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. 2023 May 18;299(7):104841. doi: 10.1016/j.jbc.2023.104841

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© 2023 The Authors

This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

PMC Copyright notice

Steady-state kinetics of oxidation of dihydrolanosterol and its 14α-CH₂OH and 14α-CHO derivatives to dihydro FF-MAS. The individual substrates were incubated with a reconstituted P450 51A1 system, and the product dihydro FF-MAS was extracted and analyzed by UPLC-UV. A, dihydrolanosterol: k_cat 0.50 ± 0.03 s⁻¹, K_m 5.0 ± 0.6 μM; B, 14α-CH₂OH dihydrolanosterol: k_cat 0.67 ± 0.03 s⁻¹, K_m 4.3 ± 0.4 μM; C, 14α-CHO dihydrolanosterol: k_cat 1.7 ± 0.1 s⁻¹, K_m 5.4 ± 0.7 μM. Rates are presented on the y-axis as nmol product (dihydro FF-MAS) formed min⁻¹(nmol P450)⁻¹. Incubations were run in triplicate, and the means (±SD) were calculated and plotted, The linear regression fits include the Prism error estimates for internal fitting (SE). The estimated parameters were not corrected using a quadratic equation, due to the high K_m values. FF-MAS, follicular fluid meiosis-activating sterol ((4β,5α)-4,4-dimethyl-cholesta-8,14,24-trien-3-ol); UPLC, ultra-performance liquid chromatography.