MECHANISTIC KINSHIP BETWEEN HYDROXYLATION AND DESATURATION REACTIONS - ACYL-CARBON BOND-CLEAVAGE PROMOTED BY PIG AND HUMAN CYP17 (P-450(17-ALPHA)17-ALPHA-HYDROXYLASE-17,20-LYASE)

Using homogeneous pig and recombinant human CYP17, the mechanism of the acyl-carbon bond fission involved in the direct cleavage of pregnenolone was studied. It was found that the formation of androsta-5, 16-dien-3 beta-ol (5,16-diene) and androst-5-ene-3 beta, l7 alpha-diol (17 alpha-hydroxyandrogen) from pregnenolone was catalyzed by both the isoforms and that the two conversions were dependent on the presence of cytochrome b(5) (cyt b(5)). 3 beta-Hydroxyandrost-5-ene-17 beta-carbaldehyde (aldehyde), an analogue of the physiological substrate pregnenolone, was handled as a substrate by both isoforms of CYP17. The aldehyde underwent cleavage to produce the 5,16-diene plus the 17 alpha-hydroxyandrogen, at rates approximately 8- and 3-fold higher than any physiological reaction catalyzed, in the absence of cytochrome b(5), by the pig and human CYP17 isoforms, respectively. The stereochemistry of the reaction was studied using the aldehyde labeled with H-2 at three strategic positions, 16 alpha, 16 beta, and 17 alpha, with incubations performed under both O-16(2) and O-18(2) The results showed that the formation of the 5,16-diene is attended by the removal of the (16)alpha-hydrogen atom; all three H-2 atoms are retained in the formation of 17 alpha-hydroxyandrogen and its 17 alpha-hydroxyl oxygen originates- from O-2. Irrespective of the nature of the substrate, or the enzymic conditions used, the 5,16-diene and 17 alpha-hydroxyandrogen were produced in similar ratios, suggesting that their genesis is closely linked. Both the compounds may be envisaged to arise from a peroxy adduct that fragments to give a carbon radical that then undergoes either a disproportionation or an oxygen-rebound reaction. The conclusion was supported by isotope-partitioning experiments when the conversion of a mixture of the unlabeled aldehyde and its isotopomer, containing H-2 at 16 alpha as well as 16 beta, led to the enrichment of H-2 in 17 alpha-hydroxyandrogen. It is suggested that the mechanistic kinship between hydroxylation and olefin formation, revealed by the present study, also apples to conventional hydroxylation and desaturation reactions,