CITRIC ACID CYCLE

The Krebs cycle, also known as the citric acid cycle or TCA cycle, is a series of reactions that take place in
the mitochondria, resulting in the oxidation of acetyl CoA to release carbon dioxide and hydrogen atoms
that later lead to the formation of water.

This cycle is termed the citric acid cycle as the first metabolic intermediate formed in the cycle is citric
acid.

This cycle is also termed tricarboxylic acid (TCA) because it was then not certain whether citric acid or
some other tricarboxylic acid (eg., isocitric acid) was the first product of the cycle. However, now it has
been known that the first product is indeed citric acid and thus the use of this name has since been
discouraged.

This cycle only occurs under aerobic conditions as energy-rich molecules like NAD+ and FAD can only be
retrieved from their reduced form once they transfer electrons to molecular oxygen.

The citric acid cycle is the final common pathway for the oxidation of all biomolecules; proteins, fatty
acids, carbohydrates. Molecules from other cycles and pathways enter this cycle through Acetyl CoA.

The citric acid cycle is a cyclic sequence of reactions formed of 8 enzyme-mediated reactions.

This cycle is also particularly important as it provides electrons/ high-energy molecules to the electron
transport chain for the production of ATPs and water.

Pyruvate formed at the end of glycolysis is first oxidized into Acetyl CoA which then enters the citric acid
cycle.

The citric acid cycle in eukaryotes takes place in the mitochondria while in prokaryotes, it takes place in
the cytoplasm.

The pyruvate formed in the cytoplasm (from glycolysis) is brought into the mitochondria where further
reactions take place.

The different enzymes involved in the citric acid cycle are located either in the inner membrane or in the
matrix space of the mitochondria.

The overall reaction/ equation of the citric acid cycle is:

Acetyl CoA + 3 NAD+ + 1 FAD + 1 ADP + 1 Pi → 2 CO2 + 3 NADH + 3 H+ + 1 FADH2 + 1 ATP

In words, the equation is written as:

Acetyl CoA + Nicotinamide adenine dinucleotide + Flavin adenine dinucleotide + Adenosine diphosphate
+ Phosphate → Pyruvate + Water + Adenosine triphosphate + Nicotinamide adenine dinucleotide +
Hydrogen ions.

Krebs Cycle Enzymes

In eukaryotic cells, the enzymes that catalyze the reactions of the citric acid cycle are present in the
matrix of the mitochondria except for succinate dehydrogenase and aconitase, which are present in the
inner mitochondrial membrane.
One common characteristic in all the enzymes involved in the citric acid cycle is that nearly all of them
require Mg2+

The following are the enzymes that catalyze different steps throughout the process of the citric acid
cycle:

1. Citrate synthase

2. Aconitase

3. Isocitrate dehydrogenase

4. α-ketoglutarate dehydrogenase

5. Succinyl-CoA synthetase

6. Succinate dehydrogenase

7. Fumarase

8. Malate dehydrogenase

Krebs Cycle Steps

After glycolysis, in aerobic organisms, the pyruvate molecules are decarboxylated to form acetyl CoA
and CO2.

Oxidative Decarboxylation of pyruvate to Acetyl CoA

The oxidative decarboxylation of pyruvate forms a link between glycolysis and the citric acid cycle.

In this process, the pyruvate derived from glycolysis is oxidatively decarboxylated to acetyl CoA and CO2
catalyzed by the pyruvate dehydrogenase complex in the mitochondrial matrix in eukaryotes and in the
cytoplasm of the prokaryotes.

From one molecule of glucose, two molecules of pyruvate are formed, each of which forms one acetyl
CoA along with one NADH by the end of the pyruvate oxidation.

The acetyl CoA formed from pyruvate oxidation, fatty acid metabolism, and amino acid pathway then
enter the citric acid cycle.
The following are the eight enzyme-catalyzed reactions/ steps in the aerobic oxidation of glucose
through the citric acid cycle:

Step 1: Condensation of acetyl CoA with oxaloacetate

The first step of the citric acid cycle is the joining of the four-carbon compound oxaloacetate (OAA) and
a two-carbon compound acetyl CoA.

The oxaloacetate reacts with the acetyl group of the acetyl CoA and water, resulting in the formation of
a six-carbon compound citric acid, CoA.

The reaction is catalyzed by the enzyme citrate synthase that condenses the methyl group of acetyl CoA
and the carbonyl group of oxaloacetate resulting in citryl-CoA which is later cleaved to free coenzyme A
and to form citrate.

Step 2: Isomerization of citrate into isocitrate

Now, for further metabolism, citrate is converted into isocitrate through the formation of intermediate
cis-aconitase.

This reaction is a reversible reaction catalyzed by the enzyme aconitase.

This reaction takes place by a two-step process where the first step involves dehydration of citrate to
cis-aconitase, followed by the second step involving rehydration of cis-aconitase into isocitrate.

Step 3: Oxidative decarboxylations of isocitrate

The third step of the citric acid cycle is the first of the four oxidation-reduction reactions in this cycle.

Isocitrate is oxidatively decarboxylated to form a five-carbon compound, α-ketoglutarate catalyzed by

the enzyme isocitrate dehydrogenase.

This reaction, like the second reaction, is a two-step reaction.

In the first step, isocitrate is dehydrogenated to oxalosuccinate while the second step involves the
decarboxylation of oxalosuccinate to α-ketoglutarate.

Both the reactions are irreversible and catalyzed by the same enzyme.

The first step, however, results in the formation of NADH while the second step involves the release of
CO2.

Step 4: Oxidative decarboxylation of α-ketoglutarate

This step is another one of the oxidation-reduction reactions where α-ketoglutarate is oxidatively
decarboxylated to form a four-carbon compound, succinyl-CoA, and CO2.

The reaction is irreversible and catalyzed by the enzyme complex α-ketoglutarate dehydrogenase found
in the mitochondrial space.

This reaction is similar to the oxidative decarboxylation of pyruvate involving the reduction of NAD+ into
NADH.

Step 5: Conversion of succinyl-CoA into succinate

In the next step, succinyl-CoA undergoes an energy-conserving reaction in which succinyl-CoA is cleaved
to form succinate.

This reaction is accompanied by phosphorylation of guanosine diphosphate (GDP) to guanosine

triphosphate (GTP).

The GTP thus formed then readily transfers its terminal phosphate group to ADP forming an ATP
molecule.

The reaction is catalyzed by the enzyme, succinyl-CoA synthase.

Step 6: Dehydration of succinate to fumarate

Here, the succinate formed from succinyl-CoA is dehydrogenated to fumarate catalyzed by the enzyme
complex succinate dehydrogenase found in the intramitochondrial space.

This is the only dehydrogenation step in the citric acid cycle in which NAD+ doesn’t participate.

Instead, another high-energy electron carrier, flavin adenine dinucleotide (FAD) acts as the hydrogen
acceptor resulting in the formation of FADH2.

The FADH2 then enters the electron transport chain via the complex II transferring the electrons to
ubiquinone, finally forming 2ATPs.

Step 7: Hydration of fumarate to malate

The fumarate is reversibly hydrated to form L-malate in the presence of the enzyme fumarate
hydratase.

As it is a reversible reaction, the formation of L-malate involves hydration, whereas the formation of
fumarate involves dehydration.

Step 8: Dehydrogenation of L-malate to oxaloacetate

The last step of the citric acid cycle is also an oxidation-reduction reaction where L-malate is
dehydrogenated to oxaloacetate in the presence of L-malate dehydrogenase, which is present in the
mitochondrial matrix.

This is a reversible reaction involving oxidation of L-malate and reduction of NAD+ into NADH.

Oxaloacetate thus formed, allows the repetition of the cycle and NADH formed participates in the
oxidative phosphorylation.

This reaction completes the cycle.

Krebs Cycle Products

Since this is a cyclic process, the oxaloacetate is formed at the end as it condenses with acetyl CoA in the
next cycle.

Products of one turn of the citric acid cycle

At each turn of the cycle,

3 NADH,

1 FADH2,

1 GTP (or ATP),

2 CO2

Note: One NADH is formed from a molecule of pyruvate in the oxidative decarboxylation of pyruvate to
Acetyl CoA.

The Physiological Role of Anaplerosis in the Citric Acid Cycle

Anaplerotic reactions are a very important part of the citric acid cycle, also known as the TCA cycle. The
citric acid cycle is an amphibolic pathway. Amphibolic pathways are those pathways that can perform
both anabolic reactions as well as catabolic reactions. The anaplerotic reaction is also known as the
anaplerotic pathways of the citric acid cycle. They are responsible for the anabolic part of the cycle. The
primary role of the citric acid cycle is the oxidation of acetyl-CoA to carbon dioxide.

It is important to understand that TCA cycle intermediates are sufficient to sustain the oxidative carbon
flux during high energy consumption like individuals performing exercise or during lower energy
consumption like fasting. It is important to note that there is no large change in the pool size of TCA
intermediates. In several physiological states, there is a large influx of intermediates like 4- and 5-carbon
intermediates into the TCA cycle. It is notable that even with the change in the intermediate
concentrations, the citric acid cycle can not act as a carbon sink, so it maintains a dynamic balance
between incoming and outgoing intermediates by anaplerosis and cataplerosis.

Anaplerotic Reactions of the Citric Acid Cycle

There are four major anaplerotic reactions in the TCA cycle.

1. Pyruvate to oxaloacetate

2. Phosphoenolpyruvate to oxaloacetate

3. Phosphophenol pyruvate to oxaloacetate using PEP carboxykinase.

4. Pyruvate to malate