Malate-oxaloacetate shuttle

Examples of such intra cellular membrane transport mechanisms include the transfer of pyruvate, the symport (exchange) mechanism of ADP and ATP and the malate-oxaloacetate shuttle, all of which operate across the mitochondrial membranes. Compartmentalization also allows the physical separation of metabolically opposed pathways. For example, in eukaryotes, the synthesis of fatty acids (anabolic) occurs in the cytosol whilst [3 oxidation (catabolic) occurs within the mitochondria. 

NADH in the cytoplasm via a mitochondrial transhydrogenase or they could export malate which could be oxidized by the cytoplasmic malate dehydrogenase to generate NADH. This would permit the establishment of a direct malate-oxaloacetate shuttle or an indirect shuttle via aspartate. 

Corn NADP-MDH activation state is strongly dependent on the reduction state of this probably dimeric protein the reduction of all the subunits of the molecule is necessary to get an active form. This peculiarity, added to the already described inhibition of the activation by NADP (13,14) makes the enzyme very sensitive to small variations In the reducing power. As a consequence, it might be expected that NADP - MDH, which is Involved In the export of reducing equivalents from the chloroplast to the cytoplasm, via the malate / oxaloacetate shuttle, can be active only under conditions where the reducing power inside the chloroplast is very high. This situation has actually been observed in intact chloroplasts where maximal activation of the enzyme was obtained only in the presence of uncouplers (15). 

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. 

FIGURE 21.34 The malate (oxaloacetate)-aspartate shuttle, which operates across the inner mitochondrial membrane. 

The second electron shuttle system, called the malate-aspartate shuttle, is shown in Figure 21.34. Oxaloacetate is reduced in the cytosol, acquiring the electrons of NADH (which is oxidized to NAD ). Malate is transported across the inner membrane, where it is reoxidized by malate dehydrogenase, converting NAD to NADH in the matrix. This mitochondrial NADH readily enters the electron transport chain. The oxaloacetate produced in this reaction cannot cross the inner membrane and must be transaminated to form aspartate, which can be transported across the membrane to the cytosolic side. Transamination in the cytosol recycles aspartate back to oxaloacetate. In contrast to the glycerol phosphate shuttle, the malate-aspartate cycle is reversible, and it operates as shown in Figure 21.34 only if the NADH/NAD ratio in the cytosol is higher than the ratio in the matrix. Because this shuttle produces NADH in the matrix, the full 2.5 ATPs per NADH are recovered. 

Malate is not the only form in which C4 compounds are exported from mitochondria. Much oxaloacetate is combined with acetyl-CoA to form citrate the latter leaves the mitochondria and is cleaved by the ATP-dependent citrate-cleaving enzymes (Eq. 13-39). This, in effect, exports both acetyl-CoA (needed for lipid synthesis) and oxaloacetate which is reduced to malate within the cytoplasm. Alternatively, oxaloacetate may be transaminated to aspartate. The aspartate, after leaving the mitochondria, may be converted in another transamination reaction back to oxaloacetate. All of these are part of the nonequilibrium process by which C4 compounds diffuse out of the mitochondria before completing the reaction sequence of Eq. 17-46 and entering into other metabolic processes. Note that the reaction of Eq. 17-46 leads to the export of reducing equivalents from mitochondria, the opposite of the process catalyzed by the malate-aspartate shuttle which is discussed in Chapter 18 (Fig. 18-18). The two processes are presumably active under different conditions. 

The interconversion of o -ketoglutarate to glutamate involves the malate-aspartate shutde. This shuttle translocates a-ketoglutarate from mitochondria into the cytoplasm and then converts it to glutamate by the catalytic action of aspartate aminotransferase (McKenna et al., 2006). As part of the malate-aspartate shuttle, NADH is oxidized during reduction of oxaloacetate to malate. Malate diffuses across the outer mitochondrial membrane (Fig. 1.6). From the intermembrane space, the malate-a-ketoglutarate antiporter in the inner membrane transports malate into the matrix. For every malate molecule entering the matrix compartment, one molecule of... 

Fig. 1.6 Reactions of the malate-aspartate shuttle showing the transport of reducing equivalents from cytoplasm to mitochondria. a-KG, a-Ketoglutarate Asp, aspartate Glu, glutamate OAA, oxaloacetate aspartate aminotransferase (1) and malate dehydrogenase (2)...

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. 

Malate-aspartate shuttle for the transport of cytoplasmic reducing equivalents across the inner membrane of mitochondria. Malate, which carries the reducing equivalents, is oxidized to oxaloacetate with the generation of NADH in the matrix. To complete the unidirectional cycle, oxaloacetate is transported out of the matrix as aspartate. Mai = malate OAA = oxaloacetate a-KG = a-ketoglutarate Glu = glutamate ... 

Gluconeogenesis from pyruvate is not equal to the reverse process of glycolytic degradation of glucose to this 3-carbon intermediate. The glycolytic pathway and the gluconeogenetic pathway deviate at three steps. The conversion of pyruvate to PEP is not mediated by pyruvate kinase due to the irreversible nature of this metabolic step. Pyruvate, derived from either lactate or alanine, is converted to oxaloacetate in the mitochondrial matrix. This step is catalyzed by pyruvate carboxylase. Oxaloacetate per se cannot pass the mitochondrial inner membrane. However, with the use of the malate-aspartate shuttle, the 4-carbon skeleton of oxaloacetate can be transferred into the cytoplasmic compartment. Then oxaloacetate is converted to PEP by the action of PEP carboxykinase (Figure 1). 

Although the malate-aspartate shuttle (Figure 10.17b) is a more complicated mechanism than the glycerol phosphate shuttle, it is more energy efficient. The shuttle begins with the reduction of cytoplasmic oxaloacetate to malate by NADH. 

FIGURE 19-27 Malate-aspartate shuttle. This shuttle for transporting reducing equivalents from cytosolic NADH Into the mitochondrial matrix is used in liver, kidney, and heart. (T) NADH in the cytosol (intermembrane space) passes two reducing equivalents to oxaloacetate, producing malate. (2) Malate crosses the inner membrane via the malate-a-ketoglutarate transporter. In the matrix, malate passes... 

Figure 15.11b shows the malate/aspartate shuttle system, which is particularly active in liver and heart. It uses malate, aspartate, and oxaloacetate to shuttle cytoplasmic electrons from NADH into the mitochondrial matrix. In this shuttle, NADH reduces oxaloacetate to malate, which travels through an inner membrane transport system that ultimately exchanges the malate for an ot-ketoglutarate. To do... 

Fig. 22.8. Malate-aspartate shuttle. NADH produced by glycolysis reduces oxaloacetate (OAA) to malate, which crosses the mitochondrial membrane and is reoxidized to OAA. The mitochondrial NADH donates electrons to the electron transpwrt chain, with 2.5 ATPs generated for each NADH. To complete the shuttle, oxaloacetate must return to the cytosol, although it cannot be directly transported on a translocase. Instead, it is transaminated to aspartate, which is then transported out to the cytosol, where it is transaminated back to oxaloacetate. The translocators exchange compounds in such a way that the shuttle is completely balanced. TA = transamination reaction. a-KG = a-ketoglutarate.

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.

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