8-11-14-eicosatrienoic-acid and lauric-acid

The determinants of the regio- and stereoselective oxidation of fatty acids by cytochrome P450 BM-3 were examined by mutagenesis of residues postulated to anchor the fatty acid or to determine its active site substrate-accessible volume. R47, Y51, and F87 were targeted separately and in combination in order to assess their contributions to arachidonic, palmitoleic, and lauric acid binding affinities, catalytic rates, and regio- and stereoselective oxidation. For all three fatty acids, mutation of the anchoring residues decreased substrate binding affinity and catalytic rates and, for lauric acid, caused a significant increase in the enzyme's NADPH oxidase activity. These changes in catalytic efficiency were accompanied by decreases in the regioselectivity of oxygen insertion, suggesting an increased freedom of substrate movement within the active site of the mutant proteins. The formation of significant amounts of 19-hydroxy AA by the Y51A mutant and of 11,12-EET by the R47A/Y51A/F87V triple mutant, suggest that wild-type BM-3 shields these carbon atoms from the heme bound reactive oxygen by restricting the freedom of AA displacement along the substrate channel, and active site accessibility. These results indicate that binding affinity and catalytic turnover are fatty acid carbon-chain length dependent, and that the catalytic efficiency and the regioselectivity of fatty acid metabolism by BM-3 are determined by active site binding coordinates that control acceptor carbon orientation and proximity to the heme iron.

Topics: 8,11,14-Eicosatrienoic Acid; Arachidonic Acid; Bacterial Proteins; Catalytic Domain; Cytochrome P-450 Enzyme System; Fatty Acids; Fatty Acids, Monounsaturated; Isoenzymes; Lauric Acids; Mixed Function Oxygenases; Mutagenesis, Site-Directed; NADPH Oxidases; NADPH-Ferrihemoprotein Reductase; Oxidation-Reduction; Stereoisomerism; Substrate Specificity

20-Hydroxy-5,8,11,14-eicosatetraenoic acid (20-HETE), the omega-hydroxylation product of arachidonic acid, is the major metabolite produced in the kidney. It has potent biological effects on renal tubular and vascular functions and on the long-term control of arterial pressure. The synthesis of 20-HETE is catalyzed by enzymes of the CYP4A family, among which CYP4A2 is the most abundant isozyme expressed in the kidneys of rats. We have cloned and sequenced the CYP4A2 cDNA from the kidney of Lewis-Wistar rats and directed its expression using baculovirus and Sf9 insect cells. A high level of expression of CYP4A2 was evident by Northern, Western, and spectral analyses revealing a P450 content of 0.3 nmol/mg microsomal protein. To study CYP4A2-catalyzed arachidonic acid omega-hydroxylation, Sf9 cells were coinfected with CYP4A2 and NADPH cytochrome P450 oxidoreductase (OR) recombinant viruses. CYP4A2/OR membranes metabolized lauric acid at a high rate (7 and 5.5 nmol/min/nmol P450 in the presence and absence of b5, respectively). However, arachidonic acid omega-hydroxylase activity was barely detectable. When purified OR was added to the membranes expressing CYP4A2 protein, a concentration-dependent production of 20-HETE was observed. Maximal synthesis of 20-HETE of 0.89 nmol/min/nmol P450 was achieved at OR:CYP4A2 ratio of 14:1. The omega-hydroxylation of arachidonic acid was dependent on the presence of b5. Furthermore, increasing OR concentrations yielded additional arachidonic acid metabolite identified by GC/MS as 11,12-EET. Microsomes prepared from isolated renal microvessels selectively expressed CYP4A2 protein and readily metabolized arachidonic acid to two major metabolites, 20-HETE and 11,12-DHET, the hydrolytic metabolite of 11, 12-EET. It is suggested that CYP4A2 functions as the renal microvessel arachidonate omega-hydroxylase and that it can also catalyze the 11,12-epoxidation of arachidonic acid.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Arachidonic Acid; Baculoviridae; Capillaries; Catalysis; Cell Line; Cloning, Molecular; Cytochrome P-450 CYP4A; Cytochrome P-450 Enzyme System; DNA, Complementary; Gas Chromatography-Mass Spectrometry; Hydroxyeicosatetraenoic Acids; Hydroxylation; Kidney; Lauric Acids; Microsomes; Mixed Function Oxygenases; NADH, NADPH Oxidoreductases; NADPH-Ferrihemoprotein Reductase; Rats; Rats, Inbred Lew; Rats, Sprague-Dawley; Spodoptera

The regiospecificity of arachidonic acid oxygenation, catalyzed by rat liver microsomal fractions in the presence of NADPH, can be altered by animal pretreatment with a fibric acid type of hypolipidemic drug, ciprofibrate. While microsomal fractions isolated from either control or phenobarbital-treated animals oxygenate arachidonic acid to mainly epoxyeicosatrienoic acids (EETs), animal pretreatment with ciprofibrate results in an eightfold stimulation of omega and omega-1 oxidation, concomitant with a net decrease in the formation of both HETEs and EETs. The isomeric composition of the EETs and of the omega and omega-1 oxidation products formed is also dependent on the type of animal pretreatment. Associated decreases in the amounts of HETEs and the rate of hydrogen peroxide formation suggests a modification of the "uncoupler action" of arachidonic acid during the function of different cytochromes P-450.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Arachidonic Acid; Arachidonic Acids; Benzphetamine; Chromatography, High Pressure Liquid; Clofibrate; Clofibric Acid; Cytochrome P-450 Enzyme System; Fatty Acids, Unsaturated; Fibric Acids; Gas Chromatography-Mass Spectrometry; Hydroxyeicosatetraenoic Acids; Hypolipidemic Agents; Lauric Acids; Male; Microsomes, Liver; NADP; Oxidation-Reduction; Rats; Rats, Inbred Strains