Human steroid hormone biosynthesis is the result of a complex series

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. “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 carbon-carbon bond cleavage activity. These results strongly suggest that the P450 reactive intermediate involved in this latter step is an iron-bound ferric peroxoanion. Since the discovery of cytochrome P450s by Omura and Sato this large superfamily of heme containing monooxygenases has yielded a rich tapestry of substrate specificities and chemical transformations.1 2 Noteworthy is the facile hydroxylation of unactivated carbon centers with the P450s cycling through a series of iron-oxygen intermediates following electron input to a ferrous dioxygen adduct.3 Carbon center functionalization is considered to occur via the “Groves rebound mechanism”.4 First the ferric resting state of the enzyme (Figure 1) is reduced by one electron transfer from an associated redox partner with subsequent binding of atmospheric dioxygen to form the ferrous-O2 complex which is reduced by a second electron to form the key peroxoanion intermediate. Active site mediated proton transfer generates the IWP-3 iron-bound hydroperoxo which undergoes O-O bond scission to release water and generate an Fe(IV)O porphyrin cation radical “Compound 1” intermediate which then initiates hydrogen abstraction from the substrate and radical recombination to form product.3 A major accomplishment in recent years has been the isolation and spectroscopic characterization of the peroxoanion [5a] hydroperoxoferric [5b] and “Compound 1” [6] intermediates in various P450 systems. 5-8 Figure 1 The P450 catalytic IWP-3 cycle engaged in Compound 1 mediated oxidation chemistry noting unproductive pathways. More circumspect in steroid metabolism is the subsequent reaction by P450 CYP17A1 which involves scission of the 17-20 carbon-carbon bond releasing acetic acid and forming a ketone at the apex of the D-ring of the cholesterol backbone. The mechanism of this C-C “lyase” activity has been subject of considerable debate for many years and yet the reactive intermediate responsible for 17 20 lyase chemistry remains undefined.9 Early work by Ahktar suggested IWP-3 a heme-bound unprotonated peroxoanion [5a] acting through nucleophilic attack on the C-20 carbonyl of 17α-OH pregenenolone (OH-PREG) creating a hemi-acetyl that would decay through Rabbit Polyclonal to TACC3. homolytic or heterolytic scission of the iron ligated acyl peroxo to form the products of the reaction.10 Alternatively a radical mechanism involving the standard IWP-3 Compound 1 intermediate [6] has been proposed.11 These two pathways are distinguished by the involvement of protons in the standard mechanism involving Compound 1 (Cpd1) formation as seen in Figure 1. In this communication we report investigation of kinetic solvent isotope effects on the steady state turnover of CYP17A1 in both its hydroxylase and lyase functionalities. We reasoned that this technique would distinguish between the traditional Cpd1 mediated catalysis which relies on at least two protons to generate the high valent iron-oxo species and a nucleophilic reactivity of a ferric peroxoanion intermediate before proton involvement in O-O bond scission. An additional concern in comparing these two pathways of androgen formation are uncoupling reactions which release hydrogen peroxide. While the hydroxylation of pregnenolone (PREG) at the 17-position is relatively well coupled it is known that when OH-PREG is a substrate and the formation of dehydroepiandrosterone (DHEA) IWP-3 is monitored much of the pyridine nucleotide reducing equivalents appear in free hydrogen peroxide rather than carbon product.12 Uncoupling occurs from the iron-peroxide intermediates and can also involve protons. We thus have the following branching pathways (Figure 2) where the addition of two protons to the ferric peroxoanion [1] results in formation of Cpd1 which is utilized in the hydroxylation of pregnenolone [3] to OH-PREG [4] in the first rung on the ladder of CYP17 catalysis. The next uncoupled step in charge of androgen formation either proceeds productively from [1] via an acyl-peroxo intermediate [5] to create DHEA [6] or unproductively through proton reliant formation of [2] and supreme discharge of peroxide. Amount 2 private branching Isotopically.