Structure of the protein with attached cofactors
|PDB structures||RCSB PDB PDBe PDBsum|
Malate dehydrogenase (EC 184.108.40.206) (MDH) is an enzyme that reversibly catalyzes the oxidation of malate to oxaloacetate using the reduction of NAD+ to NADH. This reaction is part of many metabolic pathways, including the citric acid cycle. Other malate dehydrogenases, which have other EC numbers and catalyze other reactions oxidizing malate, have qualified names like malate dehydrogenase (NADP+).
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.
Evolution and structure
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. 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.
Malate dehydrogenase has also been shown to have a mobile loop region that plays a crucial role in the enzyme's catalytic activity. Studies have indicated that this loop region is highly conserved in malate dehydrogenase.
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 oxidizing agent. 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 stabilize 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.
Kinetic studies show that malate dehydrogenase enzymatic activity is ordered. The cofactor NAD+/NADH is bound to the enzyme before the substrate.
Effect of pH on catalytic activity
Additionally, pH levels control the direction of malate dehydrogenase catalytic activity due to proton transfer in the catalytic mechanism. A histidine moiety with a pK value of 7.5 has been identified to play a role in the pH-dependency of the enzyme. Studies have indicated that the binding of L-malate and enol-oxaloacetate with the malate dehydrogenase:NADH complex forms much more rapidly at higher pH values. Specifically, when the histidine is protonated, the His residue can form a hydrogen bond with the substrate's carbonyl oxygen, which shifts electron density away from the oxygen and makes it more susceptible to nucleophilic attack by hydride. As a result, the unprotonated form the malate dehydrogenase:NADH complex binds exclusively to L-malate and enol-oxaloacetate. Additionally, the rate of the formation of this complex occurs more quickly, alongside an increase in malate dehydrogenase in the bound complex form, in response to an increase in pH level. In contrast, D-malate and hydroxymalonate has been found to bind exclusively to the protonated form of the enzyme. As a result, at lower pH values the malate dehydrogenase:NAD+ complex binds preferentially to D-malate and hydroxymalonate.
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.
Glutamate has also been shown to inhibit malate dehydrogenase activity. Furthermore, it has been shown that alpha ketogluturate dehydrogenase can interact with mitochondrial aspartate aminotransferase to form a complex, which can then bind to malate dehydrogenase, forming a ternary complex that reverses inhibitory action on malate dehydrogenase enzymatic activity by glutamate. Additionally, the formation of this complex enables glutatmate to react with aminotransferase without interfering activity of malate dehydrogenase. The formation of this ternary complex also facilitates the release of oxalocetate from malate dehydrogenase to aminotransferase. Kinetically, the binding of malate dehydrogenase to the binary complex of alpha ketogluturate dehydrogenase and aminotrannferase has been shown to increase reaction rate of malate dehydrogenase because the Km of malate dehydrogenase is decreased when it is bound as part of this complex.
Interactive pathway map
Click on genes, proteins and metabolites below to link to respective articles. [§ 1]
- The interactive pathway map can be edited at WikiPathways: "GlycolysisGluconeogenesis_WP534".
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