω-hydroxylases; however, little is known about the role of rat CYP4F isoforms in 20-HETE formation. The rat CYP4F isoforms, CYP4F1, CYP4F4, CYP4F5, and CYP4F6, were heterologously expressed in Escherichia coli, and their substrate specificity in fatty acid metabolism was characterized. Substrate-binding assays indicated that leukotriene B4 (LTB 4) and arachidonic acid bound CYP4F1 and CYP4F4 in a type-I manner with a Ks of 25 to 59 μM, and lauric acid bound CYP4F4 poorly. Reconstituted CYP4F1 and CYP4F4 catalyzed the ω-hydroxylation of LTB 4 with a Km of 24 and 31 μM, respectively, and CYP4F5 had minor activity in LTB4, metabolism. Importantly, CYP4F1 and CYP4F4 catalyzed the ω-hydroxylation of arachidonic acid with an apparent kcat of 9 and 11 min-1, respectively. Lauric acid was a poor substrate for all of the CYP4F isoforms, and CYP4F6 had no detectable fatty acid ω-hydroxylase activity. The P450 ω-hydroxylase inhibitors 17-octadecynoic acid, 10-undecynyl sulfate, and N-methylsulfonyl-12,12-dibromododec-11-enamide showed isoform-specific inhibition of CYP4F1- and CYP4F4-catalyzed ω-hydroxylation of arachidonic acid and potency differences between the CYP4A and CYP4F isoforms. These data support a significant role for CYP4F1 and CYP4F4 in the formation of 20-HETE and identify P450 inhibitors that can be used to understand the relative contribution of the CYP4A and CYP4F isoforms to renal 20-HETE formation.

AB - Arachidonic acid is ω-hydroxylated to 20-hydroxyeicosatetraenoic acid (20-HETE), which has effects on vasoactivity and renal tubular transport and has been implicated in the regulation of blood pressure. Cytochrome P450 (P450) 4A isoforms are generally considered the major arachidonic acid ω-hydroxylases; however, little is known about the role of rat CYP4F isoforms in 20-HETE formation. The rat CYP4F isoforms, CYP4F1, CYP4F4, CYP4F5, and CYP4F6, were heterologously expressed in Escherichia coli, and their substrate specificity in fatty acid metabolism was characterized. Substrate-binding assays indicated that leukotriene B4 (LTB 4) and arachidonic acid bound CYP4F1 and CYP4F4 in a type-I manner with a Ks of 25 to 59 μM, and lauric acid bound CYP4F4 poorly. Reconstituted CYP4F1 and CYP4F4 catalyzed the ω-hydroxylation of LTB 4 with a Km of 24 and 31 μM, respectively, and CYP4F5 had minor activity in LTB4, metabolism. Importantly, CYP4F1 and CYP4F4 catalyzed the ω-hydroxylation of arachidonic acid with an apparent kcat of 9 and 11 min-1, respectively. Lauric acid was a poor substrate for all of the CYP4F isoforms, and CYP4F6 had no detectable fatty acid ω-hydroxylase activity. The P450 ω-hydroxylase inhibitors 17-octadecynoic acid, 10-undecynyl sulfate, and N-methylsulfonyl-12,12-dibromododec-11-enamide showed isoform-specific inhibition of CYP4F1- and CYP4F4-catalyzed ω-hydroxylation of arachidonic acid and potency differences between the CYP4A and CYP4F isoforms. These data support a significant role for CYP4F1 and CYP4F4 in the formation of 20-HETE and identify P450 inhibitors that can be used to understand the relative contribution of the CYP4A and CYP4F isoforms to renal 20-HETE formation.

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