P450-catalyzed in-chain desaturation of valproic acid: isoform selectivity and mechanism of formation of Delta 3-valproic acid generated by baculovirus-expressed CYP3A1.
The mechanism of formation of the in-chain, unsaturated fatty acid metabolite, Delta3-valproic acid (Delta3-VPA) by rat liver microsomes was examined. Microsomal rates of formation of Delta3-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-16alpha-carbonitrile. Microsomal incubations conducted with 3-hydroxy-VPA or [2-2H1]VPA demonstrated that Delta3-VPA did not arise by dehydration of preformed alcohol nor was it reversibly isomerized to Delta2-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-, Delta3-, and Delta4-VPA in ratios of 160:35:6:3:1. Intramolecular deuterium isotope effects on metabolite formation, determined with cDNA-expressed CYP3A1 and either [3,3-2H2]VPA or [4,4-2H2]VPA, yielded kH/kD values for Delta3-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-D2-VPA (kH/kD = 6.04 +/- 0.08), or for 4-OH- and Delta4-VPA formation from 4, 4-D2-VPA (kH/kD > 5). Collectively, these data demonstrate the existence of a microsomal P450-dependent in-chain fatty acid desaturase system distinct from the well-documented cytochrome b5-linked CoA desaturases and suggest further that CYP3A1-dependent formation of Delta3-VPA arises via nonselective, initial hydrogen atom abstraction from either the C-3 or the C-4 position.
Department of Medicinal Chemistry, University of Washington, Seattle, Washington, 98195, USA.
M B
MB
Fisher
S J
SJ
Thompson
V
V
Ribeiro
M C
MC
Lechner
A E
AE
Rettie
D000818:Animals; D016367:Baculoviridae / Q000201:enzymology* / Q000235:genetics*; D002384:Catalysis; D002460:Cell Line; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism; D018076:DNA, Complementary / Q000096:biosynthesis; D004789:Enzyme Activation / Q000235:genetics; D005260:Female; D015870:Gene Expression; D005822:Genetic Vectors / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000235:genetics* / Q000378:metabolism; D008862:Microsomes, Liver / Q000201:enzymology; D006899:Mixed Function Oxygenases / Q000235:genetics* / Q000378:metabolism; D009036:Moths / Q000201:enzymology / Q000235:genetics; D010421:Pentanoic Acids / Q000378:metabolism*; D011817:Rabbits; D051381:Rats; D017207:Rats, Sprague-Dawley; D017208:Rats, Wistar; D013230:Stearoyl-CoA Desaturase / Q000378:metabolism; D013237:Stereoisomerism; D013379:Substrate Specificity; D014635:Valproic Acid / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism
D018076:DNA, Complementary; D007527:Isoenzymes; D010421:Pentanoic Acids; C113682:delta(3) valproic acid; D014635:Valproic Acid; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C104464:CYP3A protein, human; D051544:Cytochrome P-450 CYP3A; D013230:Stearoyl-CoA Desaturase