Oxaloacetate cytoplasm

This enzyme, similar to all C02 assimilating enzymes, contains biotin for a cofactor. Oxaloacetate is released from the mitochondria into the cytoplasm to enter gluconeogenesis. In the cytoplasm, oxaloacetate converts to phosphoenolpyruvate via a reaction catalyzed by phosphoenolpyruvate carboxylase ... 

Pyruvate carboxylase is a mitochondrial enzyme requiring biotin. It is activated by acetyl CoA (fiom p oxidation). The product oxaloacetate (OAA), a citric add cyde intermediate, cannot leave the mitochondria but is reduced to malate that can leave via the malate shuttle. In the cytoplasm, malate is reoxidized to OAA. 

The citrate shuttle transports acetyl CoA groups from the mitochondria to the cytoplasm for fatty acid synthesis. Acetyl CoA combines with oxaloacetate in the mitochondria to form citrate, but rather than continuing in the citric add cycle, citrate is transported into the cytoplasm. Factors that indirectly promote this process indude insuKn and high-energy status. 

In the cytoplasm, citrate lyase splits citrate back into acetyl CoA and oxaloacetate. The oxaloacetate returns to the mitochondria to transport additional acetyl CoA. This process is shown in Figure I-15-I and includes the important malic enzyme. This reaction represents an additional source of cytoplasmic NAD PH in liver and adipose tissue, supplementing that from the HMP shunt. 

The tricarboxylic acid cycle not only takes up acetyl CoA from fatty acid degradation, but also supplies the material for the biosynthesis of fatty acids and isoprenoids. Acetyl CoA, which is formed in the matrix space of mitochondria by pyruvate dehydrogenase (see p. 134), is not capable of passing through the inner mitochondrial membrane. The acetyl residue is therefore condensed with oxaloacetate by mitochondrial citrate synthase to form citrate. This then leaves the mitochondria by antiport with malate (right see p. 212). In the cytoplasm, it is cleaved again by ATP-dependent citrate lyase [4] into acetyl-CoA and oxaloacetate. The oxaloacetate formed is reduced by a cytoplasmic malate dehydrogenase to malate [2], which then returns to the mitochondrion via the antiport already mentioned. Alternatively, the malate can be oxidized by malic enzyme" [5], with decarboxylation, to pyruvate. The NADPH+H formed in this process is also used for fatty acid biosynthesis. 

In the cytoplasm, oxaloacetate is reformed and then converted into phospho-enol pyruvate by a GTP-dependent PEP car-bo)Q/kinase. The subsequent steps up to fructose 1,6-bisphosphate represent the reverse of the corresponding reactions involved in glycolysis. One additional ATP per C3 fragment is used for the synthesis of 1,3-bisphos-phoglycerate. 

The fumarate produced in step [4] is converted via malate to oxaloacetate [6, 7], from which aspartate is formed again by transamination [9]. The glutamate required for reaction [9] is derived from the glutamate dehydrogenase reaction [8], which fixes the second NH4 " in an organic bond. Reactions [6] and [7] also occur in the tricarboxylic acid cycle. However, in urea formation they take place in the cytoplasm, where the appropriate isoenzymes are available. 

In the malate shuttle (left)—which operates in the heart, liver, and kidneys, for example-oxaloacetic acid is reduced to malate by malate dehydrogenase (MDH, [2a]) with the help of NADH+HT In antiport for 2-oxogluta-rate, malate is transferred to the matrix, where the mitochondrial isoenzyme for MDH [2b] regenerates oxaloacetic acid and NADH+HT The latter is reoxidized by complex I of the respiratory chain, while oxaloacetic acid, for which a transporter is not available in the inner membrane, is first transaminated to aspartate by aspartate aminotransferase (AST, [3a]). Aspartate leaves the matrix again, and in the cytoplasm once again supplies oxalo-acetate for step [2a] and glutamate for return transport into the matrix [3b]. On balance, only NADH+H"" is moved from the cytoplasm into the matrix ATP is not needed for this. 

C. Oxaloacetate can also be converted to malate and transported to the cytoplasm for gluconeogenesis under fasting conditions (see Chapter 6). 

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)...

The activities of cytoplasmic enzymes, such as aspartase, glutamate, oxaloacetate, malic enzyme, and alcohol dehydrogenase (ADH) decrease at the onset of anaerobiosis, with occasional transient increases on the... 

Oxaloacetate, the product of the first step in gluconeogenesis, must leave the mitochondrion and enter the cytosol where the subsequent enzyme steps take place. Since the inner mitochondrial membrane is impermeable to oxaloacetate, it is converted to malate by mitochondrial malate dehydrogenase. This leaves the mitochondrion and is converted back to oxaloacetate in the cytosol by cytoplasmic malate dehydrogenase. 

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 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. 

The next reaction occurs in the cytoplasm. Malate is transported to the cytoplasm by a dicarboxylate carrier which is specific for malate, succinate, and fumarate and which requires the entry of P, or one of these dicarboxylate anions. Cytoplasmic malate is then converted to oxaloacetate by cytoplasmic malate dehydrogenase ... 

The preceding two reactions are necessary to achieve the overall result of transporting oxaloacetate to the cytoplasm from the mitochondria, as there is no direct mechanism for this. Cytoplasmic oxaloacetate is then converted irreversibly to phosphoenolpyruvate by way of phosphoenolpyruvate carboxykinase, a cytoplasmic enzyme that operates only when the ATP concentration is high ... 

The enzymes are, respectively (1) mitochondrial malate dehydrogenase (2) cytoplasmic malate dehydrogenase (3) phosphoenolpyruvate carboxykinase (4) pyruvate kinase and pyruvate dehydrogenase. The acetyl-CoA could then condense with oxaloacetate (produced from a second molecule of aspartate) to yield citrate. Aspartate could, therefore, continue to supply acctyl-CoA, which would continue to fuel the citric acid cycle. 

Pyruvate carboxylase is a mitochondrial enzyme, vhereas the other enzymes of gluconeogenesis are cytoplasmic. Oxaloacetate, the product of the pyruvate carboxylase reaction, is reduced to malate inside the mitochondrion for transport to the cytosol. The reduction is accomplished by an NADH-linked malate dehydrogenase. When malate has been transported across the mitochondrial membrane, it is reoxidized to oxaloacetate by an NAD+-linked malate dehydrogenase in the cytosol (Figure 16.28). 

I- oxaloacetate + ADP + Pj. This reaction takes place in the cytoplasm and is a source of acetyl-CoA for fatty acid biosynthesis. 

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 ... 

Oxaloacetate Is Shuttled into the Cytoplasm and Converted into Phosphoenolpyruvate... 

Pyruvate carboxylase is a mitochondrial enzyme, whereas the other enzymes of gluconeogenesis are present primarily in the cytoplasm. Oxaloacetate, the product of the pyruvate carboxylase reaction, must thus be transported to the cytoplasm to complete the pathway. Oxaloacetate is transported from a mitochondrion in the form of malate oxaloacetate is reduced to malate inside the mitochondrion by an NADH-linked malate dehydrogenase. After malate has been transported across the mitochondrial membrane, it is reoxidized to oxaloacetate by an NAD -linked malate dehydrogenase in the cytoplasm (Figure 16.26). The formation of oxaloacetate from malate also provides NADH for use in subsequent steps in gluconeogenesis. Finally, oxaloacetate is simultaneously decarboxylated and phospho-ry lated by phosphoenolpyruvate carboxy kinase to generate phosphoenol pyruvate. The phosphoryl donor is GTP. The GO2 that was added to pyruvate by pyruvate carboxylase comes off in this step. 

Figure 16.26 Compartmental cooperation. Oxaloacetate used in the cytoplasm for gluconeogenesis is formed in the mitochondrial matrix by the carboxylation of pyruvate. Oxaloacetate leaves the mitochondrion by a specific transport system not shown) in the form of malate, which is reoxidized to oxaloacetate in the cytoplasm.

Thus, acetyl CoA and oxaloacetate are transferred from mitochondria to the cytoplasm at the expense of the hydrolysis of a molecule of ATP-... 

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