Oxaloacetate synthesis

OVERSATURATION OXALOACETATE DECARBOXYLASE Oxaloacetate, synthesis in gluconeogenesis, PYRUVATE CARBOXYLASE PHOSPHOENOLPYRUVATE CARBOXYKI-NASE (PYROPHOSPHATE)... 

B. Oxaloacetate synthesis is also needed when mitochondria are formed during cell growth and division. 

High acetyl CoA levels from 3-oxidation of fatty acids in liver cells inhibit the pyruvate dehydrogenase complex and activate pyruvate carboxylase, which increases oxaloacetate synthesis. 

The solution to this problem is to have an alternative pathway for oxaloacetate synthesis that can produce enough oxaloacetate to supply the anabolic and catabolic requirements of the cell. Although bacteria and plants have several mechanisms, the only way that mammalian cells can produce more oxaloacetate is by the carboxylation of pyruvate, a reaction that is also important in gluconeogenesis. This reaction is... 

Since Corynebacterium glutamicum does not possess a PEPsynthetase, no [3- CjPEP isotopomers can be formed from pyruvate. Thus, any [3- C]oxaloac-etate isotopomers must result from the action of PyrCx in vivo and their relative abundance allows to quantitate the relative contributions of PEPCx and PyrCx to oxaloacetate synthesis. In the aspartate derived from oxaloacetate a content of isotopomers labelled in C-3 but not in C-2 similarly high as that in pyruvate was found (Fig. 8 c), suggesting synthesis of oxaloacetate from pym-... 

In 1937 Krebs found that citrate could be formed in muscle suspensions if oxaloacetate and either pyruvate or acetate were added. He saw that he now had a cycle, not a simple pathway, and that addition of any of the intermediates could generate all of the others. The existence of a cycle, together with the entry of pyruvate into the cycle in the synthesis of citrate, provided a clear explanation for the accelerating properties of succinate, fumarate, and malate. If all these intermediates led to oxaloacetate, which combined with pyruvate from glycolysis, they could stimulate the oxidation of many substances besides themselves. (Kreb s conceptual leap to a cycle was not his first. Together with medical student Kurt Henseleit, he had already elucidated the details of the urea cycle in 1932.) The complete tricarboxylic acid (Krebs) cycle, as it is now understood, is shown in Figure 20.4. 

FIGURE 20.23 Export of citrate from mitochondria and cytosolic breakdown produces oxaloacetate and acetyl-CoA. Oxaloacetate is recycled to malate or pyruvate, which re-enters the mitochondria. This cycle provides acetyl-CoA for fatty acid synthesis in the cytosol. 

Succinyl-CoA derived from propionyl-CoA can enter the TCA cycle. Oxidation of succinate to oxaloacetate provides a substrate for glucose synthesis. Thus, although the acetate units produced in /3-oxidation cannot be utilized in glu-coneogenesis by animals, the occasional propionate produced from oxidation of odd-carbon fatty acids can be used for sugar synthesis. Alternatively, succinate introduced to the TCA cycle from odd-carbon fatty acid oxidation may be oxidized to COg. However, all of the 4-carbon intermediates in the TCA cycle are regenerated in the cycle and thus should be viewed as catalytic species. Net consumption of succinyl-CoA thus does not occur directly in the TCA cycle. Rather, the succinyl-CoA generated from /3-oxidation of odd-carbon fatty acids must be converted to pyruvate and then to acetyl-CoA (which is completely oxidized in the TCA cycle). To follow this latter route, succinyl-CoA entering the TCA cycle must be first converted to malate in the usual way, and then transported from the mitochondrial matrix to the cytosol, where it is oxida-... 

The acetyl-CoA derived from amino acid degradation is normally insufficient for fatty acid biosynthesis, and the acetyl-CoA produced by pyruvate dehydrogenase and by fatty acid oxidation cannot cross the mitochondrial membrane to participate directly in fatty acid synthesis. Instead, acetyl-CoA is linked with oxaloacetate to form citrate, which is transported from the mitochondrial matrix to the cytosol (Figure 25.1). Here it can be converted back into acetyl-CoA and oxaloacetate by ATP-citrate lyase. In this manner, mitochondrial acetyl-CoA becomes the substrate for cytosolic fatty acid synthesis. (Oxaloacetate returns to the mitochondria in the form of either pyruvate or malate, which is then reconverted to acetyl-CoA and oxaloacetate, respectively.)... 

Glycolysis, the pentose phosphate pathway, and fatty acid synthesis are all found in the cytosol. In gluconeo-genesis, substrates such as lactate and pyruvate, which are formed in the cytosol, enter the mitochondrion to yield oxaloacetate before formation of glucose. 

Citrate is isomerized to isocitrate by the enzyme aconitase (aconitate hydratase) the reaction occurs in two steps dehydration to r-aconitate, some of which remains bound to the enzyme and rehydration to isocitrate. Although citrate is a symmetric molecule, aconitase reacts with citrate asymmetrically, so that the two carbon atoms that are lost in subsequent reactions of the cycle are not those that were added from acetyl-CoA. This asymmetric behavior is due to channeling— transfer of the product of citrate synthase directly onto the active site of aconitase without entering free solution. This provides integration of citric acid cycle activity and the provision of citrate in the cytosol as a source of acetyl-CoA for fatty acid synthesis. The poison fluo-roacetate is toxic because fluoroacetyl-CoA condenses with oxaloacetate to form fluorocitrate, which inhibits aconitase, causing citrate to accumulate. 

Aminotransferase (transaminase) reactions form pymvate from alanine, oxaloacetate from aspartate, and a-ketoglutarate from glutamate. Because these reactions are reversible, the cycle also serves as a source of carbon skeletons for the synthesis of these amino acids. Other amino acids contribute to gluconeogenesis because their carbon skeletons give rise to citric acid cycle... 

The synthesis of this aminoquinoline starts with one of the standard sequences for preparation of 4-hydroxyquinolines, i.e., with the formation of the Shiff base (5) from the appropriately substituted aniline and diethyl oxaloacetate. Thermal cycliza-tion gives the quinolone (6) this then spontaneously tautomerizes to the enol form (7). Saponification followed by decarboxylation gives the desired quinolol... 

The Jirst indirect route in glucose synthesis involves the formation of phosphoenolpyruvate from pyruvate without the intervention of pyruvate kinase. This route is catalyzed by two enzymes. At first, pyruvate is converted into oxaloacetate. This reaction occurs in the mitochondria as the pyruvate molecules enter them, and is catalyzed by pyruvate carboxylase according to the scheme... 

It was observed that glutamate and aspartate are diverted predominantly to the synthesis of cell substance rather than to the formation of oxalate. It is not inconsistent to see oc-ketoglutarate being formed from glutamate, while no oxaloacetic acid can be detected in the medium containing aspartate, as the oxaloacetic acid is known to be extremely unstable (2), (62), (Hi). The relatively low yields of oxalic acid, derived... 

Another observation on oxalate formation is that other a-keto acids, such as oxalosuccinic acid (74) and a-ketoglutaric acid (106) do not seem to yield oxalate directly but indirectly (123). This appears to be due to the fact that only oxaloacetic acid can function as an acetate donor. In this connection the intervention of Coenzyme A may be considered, since it is reported to function in the acetylation of sulfanilamide and choline (73) and recently was shown to take part in the enzymatic synthesis of citric acid. This concept may be illustrated as follows ... 

There are two unusual aspects to the regulation of gluconeogenesis. The first step in the reaction, the formation of oxaloacetate from pyruvate, requires the presence of acetyl-CoA. This is a check to make sure that the TCA cycle is adequately fueled. If there s not enough acetyl-CoA around, the pyruvate is needed for energy and gluconeogenesis won t happen. However, if there s sufficient acetyl-CoA, the pyruvate is shifted toward the synthesis of glucose. 

Calculating energy costs for the synthesis of a CK, fatty acid from acetyl-CoA is not as simple as you might first think. The major complication is that acetyl-CoA is made in the mitochondria, but fatty acid synthesis occurs in the cytosol—acetyl-CoA can t cross the mitochondrial membrane. Acetyl-CoA gets out of the mitochondria disguised as citrate. The acetyl-CoA is condensed with oxaloacetate to give citrate, and the citrate leaves the mitochondria. In the cytosol, the citrate is cleaved by an ATP-dependent citrate lyase into acetyl-CoA and oxaloacetate ... 

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. 

Substrate availability for certain reactions can be optimized by anaplerotic ( topping-up ) reactions. For example, citrate synthase is a key control point of the TCA cycle. The co-substrates of citrate synthase are acetyl-CoA and oxaloacetate (OAA) and clearly, restriction in the availability of either substrate will decrease the rate of the citrate synthase reaction. Suppose, for example, a situation arises when acetyl-CoA concentration is significantly higher than that of OAA, the concentration of the latter can be topped-up and the concentration of acetyl-CoA simultaneously reduced by diverting some of the pyruvate away from acetyl-CoA synthesis (via pyruvate dehydrogenase) to OAA synthesis (via pyruvate carboxylase) as shown in Figure 3.1. The net effect is to balance the relative concentrations of the two co-substrates and thus to promote citrate synthase activity. 

We should note at this point that the TCA cycle is more than just a means of producing NADH for oxidative phosphorylation. The pathway also provides a number of useful intermediates for other, often synthetic, pathways. For example, citrate is the starting substance for fat synthesis (Chapter 9) succinyl-CoA is required for haem production and 2-oxoglutarate and oxaloacetate in particular are involved with amino acid and pyrimidine metabolism. Pathways which have dual catabolic/anabolic functions are referred to as amphibolic . 

In common with cholesterol synthesis described in the next section, fatty acids are derived from glucose-derived acetyl-CoA. In the fed state when glucose is plentiful and more than sufficient acetyl-CoA is available to supply the TCA cycle, carbon atoms are transported out of the mitochondrion as citrate (Figure 6.8). Once in the cytosol, citrate lyase forms acetyl-CoA and oxaloacetate (OAA) from the citrate. The OAA cannot re-enter the mitochondrion but is converted into malate by cytosolic malate dehydrogenase (cMDH) and then back into OAA by mitochondrial MDH (mMDH) Acetyl-CoA remains in the cytosol and is available for fatty acid synthesis. 

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