Catalytic activity and isoform-specific inhibition of rat cytochrome p450 4F enzymes.
Arachidonic acid is omega-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 omega-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 B(4) (LTB(4)) and arachidonic acid bound CYP4F1 and CYP4F4 in a type-I manner with a K(s) of 25 to 59 microM, and lauric acid bound CYP4F4 poorly. Reconstituted CYP4F1 and CYP4F4 catalyzed the omega-hydroxylation of LTB(4) with a K(m) of 24 and 31 microM, respectively, and CYP4F5 had minor activity in LTB(4) metabolism. Importantly, CYP4F1 and CYP4F4 catalyzed the omega-hydroxylation of arachidonic acid with an apparent k(cat) 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 omega-hydroxylase activity. The p450 omega-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 omega-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.
Xu
Fengyun
F
Department of Biopharmaceutical Sciences, University of California, San Francisco, CA 94143-0446, USA.
Falck
John R
JR
Ortiz de Montellano
Paul R
PR
Kroetz
Deanna L
DL
D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism; D002384:Catalysis; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D000072470:Cytochrome P450 Family 4; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005227:Fatty Acids / Q000378:metabolism; D006893:Hydroxyeicosatetraenoic Acids / Q000494:pharmacology*; D006900:Hydroxylation / Q000187:drug effects; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism
D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors; D005227:Fatty Acids; D006893:Hydroxyeicosatetraenoic Acids; D007527:Isoenzymes; D011994:Recombinant Proteins; D016718:Arachidonic Acid; C055987:20-hydroxy-5,8,11,14-eicosatetraenoic acid; D003577:Cytochrome P-450 Enzyme System; C465348:Cyp4f1 protein, rat; C465481:Cyp4f4 protein, rat; C465482:Cyp4f5 protein, rat; D000072470:Cytochrome P450 Family 4