Kinetic solvent isotope effect in human P450 CYP17A1-mediated androgen formation: evidence for a reactive peroxoanion intermediate

Abstract

Human steroid hormone biosynthesis is the result of a complex series of chemical transformations operating on cholesterol, with key steps mediated by members of the cytochrome P450 superfamily. In the formation of the male hormone dehydroepiandrosterone, pregnenolone is first hydroxylated by P450 CYP17A1 at the 17-carbon, followed a second round of catalysis by the same enzyme that cleaves the C17-C20 bond, releasing acetic acid and the 17-keto product. In order to explore the mechanism of this C-C "lyase" activity, we investigated the kinetic isotope effect on the steady-state turnover of Nanodisc-incorporated CYP17A1. Our experiments revealed the expected small positive (~1.3) isotope effect for the hydroxylase chemistry. However, a surprising result was the large inverse isotope effect (~0.39) observed for the C-C bond cleavage activity. These results strongly suggest that the P450 reactive intermediate involved in this latter step is an iron-bound ferric peroxoanion.

Figure 1

The P450 catalytic cycle engaged in Compound 1 mediated oxidation chemistry, noting unproductive pathways.

Figure 2

Isotopically sensitive branching between the peroxo- and hydroperoxoferric species during CYP17 mediated catalysis.

Figure 3

Steady state kinetic solvent isotope effects observed for hydroxylase and lyase CYP17 catalysis. Rates are expressed in nmol product/min/nmol P450.