Mitochondria malate-oxaloacetate shuttle

Another way in which reducing power may be transferred across the mitochondrial membrane is by means of the malate-oxaloacetate shuttle. The cytoplasmic NADH is used to reduce oxaloacetate to malate which, unlike oxaloacetate, is able to pass into the mitochondrion. Having done so it is reconverted to oxaloacetate with the formation of mitochondrial NADH. The oxaloacetate is returned to the cytoplasm in the form of aspartate. 

A similar shuttle, the malate-aspartate shuttle, operates in heart and liver (Fig. 6). Oxaloacetate in the cytosol is converted to malate by cytoplasmic malate dehydrogenase, reoxidizing NADH to NAD+ in the process. The malate enters the mitochondrion via a malate-a-ketoglutarate carrier in the inner mitochondrial membrane. In the matrix the malate is reoxidized to oxaloacetate by NAD+ to form NADH. Oxaloacetate does not easily cross the inner mitochondrial membrane and so is transaminated to form aspartate which then exits from the mitochondrion... 

Answer Malate dehydrogenase catalyzes the conversion of malate to oxaloacetate in the citric acid cycle, which takes place in the mitochondrion, and also plays a key role in the transport of reducing equivalents across the inner mitochondrial membrane via the malate-aspartate shuttle (Fig. 19-29). This shuttle requires the presence of malate dehydrogenase in the cytosol and the mitochondrial matrix. 

Answer NADH produced in the cytosol cannot cross the inner mitochondrial membrane, but must be oxidized if glycolysis is to continue. Reducing equivalents from NADH enter the mitochondrion by way of the malate-aspartate shuttle. NADH reduces oxaloacetate to form malate and NAD+, and the malate is transported into the mitochondrion. Cytosolic oxidation of glucose can continue, and the malate is converted back to oxaloacetate and NADH in the mitochondrion (see Fig. 19-29). 

Answer The malate-aspartate shuttle transfers electrons and protons from the cytoplasm into the mitochondrion. Neither NAD+ nor NADH passes through the inner membrane, thus the labeled NAD moiety of [7-14C]NADH remains in the cytosol. The 3H on [4-3H]NADH enters the mitochondrion via the malate-aspartate shuttle (see Fig. 19-29). In the cytosol, [4-3H]NADH transfers its 3H to oxaloacetate to form [3H]malate, which enters the mitochondrion via the malate-a-ketoglutarate transporter, then donates the 3H to NAD+ to form [4-3H]NADH in the matrix. 

Fig. 31.5. Conversion of pyruvate to phosphoenolpyruvate (PEP). Follow the shaded circled numbers on the diagram, starting with the precursors alanine and lactate. The first step is the conversion of alanine and lactate to pyruvate. Pyruvate then enters the mitochondria and is converted to OAA (circle 2) by pyruvate carboxylase. Pyruvate dehydrogenase has been inactivated by both the NADH and acetyl-CoA generated from fatty acid oxidation, which allows oxaloacetate production for gluconeogenesis. The oxaloacetate formed in the mitochondria is converted to either malate or aspartate to enter the cytoplasm via the malate/aspartate shuttle. Once in the cytoplasm the malate or aspartate is converted back into oxaloacetate (circle 3), and phosphoenolpyruvate carboxykinase will convert it to PEP (circle 4). The white circled numbers are alternate routes for exit of carbon from the mitochondrion using the malate/aspartate shuttle. OAA = oxaloacetate FA = fatty acid TG = triacylglycerol.

A more complex and more efficient shutde mechanism is the malate-aspartate shuttle, which has been found in mammalian kidney, liver, and heart. This shuttle uses the fact that malate can cross the mitochondrial membrane, while oxaloacetate cannot. The noteworthy point about this shuttle mechanism is that the transfer of electrons from NADH in the cytosol produces NADH in the mitochondrion. In the cytosol, oxaloacetate is reduced to malate by the cytosolic malate dehydrogenase, accompanied by the oxidation of cytosolic NADH to NAD+ (Figure 20.24). The malate then crosses the mitochondrial membrane. In the mitochondrion, the conversion of malate back to oxaloacetate is catalyzed by the mitochondrial malate dehydrogenase (one of the enzymes of the citric acid cycle). Oxaloacetate is converted to aspartate, which can also cross the mitochondrial membrane. Aspartate is converted to oxaloacetate in the cytosol, completing the cycle of reactions. 

Unlike glycolysis, which occurs strictly in the cell cytosol, gluconeogen-esis involves a complex interaction between the mitochondrion and the cytosol. This interaction is necessitated by the irreversibility of the pyruvate kinase reaction, by the relative impermeability of the inner mitochondrial membrane to oxaloacetate, and by the specific mitochondrial location of pyruvate carboxylase. Compartmentation within the cell has led to the distribution of a number of enzymes (aspartate and alanine aminotransferases, and NAD -malate dehydrogenase) in both the mitochondria and the cytosol. In the classical situation represented by the rat, mouse, or hamster hepatocyte, the indirect "translocation" of oxaloacetate—the product of the pyruvate carboxylase reaction—into the cytosol is effected by the concerted action of these enzymes. Within the mitochondria oxaloacetate is converted either to malate or aspartate, or both. Following the exit of these metabolites from the mitochondria, oxaloacetate is regenerated by essentially similar reactions in the cytosol and is subsequently decarboxylated to P-enolpyruvate by P-enol-pyruvate carboxykinase. Thus the presence of a membrane barrier to oxaloacetate leads to the functioning of the malate-aspartate shuttle as an important element in gluconeogenesis. 

In animals and fungi there is a similar dichotomy. NADPH can be generated by cytosolic malic enzyme which catalyses the reaction malate + NADP+ — pyruvate + COg + NADPH. Cytosolic malate derives from the following successive reactions the pyruvate/ citrate shuttle on the mitochondrial inner membrane takes pyruvate to the mitochondrion in exchange for citrate cytosolic ATP citrate lyase catalyses ATP + citrate + CoA-SH —> acetylCoA (CH3CO-S-C0A) + oxaloacetate and cytosolic malate dehydrogenase, which catalyses NADH + oxaloacetate NAD+ + malate. This scheme provides both acetylCoA and NADPH for subsequent long chain fatty acid synthesis (see section on Fatty acid synthesis ). 

In humans, oxaloacetate must be transported out of the mitochondrion to supply the cytosolic PEPCK. Because there is no mitochondrial carrier for oxaloacetate and its diffusion across the mitochondrial membrane is slow, it is transported as malate or asparate (Figure 15-2). The malate shuttle carries oxaloacetate and reducing equivalents, whereas the aspartate shuttle, which does not require a preliminary reduction step, depends on the availability of glutamate and a-ketoglutarate in excess of tricarboxylic acid (TCA) cycle requirements. 

Aspartate can be transaminated to form oxaloacetate, an intermediate of the citric-acid cycle. As with most transaminations, this is a reversible reaction, and aspartate can also be synthesized by a transamination reaction with glutamate and oxaloacetate to form aspartate and a-ketoglutarate. Therefore, aspartate is a nonessential amino acid. The aminotransferase with aspartate and a-ketoglutarate is particularly active in most tissues and occurs both in the mitochondria and the cytosol. The importance of this reaction is greater than simply forming the oxaloacetate or aspartate. Aspartate aminotransferase is an important reaction in the malate shuttle (see Chapter 11) wherein, reducing power can be transferred from the cytosol to the mitochondrion. Aspartate also plays a role in purine and pyrimidine synthesis and is particularly important in pyrimidine synthesis, where it donates both carbon and... 

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