Mechanism of androstenedione formation from testosterone and epitestosterone catalyzed by purified cytochrome P-450b

  • Wood A
  • Swinney D
  • Thomas P
 et al. 
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Abstract

A purified rat hepatic monooxygenase system containing cytochrome P-450b oxidizes testosterone to androstenedione and 16 alpha- and 16 beta-hydroxytestosterone at approximately equal rates. The metabolism of epitestosterone by the same system is characterized by a marked stereoselectivity in favor of 16 beta-hydroxylation (4- to 5-fold relative to 16 alpha-hydroxylation), formation of 15 alpha-hydroxyepitestosterone, and a rate of androstenedione formation which is three to five times higher than that observed with testosterone. Apparent Km values for 16 alpha- and 16 beta-hydroxylation and androstenedione formation are 20-30 microM with either substrate. Mass spectral analysis of the androstenedione formed from [16,16-2H2]testosterone and [16,16-2H2] epitestosterone indicates essentially complete retention of deuterium, thereby ruling out a mechanism of androstenedione formation via C-16 hydroxylation followed by loss of water and rearrangement. Mass spectral analysis of the C-16 hydroxylation products from incubations of testosterone or epitestosterone in 18O2 shows essentially complete incorporation of 18O (greater than 95%). Androstenedione formed from testosterone is enriched in 18O only 2-fold (5-8%) over background, while the androstenedione formed from epitestosterone shows 84% enrichment. Kinetic experiments utilizing [17-2H]testosterone and [17-2H]epitestosterone as substrates indicate that cleavage of the C-17 carbon-hydrogen bond is involved in a rate-limiting step in the formation of androstenedione from both substrates. Taken together, our results indicate that androstenedione formation from epitestosterone proceeds exclusively through the gem-diol pathway, while androstenedione formation from testosterone may proceed through a combination of gem-diol and dual hydrogen abstraction pathways.

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  • SGR: 0024232973
  • PMID: 3053709
  • ISBN: 0021-9258
  • PUI: 19029322
  • SCOPUS: 2-s2.0-0024232973
  • ISSN: 00219258

Authors

  • A. W. Wood

  • D. C. Swinney

  • P. E. Thomas

  • D. E. Ryan

  • P. F. Hall

  • W. Levin

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