Oxaloacetate synthesis from pyruvate

Biotin (5) is the coenzyme of the carboxylases. Like pyridoxal phosphate, it has an amide-type bond via the carboxyl group with a lysine residue of the carboxylase. This bond is catalyzed by a specific enzyme. Using ATP, biotin reacts with hydrogen carbonate (HCOa ) to form N-carboxybiotin. From this activated form, carbon dioxide (CO2) is then transferred to other molecules, into which a carboxyl group is introduced in this way. Examples of biotindependent reactions of this type include the formation of oxaloacetic acid from pyruvate (see p. 154) and the synthesis of malonyl-CoA from acetyl-CoA (see p. 162). 

Thus, citrate not only modulates the rate of fatty acid synthesis but also provides carbon atoms for the synthesis. The oxaloacetate formed from pyruvate may eventually be converted (via malate) to glucose by the gluconeogenic pathway. The glucose oxidized via the pentose phosphate pathway augments fatty acid synthesis by providing NADPH. Pyruvate generated from oxaloacetate can enter mitochondria and be converted to oxaloacetate, which is required for the formation of citrate. 

Synthesis of PEP. PEP synthesis from pyruvate requires two enzymes pyruvate carboxylase and PEP carboxykinase. Pyruvate carboxylase, found within mitochondria, converts pyruvate to oxaloacetate (OAA) ... 

Kraeva NI and Vorobjeva LI (1981b) Superoxide dismutase, catalase, and peroxidase of propionic acid bacteria. Mikrobiologiya 50 813-817 Krebs flA and Eggleston LV (1941) Biological synthesis of oxaloacetic acid from pyruvic acid and carbon dioxide. II. The mechanism of carbon dioxide fixation in propionic acid bacteria. Biochem J 35 676-687... 

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. 

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... 

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. 

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. 

Pyruvate carboxylase catalyzes the synthesis of oxaloacetate from pyruvate and CO2. 

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 first "roadblock" to overcome in the synthesis of glucose from pyruvate is the irreversible conversion in glycolysis of pyruvate to phosphoenolpyruvate (PEP) by pyruvate kinase. In gluconeogenesis, pyruvate is first carboxylated by pyruvate carboxylase to oxaloacetate (OAA), which is then converted to PEP by the action of PEP-carboxykinase (Figure 10.3). 

Claisen reactions involving acetyl-CoA are made even more favourable by first converting acetyl-CoA into malonyl-CoA by a carboxylation reaction with CO2 using ATP and the coenzyme biotin (Figure 2.9). ATP and CO2 (as bicarbonate, HC03-) form the mixed anhydride, which car-boxy lates the coenzyme in a biotin-enzyme complex. Fixation of carbon dioxide by biotin-enzyme complexes is not unique to acetyl-CoA, and another important example occurs in the generation of oxaloacetate from pyruvate in the synthesis of glucose from non-carbohydrate sources... 

Answer Anaplerotic reactions replenish intermediates in the citric acid cycle. Net synthesis of a-ketoglutarate from pyruvate occurs by the sequential actions of (1) pyruvate carboxylase (which makes extra molecules of oxaloacetate), (2) pyruvate dehydrogenase, and the citric acid cycle enzymes (3) citrate synthase, (4) aconitase, and (5) isocitrate dehydrogenase ... 

The formation of acetyl-CoA from pyruvate in animals is via the pyruvate dehydrogenase complex, which catalyzes the irreversible decarboxylation reaction. Carbohydrate is synthesized from oxaloacetate, which in turn is synthesized from pyruvate via pyruvate carboxylase. Since the pyruvate dehydrogenase reaction is irreversible, acetyl-CoA cannot be converted to pyruvate, and hence animals cannot realize a net gain of carbohydrate from acetyl-CoA. Because plants have a glyoxylate cycle and animals do not, plants synthesize one molecule of succinate and one molecule of malate from two molecules of acetyl-CoA and one of oxaloacetate. The malate is converted to oxaloacetate, which reacts with another molecule of acetyl-CoA and thereby continues the reactions of the glyoxylate cycle. The succinate is also converted to oxaloacetate via the enzymes of the citric acid cycle. Thus, one molecule of oxaloacetate is diverted to carbohydrate synthesis and, therefore, plants are able to achieve net synthesis of carbohydrate from acetyl-CoA. 

Fatty acids are predominantly formed in the liver and adipose tissne, as well as the mammary glands during lactation. Fatty acid synthesis occurs in the cytosol (fatty acid oxidation occurs in the mitochondria compartmentalisation of the two pathways allows for distinct regulation of each). Oxidation or synthesis of fats utilises an activated two-carbon intermediate, acetyl-CoA, but the acetyl-CoA in fat synthesis exists temporarily bound to the enzyme complex as malonyl-CoA. Acetyl-CoA is mostly produced from pyruvate (pyruvate dehydrogenase) in the mitochondria it is condensed with oxaloacetate to form citrate, which is then transported into the cytosol and broken down to yield acetyl-CoA and oxaloacetate (ATP citrate lyase). 

Pyruvate Carboxylase Pyruvate carboxylase catalyzes the car-boxylation of pyruvate to oxaloacetate - both the first committed step of gluconeogenesis from pyruvate and also an important anaplerotic reaction, permitting repletion of tricarboxylic acid cycle intermediates and hence fatty acid synthesis. The mammalian enzyme is activated aUosterically by acetyl CoA, which accumulates when there is a need for increased activity of pyruvate carboxylase to synthesize oxaloacetate to permit increased citric acid cycle activity or for gluconeogenesis (Attwood, 1995 Jitrapakdee and Wallace, 1999). 

The synthesis of palmitate requires the input of 8 molecules of acetyl CoA, 14 molecules of NADPH, and 7 molecules of ATP. Fatty acids are synthesized in the cytosol, whereas acetyl CoA is formed from pyruvate in mitochondria. Hence, acetyl CoA must be transferred from mitochondria to the cytosol. Mitochondria, however, are not readily permeable to acetyl CoA. Recall that carnitine carries only long-chain fatty acids. The barrier to acetyl CoA is bypassed by citrate, which carries acetyl groups across the inner mitochondrial membrane. Citrate is formed in the mitochondrial matrix by the condensation of acetyl CoA with oxaloacetate (Figyu-e 22.25). When present at high levels, citrate is transported to the cytosol, where it is cleaved hy ATP-citrate lyase. 

The nonessential amino acids are synthesized by quite simple reactions, whereas the pathways for the formation of the essential amino acids are quite complex. For example, the nonessential amino acids alanine and aspartate are synthesized in a single step from pyruvate and oxaloacetate, respectively. In contrast, the pathways for the essential amino acids require from 5 to 16 steps (Figure 24.8). The sole exception to this pattern is arginine, inasmuch as the synthesis of this nonessential amino acid de novo requires 10 steps. Typically, though, it is made in only 3 steps from ornithine as part of the urea cycle. Tyrosine, classified as a nonessential amino acid because it can be synthesized in 1 step from phenylalanine, requires 10 steps to be synthesized from scratch and is essential if phenylalanine is not abundant. We begin with the biosynthesis of nonessential amino acids. 

A third fate of pyruvate is its carboxylation to oxaloacetate inside mitochondria, the first step in gluconeogenesis. This reaction and the subsequent conversion of oxaloacetate into phosphoenolpyruvate bypass an irreversible step of glycolysis and hence enable glucose to be synthesized from pyruvate. The carboxylation of pyruvate is also important for replenishing intermediates of the citric acid cycle. Acetyl CoA activates pyruvate carboxylase, enhancing the synthesis of oxaloacetate, when the citric acid cycle is slowed by a paucity of this intermediate. 

It is important to note that animals are unable to effect the net synthesis o/glu-cose from fatty acids. Specifically, acetyl Go A cannot be converted into pyruvate or oxaloacetate in animals. Recall that the reaction that generates acetyl CoA from pyruvate is irreversible (p. 477). The two carbon atoms of the acetyl group of acetyl CoA enter the citric acid cycle, but two carbon atoms leave the cycle in the decarboxylations catalyzed by isocitrate dehydrogenase and a-ke-toglutarate dehydrogenase. Consequently, oxaloacetate is regenerated, hut it is not formed de novo when the acetyl unit of acetyl CoA is oxidized by the citric acid cycle. In contrast, plants have two additional enzymes enabling them to convert the carbon atoms of acetyl CoA into oxaloacetate (Section 18.4.). 

It is likely that nature has found more ways to catalyze the caiboxylation of pyruvate and its reverse, the decarboxylation of oxaloacetate, than any other reaction. In spite of the fact that the overall equilibrium strongly favors decarboxylation, a variety of enzymes catalyze the synthesis of oxaloacetate from pyruvate. We will examine a number of these enzymes in the following sections. 

Finally, oxaloacetate is simultaneously decarboxylated and phosphorylated by phosphoenolpyruvate carboxykinase in the cytosol. The CO2 that was added to pyruvate by pyruvate carboxylase comes off in this step. Recall that, in glycolysis, the presence of a phosphoryl group traps the unstable enol isomer of pyruvate as phosphoenolpyruvate (Section 16.1.7). In gluconeogenesis, the formation of the unstable enol is driven by decarboxylation—the oxidation of the carboxylic acid to CO2—and trapped by the addition of a phosphate to carbon 2 from GTP. The two-step pathway for the formation of phosphoenolpyruvate from pyruvate has a AG° of + 0.2 kcal mol ( + 0.13 kj moP ) in contrast with +7.5 kcal mol ( + 31 kj mol ) for the reaction catalyzed by pyruvate kinase. The much more favorable AG° for the two-step pathway results from the use of a molecule of ATP to add a molecule of CO2 in the carboxylation step that can be removed to power the formation of phosphoenolpyruvate in the decarboxylation step. Decarboxylations often drive reactions otherwise highly endergonic. This metabolic motif is used in the citric acid cycle (Section IS.x.x), the pentose phosphate pathway (Section 17.x.x), and fatty acid synthesis (Section 22.x.x). 

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