Products Cited in Publications Worldwide Processive kinetics in the three-step lanosterol 14α-demethylation reaction catalyzed by human cytochrome P450 51A1

Processive kinetics in the three-step lanosterol 14α-demethylation reaction catalyzed by human cytochrome P450 51A1

McCarty, Kevin D.; Sullivan, Molly E.; Tateishi, Yasuhiro; Hargrove, Tatiana Y.; Lepesheva, Galina I.; Guengerich, F. Peter

DOI: PMID:

Abstract

Cytochrome P 450 (P 450, CYP) family 51 enzymes catalyze the 14α-demethylation of sterols, leading to critical products used for membranes and the production of steroids, as well as signaling mols. In mammals, P 450 51 catalyzes the 3-step, 6-electron oxidation of lanosterol to form (4β,5α)-4,4-dimethyl-cholestra-8,14,24-trien-3-ol (FF-MAS). P 450 51A1 can also use 24,25-dihydrolanosterol (a natural substrate in the Kandutsch-Russell cholesterol pathway). 24,25-Dihydrolanosterol and the corresponding P 450 51A1 reaction intermediates, the 14α-alc. and -aldehyde derivatives of dihydrolanosterol, were synthesized to study the kinetic processivity of the overall 14α-demethylation reaction of human P 450 51A1. A combination of steady-state kinetic parameters, steady-state binding constants, dissociation rates of P 450-sterol complexes, and kinetic modeling of the time course of oxidation of a P 450-dihydrolanosterol complex showed that the overall reaction is highly processive, with koff rates of P 450 51A1-dihydrolanosterol and the 14α-alc. and 14α-aldehyde complexes being 1 to 2 orders of magnitude less than the forward rates of competing oxidations epi-Dihydrolanosterol (the 3α-hydroxy analog) was as efficient as the common 3β-hydroxy isomer in the binding and formation of dihydro FF-MAS. The common lanosterol contaminant dihydroagnosterol was found to be a substrate of human P 450 51A1, with roughly one-half the activity of dihydrolanosterol. Steady-state experiments with 14α-Me deuterated dihydrolanosterol showed no kinetic isotope effect, indicating that C-14α C-H bond breaking is not rate-limiting in any of the individual steps. The high processivity of this reaction generates higher efficiency and also renders the reaction less sensitive to inhibitors.

Purchased from AmBeed

Lanosterol

A37170679-63-060%

Lanosterol

11.0/100mg

Detail