19.4 The Citric Acid Cycle Is Regulated

✓ 5 Describe how the citric acid cycle is regulated.

✓ 6 Describe the role of the citric acid cycle in anabolism.

The citric acid cycle is the final common pathway for the aerobic oxidation of fuel molecules. Moreover, as we will see shortly and repeatedly elsewhere in our study of biochemistry, the cycle is an important source of building blocks for a host of biomolecules. As befits its role as the metabolic hub of the cell, entry into the cycle and the rate of the cycle itself are controlled at several stages. We have seen that the pyruvate dehydrogenase complex, the link between glycolysis and the citric acid cycle, is a regulatory site controlling the metabolism of pyruvate by the citric acid cycle.

The Citric Acid Cycle Is Controlled at Several Points

Figure 19.7: Control of the citric acid cycle. The citric acid cycle is regulated primarily by the concentrations of ATP and NADh. The key control points are the enzymes isocitrate dehydrogenase and α-ketoglutarate dehydrogenase.

The regulation of pyruvate dehydrogenase is a crucial site of control, modulating the conversion of glucose-derived pyruvate into acetyl CoA. However, pyruvate is not the only source of acetyl CoA. Indeed, acetyl CoA derived from fat breakdown enters the cycle directly (Chapter 27). Consequently, the rate of the citric acid cycle itself also must be precisely controlled to meet an animal cell’s needs for ATP (Figure 19.7). The primary control points are the allosteric enzymes isocitrate dehydrogenase and α-ketoglutarate dehydrogenase.

For many tissues, such as the liver, the ATP needs are approximately constant on the time scale of minutes to hours. Thus, for these tissues, the citric acid cycle is operating at a constant rate. But, just as driving at a constant speed usually requires touching the gas pedal and the break occasionally, the rate of the citric acid cycle must be “tuned” with increases and decreases. What signals regulate the rate of the cycle? Isocitrate dehydrogenase is allosterically stimulated by ADP, which signifies the need for more energy. In contrast, NADH, which signals the presence of high-transfer-potential electrons, inhibits isocitrate dehydrogenase. Likewise, ATP, the ultimate end product of fuel catabolism, is inhibitory.

A second control site in the citric acid cycle is α-ketoglutarate dehydrogenase, which catalyzes the rate-limiting step in the citric acid cycle. Some aspects of this enzyme’s control are like those of the pyruvate dehydrogenase complex, as might be expected from the similarity of the two enzymes. α-Ketoglutarate dehydrogenase is inhibited by the products of the reaction that it catalyzes, succinyl CoA and NADH. In addition, α-ketoglutarate dehydrogenase is inhibited by high levels of ATP. Thus, the rate of the cycle is decreased when the cell has high levels of ATP and NADH. α-Ketoglutarate dehydrogenase deficiency is observed in a number of neurological disorders, including Alzheimer disease. Importantly, several other enzymes in the cycle require NAD+ or FAD. These enzymes will function only when the energy charge is low, because only then will NAD+ and FAD be available.

Although different regulatory mechanisms might be expected in skeletal muscle, where the energy needs can vary immensely and rapidly, this does not appear to be the case. While the rate of the citric acid cycle will increase 100-fold during intense exercise, the rate of the cycle in muscle is under the control of the same two enzymes as in liver.

The use of isocitrate dehydrogenase and α-ketoglutarate dehydrogenase as control points integrates the citric acid cycle with other pathways and highlights the central role of the citric acid cycle in metabolism. For instance, the inhibition of isocitrate dehydrogenase leads to a buildup of citrate, because the interconversion of isocitrate and citrate is readily reversible under intracellular conditions. Citrate can be transported to the cytoplasm where it signals phosphofructokinase to halt glycolysis and where it can serve as a source of acetyl CoA for fatty acid synthesis (Chapter 28). The α-ketoglutarate that accumulates when α-ketoglutarate dehydrogenase is inhibited can be used as a precursor for several amino acids and the purine bases (Chapter 30).

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In many bacteria, the funneling of two-carbon fragments into the cycle also is controlled. The synthesis of citrate from oxaloacetate and acetyl CoA carbon units is a key control point in these organisms. ATP is an allosteric inhibitor of citrate synthase. The effect of ATP is to increase the value of KM for acetyl CoA. Thus, as the level of ATP increases, less of this enzyme reacts with acetyl CoA and so less citrate is formed.

The Citric Acid Cycle Is a Source of Biosynthetic Precursors

Thus far, we have focused on the citric acid cycle as the major degradative pathway for the generation of ATP. As a major metabolic hub of the cell, the citric acid cycle also provides intermediates for biosyntheses (Figure 19.8). For example, most of the carbon atoms in porphyrins, the precursors to the heme groups in hemoglobin and myoglobin, come from succinyl CoA. Many of the amino acids are derived from α-ketoglutarate and oxaloacetate. These biosynthetic processes will be considered in subsequent chapters.

Figure 19.8: Biosynthetic roles of the citric acid cycle. Intermediates are drawn off for biosyntheses (shown by red arrows) when the energy needs of the cell are met. Intermediates are replenished by the formation of oxaloacetate from pyruvate (green arrow).

The Citric Acid Cycle Must Be Capable of Being Rapidly Replenished

The citric acid cycle is crucial for generating biological energy and is a source of building blocks for biosynthetic reactions. This dual use presents a problem. Suppose that much oxaloacetate is converted into glucose and, subsequently, the energy needs of the cell rise. The citric acid cycle will operate at a reduced capacity unless new oxaloacetate is formed, because acetyl CoA cannot enter the cycle unless it condenses with oxaloacetate. Even though oxaloacetate is recycled, a minimal level must be maintained to allow the cycle to function. Citric acid cycle intermediates must be replenished if any are drawn off for biosyntheses.

Figure 19.9: Pyruvate carboxylase replenishes the citric acid cycle. The rate of the citric acid cycle increases during exercise, requiring the replenishment of oxaloacetate and acetyl CoA. Oxaloacetate is replenished by its formation from pyruvate (green arrow). Acetyl CoA can be produced from the metabolism of both pyruvate and fatty acids.

How is oxaloacetate replenished? Mammals lack the enzymes for the net conversion of acetyl CoA into oxaloacetate or any other citric acid cycle intermediate. Rather, oxaloacetate is formed by the carboxylation of pyruvate, in a reaction catalyzed by the biotin-dependent enzyme pyruvate carboxylase (Figure 19.9):

Recall that this enzyme plays a crucial role in gluconeogenesis. It is active only in the presence of acetyl CoA, which signifies the need for more oxaloacetate. If the energy charge is high, oxaloacetate is converted into glucose. If the energy charge is low, oxaloacetate replenishes the citric acid cycle. The synthesis of oxaloacetate by the carboxylation of pyruvate is an example of an anaplerotic reaction (of Greek origin, meaning to “fill up”), a reaction that leads to the net synthesis, or replenishment, of pathway components. Note that, because the citric acid cycle is a cycle, it can be replenished by the generation of any of the intermediates. For instance, the removal of the nitrogen groups from glutamate and aspartate yield α-ketoglutarate and oxaloacetate, respectively. Glutamine is an especially important source of citric acid cycle intermediates in rapidly growing cells, including cancer cells. Glutamine is converted into glutamate and then into α-ketoglutarate.

!quickquiz! QUICK QUIZ 2

Why is acetyl CoA an especially appropriate activator for pyruvate carboxylase?

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!clinic! CLINICAL INSIGHT: Defects in the Citric Acid Cycle Contribute to the Development of Cancer

Four enzymes crucial to cellular respiration are known to contribute to the development of cancer: succinate dehydrogenase, fumarase, pyruvate dehydrogenase kinase, and isocitrate dehydrogenase Mutations that alter the activity of the first three of these enzymes enhance aerobic glycolysis. In aerobic glycolysis, cancer cells preferentially metabolize glucose to lactate even in the presence of oxygen. Defects in all of these enzymes have a common biochemical link: the transcription factor hypoxia inducible factor 1 (HIF-1).

Normally, HIF-1 upregulates the enzymes and transporters that enhance glycolysis only when oxygen concentration falls, a condition called hypoxia. Under normal conditions, HIF-1 is hydroxylated by prolyl hydroxylase 2 and is subsequently destroyed by a large complex of proteolytic enzymes. The degradation of HIF-1 prevents the enhanced synthesis of glycolytic proteins. Prolyl hydroxylase 2 requires α-ketoglutarate, ascorbate (Vitamin C), and oxygen for activity. Thus, when oxygen concentration falls, prolyl hydroxylase 2 is inactive, HIF-1 is not hydroxylated and not degraded, and the synthesis of proteins required for glycolysis is stimulated. As a result, the rate of glycolysis is increased.

Defects in the enzymes of the citric acid cycle can significantly alter the regulation of prolyl hydroxylase 2. When either succinate dehydrogenase or fumarase is defective, succinate and fumarate accumulate in the mitochondria and spill over into the cytoplasm. Both succinate and fumarate are competitive inhibitors of prolyl hydroxylase 2. The inhibition of prolyl hydroxylase 2 results in the stabilization of HIF-1 because HIF-1 is no longer hydroxylated. Lactate, the end product of glycolysis, also appears to inhibit prolyl hydroxylase 2 by interfering with the action of ascorbate. In addition to increasing the amount of the proteins required for glycolysis, HIF-1 stimulates the production of pyruvate dehydrogenase kinase (PDH kinase), as discussed earlier.

Mutations in isocitrate dehydrogenase result in the generation of an oncogenic metabolite, 2-hydroxyglutarate. The mutant enzyme catalyzes the conversion of isocitrate to α-ketoglutarate, but then reduces α-ketoglutarate to form 2-hydroxyglutarate. 2-Hydroxyglutarate alters the methylation patterns in DNA and reduces dependence on growth factors for proliferation. These changes alter gene expression and promote unrestrained cell growth.

These observations linking citric acid cycle enzymes to cancer suggest that cancer is also a metabolic disease, not simply a disease of mutant growth factors and cell-cycle-control proteins. The realization that there is a metabolic component to cancer opens the door to new thinking about the control of the disease. Indeed, preliminary experiments suggest that, if cancer cells undergoing aerobic glycolysis are forced by pharmacological manipulation to use oxidative phosphorylation, the cancer cells lose their malignant properties. It is also interesting to note that the citric acid cycle, which has been studied for decades, still has secrets to be revealed by future biochemists.

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