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Image : “Respiration poster.” by Duncan Hull. License: CC BY 2.0

Definition of the Citric Acid Cycle

The citric acid cycle is also referred to as the Krebs cycle or tricarboxylic acid cycle (TCA), and it is a cyclic metabolic process. It takes place in the matrix of the mitochondria and plays an important role for anabolism and catabolism.

Per reaction cycle, 1 acetyl-CoA is transformed into 2 CO2. The resulting energy is fixated as 3 NADH + H+, 1 FADH2, and 1 GTP. In the respiratory chain, the electrons of NADH + H+ and FADH2 are used for ATP synthesis.

Note: ATP is not produced in the citric acid cycle itself.

Functions of the Citric Acid Cycle

The citric acid cycle is referred to as the “hub of the intermediate metabolism” because it has a central role for a lot of metabolic pathways. Its most important function, however, is the retrieval of electrons for the respiratory chain by oxidation of acetyl-CoA.

The needed acetyl-CoA is created in the beta-oxidation of fatty acids and the oxidative decarboxylation of pyruvate, which forms during glycolysis. Also, some amino acids like isoleucine, leucine, and tryptophan can be degraded to acetyl-CoA.

In addition, the citric acid cycle has the following functions:

  • It is the final path of the degradation of amino acids, which cannot be degraded to acetyl-CoA or pyruvate.
  • It produces substances for resynthesis of amino acids (e.g., oxaloacetate for aspartate).
  • Reaction products from the citric acid cycle are diverted and fed into other metabolic pathways: citrate for fatty acid synthesis, oxaloacetate for gluconeogenesis, or succinyl-CoA for the formation of delta-aminolevulinic acid as the basic substance for heme synthesis.

Electrochemical Gradient

Individual Reaction Steps of the Citric Acid Cycle

Even if it is hard to learn the individual steps and the respective structural formulas in detail, it is definitely worth it because they are often asked for in oral and written exams.


Image: “The reductive TCA cycle. Dotted lines denote alternative enzymes pathways.” by Andrewgross. License: CC BY-SA 3.0

Step 1: Acetyl-CoA + Oxaloacetate → Citrate

Citrate synthase catalyzes the transfer of acetyl-CoA to oxaloacetate with the formation of citrate. H2O is inserted and coenzyme A is split off. As a result, the high-energy thioester-bond of acetyl-CoA is hydrolyzed.

Step 2: Citrate → Isocitrate

Acinotate hydratase, also called aconitase, transforms citrate to isocitrate. Via shifting of one OH group, the tertiary alcohol becomes a secondary one. The intermediate substance formed by this isomerization is referred to as cis-aconitate.

Step 3: Isocitrate → α-Ketoglutarate

Isocitrate dehydrogenase catalyzes the NAD+-dependent oxidation of isocitrate. In this process, the unstable intermediate substance oxalosuccinate forms, which then spontaneously decarboxylases to become succinyl-CoA. In this reaction step, the first oxidation reaction and the first decarboxylation of the citric acid cycle occur – with the formation of 1 NADH + H+ and the release of CO2.

Step 4: α-Ketoglutarate → Succinyl-CoA

α -ketoglutarate dehydrogenase is a large enzymatic complex that is very similar to pyruvate dehydrogenase. For the oxidative decarboxylation of α-ketoglutarate to succinyl-CoA, the following cofactors are needed: thiamine pyrophosphate, liponamide, coenzyme A, FAD, and NAD+. Again, CO2 and 1 more NADH + H+ form for the respiratory chain.

In case of a lack of thiamine (e.g., caused by malnutrition due to alcoholism), Wernicke’s encephalopathy occurs because both α-ketoglutarate and pyruvate dehydrogenases rely on thiamine as a cofactor. If both enzymes do not work correctly, glutamate can accumulate, and the utilization of glucose is diminished, which then results in cerebral cell damage.

Step 5: Succinyl-CoA → Succinate + CoA + GTP

The enzyme succinyl-CoA synthetase catalyzes the hydrolysis of the high-energy thioester-bond of succinyl-CoA. Coenzyme A is split off, which leads to the formation of succinate. The released energy is used to synthesize 1 GTP – also referred to as phosphate-level phosphorylation.

If one phosphate group of GTP is transferred to ADP, ATP results: GTP + ADP → GDP + ATP. However, this reaction itself is not part of the citric acid cycle.

Step 6: Succinate → Fumarate + FADH2

The FAD-dependent succinate dehydrogenase performs the oxidation of succinate to fumarate. This happens with the formation of a double bond and the release of 1 FADH2.

An important aspect is that the succinate dehydrogenase is the only enzyme of the citric acid cycle that does not freely float in the matrix space but is anchored to the inner membrane of the mitochondrion. Thus, it can directly supply the respiratory chain with the electrons of FADH2 and is therefore called complex II.

Step 7: Fumarate + H2O → Malate

Fumarate hydratase – also called fumarase – catalyzes the addition of water to fumarate, which results in malate.

Step 8: Malate → Oxaloacetate

The NAD+-dependent malate dehydrogenase oxidizes malate to oxaloacetate, which can be used as a substrate for step 1 of the citric acid cycle. In this process, 1 NADH + H+ is formed for the respiratory chain.

All in all, the energetic yield of one cycle is 3 NADH + H+, 1 FADH2, 1 GTP. The two 2 CO2 molecules that are also formed during the cycle are, in this context, useless waste products.

Energy Balance of the Citric Acid Cycle

In the respiratory chain, the mentioned yield of the citric acid cycle results in the following energy values:

  • 1 NADH + H+ is transformed to approximately 2.5 ATP.
  • 1 FADH2 is transformed to approximately 1.5 ATP.

So, per cycle of the citric acid cycle, the following fixated energy is produced: 7.5 ATP out of 3x NADH + H+ + 1.5 ATP out of 1 FADH2 + 1 ATP out of 1 GTP (since they are energetically interchangeable) – this results in a sum of about 10 ATP.

In the literature, the energetic yield of NADH + H+ and FADH2 used to be overestimated so one should not be confused when there is talk of a sum yield of 12 molecules of ATP.

Mnemonic for the Citric Acid Cycle

The following mnemonic helps one remember the steps of the citric acid cycle:

Can intelligent Karen solve some foreign Mafia operations?
  • Can = Citrate
  • Intelligent = Isocitrate
  • Karen = Alpha-Ketoglutarate
  • Solve = Succinyl-CoA
  • Some = Succinate
  • Foreign = Fumarate
  • Mafia = Malate
  • Operations = Oxaloacetate

Regulation of the Citric Acid Cycle

The citric acid cycle is mainly regulated by the following three factors:

  1. The supply of the substrates, which include the cofactors NAD+ and FAD
  2. The formation of products
  3. The inhibition via feedback

The following table illustrates how the individual enzymes of the citric acid cycle are activated or inhibited.

Enzyme Activation by Inhibition by
Citrate synthase ADP, oxaloacetate, acetyl-CoA (thus, high activity of pyruvate dehydrogenase) Citrate, NADH + H+, ATP, succinyl-CoA
Isocitrate dehydrogenase ADP, Ca2+ ATP, NADH + H+
Alpha-ketoglutarate dehydrogenase Ca2+ Succinyl-CoA, NADH + H+
Succinate dehydrogenase Succinate Oxaloacetate

Besides pyruvate dehydrogenase as a link between glycolysis and the citric acid cycle, isocitrate dehydrogenase seems to have the greatest influence on the activity of the citric acid cycle.

Because the citric acid cycle also synthesizes intermediate products that are needed for other metabolic pathways, it has to be ensured that individual reactions of the cycle occur, even if the whole cycle is inhibited. Thus, there is no key enzyme for the citric acid cycle. Hormones have no direct influence on the enzymes of the citric acid cycle.

Anaplerotic Reactions

Anaplerotic reactions are metabolic pathways that supply the citric acid cycle so it does not lack the necessary substrates. For illustration: If the body heavily performs gluconeogenesis and thus needs a lot of oxaloacetate from the citric acid cycle, the cycle would lack oxaloacetate for the first reaction step (acetyl-CoA + oxaloacetate → citrate).

For this life-threatening situation not to occur, there are the anaplerotic reactions. For the exams, the pyruvate carboxylation reaction is relevant: pyruvate + CO2 + ATP ↔ oxaloacetate + ADP + P. So, the enzyme pyruvate carboxylase catalyzes the transformation of pyruvate and carbon dioxide to oxaloacetate under ATP usage. This ensures that oxaloacetate is constantly present as a substrate for the citric acid cycle.

Glyoxylate Cycle

Overview of the glyoxylate cycle

Isocitrate lyase

Malate synthase

Glyoxylate cycle summary

  • Input 4 carbons (2-acetyl-CoA)
  • Releases 0 CO2 molecules
  • Produces one extra oxaloacetate
  • Two oxidations
  • 1 NADH, 1 FADH2, 1 (extra) oxaloacetate per turn of cycle
  • Net synthesis of glucose from acetyl-CoA

The Citric Acid Cycle as the Amphibolic Center of the Intermediate Metabolism

First of all, we must ask: “What does amphibolic mean?”. Amphibolic is a term used to refer to metabolic pathways that are both catabolic and anabolic.

citric acid cycle

Amino acid metabolism: A lot of amino acids are degraded to substrates of the citric acid cycle. In the same way, they serve as substrates for the synthesis of other amino acids (e.g., for the synthesis of nonessential amino acids like glutamate and aspartate). Glutamate is created via a transamination of alpha-ketoglutarate and aspartate via transamination of oxaloacetate.

Carbohydrate metabolism: On one hand, glycolysis leads to the citric acid cycle via the link of the pyruvate dehydrogenase. On the other, oxaloacetate is a substrate for gluconeogenesis. Here, one should never forget that, due to the irreversibility of the pyruvate dehydrogenase reaction, acetyl-CoA itself can never serve as a substrate for gluconeogenesis.

Fatty acids and steroids: With acetyl-CoA, beta-oxidation produces the basic substrate of the citric acid cycle and, simultaneously, citrate serves for the synthesis of fatty acids and cholesterol or steroids.

One must not forget that the synthesis of porphyrins or heme also depends on the citric acid cycle (i.e. its intermediate product succinyl-CoA).

Cell Respiration

Most ATP is made during chemiosmosis, actual yields vary.

Cellular respiration occurs in 4 phases

Mitochondria are considered the power plant of the cell.

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