Citric acid cycle

The citric acid cycle – also known as the tricarboxylic acid (TCA) cycle or the Krebs cycle^[1][2] – is a series of chemical reactions used by all aerobic organisms to generate energy through the oxidation of acetate derived from carbohydrates, fats and proteins into carbon dioxide and chemical energy in the form of adenosine triphosphate (ATP). In addition, the cycle provides precursors of certain amino acids as well as the reducing agent NADH that is used in numerous other biochemical reactions. Its central importance to many biochemical pathways suggests that it was one of the earliest established components of cellular metabolism and may have originated abiogenically.^[3][4]

The name of this metabolic pathway is derived from citric acid (a type of tricarboxylic acid) that is consumed and then regenerated by this sequence of reactions to complete the cycle. In addition, the cycle consumes acetate (in the form of acetyl-CoA) and water, reduces NAD⁺ to NADH, and produces carbon dioxide as a waste byproduct. The NADH generated by the TCA cycle is fed into the oxidative phosphorylation (electron transport) pathway. The net result of these two closely linked pathways is the oxidation of nutrients to produce usable chemical energy in the form of ATP.

In eukaryotic cells, the citric acid cycle occurs in the matrix of the mitochondrion. In prokaryotic cells, such as bacteria which lack mitochondria, the TCA reaction sequence is performed in the cytosol with the proton gradient for ATP production being across the cell's surface (plasma membrane) rather than the inner membrane of the mitochondrion.

Discovery

Several of the components and reactions of the citric acid cycle were established in the 1930s by the research of the Nobel laureate Albert Szent-Györgyi, for which he received the Nobel Prize in 1937 for his discoveries pertaining to fumaric acid, a key component of the cycle.^[5] The citric acid cycle itself was finally identified in 1937 by Hans Adolf Krebs while at the University of Sheffield, for which he received the Nobel Prize for Physiology or Medicine in 1953.^[6]

Evolution

Components of the TCA cycle were derived from anaerobic bacteria, and the TCA cycle itself may have evolved more than once.^[7] Theoretically there are several alternatives to the TCA cycle; however, the TCA cycle appears to be the most efficient. If several TCA alternatives had evolved independently, they all appear to have converged to the TCA cycle.^[8][9]

Overview

The citric acid cycle is a key metabolic pathway that unifies carbohydrate, fat, and protein metabolism. The reactions of the cycle are carried out by 8 enzymes that completely oxidize acetyl-CoA into two molecules of carbon dioxide. Through catabolism of sugars, fats, and proteins, a two-carbon organic product acetate in the form of acetyl-CoA is produced which enters the citric acid cycle. The reactions of the cycle also converts three equivalents of nicotinamide adenine dinucleotide (NAD⁺) into three equivalents of reduced NAD⁺ (NADH), one equivalent of flavin adenine dinucleotide (FAD) into one equivalent of FADH₂, and one equivalent each of guanosine diphosphate (GDP) and inorganic phosphate (P_i) into one equivalent of guanosine triphosphate (GTP). The NADH and FADH₂ generated by the citric acid cycle are in turn used by the oxidative phosphorylation pathway to generate energy-rich adenosine triphosphate (ATP).

One of the primary sources of acetyl-CoA is from the breakdown of sugars by glycolysis which yield pyruvate that in turn is decarboxylated by the enzyme pyruvate dehydrogenase generating acetyl-CoA according to the following reaction scheme:

• CH₃C(=O)C(=O)O⁻ (pyruvate) + HSCoA + NAD⁺ → CH₃C(=O)SCoA (acetyl-CoA) + NADH + CO₂

The product of this reaction, acetyl-CoA, is the starting point for the citric acid cycle. Acetyl-CoA may also be obtained from the oxidation of fatty acids. Below is a schematic outline of the cycle:

• The citric acid cycle begins with the transfer of a two-carbon acetyl group from acetyl-CoA to the four-carbon acceptor compound (oxaloacetate) to form a six-carbon compound (citrate).
• The citrate then goes through a series of chemical transformations, losing two carboxyl groups as CO₂. The carbons lost as CO₂ originate from what was oxaloacetate, not directly from acetyl-CoA. The carbons donated by acetyl-CoA become part of the oxaloacetate carbon backbone after the first turn of the citric acid cycle. Loss of the acetyl-CoA-donated carbons as CO₂ requires several turns of the citric acid cycle. However, because of the role of the citric acid cycle in anabolism, they might not be lost, since many TCA cycle intermediates are also used as precursors for the biosynthesis of other molecules.^[10]
• Most of the energy made available by the oxidative steps of the cycle is transferred as energy-rich electrons to NAD⁺, forming NADH. For each acetyl group that enters the citric acid cycle, three molecules of NADH are produced.
• Electrons are also transferred to the electron acceptor Q, forming QH₂.
• At the end of each cycle, the four-carbon oxaloacetate has been regenerated, and the cycle continues.

Steps

Two carbon atoms are oxidized to CO₂, the energy from these reactions being transferred to other metabolic processes by GTP (or ATP), and as electrons in NADH and QH₂. The NADH generated in the TCA cycle may later donate its electrons in oxidative phosphorylation to drive ATP synthesis; FADH₂ is covalently attached to succinate dehydrogenase, an enzyme functioning both in the TCA cycle and the mitochondrial electron transport chain in oxidative phosphorylation. FADH₂, therefore, facilitates transfer of electrons to coenzyme Q, which is the final electron acceptor of the reaction catalyzed by the Succinate:ubiquinone oxidoreductase complex, also acting as an intermediate in the electron transport chain.^[11]

The citric acid cycle is continuously supplied with new carbon in the form of acetyl-CoA, entering at step 0 below.^[12]

	Substrates	Products	Enzyme	Reaction type	Comment
0 / 10	Oxaloacetate + Acetyl CoA + H2O	Citrate + CoA-SH	Citrate synthase	Aldol condensation	irreversible, extends the 4C oxaloacetate to a 6C molecule
1	Citrate	cis-Aconitate + H2O	Aconitase	Dehydration	reversible isomerisation
2	cis-Aconitate + H2O	Isocitrate		Hydration
3	Isocitrate + NAD+	Oxalosuccinate + NADH + H +	Isocitrate dehydrogenase	Oxidation	generates NADH (equivalent of 2.5 ATP)
4	Oxalosuccinate	α-Ketoglutarate + CO2		Decarboxylation	rate-limiting, irreversible stage, generates a 5C molecule
5	α-Ketoglutarate + NAD+ + CoA-SH	Succinyl-CoA + NADH + H+ + CO2	α-Ketoglutarate dehydrogenase	Oxidative decarboxylation	irreversible stage, generates NADH (equivalent of 2.5 ATP), regenerates the 4C chain (CoA excluded)
6	Succinyl-CoA + GDP + Pi	Succinate + CoA-SH + GTP	Succinyl-CoA synthetase	substrate-level phosphorylation	or ADP→ATP instead of GDP→GTP,[11] generates 1 ATP or equivalent Condensation reaction of GDP + Pi and hydrolysis of Succinyl-CoA involve the H2O needed for balanced equation.
7	Succinate + ubiquinone (Q)	Fumarate + ubiquinol (QH2)	Succinate dehydrogenase	Oxidation	uses FAD as a prosthetic group (FAD→FADH2 in the first step of the reaction) in the enzyme,[11] generates the equivalent of 1.5 ATP
8	Fumarate + H2O	L-Malate	Fumarase	Hydration
9	L-Malate + NAD+	Oxaloacetate + NADH + H+	Malate dehydrogenase	Oxidation	reversible (in fact, equilibrium favors malate), generates NADH (equivalent of 2.5 ATP)
10 / 0	Oxaloacetate + Acetyl CoA + H2O	Citrate + CoA-SH	Citrate synthase	Aldol condensation	irreversible, extends the 4C oxaloacetate to a 6C molecule

Mitochondria in animals, including humans, possess two succinyl-CoA synthetases: one that produces GTP from GDP, and another that produces ATP from ADP.^[13] Plants have the type that produces ATP (ADP-forming succinyl-CoA synthetase).^[12] Several of the enzymes in the cycle may be loosely associated in a multienzyme protein complex within the mitochondrial matrix.^[14]

The GTP that is formed by GDP-forming succinyl-CoA synthetase may be utilized by nucleoside-diphosphate kinase to form ATP (the catalyzed reaction is GTP + ADP → GDP + ATP).^[11]

Mnemonics

Citrate Alone Is Often Krebs's Starting Substrate For Making Oxaloacetate

Products

Products of the first turn of the cycle are: one GTP (or ATP), three NADH, one QH₂, two CO₂.

Because two acetyl-CoA molecules are produced from each glucose molecule, two cycles are required per glucose molecule. Therefore, at the end of two cycles, the products are: two GTP, six NADH, two QH₂, and four CO₂

Description	Reactants	Products
The sum of all reactions in the citric acid cycle is:	Acetyl-CoA + 3 NAD+ + Q + GDP + Pi + 2 H2O	→ CoA-SH + 3 NADH + 3 H+ + QH2 + GTP + 2 CO2
Combining the reactions occurring during the pyruvate oxidation with those occurring during the citric acid cycle, the following overall pyruvate oxidation reaction is obtained:	Pyruvate ion + 4 NAD+ + Q + GDP + Pi + 2 H2O	→ 4 NADH + 4 H+ + QH2 + GTP + 3 CO2
Combining the above reaction with the ones occurring in the course of glycolysis, the following overall glucose oxidation reaction (excluding reactions in the respiratory chain) is obtained:	Glucose + 10 NAD+ + 2 Q + 2 ADP + 2 GDP + 4 Pi + 2 H2O	→ 10 NADH + 10 H+ + 2 QH2 + 2 ATP + 2 GTP + 6 CO2

The above reactions are balanced if P_i represents the H₂PO₄⁻ ion, ADP and GDP the ADP²⁻ and GDP²⁻ ions, respectively, and ATP and GTP the ATP³⁻ and GTP³⁻ ions, respectively.

The total number of ATP obtained after complete oxidation of one glucose in glycolysis, citric acid cycle, and oxidative phosphorylation is estimated to be between 30 and 38.^[15]

Efficiency

The theoretical maximum yield of ATP through oxidation of one molecule of glucose in glycolysis, citric acid cycle, and oxidative phosphorylation is 38 (assuming 3 molar equivalents of ATP per equivalent NADH and 2 ATP per FADH₂). In eukaryotes, two equivalents of NADH are generated in glycolysis, which takes place in the cytoplasm. Transport of these two equivalents into the mitochondria consumes two equivalents of ATP, thus reducing the net production of ATP to 36. Furthermore, inefficiencies in oxidative phosphorylation due to leakage of protons across of the mitochondrial membrane and slippage of the ATP synthase/proton pump commonly reduces the ATP yield from NADH and FADH₂ to less than the theoretical maximum yield.^[15] The observed yields are, therefore, closer to ~2.5 ATP per NADH and ~1.5 ATP per FADH₂, further reducing the total net production of ATP to approximately 30.^[16] An assessment of the total ATP yield with newly revised proton-to-ATP ratios provides an estimate of 29.85 ATP per glucose molecule.^[17]

Variation

While the TCA cycle is in general highly conserved, there is significant variability in the enzymes found in different taxa^[18] (note that the diagrams on this page are specific to the mammalian pathway variant).

Some differences exist between eukaryotes and prokaryotes. The conversion of D-threo-isocitrate to 2-oxoglutarate is catalyzed in eukaryotes by the NAD⁺-dependent EC 1.1.1.41, while prokaryotes employ the NADP⁺-dependent EC 1.1.1.42.^[19] Similarly, the conversion of (S)-malate to oxaloacetate is catalyzed in eukaryotes by the NAD⁺-dependent EC 1.1.1.37, while most prokaryotes utilize a quinone-dependent enzyme, EC 1.1.5.4.^[20]

A step with significant variability is the conversion of succinyl-CoA to succinate. Most organisms utilize EC 6.2.1.5, succinate–CoA ligase (ADP-forming) (despite its name, the enzyme operates in the pathway in the direction of ATP formation). In mammals a GTP-forming enzyme, succinate–CoA ligase (GDP-forming) (EC 6.2.1.4) also operates. The level of utilization of each isoform is tissue dependent.^[21] In some acetate-producing bacteria, such as Acetobacter aceti, an entirely different enzyme catalyzes this conversion – EC 2.8.3.18, succinyl-CoA:acetate CoA-transferase. This specialized enzyme links the TCA cycle with acetate metabolism in these organisms.^[22] Some bacteria, such as Helicobacter pylori, employ yet another enzyme for this conversion – succinyl-CoA:acetoacetate CoA-transferase (EC 2.8.3.5).^[23]

Some variability also exists at the previous step – the conversion of 2-oxoglutarate to succinyl-CoA. While most organisms utilize the ubiquitous NAD⁺-dependent 2-oxoglutarate dehydrogenase, some bacteria utilize a ferredoxin-dependent 2-oxoglutarate synthase (EC 1.2.7.3).^[24] Other organisms, including obligately autotrophic and methanotrophic bacteria and archaea, bypass succinyl-CoA entirely, and convert 2-oxoglutarate to succinate via succinate semialdehyde, using EC 4.1.1.71, 2-oxoglutarate decarboxylase, and EC 1.2.1.79, succinate-semialdehyde dehydrogenase.^[25]

Regulation

The regulation of the TCA cycle is largely determined by product inhibition and substrate availability. If the cycle were permitted to run unchecked, large amounts of metabolic energy could be wasted in overproduction of reduced coenzyme such as NADH and ATP. The major eventual substrate of the cycle is ADP which gets converted to ATP. A reduced amount of ADP causes accumulation of precursor NADH which in turn can inhibit a number of enzymes. NADH, a product of all dehydrogenases in the TCA cycle with the exception of succinate dehydrogenase, inhibits pyruvate dehydrogenase, isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, and also citrate synthase. Acetyl-coA inhibits pyruvate dehydrogenase, while succinyl-CoA inhibits alpha-ketoglutarate dehydrogenase and citrate synthase. When tested in vitro with TCA enzymes, ATP inhibits citrate synthase and α-ketoglutarate dehydrogenase; however, ATP levels do not change more than 10% in vivo between rest and vigorous exercise. There is no known allosteric mechanism that can account for large changes in reaction rate from an allosteric effector whose concentration changes less than 10%.^[26]

Calcium is used as a regulator. Mitochondrial matrix calcium levels can reach the tens of micromolar levels during cellular activation.^[27] It activates pyruvate dehydrogenase phosphatase which in turn activates the pyruvate dehydrogenase complex. Calcium also activates isocitrate dehydrogenase and α-ketoglutarate dehydrogenase.^[28] This increases the reaction rate of many of the steps in the cycle, and therefore increases flux throughout the pathway.

Citrate is used for feedback inhibition, as it inhibits phosphofructokinase, an enzyme involved in glycolysis that catalyses formation of fructose 1,6-bisphosphate,a precursor of pyruvate. This prevents a constant high rate of flux when there is an accumulation of citrate and a decrease in substrate for the enzyme.

Recent work has demonstrated an important link between intermediates of the citric acid cycle and the regulation of hypoxia-inducible factors (HIF). HIF plays a role in the regulation of oxygen homeostasis, and is a transcription factor that targets angiogenesis, vascular remodeling, glucose utilization, iron transport and apoptosis. HIF is synthesized consititutively, and hydroxylation of at least one of two critical proline residues mediates their interaction with the von Hippel Lindau E3 ubiquitin ligase complex, which targets them for rapid degradation. This reaction is catalysed by prolyl 4-hydroxylases. Fumarate and succinate have been identified as potent inhibitors of prolyl hydroxylases, thus leading to the stabilisation of HIF.^[29]

Major metabolic pathways converging on the TCA cycle

Several catabolic pathways converge on the TCA cycle. Reactions that form intermediates of the TCA cycle in order to replenish them (especially during the scarcity of the intermediates) are called anaplerotic reactions.

The citric acid cycle is the third step in carbohydrate catabolism (the breakdown of sugars). Glycolysis breaks glucose (a six-carbon-molecule) down into pyruvate (a three-carbon molecule). In eukaryotes, pyruvate moves into the mitochondria. It is converted into acetyl-CoA by decarboxylation and enters the citric acid cycle.

In protein catabolism, proteins are broken down by proteases into their constituent amino acids. The carbon backbones of some of these amino acids can become a source of energy by being converted to acetyl-CoA and entering into the citric acid cycle.

In fat catabolism, triglycerides are hydrolyzed to break them into fatty acids and glycerol. In the liver the glycerol can be converted into glucose via dihydroxyacetone phosphate and glyceraldehyde-3-phosphate by way of gluconeogenesis. In many tissues, especially heart tissue, fatty acids are broken down through a process known as beta oxidation, which results in acetyl-CoA, which can be used in the citric acid cycle. Beta oxidation of fatty acids with an odd number of methylene bridges produces propionyl CoA, which is then converted into succinyl-CoA and fed into the citric acid cycle.^[30]

The total energy gained from the complete breakdown of one molecule of glucose by glycolysis, the citric acid cycle, and oxidative phosphorylation equals about 30 ATP molecules, in eukaryotes. The citric acid cycle is called an amphibolic pathway because it participates in both catabolism and anabolism.

Interactive pathway map

Click on genes, proteins and metabolites below to link to respective articles. ^{[§ 1]}

See also

• Calvin cycle
• Glyoxylate cycle
• Reverse (Reductive) Krebs cycle

References

External links

• An animation of the citric acid cycle at Smith College
• Citric acid cycle variants at MetaCyc
• Pathways connected to the citric acid cycle at Kyoto Encyclopedia of Genes and Genomes
• Introduction at Khan Academy
• metpath: Interactive representation of the TCA cycle

Metabolism (catabolism, anabolism) General Metabolic pathway Metabolic network Primary nutritional groups Energy metabolism Aerobic respiration Glycolysis → Pyruvate Decarboxylation → Citric acid cycle → Oxidative phosphorylation (electron transport chain + ATP synthase) Anaerobic respiration Electron acceptors are other than oxygen Fermentation Glycolysis → Substrate-level phosphorylation ABE Ethanol Lactic acid Specific paths Protein metabolism Protein synthesis Catabolism Carbohydrate metabolism (carbohydrate catabolism and anabolism) Human Glycolysis ⇄ Gluconeogenesis Glycogenolysis ⇄ Glycogenesis Pentose phosphate pathway Fructolysis Galactolysis Glycosylation N-linked O-linked Nonhuman Photosynthesis Anoxygenic photosynthesis Chemosynthesis Carbon fixation Xylose metabolism Radiotrophism Lipid metabolism (lipolysis, lipogenesis) Fatty acid metabolism Fatty acid degradation (Beta oxidation) Fatty acid synthesis Other Steroid metabolism Sphingolipid metabolism Eicosanoid metabolism Ketosis Reverse cholesterol transport Amino acid Amino acid synthesis Urea cycle Nucleotide metabolism Purine metabolism Nucleotide salvage Pyrimidine metabolism Other Metal metabolism Iron metabolism Ethanol metabolism Index of inborn errors of metabolism Description Metabolism Enzymes and pathways: citric acid cycle enzymes pentose phosphate intermediates glycoproteins enzymes glycosaminoglycans proteoglycan enzymes phospholipid metabolism cholesterol and steroid intermediates sphingolipids enzymes eicosanoids enzymes amino acid intermediates urea cycle enzymes nucleotide intermediates Disorders Citric acid cycle and electron transport chain Glycoprotein Proteoglycan Fatty-acid Phospholipid Cholesterol and steroid Eicosanoid Amino acid Purine-pyrimidine Heme metabolism Symptoms and signs Treatment Drugs other Index of biochemical families Carbohydrates Alcohols Glycoproteins Glycosides Lipids Eicosanoids Fatty acids intermediates Glycerides Phospholipids Sphingolipids Steroids Nucleic acids Constituents intermediates Proteins Amino acids intermediates Other Tetrapyrroles intermediates

Citric Acid Cycle Metabolic Pathway
			Oxaloacetate			Malate			Fumarate			Succinate			Succinyl-CoA
Acetyl-CoA				NADH + H+	NAD+			H2O		FADH2	FAD		CoA + ATP(GTP)	Pi + ADP(GDP)
	+	H2O														NADH + H+ + CO2
		CoA														NAD+
					H2O		H2O			NAD(P)+	NAD(P)H + H+			CO2
			Citrate			cis-Aconitate			Isocitrate			Oxalosuccinate			α-Ketoglutarate
Index of inborn errors of metabolism Description Metabolism Enzymes and pathways: citric acid cycle enzymes pentose phosphate intermediates glycoproteins enzymes glycosaminoglycans proteoglycan enzymes phospholipid metabolism cholesterol and steroid intermediates sphingolipids enzymes eicosanoids enzymes amino acid intermediates urea cycle enzymes nucleotide intermediates Disorders Citric acid cycle and electron transport chain Glycoprotein Proteoglycan Fatty-acid Phospholipid Cholesterol and steroid Eicosanoid Amino acid Purine-pyrimidine Heme metabolism Symptoms and signs Treatment Drugs other

Metabolism map Glucuronate metabolism Pentose interconversion Inositol metabolism Cellulose and sucrose metabolism Starch and glycogen metabolism Other sugar metabolism Pentose phosphate pathway Glycolysis and Gluconeogenesis Amino sugars metabolism Small amino acid synthesis Branched amino acid synthesis Purine biosynthesis Histidine metabolism Aromatic amino acid synthesis Pyruvate decarboxylation Fermentation Fatty acid metabolism Urea cycle Aspartate amino acid group synthesis Porphyrins and corrinoids metabolism Citric acid cycle Glutamate amino acid group synthesis Pyrimidine biosynthesis All pathway labels on this image are links, simply click to access the article. A high resolution labeled version of this image is available here.

Metabolism: Citric acid cycle enzymes Cycle Citrate synthase Aconitase Isocitrate dehydrogenase Oxoglutarate dehydrogenase Succinyl CoA synthetase Succinate dehydrogenase (SDHA) Fumarase Malate dehydrogenase and ETC Anaplerotic to acetyl-CoA Pyruvate dehydrogenase complex (E1, E2, E3) (regulated by Pyruvate dehydrogenase kinase and Pyruvate dehydrogenase phosphatase) to α-ketoglutaric acid Glutamate dehydrogenase to succinyl-CoA Methylmalonyl-CoA mutase to oxaloacetate Pyruvate carboxylase Aspartate transaminase Mitochondrial electron transport chain/ oxidative phosphorylation Primary Complex I/NADH dehydrogenase Complex II/Succinate dehydrogenase Coenzyme Q Complex III/Coenzyme Q - cytochrome c reductase Cytochrome c Complex IV/Cytochrome c oxidase Coenzyme Q10 synthesis: COQ2 COQ3 COQ4 COQ5 COQ6 COQ7 COQ9 COQ10A COQ10B PDSS1 PDSS2 Other Alternative oxidase Electron-transferring-flavoprotein dehydrogenase Index of inborn errors of metabolism Description Metabolism Enzymes and pathways: citric acid cycle enzymes pentose phosphate intermediates glycoproteins enzymes glycosaminoglycans proteoglycan enzymes phospholipid metabolism cholesterol and steroid intermediates sphingolipids enzymes eicosanoids enzymes amino acid intermediates urea cycle enzymes nucleotide intermediates Disorders Citric acid cycle and electron transport chain Glycoprotein Proteoglycan Fatty-acid Phospholipid Cholesterol and steroid Eicosanoid Amino acid Purine-pyrimidine Heme metabolism Symptoms and signs Treatment Drugs other