RT Journal Article
SR Electronic
T1 Catalytic Activity and Isoform-Specific Inhibition of Rat Cytochrome P450 4F Enzymes
JF Journal of Pharmacology and Experimental Therapeutics
JO J Pharmacol Exp Ther
FD American Society for Pharmacology and Experimental Therapeutics
SP 887
OP 895
DO 10.1124/jpet.103.059626
VO 308
IS 3
A1 Fengyun Xu
A1 John R. Falck
A1 Paul R. Ortiz de Montellano
A1 Deanna L. Kroetz
YR 2004
UL http://jpet.aspetjournals.org/content/308/3/887.abstract
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 (LTB4) 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 LTB4 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. The American Society for Pharmacology and Experimental Therapeutics