The active site of malate dehydrogenase is a hydrophobic cavity within the protein complex that has specific binding sites for the substrate and its coenzyme, NAD. In its active state, MDH undergoes a conformational change that encloses the substrate to minimize solvent exposure and to position key residues in closer proximity to the substrate. The three residues in particular that comprise a catalytic triad are histidine (His-195), aspartate (Asp-168), both of which work together as a proton transfer system, and arginines (Arg-102, Arg-109, Arg-171), which secure the substrate.
Mechanistically, malate dehydrogenase catalyzes the oxidation of the hydroxyl group of malate by utilizing NAD as an electron acceptor. This oxidation step results in the elimination of a proton and a hydride ion from the substrate. NAD receives the hydride ion (specifically, the hydride ion is transferred to the nicotinamide ring of the NAD) and becomes reduced to NADH while concomitantly, the His-195 residue on the enzyme accepts the proton. The positively charged His-195 residue, which is involved in base catalysis of the substrate, is stabilized by the adjacent, negatively charged Asp-168 residue. This electrostatic stabilization helps facilitate the transfer of the proton. Arg-102, Arg-109, and Arg-171 (which are protonated, and thus positively charged) participate in electrostatic catalysis and help to bind the negatively charged carboxylates on the substrate. Additionally, the Arginine residues on the enzyme provide additional substrate specificity and binding through hydrogen bonding between the guanidinium side chain of the Arginine amino acid residues and the carboxylates of the substrate.
Studies have also identified a mobile loop in malate dehydrogenase that participates in the catalytic activity of the enzyme. The loop undergoes a conformational change to shield the substrate and catalytic amino acids from the solvent in response to the binding of the malate dehydrogenase:coenzyme complex to substrate. This flipping of the loop to the up position to cover the active site also promotes enhanced interaction of the catalytically important amino residues on the enzyme with the substrate. Additionally, the movement of the loop has been shown to correlate with the rate determining step of the enzyme.