Autor: |
McCarty KD; Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee, USA., Liu L; Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee, USA., Tateishi Y; Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee, USA., Wapshott-Stehli HL; Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee, USA., Guengerich FP; Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee, USA. Electronic address: f.guengerich@vanderbilt.edu. |
Jazyk: |
angličtina |
Zdroj: |
The Journal of biological chemistry [J Biol Chem] 2024 Jan; Vol. 300 (1), pp. 105495. Date of Electronic Publication: 2023 Nov 24. |
DOI: |
10.1016/j.jbc.2023.105495 |
Abstrakt: |
Cytochrome P450 (P450, CYP) 11A1 is the classical cholesterol side chain cleavage enzyme (P450 scc ) that removes six carbons of the side chain, the first and rate-limiting step in the synthesis of all mammalian steroids. The reaction is a 3-step, 6-electron oxidation that proceeds via formation of 22R-hydroxy (OH) and 20R,22R-(OH) 2 cholesterol, yielding pregnenolone. We expressed human P450 11A1 in bacteria, purified the enzyme in the absence of nonionic detergents, and assayed pregnenolone formation by HPLC-mass spectrometry of the dansyl hydrazone. The reaction was inhibited by the nonionic detergent Tween 20, and several lipids did not enhance enzymatic activity. The 22R-OH and 20R,22R-(OH) 2 cholesterol intermediates were bound to P450 11A1 relatively tightly, as judged by steady-state optical titrations and k off rates. The electron donor adrenodoxin had little effect on binding; the substrate cholesterol showed a ∼5-fold stimulatory effect on the binding of adrenodoxin to P450 11A1. Presteady-state single-turnover kinetic analysis was consistent with a highly processive reaction with rates of intermediate oxidation steps far exceeding dissociation rates for products and substrates. The presteady-state kinetic analysis revealed a second di-OH cholesterol product, separable by HPLC, in addition to 20R,22R-(OH) 2 cholesterol, which we characterized as a rotamer that was also converted to pregnenolone at a similar rate. The first oxidation step (at C-22) is the slowest, limiting the overall rate of cleavage. d 3 -Cholesterol showed no kinetic deuterium isotope effect on C-22, indicating that C-H bond cleavage is not rate-limiting in the first hydroxylation step. Competing Interests: Conflict of interest All of the authors declare that they have no conflict of interest with the contents of this article. (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.) |
Databáze: |
MEDLINE |
Externí odkaz: |
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