Biological Chemistry - Berezov, T. T., & Korovkin, B. F. 1998

Carbohydrate Metabolism
Tricarboxylic Acid Cycle (Krebs Cycle)

The Tricarboxylic Acid Cycle was first discovered by the English biochemist H. Krebs *.

He was the first to postulate The Significance of this cycle for the complete combustion of Pyruvate, the main source of which is the glycolytic breakdown of CARBOHYDRATES. It was later shown that the tricarboxylic acid cycle serves as the central hub where virtually all metabolic pathways converge. Thus, the Krebs cycle is the common final pathway for The oxidation of acetyl groups (in the form of acetyl-CoA)—into which the majority of organic molecules acting as "cellular fuel" (carbohydrates, Fatty acids, and Amino Acids) are converted during Catabolism.

The acetyl-CoA formed in the Cell/35.html">Mitochondria As a result of The oxidative decarboxylation of pyruvate enters the Krebs cycle. This cycle takes place within the mitochondrial matrix and consists of eight sequential reactions (Fig. 10.9). The cycle begins with the attachment of acetyl-CoA to oxaloacetate to form citric acid (citrate). Subsequently, citric acid (a six-carbon compound) undergoes a series of dehydrogenations (removal of hydrogen) and two decarboxylations (release of CO₂), losing two carbon atoms and ultimately reverting to oxaloacetate (a four-carbon compound) within the Krebs cycle. In other words, as a result of one full turn of the cycle, a single molecule of acetyl-CoA is burned down to CO₂ and H₂O, while an oxaloacetate molecule is regenerated. Let us examine all eight sequential reactions (stages) of the Krebs cycle.

* For this outstanding discovery, H. Krebs was awarded the Nobel Prize in 1953 (shared with F. Lipmann). The tricarboxylic acid cycle is frequently named after him as the Krebs cycle (Krebs Citric Acid Cycle).

Class="center">

Fig. 10.9. The tricarboxylic acid cycle (Krebs cycle).

The first reaction is catalyzed by the enzyme citrate synthase, wherein the acetyl group of acetyl-CoA condenses with oxaloacetate to yield citric acid:

Apparently, an enzyme-bound citryl-CoA is formed as an intermediate in this reaction. The latter is then spontaneously and irreversibly hydrolyzed to produce citrate and HS-CoA.

In the second reaction, the resulting citric acid undergoes dehydration to form cis-aconitic acid, which then adds a molecule of Water to convert into isocitric acid (isocitrate). These reversible Hydration-dehydration reactions are catalyzed by the enzyme aconitate hydratase (aconetase). As a result, the H and OH groups within the citrate molecule are interchanged:

The third reaction is presumably rate-limiting for the Krebs cycle. Isocitric acid is dehydrogenated in the presence of NAD-dependent isocitrate dehydrogenase*.

During the isocitrate dehydrogenase reaction, isocitric acid undergoes simultaneous decarboxylation. NAD-dependent isocitrate dehydrogenase is an allosteric enzyme that requires ADP as a specific activator. Additionally, the enzyme requires Mg²⁺ or Mn²⁺ ions to express its catalytic activity.

The fourth reaction involves the Oxidative Decarboxylation of α-ketoglutaric acid, yielding the high-energy compound succinyl-CoA. The Mechanism of this reaction is similar to that of the oxidative decarboxylation of pyruvate to acetyl-CoA, and the α-ketoglutarate dehydrogenase complex structurally resembles the pyruvate dehydrogenase complex. In both cases, 5 Coenzymes participate in the reaction: TPP, lipoamide, HS-CoA, FAD, and NAD⁺.

The fifth reaction is catalyzed by the enzyme succinyl-CoA synthetase. In the course of this reaction, succinyl-CoA is converted into succinic acid (succinate) with the participation of GTP and inorganic phosphate. Simultaneously, a high-energy GTP phosphate bond** is formed at the expense of the high-energy thioester bond of succinyl-CoA:

* There are 2 types of isocitrate dehydrogenases in mitochondria: NAD- and NADP-dependent; the first type is found exclusively in mitochondria, whereas the second occurs in both mitochondria and the Cytosol.

** The resulting GTP subsequently transfers its terminal phosphate group to ADP, thereby generating ATP. The synthesis of a high-energy nucleoside triphosphate during the succinyl-CoA synthetase reaction serves as yet another example of substrate-level phosphorylation.

As a result of the sixth reaction, succinate is dehydrogenated to fumaric acid. The oxidation of succinate is catalyzed by succinate dehydrogenase, whose protein molecule is tightly (covalently) bound to the FAD coenzyme. In turn, succinate dehydrogenase is firmly anchored to The inner mitochondrial membrane:

The seventh reaction is carried out under the Influence of the enzyme fumarate hydratase (fumarase). The resulting fumaric acid is hydrated, yielding malic acid (malate) as the reaction product. It should be noted that fumarate hydratase exhibits stereospecificity (see Chapter 4)—the reaction exclusively produces L-malic acid:

Finally, during the eighth reaction of the tricarboxylic acid cycle, L-malate is oxidized to oxaloacetate under the action of mitochondrial NAD-dependent malate dehydrogenase:

As can be seen, a single turn of the cycle, consisting of eight enzymatic reactions, results in the complete oxidation ("combustion") of one molecule of acetyl-CoA. For the cycle to run continuously, a constant supply of acetyl-CoA is required, while the coenzymes (NAD⁺ and FAD) converted to their reduced state must be repeatedly reoxidized. This oxidation is carried out by The electron transport System of the Respiratory Chain (the chain of respiratory Enzymes) localized in the mitochondrial membrane. The resulting FADH₂ is tightly bound to SDH; therefore, it transfers hydrogen atoms via KoQ. The energy released as a result of acetyl-CoA oxidation is largely concentrated in the high-energy phosphate bonds of ATP. Of the 4 pairs of hydrogen atoms, 3 pairs are transferred by NADH to the electron transport system; in this process, for each pair, 3 molecules of ATP are formed in the Biological Oxidation system (via coupled Oxidative Phosphorylation), totaling 9 molecules of ATP (see Chapter 9). One pair of atoms from succinate dehydrogenase-FADH₂ enters the electron transport system via KoQ, resulting in The formation of only 2 molecules of ATP. The Krebs cycle also synthesizes one molecule of GTP (substrate-level phosphorylation), which is equivalent to one molecule of ATP. Thus, the oxidation of one molecule of acetyl-CoA in the Krebs cycle and the oxidative phosphorylation system can yield 12 molecules of ATP.

If we calculate the total energy yield of the glycolytic breakdown of glucose and the subsequent oxidation of the two resulting pyruvate molecules to CO₂ and H₂O, it turns out to be significantly higher.

As noted, one molecule of NADH (3 molecules of ATP)* is formed during the oxidative decarboxylation of pyruvate to acetyl-CoA. The breakdown of one molecule of glucose yields 2 molecules of pyruvate, and upon their oxidation to 2 molecules of acetyl-CoA followed by 2 turns of the tricarboxylic acid cycle, 30 molecules of ATP are synthesized (consequently, the oxidation of one pyruvate molecule to CO₂ and H₂O yields 15 molecules of ATP). To this amount, we must add 2 molecules of ATP formed during aerobic Glycolysis and 6 molecules of ATP synthesized via the oxidation of 2 extramitochondrial NADH molecules, which are generated during the oxidation of 2 molecules of glyceraldehyde-3-phosphate in the dehydrogenase reaction of glycolysis. Consequently, the breakdown of one glucose molecule in Tissues According to the equation C₆H₁₂O₆ + 6O₂ —> 6CO₂ + 6H₂O synthesizes 38 molecules of ATP. Undoubtedly, in energetic terms, the complete breakdown of glucose is a more efficient process than anaerobic glycolysis.

It should be noted that the 2 molecules of NADH formed during The conversion of glyceraldehyde-3-phosphate may subsequently yield not 6 molecules of ATP, but only 4. The fact is that extramitochondrial NADH molecules themselves are unable to cross the mitochondrial membrane. However, the electrons they donate can be introduced into the mitochondrial biological oxidation chain via the so-called glycerol phosphate shuttle mechanism (Fig. 10.10). Cytoplasmic NADH first reacts with cytoplasmic dihydroxyacetone phosphate to form glycerol-3-phosphate. The reaction is catalyzed by NAD-dependent cytoplasmic glycerol-3-phosphate dehydrogenase:

Dihydroxyacetone phosphate + NADH + H⁺ <=> Glycerol-3-phosphate + NAD⁺.

* Recall that as reducing equivalents of NADH pass through the respiratory enzyme chain, they generate three high-energy phosphate bonds via the formation of ATP from ADP during oxidative phosphorylation (see Chapter 9).

Fig. 10.10. Glycerol phosphate shuttle mechanism. Explanations in the text.

The resulting glycerol-3-phosphate readily penetrates the mitochondrial membrane. Inside the mitochondrion, another (mitochondrial) glycerol-3-phosphate dehydrogenase (a flavin enzyme) reoxidizes glycerol-3-phosphate to dihydroxyacetone phosphate:

Glycerol-3-phosphate + FAD <=> Dihydroxyacetone phosphate + FADH₂.

The reduced flavoprotein (enzyme-FADH₂) feeds the electrons it has acquired at the level of KoQ into the chain of biological oxidation and coupled oxidative phosphorylation, while dihydroxyacetone phosphate exits the mitochondria into the Cytoplasm and can again react with cytoplasmic NADH + H⁺. Thus, a pair of electrons (from a single molecule of cytoplasmic NADH + H) introduced into the respiratory chain via the glycerol phosphate shuttle mechanism yields 2 ATP molecules instead of 3.

Fig. 10.11. The malate-aspartate shuttle for transferring reducing equivalents from cytosolic NADH to the mitochondrial matrix. See text for details.

It was subsequently demonstrated that this shuttle mechanism is used to transfer reducing equivalents from cytosolic NADH + H⁺ into the mitochondria exclusively in Skeletal Muscle and the Brain.

In Liver, Kidney, and Heart Cells, a more complex malate-aspartate shuttle operates. This shuttle mechanism Functions due to the presence of both malate dehydrogenase and aspartate aminotransferase in both the cytosol and the mitochondria.

It has been established that reducing equivalents from cytosolic NADH + H⁺ are first transferred to cytosolic oxaloacetate via the enzyme malate dehydrogenase (Fig. 10.11). This yields malate, which crosses the inner mitochondrial membrane into the matrix via a dicarboxylate transport system. Inside the matrix, malate is oxidized to oxaloacetate, while matrix NAD⁺ is reduced to NADH + H⁺, which can now donate its electrons to the respiratory enzyme chain located on the inner mitochondrial membrane. In turn, the resulting oxaloacetate* undergoes Transamination in the presence of glutamate and the enzyme ASAT. The resulting aspartate and a-ketoglutarate are able to cross the mitochondrial membrane via specialized transport systems.

Transport in the cytosol regenerates oxaloacetate, which drives the next cycle. Overall, the process involves readily reversible reactions and proceeds without energy consumption; its driving force is the continuous reduction of NAD⁺ in the cytosol by glyceraldehyde-3-phosphate generated during Glucose Catabolism.

Thus, when the malate-aspartate mechanism is active, the Complete oxidation of a single glucose molecule can yield 38 ATP molecules rather than 36 (Table 10.1).

* The resulting oxaloacetate cannot directly return to the cytosol across the membrane.

Table 10.1. Formation of high-energy phosphate bonds during glucose catabolism

* It is believed that NADH generated during glycolysis enters the mitochondria via the malate shuttle mechanism (see p. 351). If the glycerophosphate shuttle mechanism is used, only 2 ATP are produced per 1 mol of NADH, and the total number of high-energy phosphate bonds formed will be 36 instead of 38.

Table 10.1 lists the reactions resulting in the formation of high-energy phosphate bonds during glucose catabolism, indicating the efficiency of the process under aerobic and anaerobic conditions.