Isotope-Labeling Studies Support the Electrophilic Compound Ⅰ Iron Active Species, FeO~(3+), for the Carbon-Carbon Bond Cleavage Reaction of the Cholesterol Side-Chain Cleavage Enzyme, Cytochrome P450 11A1

摘要

The enzyme cytochrome P450 11A1 cleaves the C20-C22 carbon-carbon bond of cholesterol to form pregnenolone, the first 21-carbon precursor of all steroid hormones. Various reaction mechanisms are possible for the carbon-carbon bond cleavage step of P450 11A1, and most current proposals involve the oxoferryl active species, Compound Ⅰ (FeO~(3+)). Compound Ⅰ can either (ⅰ) abstract an O-H hydrogen atom or (ⅱ) be attacked by a nucleophilic hydroxy group of its substrate, 20R,22R-dihydroxycholesterol. The mechanism of this carbon-carbon bond cleavage step was tested using ~(18)O-labeled molecular oxygen and purified P450 11A1. P450 11A1 was incubated with 20R,22R-dihydroxycholesterol in the presence of molecular oxygen (~(18)O_2), and coupled assays were used to trap the labile ~(18)O atoms in the enzymatic products (i.e., isocaproaldehyde and pregnenolone). The resulting products were derivarized and the ~(18)O content was analyzed by high-resolution mass spectrometry. P450 11A1 showed no incorporation of an ~(18)O atom into either of its carbon-carbon bond cleavage products, pregnenolone and isocaproaldehyde . The positive control experiments established retention of the carbonyl oxygens in the enzymatic products during the trapping and derivatization processes. These results reveal a mechanism involving an electrophilic Compound Ⅰ species that reacts with nucleophilic hydroxy groups in the 20R,22R-dihydroxycholesterol intermediate of the P450 11A1 reaction to produce the key steroid pregnenolone.