The Tricarboxylic acid cycle

The tricarboxylic acid (TCA) cycle, also known as the citric acid cycle or Krebs cycle, is a key metabolic pathway that unifies carbohydrate, fat, and protein metabolism. Commonly referred to as the Krebs cycle, it takes its name from its discoverer Hans Adolf Krebs, a German-born British medical scientist, who discovered the cycle in 1937 while working at the University of Sheffield. The discovery won him the 1953 Nobel Prize in Physiology and Medicine. The cycle is a series of biochemical reactions employed to generate energy through the oxidation of acetate in the form of acetyl-CoA. The acetyl-CoA can be derived from the degradation of sugars (glycolysis), fats (lipolysis and fatty acid β-oxidation) and proteins (proteolysis, de-amination and/or trans-amination). The cycle comprises eight sequential enzymic reactions that reduce each 2 carbon acetate moiety to two molecules of CO2. The energy released by each turn of the cycle is stored either as high energy phosphate in one molecule of GTP, or as high energy electrons in three molecules of NADH + H+ and one molecule of the reduced cofactor, coenzyme Q (QH2) via FADH2. The three NADH and one FADH2 produced by each turn of the cycle are re-oxidised and generate ATP in a process called oxidative phosphorylation. The theoretical yield is 3 ATP per molecule of NADH and 2 ATP per molecule of FADH2 making a total per cycle of 11 ATP and 1 GTP. Inefficiencies in oxidative phosphorylation reduce the actual yield to ~2.5 per NADH and 1.5 per FADH2 (10 ATP equivalents) per cycle. Energy production via the TCA cycle and oxidative phosphorylation takes place in specialized organelles called mitochondria.

Enzymes involved in the TCA cycle

Figure 1. Overview of the citric acid cycle<sup>[1]</sup> . See text for details. Click to enlarge
Figure 1. Overview of the citric acid cycle[1] . See text for details. Click to enlarge
As shown in Figure 1 there are eight enzymic steps in the TCA cycle and details of the chemical reactions involved can be found elsewhere[1][2]. The cycle begins with an irreversible aldol condensation reaction where the two-carbon acetyl group from acetyl-CoA is transferred to the four carbon dicarboxylic acid oxaloacetate, to form the six-carbon tricarboxylic acid citrate. This reaction is catalyzed by the enzyme citrate synthase[1][2]. In the second reaction, citrate is converted to isocitrate in a two step process catalyzed by the enzyme acontinase. Here a water molecule is removed (dehydration reaction) from the citric acid forming the intermediate cis-aconitate which is then rehydrated to form isocitrate. The overall effect of this conversion is that the –OH group is moved from the 3' position (citrate) to the 4' position (isocitrate) of the molecule[2]. In the third reaction, another two step process, the enzyme isocitrate dehydrogenase catalyzes the oxidation of the –OH group at the 4' position of isocitrate to yield a molecule of NADH + H+ and the tricarboxylic acid intermediate oxalosuccinate which is then decarboxylated to yield the 5-carbon dicarboxylic acid α-ketoglutarate[1][2]. This is another rate-limiting, irreversible stage in the cycle[1]. In the fourth reaction, an oxidative decarboxylation catalyzed by the enzyme α-ketoglutarate dehydrogenase, the α-ketoglutarate is converted to succinyl-CoA. This involves the reduction of NAD+ to NADH + H+ and the production of a 4 carbon dicarboxylic acid CoA derivative (succinyl-CoA).

The fifth reaction of the TCA cycle is a substrate-level phosphorylation catalyzed by the enzyme succinyl-CoA synthetase. In this reaction a free phosphate group attacks the succinyl-CoA molecule releasing the CoA (hydrolysis) and generating a succinyl phosphate intermediate. In a condensation reaction, the phosphate is then transferred to GDP to form GTP and the 4-carbon dicarboxylic acid, succinate[1][2]. In the sixth reaction succinate is oxidized to fumarate by the enzyme succinate dehydrogenase an enzyme that functions in both the TCA cycle and the electron transport chain[1]. Again this is a two step process where the prosthetic group FAD of succinate dehydrogenase is reduced to FADH2 before the reducing equivalents are transferred to coenzyme (Q) forming QH2[1][2]. Coenzyme Q, also referred to as ubiquinone, is a component of the electron transport chain which generates ATP from FADH2 and NADH + H+[3]. The last two (seventh and eighth) steps of the TCA cycle involve the hydration of fumarate to malate by the enzyme fumarase and the oxidation of malate to oxaloacetate by the enzyme malate dehydrogenase. Both of these reactions are readily reversible and a new molecule of oxaloacetate has been generated for the next round of the cycle.

In summary, one round of the TCA cycle results in one molecule of acetyl-CoA being oxidized to two molecules of CO2 with the generation of one molecule of GTP (step 5), three molecules of NADH + H+ (steps 3, 4 and 8) and one molecule of QH2 (step 6). The NADH + H+ and QH2 molecules act as electron carriers and are used to generate ATP in the process of oxidative phosphorylation. The carbons lost as CO2 in the each turn of the TCA cycle originate from the oxaloacetate moiety, not the acetyl-CoA[2]. The carbons donated by acetyl-CoA become part of the oxaloacetate carbon backbone used in the next turn of the cycle. Loss of the acetyl-CoA-donated carbons as CO2 requires several turns of the citric acid cycle[2]. The TCA cycle occurs in the mitochondrial matrix while the process of oxidative phosphorylation occurs at the inner mitochondrial membrane[4].

Discovery of the TCA cycle

Hans Adolf Krebs[5][6]
Hans Adolf Krebs[5][6]
Hans Krebs[5][6] was born on 25 August 1900 in Hildesheim, Germany. Trained as a physician he switched to research in 1924 training in chemistry and biochemistry at the Department of Chemistry at the Pathological Institute of the Charité Hospital, Berlin[5]. After periods with Otto Warburg in Berlin (1926-30) and the Department of Medicine at the Municipal Hospital in Altona (1931) he joined the Medical Clinic of the University of Freiburg where he was in-charge of about 40 patients, and was at liberty to do his own research. Before a year was over, he, with a research student Kurt Henseleit, postulated the metabolic pathway for urea formation, now known as the ornithine cycle of urea synthesis. In 1932 he worked out the basic chemical reactions of urea cycle, which established his scientific reputation[5].

Krebs life as a reputed German scientist ended because of his Jewish ancestry on 1 July 1933 and he moved to the Department of Biochemistry, University of Cambridge, UK at the invitation of Sir Frederick Hopkins[5]. In 1935 he moved to the University of Sheffield, UK and remained there for 19 years. In 1938 the University of Sheffield opened a Department of Biochemistry with Krebs as its first Head[5].

It was at the University of Sheffield that Krebs established the biochemical cycle that bears his name. There with William Johnson he began the studies of cellular respiration by which oxygen was consumed to produce energy from the breakdown of glucose[5][7]. Krebs had earlier suggested to Warburg, when they worked together in Germany, that the manometer could be used to detect oxygen consumption and identify the chemical reactions involved in glucose oxidation an idea which Warburg flatly rejected. In Sheffield, Krebs vigorously looked for possible chemical reactions and came up with numerous hypothetical pathways. Using the manometer he tested these hypotheses one by one[5][7]. One hypothesis involving succinate, fumarate and malate proved to be useful because all these molecules increased oxygen consumption in pigeon breast muscle. Following the demonstration in 1937 by German biochemists Franz Koop and Carl Martinus in 1937, of a series of reactions using citrate that produced oxaloacetate, Krebs realised his chemicals succinate, fumarate and malate could be the missing intermediates for such reaction. After four months of experimental work, Krebs and Johnson succeeded in establishing the sequence of the chemical cycle, which they called the "citric acid cycle" [5][7]. Interestingly Krebs had submitted a short manuscript of the discovery to Nature on 10 June 1937 and received a rejection letter from the editor on 14 June. The editor advised that the journal had "already sufficient letters to fill correspondence columns of Nature for seven or eight weeks" and encouraged Krebs to "submit it for early publication to another periodical"[5] which he did[8][9].

In 1953 Krebs and Fritz Lipmann shared the Nobel Prize for Physiology and Medicine for their discoveries of ‘the citric acid cycle’ and ‘co-enzyme A and its importance for intermediary metabolism" respectively [6]. Krebs gives a detailed account of his discovery in his 1953 Nobel lecture[7].

A variant – the glyoxylate cycle

In 1957 Krebs and Hans Kornberg discovered a variant of the TCA cycle called the glyoxylate cycle which converts acetyl-CoA to succinate which can then be used to synthesis carbohydrates via gluconeogenesis [10]. The glyoxylate cycle utilizes three of the five enzymes associated with the TCA cycle and shares many of its intermediate steps. The two cycles vary as follows: in the glyoxylate cycle, the enzyme isocitrate lyase converts isocitrate into glyoxylate and succinate instead of α-ketoglutarate as seen in the TCA cycle. This bypasses the decarboxylation steps that take place in the TCA cycle, allowing simple carbon compounds to be used in the later synthesis of macromolecules, including glucose. The glyoxylate cycle continues on using glyoxylate and second molecule of acetyl-CoA to produce malate which is oxidised to oxaloacetate for the next round of the cycle[10]. The end result of the glyoxylate cycle is that two molecules of acetate (in the form of acetyl-CoA) have been combined to form one molecule of the C4 dicarboxylic acid succinate which can be metabolised to fumarate, malate and oxaloacetate via steps 6, 7 and 8 of the TCA cycle. Oxaloacetate is the starting point for glucose synthesis by the pathway of gluconeogenesis.

The glyoxylate cycle is found in plants, fungi and bacteria where it is used to synthesise carbohydrate from lipids via acetyl-CoA that has been generated by β-oxidation of fatty acids[10]. While absent from tissues of higher animals it is an important pathway in the early embryonic stages of parasitic nematodes[11], the first evidence of this being the publication, also in 1957, of the chemical changes associated with the development of infectious larvae of the large parasitic nematode, Ascaris lumbricoides [12]. Interestingly parasitic helminths, like propionic acid bacteria, generate volatile fatty acids as a major end product of glycolysis and employ PEP carboxykinase and malate dehydrogenase rather than pyruvate kinase and lactate dehydrogenase as the terminal steps of glycolysis. Malate can then be converted via a reverse TCA cycle to fumarate and succinate and metabolised further into propionic and other volatile fatty acids[13].

Regulation of the TCA cycle

The regulation of the TCA cycle is largely determined by product inhibition and substrate availability. 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 isocitrate dehydrogenase (step 3), α-ketoglutarate dehydrogenase (step 4) and malate dehydrogenase (step 8), and acetyl-CoA both inhibit pyruvate dehydrogenase and hence the production of the starting molecule acetyl-CoA. NADH also inhibits isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, and citrate synthase the latter two enzymes also being inhibited by succinyl-CoA[1].


  1. ^ Citric acid cycle -

  2. ^ The citric acid cycle -

  3. ^ Coenzyme Q10 -

  4. ^ Cell Respiration: Krebs Cycle -

  5. ^ Hans Adolf Krebs -

  6. ^ The Nobel Prize in Physiology or Medicine 1953 -

  7. ^ Krebs HA, The citric acid cycle. Nobel Lecture, December 11, 1953

  8. ^ Krebs HA, The intermediate metabolism of carbohydrates. The Lancet , 1937; 230 (5952): 736–8. doi:10.1016/S0140-6736(00)88690-0.

  9. ^ Krebs HA, Salvin E, Johnson WA, "The formation of citric and alpha-ketoglutaric acids in the mammalian body". The Biochemical Journal, 1938; 32 (1):113–7. PMC 1264001. PMID 16746585.

  10. ^ Glyoxylate cycle -

  11. ^ Barrett J, Ward CW, Fairbairn D, The glyoxylate cycle and the conversion of triglycerides to carbohydrates in developing eggs of Ascaris lumbricoides. Comp Biochem Physiol, 1970; 35:577-86.

  12. ^ Passey RF, Fairbairn D, The conversion of fat to carbohydrate during embryonation of Ascaris eggs. Can J Biochem Physiol, 1957; 35:511-25

  13. ^ Ward CW, Castro GA, Fairbairn D, Carbon dioxide fixation and phospoenolpyruvate metabolism in Trichinella spiralis larvae. J. Parasitol, 1969; 55:67-71


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