Expressed CYP4A4 metabolism of prostaglandin E-1 and arachidonic acid

Cytochrome P4504A4 (CYP4A4) is a hormonally induced pulmonary cytochrome P4 50 which metabolizes prostaglandins and arachidonic acid (AA) to their omeg a -hydroxylated products. Although the physiological function of this enzym e is unknown, prostaglandins play an important role in the regulation of re productive, vascular, intestinal, and inflammatory systems and 20-hydroxyei cosatetraenoic acid, the to-hydroxylated product of arachidonate, is a pote nt vasoconstrictor. Therefore, it is important to obtain sufficient quantit ies of the protein for kinetic and biophysical characterization. A CYP4A4 c onstruct was prepared and expressed in Escherichia coli. The enzyme was pur ified, and its activity with substrates prostaglandin E, (PGE,) 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 b y 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 k inetic measurement of CYP4A4 electron transfer rates confirmed this result. Both flavin and heme reduction rates were constant in the absence and pres ence of ct b(5) or apo b(5). CD spectroscopy demonstrated that a conformati onal change occurred in CYP4A4 protein upon binding of cyt b(5) or apo b(5) . Finally, acetylenic fatty acid inhibitors 17-octadecynoic acid, 12-hydrox y-16-heptadecynoic acid, 15-hexadecynoic acid, and 10-undecynoic acid (10-U DYA) were used to probe the substrate-binding pocket of CYP4A4. The short-c hain fatty acid inhibitor 10-UDYA was unable to inhibit either PGE, or AA m etabolism. All but 10-UDYA were effective inhibitors of CYP4A4. (C) 2001 Ac ademic Press.