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{{Short description|Interconnected biochemical reactions releasing energy}}
[[File:Citric acid cycle with aconitate 2.svg|thumb|upright=2|Overview of the citric acid cycle]]
The '''citric acid cycle'''—also known as the '''Krebs cycle''', '''Szent-Györgyi-Krebs cycle''' or the '''TCA cycle (tricarboxylic acid cycle)'''—is a series of [[chemical reaction|biochemical reactions]] to release the energy stored in [[nutrient]]s through the [[Redox|oxidation]] of [[acetyl-CoA]] derived from [[carbohydrate]]s, [[fat]]s, and [[protein]]s. The chemical energy released is available under the form of [[Adenosine triphosphate|ATP]]. The [[Hans Krebs (biochemist)|Krebs]] cycle is used by [[organism]]s that [[Cellular respiration|respire]] (as opposed to organisms that [[Fermentation|ferment]]) to generate energy, either by [[anaerobic respiration]] or [[aerobic respiration]]. In addition, the cycle provides [[precursor (chemistry)|precursors]] of certain [[amino acid]]s, as well as the [[reducing agent]] [[nicotinamide adenine dinucleotide|NADH]], that are used in numerous other reactions. Its central importance to many [[Metabolic pathway|biochemical pathways]] suggests that it was one of the earliest components of [[metabolism]]. Even though it is branded as a 'cycle', it is not necessary for [[metabolite]]s to follow only one specific route; at least three alternative segments of the citric acid cycle have been recognized.

The name of this metabolic pathway is derived from the [[citric acid]] (a [[tricarboxylic acid]], often called citrate, as the ionized form predominates at biological pH) that is consumed and then regenerated by this sequence of reactions to complete the cycle. The cycle consumes acetate (in the form of [[acetyl-CoA]]) and [[water]], reduces NAD<sup>+</sup> to NADH, releasing carbon dioxide. The NADH generated by the citric acid cycle is fed into the [[oxidative phosphorylation]] (electron transport) pathway. The net result of these two closely linked pathways is the oxidation of [[nutrient]]s to produce usable chemical energy in the form of [[Adenosine triphosphate|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 citric acid cycle reaction sequence is performed in the cytosol with the proton gradient for [[Chemiosmosis|ATP production]] being across the cell's surface ([[Cell membrane|plasma membrane]]) rather than the inner membrane of the [[mitochondrion]].

For each pyruvate molecule (from glycolysis), the overall yield of energy-containing compounds from the citric acid cycle is three NADH, one [[Flavin adenine dinucleotide|FADH<sub>2</sub>]], and one [[guanosine triphosphate|GTP]].

== Discovery ==
Several of the components and reactions of the citric acid cycle were established in the 1930s by the research of [[Albert Szent-Györgyi]], who received the [[Nobel Prize in Physiology or Medicine]] in 1937 specifically for his discoveries pertaining to [[fumaric acid]], a component of the cycle. He made this discovery by studying pigeon breast muscle. Because this tissue maintains its oxidative capacity well after breaking down in the Latapie mill and releasing in aqueous solutions, breast muscle of the pigeon was very well qualified for the study of oxidative reactions. The citric acid cycle itself was finally identified in 1937 by [[Hans Adolf Krebs]] and [[William Arthur Johnson (biochemist)|William Arthur Johnson]] while at the [[University of Sheffield]], for which the former received the [[Nobel Prize for Physiology or Medicine]] in 1953, and for whom the cycle is sometimes named the "Krebs cycle".

== Overview ==
[[File:Acetyl-CoA-2D_colored.svg|thumb|upright=1.6|Structural diagram of acetyl-CoA: The portion in blue, on the left, is the [[Acetyl|acetyl group]]; the portion in black is [[coenzyme A]].]]
The citric acid cycle is a  [[metabolic pathway]] that connects [[carbohydrate]], [[fat]], and [[protein]] [[metabolism]]. The [[Chemical reaction|reaction]]s of the cycle are carried out by eight [[enzymes]] that completely oxidize [[acetate]] (a two carbon molecule), in the form of acetyl-CoA, into two molecules each of carbon dioxide and water. Through [[catabolism]] of sugars, fats, and proteins, the two-carbon organic product acetyl-CoA is produced which enters the citric acid cycle. The reactions of the cycle also convert three equivalents of [[nicotinamide adenine dinucleotide]] (NAD<sup>+</sup>) into three equivalents of reduced [[Nicotinamide adenine dinucleotide|NAD<sup>+</sup>]] (NADH), one equivalent of [[flavin adenine dinucleotide]] (FAD) into one equivalent of [[Flavin adenine dinucleotide|FADH<sub>2</sub>]], and one equivalent each of [[guanosine diphosphate]] (GDP) and inorganic [[phosphate]] (P<sub>i</sub>) into one equivalent of [[guanosine triphosphate]] (GTP). The NADH and FADH<sub>2</sub> generated by the citric acid cycle are, in turn, used by the [[oxidative phosphorylation]] pathway to generate energy-rich ATP.

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

{{block indent|{{underset|pyruvate|2=CH<sub>3</sub>C(=O)C(=O)O<sup>−</sup>}} + [[Coenzyme A|HSCoA]] + NAD<sup>+</sup> → {{underset|acetyl-CoA|2=CH<sub>3</sub>C(=O)SCoA}} + NADH + CO<sub>2</sub>}}

The product of this reaction, acetyl-CoA, is the starting point for the citric acid cycle. [[Acetyl-CoA carboxylase|Acetyl-CoA]] may also be obtained from the oxidation of [[fatty acid]]s. 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 [[Carbon dioxide|CO<sub>2</sub>]]. The carbons lost as CO<sub>2</sub> 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 CO<sub>2</sub> requires several turns of the citric acid cycle. However, because of the role of the citric acid cycle in [[anabolic|anabolism]], they might not be lost, since many citric acid cycle intermediates are also used as precursors for the [[biosynthesis]] of other molecules.
* Most of the electrons made available by the oxidative steps of the cycle are transferred to NAD<sup>+</sup>, forming NADH. For each acetyl group that enters the citric acid cycle, three molecules of NADH are produced. The citric acid cycle includes a series of oxidation reduction reaction in mitochondria.
* In addition, electrons from the succinate oxidation step are transferred first to the [[FAD]] cofactor of succinate dehydrogenase, reducing it to FADH<sub>2</sub>, and eventually to [[ubiquinone]] (Q) in the [[Mitochondrion|mitochondrial membrane]], reducing it to [[ubiquinol]] (QH<sub>2</sub>) which is a substrate of the [[Electron transport chain|electron transfer chain]] at the level of [[Complex III]].
* For every NADH and FADH<sub>2</sub> that are produced in the citric acid cycle, 2.5 and 1.5 ATP molecules are generated in oxidative [[phosphorylation]], respectively.
* At the end of each cycle, the four-carbon [[Oxaloacetic acid|oxaloacetate]] has been regenerated, and the cycle continues.

== Steps ==
There are ten basic steps in the citric acid cycle, as outlined below. The cycle is continuously supplied with new carbon in the form of [[acetyl-CoA]], entering at step 0 in the table.
{| class="wikitable"
!
!Reaction type
!Substrates
!Enzyme
!Products
!Comment
|-
|{{nowrap|0 / 10}}
|[[Aldol condensation]]
|[[Oxaloacetic acid|Oxaloacetate]] + [[Acetyl CoA]] + H<sub>2</sub>O
|[[Citrate synthase]]
|[[Citric acid|Citrate]] + [[Coenzyme A|CoA-SH]]
|irreversible, extends the 4C [[Oxaloacetic acid|oxaloacetate]] to a 6C molecule
|-
|1
| [[Dehydration reaction|Dehydration]]
|[[Citrate]]
| rowspan="2" |[[Aconitase]]
|''[[Cis–trans isomerism|cis]]''-[[Aconitic acid|Aconitate]] + H<sub>2</sub>O
|rowspan=2|reversible [[Isomerization|isomerisation]]
|-
|2
|[[Hydration reaction|Hydration]]
|[[Aconitate|''cis''-Aconitate]] + H<sub>2</sub>O
|[[Isocitric acid|Isocitrate]]
|-
|3
|[[Oxidation]]
|[[Isocitrate]] + [[Nicotinamide adenine dinucleotide|NAD]]<sup>+</sup>
| rowspan="2" |[[Isocitrate dehydrogenase]]
|[[Oxalosuccinic acid|Oxalosuccinate]] + [[Nicotinamide adenine dinucleotide|NADH + H <sup>+</sup>]]
|generates [[NADH]] (equivalent of 2.5 ATP)
|-
|4
|[[Decarboxylation]]
|[[Oxalosuccinate]]
|[[alpha-Ketoglutaric acid|α-Ketoglutarate]] + CO<sub>2</sub>
|rate-limiting, irreversible stage, generates a 5C molecule
|-
|5
|Oxidative<br />[[decarboxylation]]
|[[alpha-Ketoglutaric acid|α-Ketoglutarate]] + NAD<sup>+</sup> + CoA-SH
|[[Alpha-ketoglutarate dehydrogenase|α-Ketoglutarate<br />dehydrogenase]], [[Thiamine pyrophosphate]], [[Lipoic acid]], Mg++,transsuccinytase
|[[Succinyl-CoA]] + [[Nicotinamide adenine dinucleotide|NADH + H <sup>+</sup>]] + CO<sub>2</sub>
|irreversible stage, generates [[Nicotinamide adenine dinucleotide|NADH]] (equivalent of 2.5 ATP), regenerates the 4C chain (CoA excluded)
|-
|6
|[[substrate-level phosphorylation|substrate-level<br />phosphorylation]]
|[[Succinyl-CoA]] + [[Guanosine diphosphate|GDP]] + [[Inorganic phosphate|P<sub>i</sub>]]
|[[Succinyl coenzyme A synthetase|Succinyl-CoA synthetase]]
|[[Succinic acid|Succinate]] + CoA-SH + [[Guanosine triphosphate|GTP]]
|or [[adenosine diphosphate|ADP]]→[[adenosine triphosphate|ATP]] instead of GDP→GTP, generates 1 ATP or equivalent.<br />[[Condensation reaction]] of [[Guanosine diphosphate|GDP]] + [[Inorganic phosphate|P<sub>i</sub>]] and [[hydrolysis]] of [[succinyl-CoA]] involve the H<sub>2</sub>O needed for balanced equation.
|-
|7
|[[Redox|Oxidation]]
|[[Succinate]] + [[ubiquinone]] (Q)
|[[Succinate dehydrogenase]]
|[[Fumaric acid|Fumarate]] + [[ubiquinol]] (QH<sub>2</sub>)
|uses [[Flavin adenine dinucleotide|FAD]] as a [[prosthetic group]] (FAD→FADH<sub>2</sub> in the first step of the reaction) in the enzyme.<br />These two electrons are later transferred to QH<sub>2</sub> during Complex II of the ETC, where they generate the equivalent of 1.5 ATP
|-
|8
|[[Hydration reaction|Hydration]]
|[[Fumarate]] + H<sub>2</sub>O
|[[Fumarase]]
|''L''-[[Malic acid|Malate]]
|Hydration of C-C double bond
|-
|9
|[[Redox|Oxidation]]
|[[Malate|<small>L</small>-Malate]] + NAD<sup>+</sup>
|[[Malate dehydrogenase]]
|[[Oxaloacetate]] + NADH + H<sup>+</sup>
|reversible (in fact, equilibrium favors malate), generates [[NADH]] (equivalent of 2.5 ATP)
|-
|10 / 0
|[[Aldol condensation]]
|[[Oxaloacetic acid|Oxaloacetate]] + [[Acetyl CoA]] + H<sub>2</sub>O
|[[Citrate synthase]]
|[[Citric acid|Citrate]] + [[Coenzyme A|CoA-SH]]
|This is the same as step 0 and restarts the cycle. The reaction is irreversible and extends the 4C oxaloacetate to a 6C molecule
|}

Two [[carbon]] atoms are [[oxidation|oxidized]] to [[carbon dioxide|CO<sub>2</sub>]], the energy from these reactions is transferred to other metabolic processes through [[Guanosine triphosphate|GTP]] (or ATP), and as electrons in [[NADH]] and [[Ubiquinol|QH<sub>2</sub>]]. The NADH generated in the citric acid cycle may later be oxidized (donate its electrons) to drive [[ATP synthase|ATP synthesis]] in a type of process called [[oxidative phosphorylation]]. [[Flavin adenine dinucleotide|FADH<sub>2</sub>]] is covalently attached to [[succinate dehydrogenase]], an enzyme which functions both in the citric acid cycle and the mitochondrial [[electron transport chain]] in oxidative phosphorylation. FADH<sub>2</sub>, 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]].

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

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).

{{clear}}

== Products ==
Products of the first turn of the cycle are one [[GTP cyclohydrolase I|GTP]] (or [[Adenosine triphosphate|ATP]]), three [[Nicotinamide adenine dinucleotide|NADH]], one [[Flavin adenine dinucleotide|FADH<sub>2</sub>]] and two [[Carbon dioxide|CO<sub>2</sub>]].

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 [[Flavin adenine dinucleotide|FADH<sub>2</sub>]], and four [[Carbon dioxide|CO<sub>2</sub>]].

{| class="wikitable"
! width="50%" | Description !! Reactants !! Products
 |-
 | The sum of all reactions in the citric acid cycle is: || Acetyl-CoA + 3 NAD<sup>+</sup> + FAD + GDP + P<sub>i</sub> + 2 H<sub>2</sub>O || → CoA-SH + 3 NADH + FADH<sub>2</sub> + 3 H<sup>+</sup> + GTP + 2 CO<sub>2</sub>
 |-
 | Combining the reactions occurring during the [[pyruvate decarboxylation|pyruvate oxidation]] with those occurring during the citric acid cycle, the following overall pyruvate oxidation reaction is obtained: || Pyruvate ion + 4 NAD<sup>+</sup> + FAD + GDP + P<sub>i</sub> + 2 H<sub>2</sub>O || → 4 NADH + FADH<sub>2</sub> + 4 H<sup>+</sup> + GTP + 3 CO<sub>2</sub>
 |-
 | 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<sup>+</sup> + 2 FAD + 2 ADP + 2 GDP + 4 P<sub>i</sub> + 2 H<sub>2</sub>O || → 10 NADH + 2 FADH<sub>2</sub> + 10 H<sup>+</sup> + 2 ATP + 2 GTP + 6 CO<sub>2</sub>
|}

The above reactions are balanced if P<sub>i</sub> represents the H<sub>2</sub>PO<sub>4</sub><sup>−</sup> ion, ADP and GDP the ADP<sup>2−</sup> and GDP<sup>2−</sup> ions, respectively, and ATP and GTP the ATP<sup>3−</sup> and GTP<sup>3−</sup> ions, respectively.

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

== Efficiency ==
The theoretical maximum yield of [[ATP synthase|ATP]] through oxidation of one molecule of glucose in glycolysis, citric acid cycle, and [[oxidative phosphorylation]] is 38 (assuming 3 [[molar equivalent]]s of ATP per equivalent NADH and 2 ATP per FADH<sub>2</sub>). In eukaryotes, two equivalents of NADH and two equivalents of ATP are generated in [[glycolysis]], which takes place in the [[cytoplasm]]. If transported using the [[glycerol phosphate shuttle]] rather than the [[malate-aspartate shuttle]], transport of two of these equivalents of NADH into the mitochondria effectively 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 the [[Mitochondrion|mitochondrial membrane]] and slippage of the [[ATP synthase]]/proton pump commonly reduces the ATP yield from NADH and [[Flavin adenine dinucleotide|FADH<sub>2</sub>]] to less than the theoretical maximum yield. The observed yields are, therefore, closer to ~2.5 ATP per NADH and ~1.5 ATP per FADH<sub>2</sub>, further reducing the total net production of ATP to approximately 30. An assessment of the total ATP yield with newly revised proton-to-ATP ratios provides an estimate of 29.85 ATP per glucose molecule.

== Variation ==
While the citric acid cycle is in general highly conserved, there is significant variability in the enzymes found in different taxa (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<sup>+</sup>-dependent [http://www.enzyme-database.org/query.php?ec=1.1.1.41 EC 1.1.1.41], while prokaryotes employ the NADP<sup>+</sup>-dependent [http://www.enzyme-database.org/query.php?ec=1.1.1.42 EC 1.1.1.42]. Similarly, the conversion of (''S'')-malate to oxaloacetate is catalyzed in eukaryotes by the NAD<sup>+</sup>-dependent [http://www.enzyme-database.org/query.php?ec=1.1.1.37 EC 1.1.1.37], while most prokaryotes utilize a quinone-dependent enzyme, [http://www.enzyme-database.org/query.php?ec=1.1.5.4 EC 1.1.5.4].

A step with significant variability is the conversion of succinyl-CoA to succinate. Most organisms utilize [http://www.enzyme-database.org/query.php?ec=6.2.1.5 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) ([http://www.enzyme-database.org/query.php?ec=6.2.1.4 EC 6.2.1.4]) also operates. The level of utilization of each isoform is tissue dependent. In some acetate-producing bacteria, such as ''Acetobacter aceti'', an entirely different enzyme catalyzes this conversion&nbsp;– [http://www.enzyme-database.org/query.php?ec=2.8.3.18 EC 2.8.3.18], succinyl-CoA:acetate CoA-transferase. This specialized enzyme links the TCA cycle with acetate metabolism in these organisms. Some bacteria, such as ''Helicobacter pylori'', employ yet another enzyme for this conversion&nbsp;– succinyl-CoA:acetoacetate CoA-transferase ([http://www.enzyme-database.org/query.php?ec=2.8.3.5 EC 2.8.3.5]).

Some variability also exists at the previous step&nbsp;– the conversion of 2-oxoglutarate to succinyl-CoA. While most organisms utilize the ubiquitous NAD<sup>+</sup>-dependent 2-oxoglutarate dehydrogenase, some bacteria utilize a ferredoxin-dependent 2-oxoglutarate [[synthase]] ([http://www.enzyme-database.org/query.php?ec=1.2.7.3 EC 1.2.7.3]).
Other organisms, including obligately autotrophic and methanotrophic bacteria and archaea, bypass succinyl-CoA entirely, and convert 2-oxoglutarate to succinate via succinate semialdehyde, using [http://www.enzyme-database.org/query.php?ec=4.1.1.71 EC 4.1.1.71], 2-oxoglutarate decarboxylase, and [http://www.enzyme-database.org/query.php?ec=1.2.1.79 EC 1.2.1.79], succinate-semialdehyde dehydrogenase.

In [[cancer]], there are substantial [[Warburg effect (oncology)|metabolic derangements]] that occur to ensure the proliferation of tumor cells, and consequently metabolites can accumulate which serve to facilitate [[tumorigenesis]], dubbed onco[[metabolites]]. Among the best characterized oncometabolites is [[2-hydroxyglutarate]] which is produced through a [[heterozygous]] [[gain-of-function mutation]] (specifically a [[Neomorphic mutation|neomorphic]] one) in [[isocitrate dehydrogenase]] (IDH) (which under normal circumstances catalyzes the [[oxidation]] of [[isocitrate]] to [[oxalosuccinate]], which then spontaneously [[Decarboxylation|decarboxylates]] to [[Alpha ketoglutarate|alpha-ketoglutarate]], as discussed above; in this case an additional [[Organic redox reaction|reduction]] step occurs after the formation of alpha-ketoglutarate via [[NADPH]] to yield 2-hydroxyglutarate), and hence IDH is considered an [[oncogene]]. Under physiological conditions, 2-hydroxyglutarate is a minor product of several metabolic pathways as an error but readily converted to alpha-ketoglutarate via hydroxyglutarate dehydrogenase enzymes ([[L2HGDH]] and [[D2HGDH]]) but does not have a known physiologic role in mammalian cells; of note, in cancer, 2-hydroxyglutarate is likely a terminal metabolite as isotope labelling experiments of colorectal cancer cell lines show that its conversion back to alpha-ketoglutarate is too low to measure. In cancer, 2-hydroxyglutarate serves as a [[Competitive inhibition|competitive inhibitor]] for a number of enzymes that facilitate reactions via alpha-ketoglutarate in alpha-ketoglutarate-dependent [[dioxygenase]]s. This mutation results in several important changes to the metabolism of the cell. For one thing, because there is an extra NADPH-catalyzed reduction, this can contribute to depletion of cellular stores of NADPH and also reduce levels of alpha-ketoglutarate available to the cell. In particular, the depletion of NADPH is problematic because NADPH is highly compartmentalized and cannot freely diffuse between the organelles in the cell. It is produced largely via the [[pentose phosphate pathway]] in the cytoplasm. The depletion of NADPH results in increased [[oxidative stress]] within the cell as it is a required cofactor in the production of [[Glutathione|GSH]], and this oxidative stress can result in DNA damage. There are also changes on the genetic and epigenetic level through the function of [[Histone code|histone lysine demethylases]] (KDMs) and [[Ten-Eleven Translocation 2|ten-eleven translocation]] (TET) enzymes; ordinarily TETs hydroxylate [[5-Methylcytosine|5-methylcytosines]] to prime them for demethylation. However, in the absence of alpha-ketoglutarate this cannot be done and there is hence hypermethylation of the cell's DNA, serving to promote [[Epithelial–mesenchymal transition|epithelial-mesenchymal transition (EMT)]] and inhibit cellular differentiation. A similar phenomenon is observed for the Jumonji C family of KDMs which require a hydroxylation to perform demethylation at the epsilon-amino methyl group.Additionally, the inability of prolyl hydroxylases to catalyze reactions results in stabilization of [[HIF1A|hypoxia-inducible factor alpha]], which is necessary to promote degradation of the latter (as under conditions of low oxygen there will not be adequate substrate for hydroxylation). This results in a [[Pseudohypoxia|pseudohypoxic]] phenotype in the cancer cell that promotes [[angiogenesis]], metabolic reprogramming, [[cell growth]], and [[Cell migration|migration]].

== Regulation ==
'''Allosteric regulation by metabolites'''. The regulation of the citric acid cycle is largely determined by product inhibition and substrate availability. If the cycle were permitted to run unchecked, large amounts of [[Metabolism|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 citric acid cycle with the exception of [[succinate dehydrogenase]], inhibits [[pyruvate dehydrogenase]], [[isocitrate dehydrogenase]], [[Alpha-ketoglutarate 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 [[Alpha-ketoglutarate dehydrogenase|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%.

'''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.

'''Regulation by calcium'''. Calcium is also used as a regulator in the citric acid cycle. Calcium levels in the mitochondrial matrix can reach up to the tens of micromolar levels during cellular activation. It activates [[pyruvate dehydrogenase phosphatase]] which in turn activates the [[pyruvate dehydrogenase complex]]. Calcium also activates [[isocitrate dehydrogenase]] and [[Alpha-ketoglutarate dehydrogenase|α-ketoglutarate dehydrogenase]]. This increases the reaction rate of many of the steps in the cycle, and therefore increases flux throughout the pathway.

'''Transcriptional regulation'''. Recent work has demonstrated an important link between intermediates of the citric acid cycle and the regulation of [[hypoxia-inducible factors]] ([[HIF1A|HIF]]). HIF plays a role in the regulation of oxygen [[homeostasis]], and is a transcription factor that targets [[angiogenesis]], [[Vascular remodelling in the embryo|vascular remodeling]], [[glucose]] utilization, iron transport and [[apoptosis]]. HIF is synthesized constitutively, 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 hydroxylase|prolyl 4-hydroxylases]]. Fumarate and succinate have been identified as potent inhibitors of prolyl hydroxylases, thus leading to the stabilisation of HIF.

== Major metabolic pathways converging on the citric acid cycle ==
Several [[catabolic]] pathways converge on the citric acid cycle. Most of these reactions add intermediates to the citric acid cycle, and are therefore known as [[anaplerotic reactions]], from the Greek meaning to "fill up". These increase the amount of acetyl CoA that the cycle is able to carry, increasing the [[Mitochondrion|mitochondrion's]] capability to carry out respiration if this is otherwise a limiting factor. Processes that remove intermediates from the cycle are termed "cataplerotic" reactions.

In this section and in the next, the citric acid cycle intermediates are indicated in ''italics'' to distinguish them from other substrates and end-products.

[[Pyruvate]] molecules produced by [[glycolysis]] are [[active transport|actively transported]] across the inner [[Mitochondrion|mitochondrial]] membrane, and into the matrix. Here they can be [[Redox|oxidized]] and combined with [[coenzyme A]] to form CO<sub>2</sub>, ''[[acetyl-CoA]]'', and [[NADH]], as in the normal cycle.

However, it is also possible for pyruvate to be [[carboxylated]] by [[pyruvate carboxylase]] to form ''oxaloacetate''. This latter reaction "fills up" the amount of ''oxaloacetate'' in the citric acid cycle, and is therefore an anaplerotic reaction, increasing the cycle's capacity to metabolize ''acetyl-CoA'' when the tissue's energy needs (e.g. in [[striated muscle tissue|muscle]]) are suddenly increased by activity.

In the citric acid cycle all the intermediates (e.g. ''[[Citric acid|citrate]]'', ''iso-citrate'', ''[[Alpha-Ketoglutaric acid|alpha-ketoglutarate]]'', ''[[Succinic acid|succinate]]'', ''[[Fumaric acid|fumarate]]'', ''[[Malic acid|malate]]'', and ''[[Oxaloacetic acid|oxaloacetate]]'') are regenerated during each turn of the cycle. Adding more of any of these intermediates to the mitochondrion therefore means that that additional amount is retained within the cycle, increasing all the other intermediates as one is converted into the other. Hence the addition of any one of them to the cycle has an anaplerotic effect, and its removal has a cataplerotic effect. These anaplerotic and cataplerotic reactions will, during the course of the cycle, increase or decrease the amount of ''oxaloacetate'' available to combine with ''acetyl-CoA'' to form ''citric acid''. This in turn increases or decreases the rate of [[Adenosine triphosphate|ATP]] production by the mitochondrion, and thus the availability of ATP to the cell.

''[[Acetyl-CoA]]'', on the other hand, derived from pyruvate oxidation, or from the [[beta-oxidation]] of [[fatty acids]], is the only fuel to enter the citric acid cycle. With each turn of the cycle one molecule of ''acetyl-CoA'' is consumed for every molecule of ''oxaloacetate'' present in the mitochondrial matrix, and is never regenerated. It is the oxidation of the acetate portion of ''acetyl-CoA'' that produces CO<sub>2</sub> and water, with the energy thus released captured in the form of ATP. The three steps of beta-oxidation resemble the steps that occur in the production of oxaloacetate from succinate in the TCA cycle. Acyl-CoA is oxidized to trans-Enoyl-CoA while FAD is reduced to FADH<sub>2</sub>, which is similar to the oxidation of succinate to fumarate. Following, [[Trans-2-enoyl-CoA reductase (NADPH)|trans-Enoyl-CoA]] is hydrated across the double bond to beta-hydroxyacyl-CoA, just like fumarate is hydrated to malate. Lastly, beta-hydroxyacyl-CoA is oxidized to beta-ketoacyl-CoA while NAD+ is reduced to NADH, which follows the same process as the oxidation of malate to [[Oxaloacetic acid|oxaloacetate]].

In the liver, the carboxylation of [[cytosol]]ic pyruvate into intra-mitochondrial ''oxaloacetate'' is an early step in the [[gluconeogenesis|gluconeogenic]] pathway which converts [[lactic acid|lactate]] and de-aminated [[alanine]] into glucose, under the influence of high levels of [[glucagon]] and/or [[epinephrine]] in the blood. Here the addition of ''oxaloacetate'' to the mitochondrion does not have a net anaplerotic effect, as another citric acid cycle intermediate (''malate'') is immediately removed from the mitochondrion to be converted into cytosolic oxaloacetate, which is ultimately converted into glucose, in a process that is almost the reverse of [[glycolysis]].

In [[protein catabolism]], [[protein]]s are broken down by [[protease]]s into their constituent amino acids. Their carbon skeletons (i.e. the de-aminated amino acids) may either enter the citric acid cycle as intermediates (e.g. ''alpha-ketoglutarate'' derived from glutamate or glutamine), having an anaplerotic effect on the cycle, or, in the case of [[leucine]], [[isoleucine]], [[lysine]], [[phenylalanine]], [[tryptophan]], and [[tyrosine]], they are converted into ''acetyl-CoA'' which can be burned to CO<sub>2</sub> and water, or used to form [[ketone bodies]], which too can only be burned in tissues other than the liver where they are formed, or excreted via the urine or breath. These latter amino acids are therefore termed "ketogenic" amino acids, whereas those that enter the citric acid cycle as intermediates can only be cataplerotically removed by entering the gluconeogenic pathway via ''malate'' which is transported out of the mitochondrion to be converted into cytosolic oxaloacetate and ultimately into [[glucose]]. These are the so-called "glucogenic" amino acids. De-aminated alanine, cysteine, glycine, serine, and threonine are converted to pyruvate and can consequently either enter the citric acid cycle as ''oxaloacetate'' (an anaplerotic reaction) or as ''acetyl-CoA'' to be disposed of as CO<sub>2</sub> and water.

In [[fat catabolism]], [[triglyceride]]s are [[hydrolysis|hydrolyzed]] to break them into [[fatty acid]]s and [[glycerol]]. In the liver the glycerol can be converted into glucose via [[dihydroxyacetone phosphate]] and [[glyceraldehyde-3-phosphate]] by way of [[gluconeogenesis]]. In skeletal muscle, glycerol is used in [[glycolysis]] by converting glycerol into [[Glycerol 3-phosphate|glycerol-3-phosphate]], then into [[Dihydroxyacetone phosphate|dihydroxyacetone phosphate (DHAP)]], then into glyceraldehyde-3-phosphate.

In many tissues, especially heart and skeletal [[muscle tissue]], [[fatty acid]]s are broken down through a process known as [[beta oxidation]], which results in the production of mitochondrial ''acetyl-CoA'', which can be used in the citric acid cycle. [[Beta oxidation]] of [[fatty acid]]s with an odd number of [[methylene bridge]]s produces [[propionyl-CoA]], which is then converted into ''[[succinyl-CoA]]'' and fed into the citric acid cycle as an anaplerotic intermediate.

The total energy gained from the complete breakdown of one (six-carbon) molecule of glucose by [[glycolysis]], the formation of 2 ''acetyl-CoA'' molecules, their catabolism in the citric acid cycle, and oxidative phosphorylation equals about 30 [[Adenosine triphosphate|ATP molecules]], in [[eukaryote]]s. The number of ATP molecules derived from the beta oxidation of a 6 carbon segment of a fatty acid chain, and the subsequent [[Redox|oxidation]] of the resulting 3 molecules of [[Acetyl-CoA carboxylase|''acetyl-CoA'' is]] 40.

== Citric acid cycle intermediates serve as substrates for biosynthetic processes ==
In this subheading, as in the previous one, the TCA intermediates are identified by ''italics''.

Several of the citric acid cycle intermediates are used for the synthesis of important compounds, which will have significant cataplerotic effects on the cycle.
''Acetyl-CoA'' cannot be transported out of the mitochondrion. To obtain cytosolic acetyl-CoA, ''citrate'' is removed from the citric acid cycle and carried across the inner mitochondrial membrane into the cytosol. There it is cleaved by [[ATP citrate lyase]] into acetyl-CoA and oxaloacetate. The oxaloacetate is returned to mitochondrion as ''malate'' (and then converted back into ''oxaloacetate'' to transfer more ''acetyl-CoA'' out of the mitochondrion). The cytosolic acetyl-CoA is used for [[fatty acid synthesis]] and the [[Mevalonate pathway|production of cholesterol]]. [[Cholesterol]] can, in turn, be used to synthesize the [[Steroid#Steroidogenesis|steroid hormones]], [[Bile acids|bile salts]], and [[vitamin D]].

The carbon skeletons of many [[Essential amino acid|non-essential amino acids]] are made from citric acid cycle intermediates. To turn them into amino acids the [[Keto acid|alpha keto-acids]] formed from the citric acid cycle intermediates have to acquire their amino groups from [[glutamate]] in a [[transamination]] reaction, in which [[Pyridoxine|pyridoxal phosphate]] is a cofactor. In this reaction the glutamate is converted into [[alpha-Ketoglutaric acid|''alpha-ketoglutarate'']], which is a citric acid cycle intermediate. The intermediates that can provide the [[Skeletal formula|carbon skeletons]] for amino acid synthesis are ''[[Oxaloacetic acid|oxaloacetate]]'' which forms [[aspartate]] and [[asparagine]]; and ''alpha-ketoglutarate'' which forms [[glutamine]], [[proline]], and [[arginine]].

Of these amino acids, aspartate and glutamine are used, together with carbon and nitrogen atoms from other sources, to form the [[purines]] that are used as the bases in [[DNA]] and [[RNA]], as well as in [[Adenosine triphosphate|ATP]], [[Adenosine monophosphate|AMP]], [[Guanosine triphosphate|GTP]], [[Nicotinamide adenine dinucleotide|NAD]], [[Flavin adenine dinucleotide|FAD]] and [[Coenzyme A|CoA]].

The [[pyrimidines]] are partly assembled from aspartate (derived from ''oxaloacetate''). The pyrimidines, [[thymine]], [[cytosine]] and [[uracil]], form the complementary bases to the purine bases in DNA and RNA, and are also components of [[Cytidine triphosphate|CTP]], [[Uridine monophosphate|UMP]], [[Uridine diphosphate|UDP]] and [[Uridine triphosphate|UTP]].

The majority of the carbon atoms in the [[porphyrin]]s come from the citric acid cycle intermediate, ''[[succinyl-CoA]]''. These molecules are an important component of the [[hemoprotein]]s, such as [[hemoglobin]], [[myoglobin]] and various [[cytochrome]]s.

During gluconeogenesis [[Gluconeogenesis#Pathway|mitochondrial ''oxaloacetate'' is reduced to ''malate'']] which is then transported out of the mitochondrion, to be oxidized back to oxaloacetate in the cytosol. Cytosolic oxaloacetate is then [[Decarboxylation|decarboxylated]] to [[phosphoenolpyruvate]] by [[phosphoenolpyruvate carboxykinase]], which is the rate limiting step in the conversion of nearly all the [[Glucogenic amino acid|gluconeogenic]] precursors (such as the glucogenic amino acids and lactate) into glucose by the [[liver]] and [[kidney]].

Because the citric acid cycle is involved in both [[catabolic]] and [[anabolic]] processes, it is known as an [[amphibolic]] pathway.
Evan M.W.Duo
{{TCACycle WP78}}

== Glucose feeds the TCA cycle via circulating lactate ==
The [[Metabolic pathway|metabolic]] role of [[Lactic acid|lactate]] is well recognized as a fuel for [[Tissue (biology)|tissue]]s, [[mitochondrial disease|mitochondrial cytopathies]] such as [[DPH Cytopathy]], and the scientific field of [[oncology]] ([[Neoplasm|tumor]]s). In the classical [[Cori cycle]], muscles produce lactate which is then taken up by the [[liver]] for [[gluconeogenesis]]. New studies suggest that lactate can be used as a source of [[carbon]] for the TCA cycle.

== Evolution ==
It is believed that components of the citric acid cycle were derived from [[anaerobic bacteria]], and that the TCA cycle itself may have evolved more than once. It may even predate biosis: the substrates appear to undergo most of the reactions spontaneously in the presence of [[persulfate]] radicals.  Theoretically, several alternatives to the TCA cycle exist; however, the TCA cycle appears to be the most efficient. If several TCA alternatives had evolved independently, they all appear to have [[convergent evolution|converged]] to the TCA cycle.

== See also ==
* [[Calvin cycle]]
* [[Glyoxylate cycle]]
* [[Reverse Krebs cycle|Reverse (reductive) Krebs cycle]]
* [[w:simple:Krebs cycle|Krebs cycle (simple English)]]

== External links ==
{{Scholia|Q27436670}}
* [https://www.science.smith.edu/departments/Biology/Bio231/krebs.html An animation of the citric acid cycle] at [[Smith College]]
* [https://biocyc.org/META/NEW-IMAGE?object=TCA-VARIANTS Citric acid cycle variants] at [[MetaCyc]]
* [https://www.genome.ad.jp/kegg/pathway/map/map00020.html Pathways connected to the citric acid cycle] {{Webarchive|url=https://web.archive.org/web/20081026150930/http://www.genome.ad.jp/kegg/pathway/map/map00020.html |date=2008-10-26 }} at [[KEGG|Kyoto Encyclopedia of Genes and Genomes]]
* [http://www.metpath.teithe.gr/?part=TCA&lang=en ''metpath'': Interactive representation of the citric acid cycle]

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[[Category:Citric acid cycle| ]]
[[Category:Biochemistry]]
[[Category:Cellular respiration]]
[[Category:Exercise physiology]]
[[Category:Metabolic pathways]]
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