The enzyme human aromatase (HA) catalyzes the conversion of androgens to estrogens via two hydroxylation reactions and a final unique aromatization step. Despite the great interest of HA as a drug target against breast cancer detailed structural and spectroscopic information on this enzyme became available only in the past few years. As such, the enigmatic mechanism of the final aromatization step is still a matter of debate. Here, we investigated the final step of the HA enzymatic cycle via hybrid quantum-classical (QM/MM) metadynamics and blue-moon ensemble simulations. Our results show that the rate-determining step of the aromatization process is the nucleophilic attack of the distal oxygen of a peroxo-ferric species on the formyl carbon of the enol-19-oxo-androstenedione, which occurs with a free energy barrier (Delta F-#) of similar to 16.7 +/- 1.9 kcal/mol, in good agreement with experimental data. This reaction is followed by a water mediated 1 beta-hydrogen abstraction (Delta F-# = 7.9 +/- 0.8 kcal/mol) and by the formation of a hydroxo-ferric moiety. This latter may be finally protonated by a hydrogen delivery channel involving Asp309 and Thr310, both residues pointed out as crucial for HA activity. In the absence of the catalytic water in the active site the substrate does not assume a position suitable to undergo the nucleophilic attack. Our data not only reveal a novel possible mechanism for the aromatization process consistent with some of the spectroscopic and kinetic data available in the literature, complementing current knowledge on the mechanism of this enzyme, but also point out a remarkable influence of the level of theory used on the calculated free energy barriers. The structural information obtained in this study may be used for the rational structure-based drug design of HA inhibitors to be employed in breast cancer therapy.

Role of Water in the Puzzling Mechanism of the Final Aromatization Step Promoted by the Human Aromatase Enzyme. Insights from QM/MM MD Simulations / Sgrignani, J; Iannuzzi, M; Magistrato, Alessandra. - In: JOURNAL OF CHEMICAL INFORMATION AND MODELING. - ISSN 1549-960X. - 55:10(2015), pp. 2218-2226. [10.1021/acs.jcim.5b00249]

Role of Water in the Puzzling Mechanism of the Final Aromatization Step Promoted by the Human Aromatase Enzyme. Insights from QM/MM MD Simulations

MAGISTRATO, ALESSANDRA
2015

Abstract

The enzyme human aromatase (HA) catalyzes the conversion of androgens to estrogens via two hydroxylation reactions and a final unique aromatization step. Despite the great interest of HA as a drug target against breast cancer detailed structural and spectroscopic information on this enzyme became available only in the past few years. As such, the enigmatic mechanism of the final aromatization step is still a matter of debate. Here, we investigated the final step of the HA enzymatic cycle via hybrid quantum-classical (QM/MM) metadynamics and blue-moon ensemble simulations. Our results show that the rate-determining step of the aromatization process is the nucleophilic attack of the distal oxygen of a peroxo-ferric species on the formyl carbon of the enol-19-oxo-androstenedione, which occurs with a free energy barrier (Delta F-#) of similar to 16.7 +/- 1.9 kcal/mol, in good agreement with experimental data. This reaction is followed by a water mediated 1 beta-hydrogen abstraction (Delta F-# = 7.9 +/- 0.8 kcal/mol) and by the formation of a hydroxo-ferric moiety. This latter may be finally protonated by a hydrogen delivery channel involving Asp309 and Thr310, both residues pointed out as crucial for HA activity. In the absence of the catalytic water in the active site the substrate does not assume a position suitable to undergo the nucleophilic attack. Our data not only reveal a novel possible mechanism for the aromatization process consistent with some of the spectroscopic and kinetic data available in the literature, complementing current knowledge on the mechanism of this enzyme, but also point out a remarkable influence of the level of theory used on the calculated free energy barriers. The structural information obtained in this study may be used for the rational structure-based drug design of HA inhibitors to be employed in breast cancer therapy.
55
10
2218
2226
Sgrignani, J; Iannuzzi, M; Magistrato, Alessandra
File in questo prodotto:
File Dimensione Formato  
Sgrignani-react-acs.jcim.5b00249.pdf

non disponibili

Tipologia: Versione Editoriale (PDF)
Licenza: Non specificato
Dimensione 4.12 MB
Formato Adobe PDF
4.12 MB Adobe PDF   Visualizza/Apri   Richiedi una copia

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/20.500.11767/33261
Citazioni
  • ???jsp.display-item.citation.pmc??? 5
  • Scopus 23
  • ???jsp.display-item.citation.isi??? 22
social impact