TCA CYCLE

III. The Tricarboxylic Acid [TCA] Cycle occurs in mitochondria.

Each turn of the cycle produces one high-energy phosphate bond in the formation of GTP (high energy phosphate transferable to ADP to form ATP) and 4 reducing equivalents (3 NADH and 1 FADH₂). After the O₂-dependent processes of electron transport and oxidative phosphorylation, the total number of ATP produced per cycle is 10 (2.5 ATP from each NADH oxidized, 1.5 ATP from oxidation of FADH₂ and 1 GTP + ADP <—> GDP + ATP ) Two molecules of pyruvate are generated from one molecule of glucose, fueling two turns of the TCA cycle. Glycolysis of 1 molecule of glucose (6 carbons) yields a net of two molecules of ATP, 2 molecules of NADH (= 5 ATP) and two molecules of pyruvate (3 carbons). Each of the two molecules of pyruvate generated per glucose molecule is converted to acetyl CoA (step # 12), producing one molecule of NADH (= 2 NADH, or 5 ATP per molecule of glucose). Acetyl CoA then enters the TCA cycle. Thus the complete oxidation of one molecule of glucose yields either 30 or 32 ATP, depending on which of two biochemical shuttles carries the electrons from the NADH produced by glycolysis across the mitochondrial membrane to the electron transport chain. Anaerobic glycolysis of glucose to lactate nets only 2 ATP per glucose molecule — no electrons from glucose are passed to the electron transport chain and no ATP is generated by oxidative phosphorylation.

12. Formation of acetyl CoA from pyruvate by pyruvate dehydrogenase:

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Pyruvate is oxidized by pyruvate dehydrogenase, which requires 4 vitamins, including thiamine, to yield acetyl CoA, which enters the TCA cycle.


In thiamine deficiency, pyruvate dehydrogenase activity is reduced, slowing the oxidation of pyruvate to acetyl CoA, thereby limiting energy production in the mitochondria.


Consequently, the concentration of pyruvate increases and the electrons collected as NADH are used to reduce the pyruvate to lactate by lactate dehydrogenase, similarly as in oxygen deficiency (anaerobic conditions), instead of being transferred to the mitochondrial electron transport chain, where they would have been used in the generation of ATP.


Wernicke's encephalopathy begins abruptly, presenting with the classic triad of encephalopathy, opthalmoplegia and ataxia. Untreated, it may progress to Korsakoff's psychosis, coma and death. Either an energy deficit or the build up of lactate — ionized lactic acid, which lowers the pH — or both induce the symptoms of Wernicke's encephalopathy.
• pyruvate moves between cytosolic and mitochondrial compartments by carrier-mediated transport.
• irreversible
• NADH produced, CO₂ released
• deactivated by phosphorylation by a protein kinase activity stimulated by high NADH/NAD⁺, acetyl CoA/CoA or ATP/ADP ratios
• pyruvate and ADP activate by inhibiting the kinase
• dephosphorylation (by a protein phosphatase), and consequently enzymatic activity, is stimulated by insulin.
• Ca²⁺ activates the phosphatase in heart muscle to increase energy for rapid contraction
• acetyl CoA, NADH are competitive inhibitors
• 4 vitamins are required as cofactors: thiamin (as thiamine pyrophosphate, TPP), riboflavin (as FAD), niacin (as NAD+) and pantothenate (as Coenzyme A). Lipoate, non-essential in the diet, is also required.

  Alcoholism, in which ethanol consumption interferes with thiamine absorption, combined with poor nutrition, can deplete thiamin, thereby blocking the TCA cycle. For such malnourished individuals, glucose administration without thiamin supplementation is harmful, because the aerobic metabolism of glucose is compromised and lactate (ionized lactic acid) and its dissociated H⁺ accumulate, potentially resulting in lactic acidosis.

13. Condensation of oxaloacetate and acetyl CoA by citrate synthase to form citrate:
• irreversible
• feed-back inhibited by citrate (the product of the enzymatic reaction)
• ordered binding of substrates: oxaloacetate binding changes the enzyme conformation to accept acetyl CoA
• Remember, citrate is an allosteric inhibitor of reaction #3, the rate-limiting step of glycolysis catalyzed by PFKI.
• Citrate can exit the mitochondria to the cytosol, where it is used for fatty acid synthesis (discussed in the lectures on the metabolism of triacylglycerol).

14. Interconversion of citrate, cis-aconitate and isocitrate by aconitase:
    • reversible
15. Conversion of isocitrate to alpha-ketoglutarate by isocitrate dehydrogenase:
    • irreversible oxidative decarboxylation
    • NADH produced, C0₂ released
    • major regulation step of the cycle responsive to the energy charge of the cell
    • the cooperative binding of isocitrate and NAD⁺ is enhanced by ADP (reduced energy)
    • competitively inhibited by NADH (increased reducing power for ATP generation)
    • Ca²⁺, released from the sarcoplasmic reticulum in contracting heart muscle, and possibly other muscle as well, lowers the Km and increases the velocity of the enzyme to increase energy for contraction
16. Conversion of alpha-ketoglutarate to succinyl CoA by alpha-ketoglutarate dehydrogenase:
    • irreversible oxidative decarboxylation
    • NADH produced, CO₂ released
    • product inhibited by succinyl CoA and NADH
    • Ca²⁺, released from sarcoplasmic reticulum in contracting heart muscle, and possibly other muscle as well, lowers the Km and increases the velocity of the enzyme to increase energy for contraction
17. Formation of succinate from succinyl CoA by succinyl CoA synthetase (succinate thiokinase):
    • substrate-level phosphorylation; GTP (= ATP) produced
    • succinyl CoA reacts with glycine in the mitochondria to form the first intermediate, δ-aminolevulinic acid, which exits the mitochondria to the cytosol, in the heme synthetic pathway
18. Conversion of succinate to fumarate by succinate dehydrogenase:
    • FADH₂ produced, a C=C double bond is formed
    • Succinate dehydrogenase is a membrane-bound protein located on the inner mitochondrial membrane, unlike the other enzymes of the TCA cycle, which are located in the mitochondrial matrix.
19. Conversion of fumarate to malate by fumarase:
    • reversible
    • hydration of the fumarate double bond
20. Oxidation of malate to form oxaloacetate by malate dehydrogenase:
    • NADH produced

Summary of the TCA cycle:

Although O₂ does not participate in the TCA cycle, the cycle cannot proceed in the absence of O₂ because NAD⁺ and FAD can be regenerated in the mitochondria only by the transfer of electrons from NADH and FADH₂ to molecular oxygen via the electron transport chain. In the absence of oxygen the concentrations of oxidized NAD⁺ and FAD are diminished and NAD⁺ and FAD become limiting for those reactions of the TCA cycle that require them as electron acceptors.

Intermediates of the TCA cycle are precursors for different biosynthetic pathways in different cell types. Because the liver is the site of synthesis of many biological molecules there is a high efflux of intermediates from the cycle. After a high carbohydrate meal citrate efflux and cleavage to acetyl CoA provides acetyl units for cytosolic fatty acid synthesis. During fasting, gluconeogenic precursors are converted to malate, which exits the mitochondria for cytosolic gluconeogenesis. The liver also uses TCA cycle intermediates to synthesize some non-essential amino acids, and succinyl CoA is used in both liver and bone marrow for the synthesis of heme. In the brain α-ketoglutarate is converted to glutamate and then to γ-aminobutyric acid (GABA), a neurotransmitter, and in skeletal muscle it is converted to glutamine, which is transported throughout the blood for use by other tissues.

Removal of any of the TCA cycle intermediates depletes the four-carbons that regenerate oxaloacetate at each turn of the cycle, diminishing the ability of the cycle to oxidize acetyl CoA. For the TCA cycle to continue functioning, enough four-carbon intermediates must be supplied from the degradation of carbohydrates or certain other molecules to compensate for the depletion. Reactions that replenish TCA cycle intermediates are called “anaplerotic”, meaning “to fill up”.

Pyruvate carboxylase (reaction #29) is a major anaplerotic enzyme, which uses ATP to catalyze the addition of CO₂ to three-carbon pyruvate, forming four-carbon oxaloacetate. Many tissues contain pyruvate carboxylase as an anapleoric enzyme, but its concentration is particularly high in the liver and the kidney cortex, where gluconeogenesis withdraws four-carbon oxaloacetate from the TCA cycle for the synthesis of glucose.

Eighteen of the common twenty amino acids degrade to carbon skeletons that enter the TCA cycle as four- or five-carbon units that can be converted to oxaloacetate, an intermediate of gluconeogenesis. The liver is the major site of gluconeogenesis and also the major site of amino acid degradation. Amino acid degradation in the liver provides a major source of carbon skeletons for the synthesis of glucose.

The TCA cycle is regulated at isocitrate dehydrogenase and α-ketoglutatate dehydrogenase. Increased β-oxidation of fatty acids, which occurs during fasting, yields a large amount of NADH and Acetyl Coal. The increased NADH slows the TCA cycle at isocitric dehydrogenase and α ketoglutarate dehydrogenase. In the liver, the Acetyl CoA is directed toward the synthesis of ketone bodies; the liver is the only tissue that syntheses ketone bodies. In cardiac muscle, Ca²⁺ increases the activity of the TCA cycle to increase the production of energy for cardiac muscle contractions.