Cellular Respiration: TriCyclic Acid Cycle

By Lynnea Wilson and Isabel Montano

  1. Introduction
  2. History
  3. Hans Krebs Discovery
  4. Overall Energy Kinetics
  5. Diagram of TCA Cycle
    1. Bridge Step
    2. Step 1: Oxaloacetate to Citrate
    3. Step 2: Citrate to Isocitrate
    4. Step 3: Isocitrate to Alpha-ketoglutarate
    5. Step 4: Alpha-ketoglutarate to succinyl-CoA
    6. Step 5: Succinyl-CoA to Succinate
    7. Step 6: Succinate to Fumarate
    8. Step 7: Fumarate to Malate
    9. Step 8: Malate to Oxaloacetate
  6. The Fate of Carbon in TCA
  7. Interactions with other metabolic pathways
  8. References


The TriCyclic Acid Cycle, or TCA cycle, rests between glycolysis and electron transport chain in the cellular respiration process. The TCA cycle occurs in the mitochondria and is amphibolic (serves in both catabolism and anabolism). It is also known as the citric acid cycle or the Krebs' cycle. The primary function of this process is to cycle pyruvate (the end product of glycolysis) and reduce coenzymes such as NADH and FADH2. When reduced, these coenzymes carry electrons into the electron transport chain, thus finishing the cellular respiration process and generating ATP. The TCA cycle also creates 2 ATP per molecule of glucose on its own.


The most common names for the TCA cycle are tricarboxylic acid cycle and citric acid cycle. This cycle is also known as the Krebs cycle, recognizing the contribution of Hans Krebs. Hans Krebs, a German scientist, called his discovery the citric acid cycle.
Previously to Hans Krebs' discovery, other scientists like T. Thunberg, F. Batelli, L.S. Stern, and Albert Szent-Gyorgyi experimented on reactions involved in the TCA cycle. Hans Krebs established the cyclic nature of the reactions.

Hans Krebs Discovery:

The most important discovery Hans Krebs made clearly explained the cyclical sequence of the reactions was the formation of citrate from oxaloacetate and pyruvate. Krebs added malonate to muscle suspensions and found the accumulation of succinate in the presence of fumarate, malate, or oxaloacetate and also citrate, isocitrate, cis-aconitate or alpha-ketoglutarate. This evidently established a cyclic sequence leading to succinate. Not until later was it known that citric acid was the first substrate formed from the reaction of pyruvate and oxaloacetate.

Overall Energy Kinetics:

Per glucose molecule, the following energy is produced
  • 2 GTP/ATP
  • 6 NADH
  • 2 FADH2

Diagram of TCA Cycle:


Image credit: http://www.sci.sdsu.edu/class/bio202/TFrey/KrebsTC%20A.gif

Bridge Step:


Image Credit: http://mcb.berkeley.edu/labs/krantz/mcb102/lect_S2008/MCB102-SPRING2008-LECTURE8-CITRIC_ACID_CYCLE.pdf

The pyruvate dehydrogenase complex (PDC) is shown above. It is composed of three different enzymes that each serve a separate function in the conversion of pyruvate to acetyl-CoA. E1, or pyruvate dehydrogenase, is the first enzyme in the complex. This enzyme requires TPP (from Vitamin B1), which is a powerful electron sink. The withdrawing of electrons is what causes the C-C bond to break. The carbanion form of TPP attaches to pyruvate, which allows the decarboxylation of CO2, which is then released. E2, or dihydrolipoyl transacetylase, takes the hydroxyethyl TPP from the previous reaction and acyl lipollysine oxidizes it. The two carbon molecule remaining binds to dihydrolipollysine and is bound to the coenzyme CoA. E3, or dihydrolipoyl dehydrogenase, oxidizes dihydrolipollysine. The electrons then are transferred to FAD, which is tightly bound to the complex. From there, the electrons are transferred to NAD+, creating NADH. This complex is regulated by the phosphorylation and dephosphorylation of the enzyme complex. It is also inhibited by high levels of acetyl-CoA or NADH

Step 1: Oxaloacetate to Citrate


Acetyl-CoA and oxaloacetate interact in what is called a Perkin condensation reaction. This is defined as a "carbon-carbon condensation between a ketone or aldehyde and an ester" (Garrett 612). The two carbons in acetyl-CoA are activated in two separate ways. The alpha carbon is deprotonated to form a carbanion. This stabilizes the alpha carbanion, and it attacks the alpha carbon of oxaloacetate, forming citryl-CoA. The carbonyl carbon is activated for nucleophilic attack.

  • Enzyme used: citrate synthase
  • Reaction type: condensation
  • Coenzymes involved: n/a
  • Intermediates: citryl-CoA
  • Thermodynamics: exergonic ΔG= -31.4 kJ/mol
  • Regulation: yes by NADH, succinyl Co-A

Step 2: Citrate to Isocitrate


Image Credit: http://mcb.berkeley.edu/labs/krantz/mcb102/lect_S2008/MCB102-SPRING2008-LECTURE8-CITRIC_ACID_CYCLE.pdf

Citrate is not easily oxidized, so it must be converted into isocitrate, which is a secondary alcohol. This is a two step process, where citrate is converted to aconitate, then the elements of water are added on opposite sides to produce isocitrate. Aconitase contains an iron-sulfur cluster, shown above. It is bound to the enzyme using three cysteine groups. The fourth corner is the one that activates aconitase. It accepts an unshared electron pair, acting as a Lewis acid.
  • Enzyme used: aconitase
  • Reaction type: sequential dehydration & hydration
  • Coenzymes involved: n/a
  • Intermediates: aconitate
  • Thermodynamics: exergonic
  • Regulation: No

Step 3: Isocitrate to Alpha-Ketoglutarate


Image Credit: http://mcb.berkeley.edu/labs/krantz/mcb102/lect_S2008/MCB102-SPRING2008-LECTURE8-CITRIC_ACID_CYCLE.pdf
This is the first oxidative decarboxylation in the cycle. It also reduces NAD+ to NADH. It also provides the first connection between TCA cycle and electron-transport pathway and oxidative phosphorylation because of the production of NADH. It is a two-step reaction which involves the oxidation of the secondary alcohol on isocitrate to form oxalosuccinate. There is then a beta-decarboxylation reaction that involves Mn+ and allows for the decarboxylation of oxalosuccinate therefore producing alpha-ketoglutarate.
  • Enzyme used: isocitrate dehydrogenase
  • Reaction type: oxidative decarboxylation
  • Substrates/Cofactors involved: Yes, NAD+ and Mn+
  • Intermediates: oxalosuccinate
  • Thermodynamics: exergonic, ΔG= -8.4 kJ/mol
  • Regulation: Yes, inhibited by NADH.

Step 4: Alpha-Ketoglutarate to Succinyl-CoA


Image Credit: http://mcb.berkeley.edu/labs/krantz/mcb102/lect_S2008/MCB102-SPRING2008-LECTURE8-CITRIC_ACID_CYCLE.pdf
Alpha-ketoglutarate dehydrogenase is a multi-enzyme complex consisting of alpha-ketoglutarate dehydrogenase, dihydolipoyl transsuccinylase, and dihydrolipoyl dehydrogenase. The reaction is also a oxidative decarboxylation like the previous step.
  • Enzyme used: alpha-ketoglutarate dehydrogenase
  • Reaction type: oxidative decarboxylation
  • Substrates/Cofactors involved: Yes, CoA-SH, NAD+, H+, FAD, Lipoic acid,, Thiamine pyrophosphate
  • Intermediates: none
  • Thermodynamics:exergonic, ΔG= -29 to -33.5 kJ/mol
  • Regulation: Yes, inhibited by NADH

Step 5: Succinyl-CoA to Succinate


Image Credit: http://mcb.berkeley.edu/labs/krantz/mcb102/lect_S2008/MCB102-SPRING2008-LECTURE8-CITRIC_ACID_CYCLE.pdf

The mechanism of succinyl-CoA synthetase involves a phosphohistidine. The displacement of CoA by phosphate forms succinyl phosphate at the active site. There is then a transfer of the phosphoryl group to an active-site histidine. This produces a phosphohistidine intermediate and ultimately releases succinate. The GDP is transferred to form GTP. The sequence of steps leads to a molecule of ATP by preserving the energy of the thioester bond of succinyl-CoA.

  • Enzyme used: succinyl-CoA synthetase
  • Reaction type: substrate-level phosphorylation
  • Substrates/Cofactors involved: Yes, GDP and phosphate
  • Intermediates: none
  • Thermodynamics: near equilibrium
  • Regulation: No

Step 6: Succinate to Fumarate

SuccinateFumarate.jpgImage Credit: http://mcb.berkeley.edu/labs/krantz/mcb102/lect_S2008/MCB102-SPRING2008-LECTURE8-CITRIC_ACID_CYCLE.pdf

Succinate dehydrogenase is an enzyme that is part of the succinate-coenzyme Q reductase of the electron transport chain. The succinate dehydrogenase reaction involves the oxidation of succinate with the reduction of FAD. The reoxidation of FADH2 transfers electrons to conenzyme Q. This oxidation reaction of succinate produces fumarate. This enzyme also appears in the electron transport pathway.

  • Enzyme used: succinate dehydrogenase
  • Reaction type: dehydrogenase or oxidation-reduction
  • Substrates/Cofactors involved: Yes, FAD+ and ubiquinone
  • Intermediates: none
  • Thermodynamics: near equilibrium
  • Regulation: No

Step 7: Fumarate to Malate

FumarateMalate.jpgImage Credit: http://mcb.berkeley.edu/labs/krantz/mcb102/lect_S2008/MCB102-SPRING2008-LECTURE8-CITRIC_ACID_CYCLE.pdf

Fumarase converts fumarate by a trans-hydration reaction into L-malate, which is a hydroxydicarboxylic acid. This reaction involves the trans-addition of water across the double bond. Although the exact mechanism is uncertain, it may produce a carbonium ion intermediate.

  • Enzyme used: fumarase
  • Reaction type: hydration
  • Substrates/Cofactors involved: No
  • Intermediates: carbanion transition state
  • Thermodynamics: near equilibrium
  • Regulation: No

Step 8: Malate to Oxaloacetate

Image Credit: http://mcb.berkeley.edu/labs/krantz/mcb102/lect_S2008/MCB102-SPRING2008-LECTURE8-CITRIC_ACID_CYCLE.pdf

Malate dehydrogenase catalyzes the final step of the TCA cycle. L-malate is oxidized using NAD+ to remake oxaloacetate. This reaction is very endergonic and is pulled forward by the citrate synthase reaction. At the end of the process it yields NADH.

  • Enzyme used: malate dehydrogenase
  • Reaction type: dehydrogenation
  • Substrates/Cofactors involved: Yes, NAD+ and H+
  • Intermediates: none
  • Thermodynamics: endergonic ΔG= +29.7 KJ/mol
  • Regulation: No

The Fate of Carbon in TCA:

The carbon atoms of acetate in successive TCA cycles can be from the carbonyl carbon or the methyl carbon. The carbonyl carbon of an acetyl-CoA is still intact through one turn of the TCA cycle, but is lost in the second turn of the cycle. The methyl carbon of an acetyl-CoA stays intact for two turns of the TCA cycle, but half of what is left exits the cyle on each turn after that.

Interactions with other metabolic pathways:

As the TCA cycle rests between glycolysis and the electron transport pathway, it obviously interacts with these two pathways. The end product of glycolysis, pyruvate, is used in the bridge step to create acetyl-CoA. The electron carriers, NADH and FADH2, are vital to the process of oxidative phosphorylationwhere these electrons help create the proton gradient used to generate ATP. Aside from these two obvious links, however, there are other pathways enabled by the TCA cycle. Acetate can be used as a source of carbon for many plants for whom photosynthesis does not suffice. There is an alternative pathway used by plants and bacteria that creates four carbon compounds from two carbon compounds such as acetate. This is known as the glyoxylate cycle. This process is enabled because the two carbon dioxide releasing steps are skipped. Instead of running through isocitrate dehydrogenase, isocitrate goes through isocitrate lyase. This reaction produces glyoxylate, which is then converted to malate via malate synthase. Acetyl-CoA is necessary to produce this reaction. The glyoxylate pathway enables certain seeds to grow underground where light is not available for photosynthesis. Instead, lipids stored in the seeds are converted to acetyl-CoA and use this pathway to commence germination. After photosynthesis is possible, this pathway no longer is used. In addition to the glyoxylate cycle, intermediates in the TCA cycle have other purposes. They sometimes function as intermediates for biosynthesis. Some precursors needed in cellular species are succinyl-CoA, fumarate, oxaloacetate, and α-ketoglurate. These precursors must be transported out of the mitochondria before participating in eukaryotic biosynthetic processes. Glutamate is produced through the transamination of α-ketoglutarate and is used as a precursor for proline, arginine, and glutamine. Porphyrins are produced using the carbons from succinyl-CoA.. Oxaloacetate is transformed to produce aspartate and decarboxylated to yield PEP. PEP is important in synthesis of aromatic amino acids, formation of 3-phosphoglycerate, and gluconeogenesis. Citrate yields oxaloacetate and acetyl-CoA by ATP-citrate lyase after it exits the mitochondria. This is a precursor for fatty acids. Oxaloacetate can then be reduced to malate, which is can be reoxidized to oxaloacetate in the mitochondria or can be oxidatively decarboxylated to pyruvate by malic enzyme.


Garrett, Reginald H, and Charles M. Grisham. Biochemistry; Updated Third Edition. Belmont: Thomson Brooks/Cole, 2007. p. 608-637