A QM/MM study of the racemization of vinylglycolate catalyzed by mandelateracemase enzyme

The experimentally postulated mechanism for the interconversion between (S) -vinylglycolate and (R)-vinylglycolate catalyzed by mandelate racemase enzy me consists of a two-step quite symmetric process through a dianionic enoli c intermediate that is formed after the abstraction of the a-proton of viny lglycolate by a basic enzymatic residue and is then reprotonated by another residue. The challenging problem behind this reaction is how the enzyme ma nages to stabilize such an intermediate, that is, how it lowers enough the high pK(a) of the alpha -proton for the reaction to take place. The QM/MM s imulations performed in this paper indicate that catalysis is based-on,the stabilization of the negative charge developed on the substrate: along the reaction. We have identified; three different reaction mechanisms starting from different quasi-degenerate structures of-the substrate-enzyme complex. In two. of them the stabilizing role is done by means of a catalytic proto n transfer that avoids: the formation of a dianionic intermediate,land they involve six steps instead of the two experimentally proposed On the contra ry, the third mechanism passes through a dianionic species stabilized by th e concerted approach of a protonated enzymatic residue during the proton ab straction. The potential energy barriers theoretically found along, these m echanisms are qualitatively in good; agreement with the experimental free;e nergy barriers determined for racemization of vinylglycolate and mandelate. The theoretical study of the effect of the mutation of Glu317 by Gln317 in the kinetics of the reaction reveals the important role in the catalysis o f the hydrogen bond formed by Glu317 in the native enzyme, as only one of t he mechanisms, the Slower one, is able to produce the racemization in the a ctive site of the mutant. However, we have found that this hydrogen bond is not an LBHB within our model.