The citric acid cycle
) – also known as the tricarboxylic acid
or the Krebs cycle
– is a series of chemical reaction
s used by all aerobic organism
s to release stored energy through the oxidation
derived from carbohydrate
s, and protein
s 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 are 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
The name of this metabolic pathway is derived from the citric acid
(a type of 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+
to NADH, and produces carbon dioxide as a waste byproduct. 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 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 citric acid cycle 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
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 key component of the cycle. The citric acid cycle itself was finally identified in 1937 by Hans Adolf Krebs
and William Arthur Johnson while at the University of Sheffield
, for which the former received the Nobel Prize for Physiology or Medicine
Components of the citric acid cycle were derived from anaerobic bacteria
, and the TCA cycle itself may have evolved more than once. 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 converged
to the TCA cycle.
The portion in blue, on the left, is the acetyl group
; the portion in black is coenzyme A
The citric acid cycle is a key metabolic pathway that connects carbohydrate, fat, and protein metabolism. The reactions of the cycle are carried out by eight enzymes that completely oxidize acetate, 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 (a form of acetate) is produced which enters the citric acid cycle. The reactions of the cycle also convert 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
(Pi) 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 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:
+ 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 citric acid cycle intermediates are also used as precursors for the biosynthesis of other molecules.
- 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 (Q = FAD+, QH2 = FADH2).
- For every NADH and FADH2 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 oxaloacetate has been regenerated, and the cycle continues.
atoms are oxidized
, the energy from these reactions is transferred to other metabolic processes through GTP
(or ATP), and as electrons in NADH
. The NADH generated in the citric acid cycle may later be oxidized (donate its electrons) to drive ATP synthesis in a type of process called oxidative phosphorylation. FADH2
is covalently attached to succinate dehydrogenase
, an enzyme which functions both in the CAC 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.
The citric acid cycle is continuously supplied with new carbon in the form of acetyl-CoA, entering at step 0 below.
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).
Products of the first turn of the cycle are one GTP (or ATP), three NADH, one QH2, and 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.
The above reactions are balanced if Pi 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 molecules obtained after complete oxidation of one glucose in glycolysis, citric acid cycle, and oxidative phosphorylation
is estimated to be between 30 and 38.
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 equivalent
s 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 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. 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. An assessment of the total ATP yield with newly revised proton-to-ATP ratios provides an estimate of 29.85 ATP per glucose molecule.
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+-dependent EC 126.96.36.199
, while prokaryotes employ the NADP+-dependent EC 188.8.131.52
. Similarly, the conversion of (S
)-malate to oxaloacetate is catalyzed in eukaryotes by the NAD+-dependent EC 184.108.40.206
, while most prokaryotes utilize a quinone-dependent enzyme, EC 220.127.116.11
A step with significant variability is the conversion of succinyl-CoA to succinate. Most organisms utilize EC 18.104.22.168
, 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 22.214.171.124
) 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 – EC 126.96.36.199
, 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 – succinyl-CoA:acetoacetate CoA-transferase ( EC 188.8.131.52
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 184.108.40.206
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 220.127.116.11
, 2-oxoglutarate decarboxylase, and EC 18.104.22.168
, succinate-semialdehyde dehydrogenase.
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 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
, α-ketoglutarate dehydrogenase
, and also citrate synthase
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%.
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 α-ketoglutarate dehydrogenase
. 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 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-hydroxylase
s. 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
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'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.
molecules produced by glycolysis
are actively transported
across the inner mitochondrial
membrane, and into the matrix. Here they can be oxidized
and combined with coenzyme A
to form CO2, 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 muscle
) are suddenly increased by activity.
In the citric acid cycle all the intermediates (e.g. citrate, iso-citrate, alpha-ketoglutarate, succinate, fumarate, malate
) 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 ATP
production by the mitochondrion, and thus the availability of ATP to the cell.
, 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 CO2 and water, with the energy thus released captured in the form of ATP.
In the liver, the carboxylation of cytosol
ic pyruvate into intra-mitochondrial oxaloacetate
is an early step in the gluconeogenic
pathway which converts lactate
and de-aminated alanine
into glucose, under the influence of high levels of glucagon
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
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 CO2 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 CO2 and water.
In fat catabolism
s are hydrolyzed
to break them into fatty acid
s and glycerol
. In the liver the glycerol can be converted into glucose via dihydroxyacetone phosphate
by way of gluconeogenesis. In many tissues, especially heart and skeletal muscle tissue, fatty acids 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 acids 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 ATP molecules, in eukaryotes. The number of ATP molecules derived from the beta oxidation of a 6 carbon segment of a fatty acid chain, and the subsequent oxidation of the resulting 3 molecules of acetyl-CoA
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.
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 production of cholesterol
. Cholesterol can, in turn, be used to synthesize the steroid hormones
, bile salts
, and vitamin D
The carbon skeletons of many non-essential amino acids
are made from citric acid cycle intermediates. To turn them into amino acids the alpha keto-acids
formed from the citric acid cycle intermediates have to acquire their amino groups from glutamate
in a transamination
reaction, in which pyridoxal phosphate
is a cofactor. In this reaction the glutamate is converted into alpha-ketoglutarate
, which is a citric acid cycle intermediate. The intermediates that can provide the carbon skeletons for amino acid synthesis are oxaloacetate
which forms aspartate
; and alpha-ketoglutarate
which forms glutamine
, 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
, as well as in ATP
are partly assembled from aspartate (derived from oxaloacetate
). The pyrimidines, thymine
, form the complementary bases to the purine bases in DNA and RNA, and are also components of CTP
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
and various cytochrome
During gluconeogenesis 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 decarboxylated to phosphoenolpyruvate
by phosphoenolpyruvate carboxykinase
, which is the rate limiting step in the conversion of nearly all the 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
processes, it is known as an amphibolic
Interactive pathway map
Glucose feeds the TCA cycle via circulating Lactate
The metabolic role of lactate is well recognized, including as a fuel for tissues and tumors. 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.
TY - JOUR
AU - Hui, Sheng
AU - Ghergurovich, Jonathan M.
AU - Morscher, Raphael J.
AU - Jang, Cholsoon
AU - Teng, Xin
AU - Lu, Wenyun
AU - Esparza, Lourdes A.
AU - Reya, Tannishtha
AU - Le Zhan,
AU - Yanxiang Guo, Jessie
AU - White, Eileen
AU - Rabinowitz, Joshua D.
TI - Glucose feeds the TCA cycle via circulating lactate
JA - Nature
PY - 2017/10/18/online
VL - advance online publication
PB - Macmillan Publishers Limited, part of Springer Nature. All rights reserved.
SN - 1476-4687
UR - http://dx.doi.org/10.1038/nature24057
L3 - 10.1038/nature24057
M3 - Letter L3 - http://www.nature.com/nature/journal/vaop/ncurrent/abs/nature24057.html#supplementary-information ER -