CYP4 ISOZYME SPECIFICITY AND THE RELATIONSHIP BETWEEN OMEGA-HYDROXYLATION AND TERMINAL DESATURATION OF VALPROIC ACID

The cytochrome P450-dependent terminal desaturation of valproic acid (VPA) is of both toxicological and mechanistic interest because the product, 4-ene-VPA, is a more potent hepatotoxin than the parent compound and its generation represents a rather novel metabolic reaction for the cytochrome P450 system. In the present study, lung microsomes from rabbits were identified as a rich source of VPA desaturase activity. Monospecific polyclonal antibodies directed against CYP4B1 (anti-4B) inhibited 82% of 4-ene-VPA formation, whereas monospecific polyclonal antibodies directed against CYP2B4 (anti-2B) inhibited only 15% of 4-ene-VPA formation. Anti-4B also inhibited 95% of the 5-hydroxy-VPA formation, but only 42% of LF-hydroxy-VPA formation. These data suggest that CYP4B1 accounts for more than 80% of the 4-ene- and 5-hydroxy-VPA metabolites generated by rabbit lung microsomes. CYP4B1 expressed in HepG2 cells metabolized VPA with a turnover number of 35 min(-1) and formed the 5-hydroxy-, 4-hydroxy-, and 4-ene-VPA metabolites in a ratio of 110:2:1, respectively. In contrast, the lauric acid omega-hydroxylases, CYP4A1 and CYP4A3, did not give rise to detectable levels of any of these VPA metabolites. Therefore, these studies demonstrate a new functional role for CYP4B1 in the terminal desaturation and omega-hydroxylation of this short, branched-chain fatty acid. Intramolecular deuterium isotope effects on the formation of 4-ene-VPA, 4-hydroxy-VPA, and 5-hydroxy-VPA by cDNA-expressed CYP4B1 and by purified, reconstituted CYP2B1 indicate that a carbon-centered free radical at C-4 serves as a common intermediate for P450-dependent formation of both 4-ene-VPA and 4-hydroxy-VPA, regardless of whether the enzyme source functions preferentially as an omega-hydroxylase or an omega-1 hydroxylase of VPA. The partition between oxygen rebound to, and desaturation proceeding from, the C-4 VPA radical was 2:1 for CYP4B1 and 37:1 for the VPA 4-hydroxylase CYP2B1. Therefore, active site constraints which promote facile omega-hydroxylation bias the partition ratio toward desaturation and significantly enhance the rate of P450-dependent 4-ene-VPA formation.