Malate dehydrogenase is also involved in gluconeogenesis, the synthesis of glucose from smaller molecules. Pyruvate in the mitochondria is acted upon by pyruvate carboxylase to form oxaloacetate, a citric acid cycle intermediate. In order to get the oxaloacetate out of the mitochondria, malate dehydrogenase reduces it to malate, and it then traverses the inner mitochondrial membrane. Once in the cytosol, the malate is oxidized back to oxaloacetate by cytosolic malate dehydrogenase. Finally, phosphoenolpyruvate carboxykinase (PEPCK) converts oxaloacetate to phosphoenolpyruvate (PEP).
Several isozymes of malate dehydrogenase exist. There are two main isoforms in eukaryotic cells. One is found in the mitochondrial matrix, participating as a key enzyme in the citric acid cycle that catalyzes the oxidation of malate. The other is found in the cytoplasm, assisting the malate-aspartate shuttle with exchanging reducing equivalents so that malate can pass through the mitochondrial membrane to be transformed into oxaloacetate for further cellular processes.
Humans and most other mammals express the following two malate dehydrogenases:
Malate dehydrogenases catalyse the interconversion of malate to oxaloacetate. The enzyme participates in the citric acid cycle. The family also contains L-lactate dehydrogenases that catalyse the conversion of L-lactate to pyruvate, the last step in anaerobic glycolysis. L-2-hydroxyisocaproate dehydrogenases are also members of the family. The N-terminus is a Rossmann NAD-binding fold and the C-terminus is an unusual alpha+beta fold.
In most organisms, malate dehydrogenase exists as a homodimeric molecule and is closely related to lactate dehydrogenase in structure. It is a large protein molecule with subunits weighing between 30 and 35 kDa. Based on the amino acid sequences, it seems that MDH has diverged into two main phylogenetic groups that closely resemble either mitochondrial isozymes or cytoplasmic/chloroplast isozymes. Because the sequence identity of malate dehydrogenase in the mitochondria is more closely related to its prokaryotic ancestors in comparison to the cytoplasmic isozyme, the theory that mitochondria and chloroplasts were developed through endosymbiosis is plausible. It is interesting to note that the amino acid sequences of archaeal MDH are more similar to that of LDH than that of MDH of other organisms. This indicates that there is a possible evolutionary linkage between lactate dehydrogenase and malate dehydrogenase.
Each subunit of the malate dehydrogenase dimer has two distinct domains that vary in structure and functionality. A parallel β-sheet structure makes up the domain, while four β-sheets and one α-helix comprise the central NAD+ binding site. The subunits are held together through extensive hydrogen-bonding and hydrophobic interactions.
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. Kinetic studies show that MDH enzymatic activity is ordered. NAD+/NADH is bound before the substrate.
Because malate dehydrogenase is closely tied to the citric acid cycle, regulation is highly dependent on TCA products. High malate concentrations stimulate MDH activity, and, in a converse manner, high oxaloacetate concentrations inhibit the enzyme.Citrate can both allosterically activate and inhibit the enzymatic activity of MDH. It inhibits the oxidation of malate when there are low levels of malate and NAD+. However, in the presence of high levels of malate and NAD+, citrate can stimulate the production of oxaloacetate. It is believed that there is an allosteric regulatory site on the enzyme where citrate can bind to and drive the reaction equilibrium in either direction.
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