title: Metabolism of all-trans, 9-cis, and 13-cis isomers of retinal by purified isozymes of microsomal cytochrome P450 and mechanism-based inhibition of retinoid oxidation by citral.
abstract: The involvement of a series of microsomal cytochrome P450 (P450) isozymes in all-trans-retinoid metabolism, including the conversion of all-trans-retinal to all-trans-retinoic acid, was previously described. In the current study, we examined the role of seven liver microsomal P450 isozymes in the oxidation of three isomers of retinal. P450 1A1, which was not tested previously, is by far the most active in the conversion of all-trans-, 9-cis-, and 13-cis-retinal to the corresponding acids, as well as in the 4-hydroxylation of all-trans- and 13-cis retinal. In contrast, P450s 2B4 and 2C3 are the most active in the 4-hydroxylation of 9-cis-retinal, with turnover numbers approximately 7 times as great as that of P450 1A1. The inclusion of cytochrome b5 in the reconstituted enzyme system is without effect or inhibitory in most cases but stimulates the 4-hydroxylation of 9-cis-retinal by P450 2B4, giving a turnover of 3.7 nmol of product/min/nmol of this isozyme, the highest for any of the retinoid conversions we have studied. Evidence was obtained for two additional catalytic reactions not previously attributed to P450 oxygenases: the oxidation of all-trans- and 9-cis-retinal to the corresponding 4-oxo derivatives by isoform 1A2, and the oxidative cleavage of the acetyl ester of vitamin A (retinyl acetate) to all-trans-retinal, also by isoform 1A2. The physiological significance of the latter reaction, with a Km for the ester of 32 microM and a Vmax of 18 pmol/min/nmol of P450, remains to be established. We also examined the effect on P450 of citral, a terpenoid alpha, beta-unsaturated aldehyde and a known inhibitor of cytosolic retinoid dehydrogenases. Evidence was obtained that citral is an effective mechanism-based inactivator of isozyme 2B4, with a KI of 44 microM as determined by the oxidation of 1-phenylethanol to acetophenone, and by isozyme 1A2 in the oxidation of all-trans-retinal to the corresponding acid and by isozyme 2B4 in the 4-hydroxylation of all-trans-retinol and retinoic acid. Thus, citral is not suitable for use in attempts to distinguish between retinoid conversions catalyzed by dehydrogenases in the cytoplasm and by P450 cytochromes in the endoplasmic reticulum.
authors.affiliation: Department of Biological Chemistry, Medical School, The University of Michigan, Ann Arbor, 48109-0606, USA.
authors.forename: G M
authors.initials: GM
authors.lastname: Raner
authors.forename: A D
authors.initials: AD
authors.lastname: Vaz
authors.forename: M J
authors.initials: MJ
authors.lastname: Coon
mesh_terms: D000080462:Acyclic Monoterpenes; D000818:Animals; D001592:Benzyl Alcohols / Q000378:metabolism; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006900:Hydroxylation; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism*; D007536:Isomerism; D007700:Kinetics; D008099:Liver; D008861:Microsomes / Q000201:enzymology; D039821:Monoterpenes; D010084:Oxidation-Reduction; D011817:Rabbits; D012172:Retinaldehyde / Q000378:metabolism*; D012176:Retinoids / Q000378:metabolism*; D013729:Terpenes / Q000494:pharmacology*
chemical_list: D000080462:Acyclic Monoterpenes; D001592:Benzyl Alcohols; D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D039821:Monoterpenes; D012176:Retinoids; D013729:Terpenes; D003577:Cytochrome P-450 Enzyme System; C002017:methylphenyl carbinol; D012172:Retinaldehyde; C007076:citral