Kinetic solvent isotope effect in human P450 CYP17A1-mediated androgen formation: evidence for a reactive peroxoanion intermediate
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Human steroid hormone biosynthesis is the result of a complex series of chemical transformations operating on cholesterol, with key steps mediated by members of the cytochrome P450 superfamily. In the formation of the male hormone dehydroepiandrosterone, pregnenolone is first hydroxylated by P450 CYP17A1 at the 17-carbon, followed a second round of catalysis by the same enzyme that cleaves the C17-C20 bond, releasing acetic acid and the 17-keto product. In order to explore the mechanism of this C-C "lyase" activity, we investigated the kinetic isotope effect on the steady-state turnover of Nanodisc-incorporated CYP17A1. Our experiments revealed the expected small positive (~1.3) isotope effect for the hydroxylase chemistry. However, a surprising result was the large inverse isotope effect (~0.39) observed for the C-C bond cleavage activity. These results strongly suggest that the P450 reactive intermediate involved in this latter step is an iron-bound ferric peroxoanion.
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CYP17A1-catalyzed 17α-hydroxylation of pregnenolone yields 17α-hydroxypregnenolone, which serves as preferred substrate for 17,20-lyase activity of CYP17A1, producing C19 steroids from C21 precursors.
“The CYP17A1-catalyzed 17α-hydroxylation of pregnenolone yields 17α-hydroxypregnenolone, which serves as the preferred substrate for the 17,20-lyase activity of CYP17A1, producing C19 steroids from C21 precursors [[30], [31], [32]].”
From Human steroid biosynthesis, metabolism and excretion are differentially reflected by serum and urine steroid metabolomes: a comprehensive review (2019) · cites this paper as [32]
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- Authors
- Gregory, M. C., Denisov, I. G., Grinkova, Y. V., Khatri, Y., & Sligar, S. G.
- Affiliations
- University of Illinois Urbana-Champaign (US)
- Corresponding author
- Sligar, Stephen G.
- Venue
- Journal of the American Chemical Society
- Publisher
- American Chemical Society (ACS)
- Published
- 2013-10-29
- Volume
- 135
- Issue
- 44
- Pages
- 16245-16247
- ISSN
- 0002-7863 (ISSN-L)
- 1520-5126
- PMC
- PMC3867208
- OpenAlex
- W2319340717
- Cited by
- 80
- References
- 21
- Citations by year
- 2026: 5 · 2025: 2 · 2024: 8 · 2023: 8 · 2022: 4 · 2021: 4 · 2020: 3 · 2019: 8 · 2018: 8 · 2017: 10 · 2016: 7 · 2015: 7 · 2014: 5 · 2013: 1
- FWCI
- 3.14
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Locations
- Journal of the American Chemical Society (publishedVersion)
- PubMed (publishedVersion)
- PubMed Central (acceptedVersion)
- Funders
- National Institute of General Medical Sciences (R01GM033775, GM 31756, R01GM031756, R37GM031756)
- Topics
- Physical Sciences › Chemistry › Inorganic Chemistry › Metal-Catalyzed Oxygenation Mechanisms
- Physical Sciences › Chemistry › Organic Chemistry › Chemical Reaction Mechanisms
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Steroid Chemistry and Biochemistry
- Keywords
- CYP17A1, kinetic isotope effects, C–C bond cleavage, steroidogenesis, nanodiscs, enzyme turnover
- MeSH terms
- Dehydroepiandrosterone, Ferric Compounds, Ferrous Compounds, Humans, Kinetics, Male, Solvents, Steroid 17-alpha-Hydroxylase, Water, Deuterium Oxide, Biocatalysis
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