Expressed CYP4A4 Metabolism of Prostaglandin E1 and Arachidonic Acid

Cytochrome P4504A4 (CYP4A4) is a hormonally induced pulmonary cytochrome P450 which metabolizes prostaglandins and arachidonic acid (AA) to their ω-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 ω-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 E1 (PGE1) and AA was examined in the presence and absence of cytochrome b5 (cyt b5) and with a heme-depleted form of cyt b5 (apo b5). The stimulatory role played by cyt b5 in this system is not dependent on electron transfer from cyt b5 to the CYP4A4 as similar stimulation was observed with apo b5. 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 b5 or apo b5. CD spectroscopy demonstrated that a conformational change occurred in CYP4A4 protein upon binding of cyt b5 or apo b5. 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 PGE1 or AA metabolism. All but 10-UDYA were effective inhibitors of CYP4A4.