Citric Acid Cycle

The Citric Acid Cycle

Pre-requisites for this article include an understanding of glycolysis and the link reaction.

• Glycolysis blogpost
• Link reaction blogpost

The citric acid cycle completes the burning of glucose that was initiated in glycolysis. It breaks down or oxidizes pyruvate, the end product of glycolysis to carbon-di-oxide.

The citric acid cycle gets its name from the first intermediate formed in the cycle: citrate or citric acid. This intermediate is a tri-carboxylic acid, that is, it has three $-CO_2^-$ groups. The cycle gets its alternative name the tri-carboxylic acid cycle (TCA) from here. The TCA was identified in 1937 by Hans Adolf Krebs while at the University of Sheffield after whom the cycle is sometimes named Krebs cycle.

The citric acid cycle is also know as the tricarboxylic acid cycle (TCA) or the Krebs cycle.

Steps of the citric acid cycle

To obtain maximum amount of energy from pyruvate, it is necessary to completely oxidize the molecule. This means that the body strives to completely oxidize every carbon of pyruvate to carbon-di-oxide which is exhaled as a waste product (energy can no longer be derived from it). Being a three carbon molecule, pyruvate undergoes three decarboxylation reactions (reactions that remove carbon-di-oxide from the substrate), the first of which is the link reaction. The product of the link reaction, acetyl-CoA is attached to another compound; oxaloacetic acid (OAA) which acts as a sort of ‘frame’ to aid in the subsequent decarboxylations. After the liberation of two CO$_2$ molecules, this ‘frame’ is regenerated so that another molecule of acetyl-CoA may be oxidized. Thus, the frame (that is oxaloacetic acid) plays a catalytic role in the decarboxylation of acetyl-CoA. A single oxaloacetic acid molecule is sufficient to oxidize many acetyl-CoA molecules causing citric acid cycle intermediates to only be present in catalytic amounts. On the addition of oxaloacetic acid, acetyl-CoA can be converted into a $\beta$-ketoacid, which undergoes decarboxylation readily. Following this, an $\alpha$-ketoacid is formed which undergoes decarboxylation with the help of the cofactor thiamine pyrophosphate (TPP).

Steps in the citric acid cycle:
a) Addition of the frame (oxaloacetate)to acetyl-CoA
b) Formation and decarboxylation of the beta ketoacid
c) Decarboxylation of the alpha ketoacid
d) Regeneration of the frame

Reactions of the TCA

In the figure above all the reactions are shown as unidirectional because its too cluttered to depict otherwise. All the reactions expect 1 (citrate synthase),3 (isocitrate dehydrogenase) and 4 (alpha ketoglutarate) are reversible under physiological conditions.

Addition of the frame

1) Formation of citric acid
Enzyme: Citrate synthase

Citrate synthase catalyzes the condensation of acetyl-CoA and oxaloacetic acid (OAA) into citrate. The cycle gets its name form the formation of this compound. This reaction is a mixed aldol Claisen condensation. The thioester, acetyl-CoA loses an $\alpha$ hydrogen and attacks the carbonyl carbon of oxaloacetate. This results in the formation of citryl-CoA. The enzyme citrate synthase lays down oxaloacetate upon itself like a blanket. It allows attack of acetyl-CoA only form above by positioning itself under the molecule effectively blocking attack. Thus, only $(s)$-citryl-CoA is formed. The high-energy thioester bond of citryl-CoA is hydrolyzed to give citrate. Consequently, a large amount of energy is dissipated rendering this reaction irreversible.

Formation and decarboxylation of the $\beta$-ketoacid

2) Formation of isocitrate
Enzyme: Aconitase

Citrate has a tertiary alcohol ($-OH$) group. This cannot be oxidized to a keto group to form the desired ketoacid. Thus, the alcohol group is transferred to the adjacent carbon forming isocitrate. Now a secondary alcohol group is present and it can undergo oxidation.

3) Formation of $\alpha$-ketoglutarate
Enzyme: Isocitrate dehydrogenase

This reaction has two parts to it:

• First, the secondary alcohol group is oxidized to a keto group forming oxalosuccinate, a $\beta$-ketoacid. This reaction requires the redox cofactor NAD$^+$ which simultaneously undergoes reduction to NADH$_2$.
• Oxalosuccinate undergoes spontaneous decarboxylation to form $\alpha$-ketoglutarate, an $\alpha$-ketoacid.

The enzyme catalyzing this reaction shares a mechanism similar to that of phosphogluconate dehydrogenase of the HMP shunt pathway. Notice the similarities between the substrates and products of the two enzymes.

Decarboxylation of the $\alpha$-ketoacid

4) Formation of succinyl-CoA
Enzyme: $\alpha$-ketoglutarate dehydrogenase

The $\alpha$-ketoacid, $\alpha$-ketoglutarate is decarboxylated by an enzyme complex similar to the pyruvate dehydrogenase complex. Among other cofactors, TPP is used in the first decarboxylation step. Additionally, NAD$^+$ is reduced to NADH$_2$ in this step. For details about this reaction refer to this blogpost. Ammonia inhibits $\alpha$-ketoglutarate dehydrogenase.

Regeneration of OAA

5) Succinyl-CoA is converted to succinate
Enzyme: Succinate thiokinase or succinyl-CoA synthetase

The thioester bond of succinyl-CoA is hydrolyzed releasing large amounts of energy. This reaction is coupled with either the synthesis of ATP or GTP. The energy released in the previous thioester hydrolysis (step 1) was not coupled with ATP or GTP synthesis but was simply dissipated. It was the price paid by the body for irreversibility. In this step however, the energy is stored and the reaction is reversible (ATP/GTP is utilized in the reverse reaction). Biologically, both ATP and GTP are energetically equivalent.
$ATP + GDP \rightleftharpoons ADP + GTP$

In cells of organs that take part in gluconeogenesis (like the liver), the isoenzyme of succinate dehydrogenase present phosphorylates GDP forming GTP whereas in cells not involved in gluconeogenesis, the isoenzyme present phosphorylates ADP forming ATP. GTP catalyzes the conversion of oxaloacetate to phosphoenolpyruvate during gluconeogenesis. Thus, regulation of this step of the citric acid cycle in turn regulates gluconeogenesis.

The GTP formed (by succinate dehydrogenase) is used for the decarboxylation of oxaloacetate to phosphoenolpyruvate in gluconeogenesis, and provides a regulatory link between citric acid cycle activity and the withdrawal of oxaloacetate for gluconeogenesis. - Harper’s Illustrated Biochemistry 31st Edition

The blue double line represents the mitochondrial membrane. This membrane is impermeable to oxaloacetate. Thus, malate enters the cytoplasm where it is converted to oxaloacetate.

The onward metabolism of succinate, leading to the regeneration of oxaloacetate, is the same sequence of chemical reactions as occurs in the
β-oxidation of fatty acids:

• dehydrogenation to form a carbon-carbon double bond ($C=C$)
• addition of water to form a hydroxyl group
• and a further dehydrogenation to yield the oxo-group (keto group) of oxaloacetate.

6) Dehydrogenation of succinate of fumarate
Enzyme: Succinate dehydrogenase

Succinate dehydrogenase catalyzes the oxidation of succinate’s central single bond to a trans double bond, yielding fumarate with the concomitant reduction of the redox coenzyme FAD to FADH$_2$. This enzyme is competitively inhibited by malonate, probably due to its structural similarities with succinate.

Dehydrogenation reactions use FAD$^+$ and not NAD$^+$. This is because FAD$^+$ is thermodynamically easier to reduce and the free-energy change of dehydrogenation reactions is insufficient to reduce NAD$^+$. Examples include the dehydrogenation (oxidation) of dihydrolipoamide and the dehydrogenation succinate.

7) Addition of water to fumarate to form malate
Enzyme: Fumarase

Fumarase also known as fumarate hydratase catalyzes the addition of water to fumarate to form malate.

8) Malate is oxidized into oxaloacetate
Enzyme: Malate dehydrogenase

In the final step of the citric acid cycle, the hydroxyl group ($-OH$) of malate is oxidized into the keto group ($=O$) of oxaloacetate with the concomitant reduction of the redox coenzyme NAD to NADH$_2$.

A total of 3 NADH$_2$ molecules (steps 2, 3 and 8), 1 FADH$_2$ molecule (step 6) and 1 ATP or 1 GTP (step 5) molecule are generated in a single turn of the citric acid cycle.

Carbon labeling in the citric acid cycle figure above
In order to follow the passage of acetyl-CoA through the cycle in the figure above, the two carbon atoms of the acetyl moiety are shown labeled on the carboxyl carbon (*) and on the methyl carbon (·). Although two carbon atoms are lost as CO$_2$ in one turn of the cycle, these atoms are not derived from the acetyl-CoA that has immediately entered the cycle, but from that portion of the citrate molecule that was derived from oxaloacetate. However, on completion of a single turn of the cycle, the oxaloacetate that is regenerated is now labeled, which leads to labeled CO$_2$ being evolved during the second turn of the cycle. Because succinate is a symmetrical compound, “randomization” of label occurs at this step so that all four carbon atoms of oxaloacetate appear to be labeled after one turn of the cycle. During gluconeogenesis, some of the label in
oxaloacetate is incorporated into glucose and glycogen.

Regulation of the citric acid cycle

The three irreversible reaction catalyzing enzymes of the citric acid cycle act as points of metabolic control. These enzymes are:

• citrate synthase
• isocitrate dehydrogenase,
• and $\alpha$-ketoglutarate dehydrogenase

NADH$_2$ produced by the citric acid cycle enters the electron transport chain resulting in ATP synthesis. Thus, the rate of ATP (energy) consumption of the cell must be coordinated with the rate of NADH$_2$ formation. Two out of the three enzymes above: isocitrate dehydrogenase and $\alpha$-ketoglutarate dehydrogenase produce NADH$_2$.

Consider a situation in which there is increased energy consumption by the cell. In such a situation, the concentration of NADH$_2$ drops and isocitrate dehydrogenase and $\alpha$-ketoglutarate dehydrogenase are liberated from feedback inhibition. Their activity is enhanced. Citrate inhibits citrate synthase (classic case of feedback inhibition). Due to the enhanced activity of isocitrate dehydrogenase, the concentration of citrate is reduced. Thus, the activity of citrate synthase also increases.

On the flip side, an increase in $\alpha$-ketoglutarate dehydrogenase activity results in increased succinyl-CoA production. Succinyl-CoA competes with acetyl-CoA to bind with citrate synthase thereby inhibiting it. Thus, the activity of the cycle as a whole is down regulated.

Unlike the rate-limiting enzymes of glycolysis and glycogen metabolism, which utilize elaborate systems of allosteric control, substrate cycles, and covalent modification as flux control mechanisms, the regulatory enzymes of the citric acid cycle seem to be controlled almost entirely in three simple ways: substrate availability, product inhibition, and competitive feedback inhibition by intermediates farther along the cycle. - Biochemistry by Donald Voet and Judith G. Voet

Citric acid cycle as the ‘Metabolic Hub’

With respect to free energy production, the citric acid cycle has a catabolic role (breakdown of pyruvate). At the same time, the intermediates of the citric acid cycle serve as useful starting points for the synthesis of many biomolecules. Being both catabolic and anabolic makes the citric acid cycle amphibolic in nature. Synthesis of biomolecules requires free energy. Thus, when intermediates from the citric acid cycle are siphoned off for synthesis reactions, they must be replenished so that the catabolic, energy producing role of the cycle is not hindered.

This diagram indicates the positions at which intermediates are cataplerotically drawn off for use in anabolic pathways (red arrows) and the points where anaplerotic reactions replenish depleted cycle intermediates (green arrows). Reactions involving amino acid transamination and deamination are reversible, so their direction varies with metabolic demand. Source: Biochemistry, 4th Edition by Donald Voet, Judith G. Voet

Pathways that utilized citric acid cycle intermediates

Oxaloacetate is utilized as the starting material for gluconeogenesis. However, oxaloacetate cannot be transported across the mitochondrial membrane but malate can be. Thus, malate is transported to the cytoplasm where it is converted back to oxaloacetate.

Acetyl-CoA is utilized for lipid biosynthesis. Like oxaloacetate, it cannot be transported across the mitochondrial membrane either. Thus, citrate is siphoned off for fatty acid synthesis into the cytoplasm where it is converted back to acetyl-CoA and oxaloacetate.

$\alpha$-ketoglutarate and oxaloacetate are used to synthesize glutamate and aspartate.

To completely oxidize amino acids into acetyl-CoA, they are degraded into their respective citric acid cycle intermediates and converted to oxaloacetate. The enzyme phosphoenolpyruvate carboxykinase converts oxaloacetate to phosphoenolpyruvate which is eventually converted to acetyl-CoA via the glycolytic pathway and the link reaction.

Succinyl-CoA is utilized for porphyrin synthesis. Heme, a part of hemoglobin is an iron containing porphyrin.

Pathways that replenish citric acid cycle intermediates

Reactions that replenish the citric acid cycle intermediates are called anaplerotic reactions. Pyruvate carboxylase is the main anaplerotic enzyme. It catalyzes the conversion of pyruvate to oxaloacetate. When there is lack of citric acid cycle intermediates, the mitochondrial concentration of acetyl-CoA builds up due to underutilization. Acetyl-CoA activates pyruvate carboxylase which replenishes oxaloacetate and consequently all the citric acid intermediates.

Apart from this,

• oxidation of odd chain fatty acids produces succinyl-CoA
• breakdown of the amino acids isoleucine, methionine,
  and valine also leads to the production of succinyl-CoA.
• Transamination and deamination of amino acids lead to the production of $\alpha$-ketoglutarate and oxaloacetate.

The citric acid cycle plays a pivotal role in metabolism. It is not only a pathway for oxidation of two carbon units, but is also a major pathway for interconversion of metabolites arising from transamination and deamination of amino acids and providing the substrates for amino acid synthesis by transamination as well as for gluconeogenesis and fatty acid synthesis. -Harper’s Illustrated Biochemistry 31st Edition

Sources

• Thumbnail: The starting letter of each world represents a Krebs cycle intermediate (in the correct order): “Can I keep selling sunlight for money officer?”
• Harper’s Illustrated Biochemistry 31st Edition
• Biochemistry by Donald Voet and Judith G. Voet
• MITOCW lecture video.
• Stack exchange answer: FAD$^+$vs NAD$^+$