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.

Identification of unique amino acids that modulate CYP4A7 activity.

A multifamily sequence alignment of the rabbit CYP4A members with the known structure of CYP102 indicates amino acid differences falling within the so-called substrate recognition site(s) (SRS). Chimeric proteins constructed between CYP4A4 and CYP4A7 indicate that laurate activity is affected by the residues within SRS1 and prostaglandin activity is influenced by SRS2-3. Site-directed mutant proteins of CYP4A7 found laurate and arachidonate activity markedly diminished in the R90W mutant (SRS1) and somewhat decreased in W93S. While PGE(1) activity was only slightly increased, the mutant proteins H206Y and S255F (SRS2-3), on the other hand, exhibited remarkable increases in laurate and arachidonate metabolism (3-fold) above wild-type substrate metabolism. Mutant proteins H206Y, S255F, and H206Y/S255F but not R90W/W93S, wild-type CYP4A4, or CYP4A7 metabolized arachidonic acid in the absence of cytochrome b(5). Stopped-flow kinetic experiments were performed in a CO-saturated environment performed to estimate interaction rates of the monooxygenase reaction components. The mutant protein H206Y, which exhibits 3-fold higher than wild-type substrate activity, interacts with CPR at a rate at least 10 times faster than that of wild-type CYP4A7. These experimental results provide insight regarding the residues responsible for modulation of substrate specificity, affinity, and kinetics, as well as possible localization within the enzyme structure based on comparisons with homologous, known cytochrome P450 structures.