Mechanistic kinship between hydroxylation and desaturation reactions: acyl-carbon bond cleavage promoted by pig and human CYP17 (P-45017.alpha.; 17.alpha.-hydroxylase-17,20-lyase)

Abstract

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,17 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 b5 (cyt b5). 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 b5, by the pig and human CYP17 isoforms, respectively. The stereochemistry of the reaction was studied using the aldehyde labeled with 2H at three strategic positions, 16 alpha, 16 beta, and 17 alpha, with incubations performed under both 16O2 and 18O2. The results showed that the formation of the 5,16-diene is attended by the removal of the 16 alpha-hydrogen atom; all three 2H atoms are retained in the formation of 17 alpha-hydroxyandrogen and its 17 alpha-hydroxyl oxygen originates from O2. 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 2H at 16 alpha as well as 16 beta, led to the enrichment of 2H in 17 alpha-hydroxyandrogen. It is suggested that the mechanistic kinship between hydroxylation and olefin formation, revealed by the present study, also applies to conventional hydroxylation and desaturation reactions.

Mechanism of 17α,20-Lyase and New Hydroxylation Reactions of Human Cytochrome P450 17A1

“…These results are consonant with the original analysis of Akhtar and co-workers on P450 17A1 (27,28) and, on their own, are consistent with the FeO 2 Ϫ mechanism presented in Fig. 3A (40, 46).…”

“…Iodosylbenzene also did not support the 17␣,20-lyase reaction in a P450 17A1 yeast microsomal system (40). (27)(28)(29)(30)(31); B, compound I mechanism with hydrogen atom abstraction from the 17␣ alcohol followed by C17-C20 bond scission to yield an acetyl radical; C, compound I mechanism with hydrogen atom abstraction from the C16 carbon; D, compound I mechanism with hydrogen atom abstraction from the 17␣ alcohol followed by C17-C20 bond scission to yield a hydrated acetyl radical (gem-diol); E, compound I mechanism with hydrogen atom abstraction from the C21 methyl group followed by C17-C20 bond scission to yield a C17 radical; F, addition of the 17␣-hydroxyl group to compound I to yield an iron peroxide-C17 complex, which can decompose via either (a) a C20 gem-diol or (b) a C17-C20 dioxetane. See text for discussion and also Fig.…”

“…The results for P450 19A1 unambiguously ruled out 18 O incorporation of 18 O label from O 2 into formic acid by distinguishing 2 H from 13 C isotope composition and are only consistent with an FeO 3ϩ mechanism for P450 19A1 (33). Because of the impact of the new studies (33), we re-examined the 18 O experiments with P450 17A1 (27)(28)(29)(30)(31) with the newer methodologies. Although our 18 O labeling results could be interpreted as support of an FeO 2 Ϫ mechanism for human P450 17A1, at least three possible FeO 3ϩ mechanisms are still consistent with the data (Fig.…”

“…One powerful approach originally used by Akhtar and co-workers (27)(28)(29)(30)(31) analyzes the actual reaction and can provide discrimination between the nucleophilic FeO 2 Ϫ and electrophilic FeO 3ϩ reactions ( Fig. 2), based on the incorporation of 18 O label from O 2 into the carboxylic acid products (Fig.…”

Cytochrome b5 modulation of 17α hydroxylase and 17–20 lyase (CYP17) activities in steroidogenesis

“…The reaction mechanism for each activity is thought to involve formation of distinct iron-oxygen complexes. For the hydroxylation mechanism, the oxointermediate, Fe v =O, is considered to be the active catalytic oxygen-bound CYP complex (Atkinson & Ingold 1993), while for the acyl-carbon bond cleavage, participation of the iron-peroxo, Fe Fe v =O, species have both been suggested as possible candidates (Akhtar et al 1994, Lee-Robichaud et al 1995. Kinetic studies of the aromatase and the 14 -demethylase reactions, which also involve similar cleavage of the acyl-carbon bonds, strongly favour the involvement of the latter complex (Akhtar et al 1993, Shyadehi et al 1996.…”