| Abstract
| - The experimentally postulated mechanism for the interconversion between (S)-vinylglycolate and(R)-vinylglycolate catalyzed by mandelate racemase enzyme consists of a two-step quite symmetric processthrough a dianionic enolic intermediate that is formed after the abstraction of the α-proton of vinylglycolateby a basic enzymatic residue and is then reprotonated by another residue. The challenging problem behindthis reaction is how the enzyme manages to stabilize such an intermediate, that is, how it lowers enough thehigh pKa of the α-proton for the reaction to take place. The QM/MM simulations performed in this paperindicate that catalysis is based on the stabilization of the negative charge developed on the substrate along thereaction. We have identified three different reaction mechanisms starting from different quasi-degeneratestructures of the substrate−enzyme complex. In two of them the stabilizing role is done by means of a catalyticproton transfer that avoids the formation of a dianionic intermediate, and they involve six steps instead of thetwo experimentally proposed. On the contrary, the third mechanism passes through a dianionic species stabilizedby the concerted approach of a protonated enzymatic residue during the proton abstraction. The potentialenergy barriers theoretically found along these mechanisms are qualitatively in good agreement with theexperimental free energy barriers determined for racemization of vinylglycolate and mandelate. The theoreticalstudy of the effect of the mutation of Glu317 by Gln317 in the kinetics of the reaction reveals the importantrole in the catalysis of the hydrogen bond formed by Glu317 in the native enzyme, as only one of themechanisms, the slower one, is able to produce the racemization in the active site of the mutant. However, wehave found that this hydrogen bond is not an LBHB within our model.
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