SIMULATION OF THE ENZYME REACTION-MECHANISM OF MALATE-DEHYDROGENASE

A hybrid numerical method, which employs molecular mechanics to descri be the bulk of the solvent-protein matrix and a semiempirical quantum- mechanical treatment for atoms near the reactive site, was utilized to simulate the minimum energy surface and reaction pathway for the inte rconversion of malate and oxaloacetate catalyzed by the enzyme malate dehydrogenase (MDH). A reaction mechanism for proton and hydride trans fers associated with MDH and cofactor nicotinamide adenine dinucleotid e (NAD) is deduced from the topology of the calculated energy surface. The proposed mechanism consists of (1) a sequential reaction with pro ton transfer preceding hydride transfer (malate to oxaloacetate direct ion), (2) the existence of two transition states with energy barriers of approximately 7 and 15 kcal/mol for the proton and hydride transfer s, respectively, and (3) reactant (malate) and product (oxaloacetate) states that are nearly isoenergetic. Simulation analysis of the calcul ated energy profile shows that solvent effects due to the protein matr ix dramatically alter the intrinsic reactivity of the functional group s involved in the MDH reaction, resulting in energetics similar to tha t found in aqueous solution. An energy decomposition analysis indicate s that specific MDH residues (Arg-81, Arg-87, Asn-119, Asp-150, and Ar g-153) in the vicinity of the substrate make significant energetic con tributions to the stabilization of proton transfer and destabilization of hydride transfer. This suggests that these amino acids play an imp ortant role in the catalytic properties of MDH.