Citrate synthase is an enzyme of key importance to human metabolic pathways, in particular the citric acid cycle. Experimental information has lead to the proposal of an enzymatic mechanism. However, this mechanism is not completely determined, with the existence of a low barrier hydrogen bond being the most controversial aspect of the mechanism. QM/FE (Quantum Mechanics/Free Energy) simulations have the advantage of allowing direct comparison between simulations on the proposed intermediates, and experimental assessments. Under the assumption that the free energy of formation of the intermediate will be similar to that of the corresponding transition state, the theoretically determined free energies can be compared to known activation energies of the enzyme. QM/FE simulations involve the use of quantum mechanics to accurately model the bond forming and bond breaking characteristics of an enzyme mechanism. The QM results are used to parameterize a molecular mechanics force field, which can then be used to determine the free energy. The free energy assessments are based on molecular dynamics trajectories in which the starting structure is gradually mutated into the proposed intermediate. The completion of these simulations should allow us to comment on the nature of the mechanism, as well as the likelihood of the existence of the low barrier hydrogen bond. We will compare the results of these simulations directly to experimental information.
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