Computational Enzymology: Simulation of the Enzyme Reaction Mechanism of Malate Dehydrogenase

Paul A. Bash, Northwestern University Medical School

A Computational Enzymology, which combines quantum and molecular mechanics (QM/MM) methods, is used to simulate the complete minimum energy surface and reaction pathway for the interconversion of malate and oxaloacetate catalyzed by the enzyme malate dehydrogenase (MDH). A reaction mechanism for the proton and hydride transfers associated with MDH is deduced from the topology of the calculated energy surface. The putative mechanism consists of (1) a sequential reaction with the proton transfer preceding the hydride transfer (malate to oxaloacetate direction), (2) the existence of two transition states with energy barriers of about 10 and 15 kcal/mol for the proton and hydride transfers, respectively, and (3) reactant (malate) and product (oxaloacetate) states that are nearly isoenergetic. A simulation analysis of the calculated energy profile shows that solvent effects due to the protein matrix dramatically alter the intrinsic reactivity of the functional groups involved in the MDH reaction. The enzyme effectively changes the reaction


imidazole + malate + NAD imidazolium + oxaloacetate + NADH


from an exothermic reaction (ΔE ≅ -54 kcal/mol) in the gas phase to a nearly isoenergetic one (ΔE ≅1 kcal/mol) in the protein_solvent environment of MDH. An energy decomposition analysis indicates that specific MDH residues (Asp-150, Arg-81, Arg-87, and Arg-153) in the vicinity of the substrate make significant energetic contributions to the stabilization of the proton transfer and destabilization of the hydride transfer. This suggests that these amino acids play an important role in the catalytic properties of MDH, which is consistent with site-directed mutagenesis experiments.

Abstract Author(s): Paul A. Bash