DFT STUDY ON THE REACTION MECHANISM OF ACETYLENE HYDRATASE FROM PELOBACTER ACETYLENICUS

Abstract

Introduction: Acetylene Hydratase (AH) is a unique tungsten containing enzyme that catalyzes a non-redox hydration reaction; where acetylene is converted to acetaldehyde under anaerobic conditions. The X-ray crystal structure of AH from Pelobactor acetylenicus provides important insight into its active site as the proposed catalytic mechanism of AH depends on the nature of oxygen specie bound to the tungsten center. Hypothetically, two possible mechanisms, electrophilic or nucleophilic, for hydration of acetylene were suggested by Seiffert et. al. Computational studies were performed by Antony and Bayse, Vincent, and Himo et al., however, they ruled out both the mechanisms suggested by Seiffert et al and proposed a reaction mechanism where hydration reaction starts with the displacement of a water molecule with the acetylene. Later on, based on the computational results for AH active site model complexes the most likely single step nucleophilic mechanism for the hydration of acetylene was suggested by Uzma et al, where initially, small model complexes (3 Å) were computed but to get more reliable results surrounding amino acid residues (6 Å) were considered. Although the relative energies for the formation of vinyl alcohol products are comparable with the results from the small model complexes, the energy barriers were considerably higher for both mechanistic options. These energy barriers were decreased when solvent molecules present in selected area (8 Å) of AH were considered. We sought to focus on identifying the importance of solvent molecules in the catalytic process of AH and 7 validating the suggested most probable nucleophilic reaction mechanism for hydration of acetylene. Method: Active site model complexes of Acetylene Hydratase (AH) (6 Å), considering the solvent molecules, based on the X-ray crystal structure were computed for hydration of acetylene at the COSMO-B3LYP/SDDp//B3LYP/Lanl2DZ(p) level of density functional theory (DFT). Activation energies have been calculated for both the electrophilic and nucleophilic reaction mechanisms in gas as well as in solvent phase. Results: Nucleophilic reaction mechanism shows energy barrier of 15.1 kcal/mol (20.8 kcal/mol in gas phase) which is in concordance with the results obtained by Uzma et al. This energy barrier is lower than those for the electrophilic pathway i.e.29.4 kcal/mol (33.4 kcal/mol) or for the other mechanisms suggested in literature Conclusion: The results of the study suggest that the water/solvent molecules play key role in the catalytic reaction of AH and it is also validated that nucleophilic mechanism is the most probable reaction mechanism for hydration of acetylene to acetaldehyde.