The citric acid cycle is the principal process for generating reduced coenzymes NADH and FADH2, which are necessary for ATP synthesis. It takes place in the mitochondrial matrix and involves eight steps catalyzed by different enzymes. Acetyl-CoA enters the cycle and is oxidized, producing carbon dioxide and the reduced coenzymes that fuel ATP production. Regulation occurs at three steps to precisely adjust the cycle's rate according to cellular energy needs. Overall, 12 ATP molecules are generated for each acetyl-CoA molecule that completes the citric acid cycle.

1. LECTURE NOTES: PHARMACEUTICAL BIOCHEMISTRY
PREPARED BY: DR. USMAN SALEEM 1
Citric Acid Cycle
Over View
Stage III in the oxidation of fuel molecules begins when the two-carbon acetyl units (of acetyl
CoA) enter the citric acid cycle. This process is called the citric acid cycle because one of the key
intermediates is citric acid. However, it is also called the tricarboxylic acid cycle (TCA) in reference
to the three carboxylic acid groups in citric acid, and the Krebs cycle in honor of Sir Hans A. Krebs,
who deduced its reaction sequence in 1937.
Functions of Citric Acid Cycle
1. The citric acid cycle is the principal process for generating the reduced coenzymes NADH and
FADH2, which are necessary for the reduction of oxygen and ATP synthesis in the electron
transport chain.
2. Another important function of the citric acid cycle as a source of intermediates for
biosynthesis of
 Fatty acids
 Heme
 Nonessential amino acids (Glutamate and Aspartate)
 Steroids

2. LECTURE NOTES: PHARMACEUTICAL BIOCHEMISTRY
PREPARED BY: DR. USMAN SALEEM 2
Location of the Citric Acid Cycle
The citric acid cycle takes place in the mitochondrial matrix, with exception that one enzyme
(succinate dehydrogenase) is located in the inner mitochondrial membrane.
Reactions of Citric Acid Cycle
The citric acid cycle is shown in schematic form in following figure.

3. LECTURE NOTES: PHARMACEUTICAL BIOCHEMISTRY
PREPARED BY: DR. USMAN SALEEM 3
The citric acid cycle has eight steps, each catalyzed by a different enzyme. Four of the eight
steps—Steps 3, 4, 6, and 8—are oxidation reactions. The oxidizing agent is NAD+ in all except Step
6, in which FAD plays the same role. In Step 5, a molecule of GDP (guanosine diphosphate) is
phosphorylated to produce GTP. This reaction is equivalent to the production of ATP because the
phosphate group is easily transferred to ADP, producing GDP and ATP.
Net Reaction
The individual reactions of the cycle can be added to give an overall net equation:
Important Features of Citric Acid Cycle
Some important features of the citric acid cycle are the following:
1. Acetyl CoA, available from the breakdown of carbohydrates, lipids, and amino acids, is the
fuel of the citric acid cycle.
2. The operation of the cycle requires a supply of the oxidizing agents NAD+
and FAD.
3. The cycle is dependent on reactions of the electron transport chain to supply the necessary
NAD+
and FAD. Because oxygen is the final acceptor of electrons in the electron transport
chain, the continued operation of the citric acid cycle depends ultimately on an adequate
supply of oxygen.
4. Two carbon atoms enter the cycle as an acetyl unit, and two carbon atoms leave the cycle as
two molecules of CO2.
5. In each complete cycle, four oxidation–reduction reactions produce three molecules of NADH
and one molecule of FADH2.
6. One molecule of the high-energy phosphate compound guanosine triphosphate (GTP) is
generated.
Regulation of the Citric Acid Cycle
The rate at which the citric acid cycle operates is precisely adjusted to meet cellular needs for
ATP. The cycle is regulated at three steps which are catalyzed by:
1. Citrate synthetase (Reaction 1)
2. Isocitrate dehydrogenase (Reaction 3)
3. -ketoglutarate dehydrogenase complex (Reaction 4)
 The citrate synthetase is an allosteric enzyme that is inhibited by ATP and NADH and activated
by ADP. When cellular needs for ATP are met, ATP interacts with citrate synthetase and

4. LECTURE NOTES: PHARMACEUTICAL BIOCHEMISTRY
PREPARED BY: DR. USMAN SALEEM 4
reduces its affinity for acetyl CoA. Thus, ATP acts as a modulator (inhibitor) of citrate
synthetase and inhibits the entry of acetyl CoA into the citric acid cycle. Similarly, NADH acts as
an inhibitor and signals the citric acid cycle to slow the production of NADH and FADH2. On the other
hand, when ATP levels are low, ADP levels are usually high. ADP, an activator of citrate
synthetase, stimulates the entry of acetyl CoA into the citric acid cycle and thus boosts the
production of NADH and FADH2.
 Isocitrate dehydrogenase, a second controlling enzyme, is an allosteric enzyme that is
activated by ADP and inhibited by NADH in very much the same way as is citrate synthetase.
 The third control point is catalyzed by the a-ketoglutarate dehydrogenase complex. This
group of enzymes is inhibited by succinyl CoA and NADH, products of the reaction it catalyzes,
and also by ATP.
These control points are indicated the following figure.
In short, the entry of acetyl CoA into the citric acid cycle and the rate at which the cycle operates
are reduced when cellular ATP levels are high. When ATP supplies are low (and ADP levels are
high), the cycle is stimulated.

5. LECTURE NOTES: PHARMACEUTICAL BIOCHEMISTRY
PREPARED BY: DR. USMAN SALEEM 5
Energetics of Citric Acid Cycle
 As a result of oxidation of one molecule of acetyl-CoA to H2O and CO2 by citric acid cycle,
three molecules of NADH and one FADH2 are produced.
 Oxidation of 3 NADH by electron transport chain results in the synthesis of 9 ATP, whereas
FADH2 generates 2 ATP molecules.
 One molecule of ATP is generated at substrate level during the conversion of succinyl-CoA
to succinate. Thus, a total of 12 ATP are generated from one molecule of acetyl-CoA.
Production of ATP in Citric Acid Cycle
Reaction Comments
Yield of ATP
(Moles)
Isocitrate  -ketoglutarate + CO2 2 mol produces 2 NADH* +6
a-ketoglutarate  Succinyl CoA + CO2 2 mol produces 2 NADH +6
Succinyl CoA  Succinate 2 mol produces 2 GTP +2
Succinate  Fumarate 2 mol produces 2 FADH2* +4
Malate  Oxaloacetate 2 mol produces 2 NADH +6
Yield of ATP Per Two Molecules of Acetyl-CoA in Citric Acid Cycle: +24
*Oxidation of 1 mole NADH produces 3 moles ATP
*Oxidation of 1 mole FADH2 produces 2 moles ATP
Note: The yield of ATP is 2 moles per molecule of glucose (or two molecules of Acetyl-CoA) as it
passes through Citric acid cycle. The remaining 22 moles of ATP are produced by the reoxidation
of reduced electron carriers (NADH and FADH2) by the electron transport chain.
Summary
 The citric acid cycle is amphibolic. It plays a role in both catabolism and anabolism. It is the
central metabolic pathway.
 The citric acid cycle is exergonic in terms of overall free-energy changes. In addition, it
produces three NADH and one FADH2 for each Acetyl-CoA that enters the cycle.
 NADH and FADH2 generated by the citric acid cycle ultimately pass their electrons to oxygen.
 Control of the citric acid cycle is exercised at three points.