Article pubs.acs.org/JPCB
Enolization as an Alternative Proton Delivery Pathway in Human Aromatase (P450 19A1) Balázs Krámos and Julianna Oláh* Department of Inorganic and Analytical Chemistry, Budapest University of Technology and Economics, Szent Gellért tér 4, H-1111 Budapest, Hungary S Supporting Information *
ABSTRACT: Human aromatase catalyzes the last step of estrogen biosynthesis, the aromatization of ring A of androstenedione (ASD) and testosterone leading to estrone and estradiol. The enolization of the substrate molecule has been suggested to play an essential role in this process. In this work using quantum mechanical and hybrid QM/MM calculations, the reaction mechanism of enolization was investigated. It is shown that the energetically unfavorable enolization of andostenedione occurs in a coupled process with the energetically favorable protonation of the ferrous superoxo complex (traditionally called ferric peroxo complex) via a low barrier of about 5 kcal/mol. This mechanism implies an alternative way for protonation of the ferrous superoxo complex to form compound 0, which occurs via the Asp309−water−ASD proton delivery pathway instead of the Asp−water−Thr pathway suggested for other P450 enzymes. It is also shown that Thr310, which is known experimentally to be important for catalysis, plays a key role in the conversion of compound 0 to compound I. C19, and 10β-hydroxyestr-4-ene-3,17-dione formation,15 have been put forward to explain this mysterious step; however, all of them were proven experimentally to be unlikely.3 Although several of these intermediates are known to be converted spontaneously10 or by aromatase16 into estrone, none of them is currently accepted as lying upon the major pathway for aromatization primarily, because they contradict the available 18 O labeling studies which indicate that the third oxygen atom is incorporated into formate.3,15 According to the most accepted mechanism a peroxohemiacetal is formed after the nucleophilic attack of the ferrous superoxo complex on the C19 carbon,15 but several unanswered questions remain in connection with this reaction. It is experimentally proven that the 1β- and 2β-hydrogen atoms are eliminated selectively.17,18 Furthermore, based on model reaction studies the formation of the 2,3-enol is a prerequisite for aromatization,19,20 as the energy barrier for 2β-hydrogen abstraction is significantly lower in the 2,3-enol form because of the increased stability of the resulting intermediate.21 The stereoselectivity of the 2βhydrogen atom removal indicates that the enzyme mediates the enolization process.22 Before the crystallization of human aromatase, homology modeling methods were used to study the enolization reaction and the roles of Asp309 as proton acceptor and Lys473 as proton donor were emphasized.23 However, based on the crystal structure of aromatase (PDB ID, 3EQM),24 the position of these side chains does not allow them
1. INTRODUCTION Human aromatase (P450 19A1) is a hemoprotein belonging to the superfamily of cytochrome P450 enzymes (P450s). The active site of P450 enzymes contains an iron ion which is tethered to the protein via a thiolate ligand derived from a cysteine residue. Human aromatase plays a key role in the regulation of sex steroids in the human body1 as it is responsible for the last and rate-limiting step of estrogen biosynthesis. It converts androstenedione (ASD) and testosterone to estrone and estradiol, respectively, by aromatizing ring A2,3 (Scheme 1). As estrogens are known to play a major role in the development of hormone-dependent cancers, e.g., breast cancer, aromatase has become a particularly attractive target for their treatment4−6 and several aromatase inhibitors (anastrozole, exemestane, letrozole) are already on the market. Understanding the catalytic mechanism of aromatase could contribute to the development of newer and more efficacious drugs to prevent estrogen production in patients suffering from breast cancer. The conversion catalyzed by aromatase consists of three catalytic subcycles. In the first and second oxidation steps hydroxylation of the 19-methyl group occurs most likely by the classical P450-mediated hydrogen atom abstraction−hydroxyl radical rebound mechanism originally proposed by Groves and co-workers.7 In the second subcycle a 19-gem-diol is generated, which may dehydrate to the 19-aldehyde.8,9 In the third subcycle the oxidative cleavage of the C10−C19 bond occurs and the aromatized steroid and one molecule of formic acid are generated. Several mechanisms including 2β-hydroxylation,10−12 4,5-epoxidation,13,14 Baeyer−Villiger oxidation of © 2013 American Chemical Society
Received: July 24, 2013 Revised: December 19, 2013 Published: December 26, 2013 390
dx.doi.org/10.1021/jp407365x | J. Phys. Chem. B 2014, 118, 390−405
The Journal of Physical Chemistry B
Article
Scheme 1. Conversion of Androstenedione to Estrone Catalyzed by Aromatase
Figure 1. Consensus catalytic cycle of P450s. The two protonation steps are highlighted and the electron configurations of the states involved are shown.
study, the mysterious third catalytic subcycle was thoroughly investigated.25 According to this multistep mechanism first a peroxo hemiacetal is formed by the nucleophilic attack of the ferrous superoxo complex on the formyl group of the substrate. The enolization occurs in the final step after the fission of the C10−C19 bond and the elimination of the 1β-hydrogen. The
to facilitate the conversion. Therefore, Ghosh and co-workers suggested that Asp309 may be the proton donor in the enolization reaction and a network, consisting of the backbone oxygen of Ala306, the side chain of the highly conserved Thr310, and a water molecule, may serve as proton acceptor.24 In a recent quantum mechanics/molecular mechanics (QM/MM) 391
dx.doi.org/10.1021/jp407365x | J. Phys. Chem. B 2014, 118, 390−405
The Journal of Physical Chemistry B
Article
P450cam enzyme, but that Asp251 could be essential.36 The highly increased kinetic solvent isotope effect (KSIE) value in the D251N mutant enzyme (wild type, 1.7; D251N, 10)37 suggested a different proton delivery pathway in the mutant, which should be longer and mediated by more water molecules. It was also hypothesized that Asp251 might work as a gate for the protons between bulk water and the active site. The newest results suggest that Asp251, which is hydrogen bonding to Arg186, can move out from this position through a low energy barrier (
