Reaction mechanism of mandelate substrate

Next, the results obtained in the study of mandelate racemization by Mandelate Racemase are presented. Concerning mec I and mec II, the first remarkable difference between mandelate and the other two substrates is found in the minimum energy structure of the (S)-enantiomer (see figure 2.8). In this minimum energy structure (which will be called S'), the His297 residue is somewhat closer to the substrate (C7-H distance of 3.21 Å). Despite the approach of His297 to the substrate, the hydrogen bond between this residue and Glu247 is maintained in S'.

Figure 2.8: (a) Structure of the active site at the stationary point S' for mandelate. The labels used to identify some atoms are the same as in figure 2.5. (b) Structure of the active site at the stationary point R for mandelate. (c) Structure of the active site at the stationary point S2 for mandelate. Distances are given in Å.

a$)$
b$)$
c$)$

Mechanism I for mandelate:
Mec I for mandelate consists of the same six steps with the same stationary points as in the corresponding mechanisms for vinylglycolate and propargylglycolate (see table 2.4 and 2.5). However, the last step of this mechanism (from I5 to R) is around 4 kcal/mol above the corresponding stationary points on mec I for the other two substrates. Consequently, the highest energy point on mec I for mandelate corresponds to the transition state of the last proton transfer, that is, from the substrate and along the catalytic hydrogen bond back to Lys164.

Mechanism II for mandelate:
Mec II for mandelate presents more significant differences when compared with the same mechanism for the two other substrates. Mec II for mandelate consists of only five steps. The change of hybridization of the $\alpha$-carbon takes place in a concerted way with the $\alpha$-proton donation from His297. This fragment of the reaction coordinate, from I3 to I5, was recalculated using a smaller step, trying to locate the I4 intermediate. However, this stationary point was not found on the reaction path of mec II for mandelate.

Other significant differences arise in mec II of mandelate: first, the barrier for the catalytic proton transfer between positions O5 and O6 is appreciably higher than for the two other substrates and the product of this proton transfer (intermediate I1) is substantially more destabilized. In contrast, step 3 (from I2 to I3) is lower in energy than for vinylglycolate and propargylglycolate, specially I3, that is around 8 kcal/mol more stabilized than the other two substrates.

The highest energy point in mec II of mandelate corresponds to the proton transfer from the substrate and along the catalytic hydrogen bond back to Glu317. The global processes (mec I and mec II) are more endoergic than for vinylglycolate and propargylglycolate.

Mechanism III for mandelate:
The minimum energy stationary point that we have called S2 for the other two substrates also exists for mandelate. In S2 for mandelate His297 is 0.28 Å closer to the substrate than in S' (see figure 2.8) and consequently this residue does not form a hydrogen bond with Glu247, similar to what happens in the S2 structure for vinylglycolate and propargylglycolate complexes. The S2 structure connects with an (R)-enantiomer (R') via mec III. This structure R' is 2.10 kcal/mol more stable than the (R)-enantiomer for mec I and mec II, although there are no significant geometrical differences in the active site^3.9.

Mec III for mandelate, similar to vinylglycolate, is a two-step energy profile rather asymmetric, with a higher barrier in the S-to-R direction that accounts for the abstraction of the $\alpha$-proton by Lys166 and the hybridization change of the $\alpha$-carbon. The values of the C-C1(C7)-O(OH)-C mandelate dihedral angle are 51.5$^o$(S2), -23.7$^o$(TS1), -44.9$^o$(I), -48.4$^o$(TS2) and -56.1$^o$ (R'). The lowest energetic barrier corresponds to the reprotonation of C7 by His297. A very shallow minimum appears in between the two barriers. The two steps of this mec III correspond to the experimentally proposed mechanism for this reaction. However, contrary to the experimental conclusions, it is a rather asymmetric mechanism.