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QM/MM study on catalytic mechanism of aspartate racemase from Pyrococcus horikoshii OT3

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Abstract

The enzyme aspartate racemase from Pyrococcus horikoshii OT3 catalyzes the interconversion between l- and d-Asp. In this work, we employed the hybrid QM/MM approach with the self-consistent charge-density functional tight binding (SCC-DFTB) model to study the catalytic mechanism for the conversion of l-Asp into d-Asp. The molecular dynamics simulation showed that the substrate l-Asp forms an extensive network of interactions with the active-site residues of the aspartate racemase through its side chain carboxylate, ammonium group, and α-carboxylate. The potential of mean force calculations confirmed that the racemization reaction involves two proton transfers (from the α-carbon to Cys194 and from Cys82 to the α-carbon), which occurs in a concerted way, although highly asynchronous. The calculated free energy of activation is 17.5 kcal/mol, which is consistent with the reaction rate measured from experiment. An electrostatic interaction analysis was performed to estimate the key role played by individual residues in stabilizing the transition state. The docking study on the binding of l-Asp and d-Asp to aspartate racemase indicates that this enzyme employs a “two-base” mechanism not a “one-base” mechanism.

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Acknowledgments

This project has been supported by the National Natural Science Foundation of China (Grant Nos. 20773089 and 21075083) and National Basic Research Program of China (973 Program) (2011CB201202). We appreciate Prof. Dingguo Xu for his many stimulating discussions. The CHARMM calculations have been carried out in Wuhan Institute of Physics and Mathematics, the Chinese Academy of Science.

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Authors and Affiliations

  1. College of Chemistry, Key Laboratory of Green Chemistry and Technology in Ministry of Education, Sichuan University, 610064, Chengdu, People’s Republic of China

    Chenghua Zhang, Yong Guo & Ying Xue

  2. State Key Laboratory of Biotherapy, Sichuan University, 610064, Chengdu, People’s Republic of China

    Ying Xue

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  1. Chenghua Zhang
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  2. Yong Guo
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Correspondence to Ying Xue.

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Zhang, C., Guo, Y. & Xue, Y. QM/MM study on catalytic mechanism of aspartate racemase from Pyrococcus horikoshii OT3. Theor Chem Acc 129, 781–791 (2011). https://doi.org/10.1007/s00214-011-0935-7

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