Discussion and conclusions

In this final chapter we have revisited the Mandelate Racemase reactivity with its natural substrate. Molecular Dynamics umbrella-sampling simulations have been used to calculate the Potential of Mean Force. The activation free energy is computed for the mechanism III previously described. In addition, a computational simulation of the mutagenesis experiment N197A has shed some light on the molecular basis that could qualitatively explain the lowering in the rate of racemization of the substrate by the mutant enzyme.

The inclusion of temperature effects is not only a clue step to compute energy barriers. Molecular Dynamics simulations include the different parallel pathways found in condensed phase systems with optimization methods. Statistical methods collect in only one magnitude the multiplicity of pathways and eliminates the uncertainty problem in optimization methods when they fall down into a certain valley that may determine in a different way the potential energy profile.

However, the location of saddle points on the PES has been very valuable for choosing an adequate reaction coordinate used for PMF calculations. The reaction coordinate labeled as R$_4$ is a combination of the four bond distances, and it is the most adequate reaction coordinate to describe the whole process. We have tried different reaction coordinates although we knew from the beginning that the whole process would not be well described by a too simple reaction coordinate because the mechanism implies two proton transfer and an inversion of configuration.

Note that the problem encountered with some reaction coordinates is not the lack of sampling in some regions, beacuase the overlap between two adjacent windows is quite adequate. The problems reside in the inadequacy of the reaction coordinate to explore some regions whereas relevant chemical changes are taking place.

This particular case of Mandelate Racemase would probably be solved by a two-dimensional PMF calculation, which despite being very expensive is still feasible. However, this discussion could be extrapolated to enzymatic reactions where more than two or even four coordinates significantly participate in the chemical step. Even in Mandelate Racemase enzyme it has also been proposed the participation of Glu317 as a candidate residue which could withdraw negative charge during the racemization process. Glu270 coordinated to His297 has also been proposed as a catalytic dyad in order to decrease the $pK_a$ of His297. These residues altogether would imply asynchronically the participation of four proton transfers and eight bond distances. A PMF of this complexity is very difficult to calculate whereas the location of the saddle points would give a very useful information in order to decide the reaction coordinate.