The multistep oxidation of cholesterol to pregnenolone by human cytochrome P450 11A1 is highly processive
Kevin D. McCarty, Lu Liu, Yasuhiro Tateishi, Hannah L. Wapshott‐Stehli, F. Peter Guengerich
Abstract
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 22 R -hydroxy (OH) and 20 R ,22 R -(OH) 2 cholesterol, yielding pregnenolone. We expressed human P450 11A1 in bacteria, purified the enzyme in the absence of non-ionic 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 22 R -OH and 20 R ,22 R -(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 (Adx) had little effect on binding; the substrate cholesterol showed a ∼5-fold stimulatory effect on the binding of Adx to P450 11A1. Pre-steady-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 pre-steady-state kinetic analysis revealed a second di-OH cholesterol product, separable by HPLC, in addition to 20 R ,22 R -(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.