QM/MM modeling of the hydroxylation of the androstenedione substrate catalyzed by cytochrome P450 aromatase (CYP19A1)

Abstract

CYP19A1 aromatase is a member of the Cytochrome P450 family of hemeproteins, and is the enzyme responsible for the final step of the androgens conversion into the corresponding estrogens, via a three‐step oxidative process. For this reason, the inhibition of this enzyme plays an important role in the treatment of hormone‐dependent breast cancer. The first catalytic subcycle, corresponding to the hydroxilation of androstenedione, has been proposed to occur through a first hydrogen abstraction and a subsequent oxygen rebound step. In present work, we have studied the mechanism of the first catalytic subcycle by means of hybrid quantum mechanics/molecular mechanics methods. The inclusion of the protein flexibility has been achieved by means of Free Energy Perturbation techniques, giving rise to a free energy of activation for the hydrogen abstraction step of 13.5 kcal/mol. The subsequent oxygen rebound step, characterized by a small free energy barrier (1.5 kcal/mol), leads to the hydroxylated products through a highly exergonic reaction. In addition, an analysis of the primary deuterium kinetic isotopic effects, calculated for the hydrogen abstraction step, reveals values (∼10) overpassing the semiclassical limit for the CH, indicating the presence of a substantial tunnel effect. Finally, a decomposition analysis of the interaction energy for the substrate and cofactor in the active site is also discussed. According to our results, the role of the enzymatic environment consists of a transition state stabilization by means of dispersive and polarization effects. © 2015 Wiley Periodicals, Inc.

Theoretical Study of the Mechanism of Exemestane Hydroxylation Catalyzed by Human Aromatase Enzyme

“…40 According to our findings, the mechanism discussed above, which is common for all cytochromes P450, is fully compatible with the androgen hydroxylation by aromatase enzyme. In this work, apart from calculating the free energy of the whole hydroxylation mechanism, an analysis of the decomposition of the interaction energy for substrate and cofactor was performed.…”

A Dehydrogenase Dual Hydrogen Abstraction Mechanism Promotes Estrogen Biosynthesis: Can We Expand the Functional Annotation of the Aromatase Enzyme?

“…Because a consensus view exists on the mechanism of the two hydroxylation reactions, the first two catalytic steps were not investigated herein, but we focused exclusively on the possible reaction paths leading to the aromatization of ring A of the substrate . We considered the keto and enol gem ‐diol tautomers ( gem ‐diol k and gem ‐diol e , respectively) and Cpd I (Scheme ) as reactive adducts for the final catalytic step.…”

“…Because ac onsensus view exists on the mechanism of the two hydroxylation reactions, the first two catalytic steps were not investigated herein, butwef ocused exclusively on the possible reaction paths leadingt ot he aromatization of ring Ao ft he substrate. [12] We considered the keto and enol gem-diol tautomers (gem-diol k and gem-diol e ,r espectively) and Cpd I (Scheme 1) as reactive adducts for the final catalytic step. After an initial equilibration of the membrane model (100 ns), 200 ns of classical molecular dynamics (MD) simulationsw ere performed to equilibrate the adduct between HA and gem-diol k .A representative snapshot extracted from the equilibrated trajectory was then relaxed for 5psb yQ M/MM MD ( Figure S1 in the Supporting Information), and an equilibrated frame from the QM/MM MD trajectory was used to identify the most likely aromatization pathway by MTD simulations.…”

Theoretical Studies on Mechanism of Inactivation of Kanamycin A by 4′-O-Nucleotidyltransferase

“…where x j, i , y j, i , and z j, i are the coordinates of the i th structure for the j th QM atom belonging to the IRC traced from the transition state structure (x j0 , y j0 , and zj 0 coordinates) and m j represents the corresponding masses of the atoms. Therefore, the free energy relative to the reactant is expressed as a function of the s coordinate as explained elsewhere (Świderek et al, 2013; Viciano et al, 2015). The MD simulations for the FEP calculation were performed at 300 K, using the NVT ensemble for the each window.…”