Computational study on the carboligation reaction of acetohidroxyacid synthase: New approach on the role of the HEThDP− intermediate |
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Authors: | Gonzalo Jaña Verónica Jiménez Jordi Villà‐Freixa Xavier Prat‐Resina Eduardo Delgado Joel Alderete |
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Institution: | 1. Grupo de Química Teórica y Computacional, Facultad de Ciencias Químicas, Universidad de Concepción, Casilla 160‐C, Concepción, Chile;2. CBB Laboratory, Research Unit on Biomedical Informatics, Universitat Pompeu Fabra, Parc de Recerca Biomèdica, Doctor Aiguader 88, Barcelona 08003, Spain;3. Department of Chemistry, University of Wisconsin, Madison, WI, USA |
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Abstract: | Acetohydroxyacid synthase (AHAS) is a thiamin diphosphate dependent enzyme that catalyses the decarboxylation of pyruvate to yield the hydroxyethyl‐thiamin diphosphate (ThDP) anion/enamine intermediate (HEThDP–). This intermediate reacts with a second ketoacid to form acetolactate or acetohydroxybutyrate as products. Whereas the mechanism involved in the formation of HEThDP– from pyruvate is well understood, the role of the enzyme in controlling the carboligation reaction of HEThDP– has not been determined yet. In this work, molecular dynamics (MD) simulations were employed to identify the aminoacids involved in the carboligation stage. These MD studies were carried out over the catalytic subunit of yeast AHAS containing the reaction intermediate (HEThDP–) and a second pyruvate molecule. Our results suggest that additional acid–base ionizable groups are not required to promote the catalytic cycle, in contrast with earlier proposals. This finding leads us to postulate that the formation of acetolactate relies on the acid–base properties of the HEThDP– intermediate itself. PM3 semiempirical calculations were employed to obtain the energy profile of the proposed mechanism on a reduced model of the active site. These calculations confirm the role of HEThDP– intermediate as the ionizable group that promotes the carboligation and product formation steps of the catalytic cycle. Proteins 2010. © 2010 Wiley‐Liss, Inc. |
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Keywords: | AHAS molecular dynamics semiempirical calculations thiamin diphosphate |
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