Expressed CYP4A4 metabolism of prostaglandin E(1) and arachidonic acid.
Cytochrome P4504A4 (CYP4A4) is a hormonally induced pulmonary cytochrome P450 which metabolizes prostaglandins and arachidonic acid (AA) to their omega-hydroxylated products. Although the physiological function of this enzyme is unknown, prostaglandins play an important role in the regulation of reproductive, vascular, intestinal, and inflammatory systems and 20-hydroxyeicosatetraenoic acid, the omega-hydroxylated product of arachidonate, is a potent vasoconstrictor. Therefore, it is important to obtain sufficient quantities of the protein for kinetic and biophysical characterization. A CYP4A4 construct was prepared and expressed in Escherichia coli. The enzyme was purified, and its activity with substrates prostaglandin E(1) (PGE(1)) and AA was examined in the presence and absence of cytochrome b(5) (cyt b(5)) and with a heme-depleted form of cyt b(5) (apo b(5)). The stimulatory role played by cyt b(5) in this system is not dependent on electron transfer from cyt b(5) to the CYP4A4 as similar stimulation was observed with apo b(5). Rapid kinetic measurement of CYP4A4 electron transfer rates confirmed this result. Both flavin and heme reduction rates were constant in the absence and presence of cyt b(5) or apo b(5). CD spectroscopy demonstrated that a conformational change occurred in CYP4A4 protein upon binding of cyt b(5) or apo b(5). Finally, acetylenic fatty acid inhibitors 17-octadecynoic acid, 12-hydroxy-16-heptadecynoic acid, 15-hexadecynoic acid, and 10-undecynoic acid (10-UDYA) were used to probe the substrate-binding pocket of CYP4A4. The short-chain fatty acid inhibitor 10-UDYA was unable to inhibit either PGE(1) or AA metabolism. All but 10-UDYA were effective inhibitors of CYP4A4.
Aitken
A E
AE
Department of Biochemistry, The University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, Texas 78284-7760, USA.
Roman
L J
LJ
Loughran
P A
PA
de la Garza
M
M
Masters
B S
BS
D000527:Alprostadil / Q000378:metabolism*; D016718:Arachidonic Acid / Q000378:metabolism*; D001189:Aryl Hydrocarbon Hydroxylases; D001665:Binding Sites; D002942:Circular Dichroism; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000378:metabolism*; D000072470:Cytochrome P450 Family 4; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli; D005229:Fatty Acids, Monounsaturated / Q000494:pharmacology; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism
D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors; D005229:Fatty Acids, Monounsaturated; D011994:Recombinant Proteins; C068165:12-hydroxy-16-heptadecynoic acid; D016718:Arachidonic Acid; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D001189:Aryl Hydrocarbon Hydroxylases; D000072470:Cytochrome P450 Family 4; C436964:cytochrome P-450 CYP4A4 (rabbit); D000527:Alprostadil