TCA Cycle (Citric acid cycle or Krebs cycle)

• The tricarboxylic acid cycle (TCA cycle), also known as the citric acid cycle or the Krebs cycle, is a major energy-producing pathway in living bodies.
• Cells obtain ATP from breakdown of glucose in the absence of oxygen as in glycolysis.
• However, most organisms normally are aerobic and oxidize their organic fuels completely to CO₂ and water.
• Foodstuffs feed into the citric acid cycle as acetyl-CoA and the acetyl-CoA is oxidized to carbon dioxide and water in order to generate energy.
• Thus, under aerobic conditions, the generation of energy from glucose is the oxidative decarboxylation of pyruvate to form acetyl CoA.
• The cycle also serves in the synthesis of fatty acids, amino acids, and glucose.

Location of Krebs Cycle

• The citric acid cycle occurs in the mitochondrial matrix in the eukaryotes.
• All the enzymes of the TCA cycle are in the mitochondrial matrix except succinate dehydrogenase, which is in the inner mitochondrial membrane.
• However, in the prokaryotes, the reaction cycle occurs in plasma membrane.

Glycolysis and the Citric acid cycle

The oxidative decaroxylation of pyruvate (end product of glycolysis) to form acetyl CoA (initiator of Kreb’s cycle) is the link between Glycolysis and the Citric acid cycle.

• In the conversion of pyruvate to acetyl CoA, each pyruvate molecule loses one carbon atom with the release of carbon dioxide.
• During the breakdown of pyruvate, electrons are transferred to NAD+ to produce NADH, which will be used by the cell to produce ATP.
• In the final step of the breakdown of pyruvate, an acetyl group is transferred to Coenzyme A to produce acetyl CoA.

Steps Involved in the Citric acid Cycle

The cycle starts with the 4-carbon compound oxaloacetate, adds two carbons from acetyl-CoA, loses two carbons as CO₂, and regenerates the 4-carbon compound oxaloacetate.

Electrons are transferred by the cycle to NAD+ and FAD. As the electrons are subsequently passed to O₂ by the electron transport chain, ATP is generated by the process of oxidative phosphorylation. ATP is also generated from GTP, produced in one reaction of the cycle by substrate-level phosphorylation.

Oxidation of the carbons of acetyl-CoA to carbon dioxide requires capturing eight electrons from the molecule.

1. Acetyl-CoA and oxaloacetate condense, forming citrate.

• Enzyme: Citrate synthase.
• Cleavage of the high-energy thioester bond in acetyl-CoA provides the energy for this condensation.
• Citrate (the product) is an inhibitor of this reaction.

2. Citrate is isomerized to isocitrate by a rearrangement of the molecule.

• Enzyme: Aconitase.
• Aconitate serves as an enzyme-bound intermediate.
• Under physiological conditions, this is an unfavorable reaction, favoring citrate formation.

3. Isocitrate is oxidized to α-ketoglutarate, in a two-step reaction in which there is first an oxidation, and then a decarboxylation. CO₂ is produced, and the electrons are passed to NAD+ to form NADH and H+. This step captures two of the eight electrons present in the carbons of acetyl-CoA.

• Enzyme: Isocitrate dehydrogenase.
• This key regulatory enzyme of the TCA cycle is allosterically activated by ADP and inhibited by NADH.

4. Alpha-Ketoglutarate is converted to succinyl-CoA in an oxidative decarboxylation reaction, CO2 is released, and succinyl-CoA, NADH, and H+ are produced. This step captures another two electrons from the carbons of acetyl-CoA.

• Enzyme: Alpha-ketoglutarate dehydrogenase.
• This enzyme requires five cofactors: thiamine pyrophosphate, lipoic acid, CoASH, FAD, and NAD+.

5. Succinyl-CoA is cleaved to succinate.

• Cleavage of the high-energy thioester bond of succinyl- CoA provides energy for the substrate level phosphorylation of GDP to GTP.
• Enzyme: succinate thiokinase (succinyl-CoA synthetase).

6. Succinate is oxidized to fumarate.

Succinate transfers two hydrogens together with their electrons to FAD, which forms FADH₂. After this step, six of the eight electrons from the carbons in acetyl-CoA have been captured.

• Enzyme: Succinate dehydrogenase.       

7. Fumarate is converted to malate by the addition of water across the double bond.

• Enzyme: Fumarase.

8. Malate is oxidized, regenerating oxaloacetate and thus completing the cycle.

• Two hydrogens along with their electrons are passed to NAD+, producing NADH and H+, and finishing the capture of the eight electrons from the carbons of acetyl-CoA.
• Enzyme: Malate dehydrogenase.

Yield of the Citric acid Cycle

Each molecule of acetyl CoA entering the citric acid cycle yields the following:

• Two CO₂
• Three NADH
• One FADH₂
• One GTP

Because each NADH will eventually produce 2.5 ATP and each FADH 2 will produce 1.5 ATP through the electron transport chain, the overall ATP yield from 1 acetyl CoA is 10 ATP (7.5 from NADH, 1.5 from FADH₂ , and 1 from GTP).

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Functions

1. Oxidation of acetyl CoA to CO₂.
2. Formation of NADH and FADH₂ for entrance into the electron transport chain and subsequent ATP generation.
3. Synthesis of several important molecules, including succinyl CoA (precursor molecule of heme), oxaloacetate (early intermediate molecule in gluconeogenesis and substrate for amino acid synthesis), α-ketoglutarate (substrate for amino acid synthesis), and citrate (substrate for fatty acid synthesis).
4. It is responsible for the major share of energy release and supply during aerobic respiration.
5. Due to the many functions of the citric acid cycle is also considered to be the “central hub of metabolism”. This is because, as most of the absorbed nutrients, the fuel molecules are oxidized ultimately within the Kreb’s Cycle and its intermediates are used for various biosynthetic pathways.

References

1. Smith, C. M., Marks, A. D., Lieberman, M. A., Marks, D. B., & Marks, D. B. (2005). Marks’ basic medical biochemistry: A clinical approach. Philadelphia: Lippincott Williams & Wilkins.
2. Lehninger, A. L., Nelson, D. L., & Cox, M. M. (2000). Lehninger principles of biochemistry. New York: Worth Publishers.
3. John W. Pelley, Edward F. Goljan (2011). Biochemistry. Third edition. Philadelphia: USA.
4. Madigan, M. T., Martinko, J. M., Bender, K. S., Buckley, D. H., & Stahl, D. A. (2015). Brock biology of microorganisms (Fourteenth edition.). Boston: Pearson.
5. Rodwell, V. W., Botham, K. M., Kennelly, P. J., Weil, P. A., & Bender, D. A. (2015). Harper’s illustrated biochemistry (30th ed.). New York, N.Y.: McGraw-Hill Education LLC.