P450-catalyzed in-chain desaturation of valproic acid:: Isoform selectivity and mechanism of formation of Δ3-valproic acid generated by baculovirus-expressed CYP3A1

The mechanism of formation of the in-chain, unsaturated fatty acid metabolite, Delta(3)-valproic acid (Delta(3)-VPA) by rat liver microsomes was examined. Microsomal rates of formation of Delta(3)-VPA were below quantifiable limits in reactions catalyzed by control female rat liver microsomes, but were induced more than 20-fold following pretreatment with triacetyloleandomycin and pregnenolone-16 alpha-carbonitrile. Microsomal incubations conducted with 3-hydroxy-VPA or [2-H-2,]VPA demonstrated that Delta(3)-VPA did not arise by dehydration of preformed alcohol nor was it reversibly isomerized to Delta(2)-VPA. CYP3A1 expression was optimized in the baculovirus expression vector system, and infected insect cell membranes which were supplemented with P450 reductase catalyzed formation of 3-OH-, 4-OH-, 5-OH-, Delta(3)-, and Delta(4)-VPA in ratios of 160:35: 6:3:1. Intramolecular deuterium isotope effects on metabolite formation, determined with cDNA-expressed CYP3A1 and either [3,3-H-2(2)]VPA or [4,4-H-2(2)]VPA, yielded k(H)/k(D) values for Delta(3)-VPA of 2.00 +/- 0.06 and 2.36 +/- 0.08, respectively. These values were significantly lower than the isotope effects observed in the same incubations for 3-OH-VPA formation from 3,3-D-2-VPA (k(H)/k(D) = 6.04 +/- 0,08), or for 4-OH- and Delta(4)-VPA formation from 4,4-D-2-VPA (k(H)/k(D) > 5). Collectively, these data demonstrate the existence of a microsomal P450-dependent in-chain fatty acid desaturase system distinct from the well-documented cytochrome b(5)-linked CoA desaturases and suggest further that CYP3A1-dependent formation of Delta(3)-VPA arises via nonselective, initial hydrogen atom abstraction from either the C-3 or the C-4 position. (C) 1998 Academic Press.