Theoretical site-directed mutagenesis. The Asp168Ala mutant of L-Lactate Dehydrogenase

The reduction of pyruvate to lactate catalyzed by the L-Lactate dehydrogenase has been studied in this paper by means of hybrid Quantum Mechanical / Molecular Mechanical simulations. A very flexible molecular model consisting on the full tetramer of the enzyme, together with the cofactor NADH, the substrate and solvent water molecules has allowed to theoretically mimic site directed mutagenesis studies, most of them previously experimentally performed. The potential energy surfaces obtained for every single mutation, compared with the one corresponding to the native enzyme, have been used to trace the possible reaction pathways and to locate and characterize the structures corresponding to the stationary points. The analysis of the results has been a very powerful tool to conclude about the role of key residues on the vacuole formed in the active site of the enzyme. Our results are in very good agreement with the previous conclusions derived from site directed mutagenesis. This strategy can be extrapolated to other enzyme systems thus opening the door of a very promising methodology that, in combination with the appropriate experimental technique, will enable us to describe enzyme catalysis phenomenon and the particular role of the residues that form the protein. This knowledge placed us in a privileged position to modify the activity of enzymes and to propose efficient inhibitor