Theoretical site-directed mutagenesis: Asp168Ala mutant of lactate dehydrogenase.
Molecular simulations based on the use of hybrid quantum mechanics/molecular mechanics methods are able to provide detailed information about the complex enzymatic reactions and the consequences of specific mutations on the activity of the enzyme. In this work, the reduction of pyruvate to lactate catalysed by wild-type and Asp168Ala mutant lactate dehydrogenase (LDH) has been studied by means of simulations using a very flexible molecular model consisting of the full tetramer of the enzyme, together with the cofactor NADH, the substrate and solvent water molecules. Our results indicate that the Asp168Ala mutation provokes a shift in the pKa value of Glu199 that becomes unprotonated at neutral pH in the mutant enzyme. This change compensates the loss of the negative charge of Asp168, rendering a still active enzyme. Thus, our methodology gives a calculated barrier height for the Asp168Ala mutant 3 kcal mol-1 higher than that for wild-type LDH, which is in very good agreement with the experiment. The computed potential energy surfaces reveal the reaction pathways and transition structures for the wild-type and mutant enzymes. Hydride transfer is less advanced and the proton transfer is more advanced in the Asp168Ala mutant than in the wild type. This approach provides a very powerful tool for the analysis of the roles of key active-site residues.
Ferrer
Silvia
S
Departamento de Química Física, Universidad de Valencia, Burjassot, Valencia, Spain.
Tuñón
Iñaki
I
Moliner
Vicent
V
Williams
Ian H
IH
D019295:Computational Biology / Q000379:methods*; D003198:Computer Simulation; D007770:L-Lactate Dehydrogenase / Q000235:genetics*; D008958:Models, Molecular; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D020125:Mutation, Missense / Q000235:genetics*
D007770:L-Lactate Dehydrogenase