Slide 1

Samantha Soriano-Castañeda,

THECITRICACIDCYCLE MD, FPSOHNS

September 2021

This lecture is based on Chapter 16 The Citric Acid Cycle :The Central Pathway of
Carbohydrate, Lipid, & Amino Acid Metabolism Harper’s Illustrated Biochemistry 31 st ed. This
is one of the lectures included in the TBL on Sept 15

Slide 2

OUTCOMES
At the end of the session, the student should be able to:
❖Describe the citric acid cycle
❖Explain the energetics of the citric acid cycle
❖Know the inhibitors of the citric acid cycle & their biomedical
role
❖Know the role of amino acids in the citric acid cycle
Slide 3

CITRICACIDCYCLE
❖Krebs cycle or tricarboxylic
acid cycle
❖Occurs in the matrix of the
mitochondria
❖Final common pathway for
the oxidation of
carbohydrates, lipids & fats
❖Aerobic process

researchgate.net

During digestion, fats, carbohydrates and proteins are metabolized to fatty acids +
glycerol, glucose and amino acids respectively. All of these are metabolized to a
common product, acetyl Co-A which is then oxidized by the citric acid cycle (CAC)
in the matrix of the mitochondrion. All of the enzymes of the citric acid cycle is
present in the mitochondrion. All cells contain a mitochondrion except the red
blood cell (RBC). The RBC thus generates its energy thru glycolysis. The co-
enzymes (co-factors) produced in the CAC is re-oxidized in the respiratory chain
and linked to phosphorylation. Thus the CAC is an aerobic process requiring
oxygen
Slide 4

Biology Notes for A level

The cycle starts with the reaction between the 2-carbon acetyl Co-A and
the 4 carbon dicarboxylic acid oxaloacetate, forming a 6 carbon
tricarboxylic acid, citrate. There are several reactions in between but
oxaloacetate is regenerated thus it has a catalytic role. Only a small
quantity of oxaloacetate is needed to oxidize a large quantity of acetyl
CoA. The CAC is regulated by the energy levels within the matrix of the
mitochondrion. When ATP and NADH levels are high, the cycle slows
and vise versa.
Slide 5

SlideShare

1st step : Acetyl Co-A and Oxaloacetate catalyzed by citrate synthase to form Citrate and
releasing CoASH
2nd step : Citrate is isomerized to Isocitrate by the enzyme aconitase. Channeling – transfer of
the product of citrate synthase directly onto the active site of aconitase without entering free
solution. This ensures citrate is available for the CAC. Citrate is only available for transport out of
the mitochondria to the cytosol for fatty acid synthesis when aconitase is inhibited by
accumulation of its product, Isocitrate.
3rd step: Isocitrate undergoes dehydrogenation then decarboxylation catalyzed by Isocitrate
dehydrogenase to α-ketoglutarate. The decarboxylation requires Mg2+ and Mn2+ ions. This step
also reduces NAD+ to NADH
4th step : α-ketoglutarate undergoes oxidative decarboxylation catalyzed by a multienzyme
complex, α-ketoglutarate dehydrogenase, to produce Succinyl CoA. This enzyme requires the
following co-factors : thiamin diphosphate, lipoate, NAD+, FAD and Co-A. This is physiologically
unidirectional since this reaction favors the formation of Succinyl CoA. This step reduces NAD+
to NADH
5th step: Succinyl CoA is converted to succinate by succinate thiokinase (succinyl CoA
synthetase). This produces GTP (tissues in which gluconeogenesis occurs such as liver & kidney)
or ATP (non-gluconeogenic tissues such as skeletal muscles). This is the only substrate level
phosphorylation in the CAC.
6th step: Succinate undergoes dehydrogenation catalyzed by succinate dehydrogenase to form
fumarate. Succinate dehydrogenase is bound to the inner surface of the inner mitochondrial
membrane. This enzyme contains FAD and Fe-S proteins and directly reduces ubiquinone in the
ETC. (Complex II)
7th step: Fumarate is catalyzed by Fumarase to produce malate.
8th step: Malate is oxidized by malate dehydrogenase linked to the reduction of NAD+ to NADH
to form oxaloacetate. The net flux is towards oxaloacetate because of the continual removal of
oxaloacetate and the reoxidation of NADH.
Slide 6

creative-proteomics.com

Slide 7

RESULTOFCACFROM1 ACETYLCOA
▪3 NADH : isocitrate dehydrogenase, α-ketoglutarate
dehydrogenase, malate dehydrogenase
▪3 x 2.5 ATP = 7.5 ATP
▪1 FADH2 : succinate dehydrogenase
▪1 x 1.5 ATP = 1.5 ATP
▪Substrate-level phosphorylation : succinate thiokinase
▪ 1 ATP (non-gluconeogenic tissues) or GTP (gluconeogenic
tissues)
▪Total : 10 ATP
Slide 8

VITAMINB
❖Riboflavin : FAD
❖Niacin : NAD
❖Thiamin (Vit B1) : thiamin diphosphate
❖Pantothenic acid : part of co-enzyme A

Vitamins play an important role in the CAC

Slide 9

The CAC is not only a pathway for oxidation of 2 carbon unit but is also a major pathway for
interconversion of metabolites arising from transamination and deamination of amino acids,
providing substrates for amino acid synthesis as well as for gluconeogenesis and fatty acid
synthesis. Because it functions in both oxidative and synthetic processes, it is amphibolic.

All the intermediates of the CAC are potentially glucogenic since they can give rise to
oxaloacetate which is used for the production of glucose. Oxaloacetate is catalyzed by
phosphoenolpyruvate carboxykinase ,with GTP as the phosphate donor, to
phosphoenolpyruvate. The GTP is from succinate thiokinase step thus this ensures that
oxaloacetate is not used for gluconeogenesis if it will lead to depletion of CAC intermediates.

Transfer into the CAC can occur thru

- Carboxylation of pyruvate catalyzed by pyruvate carboxylase to form oxaloacetate. This is
important to maintain adequate concentration of oxaloacetate. This step is activated by
excess acetyl CoA. This is an ATP dependent reaction in which Biotin is a coenzyme
- Lactate, an important substrate for gluconeogenesis , is oxidized to pyruvate.
- Variety of compounds that are metabolized to yield propionyl CoA which can be carboxylated
and isomerized to succinyl CoA
- Aminotransferase reactions from amino acids to CAC intermediates and vice versa
- Alanine, tryptophan, cysteine, glycine, hydroxyproline, serine, threonine to pyruvate
- Aspartate to oxaloacetate
- Glutamine, arginine and histidine to form α-ketoglutarate
- Isoleucine, methionine and valine yield succinyl CoA
- Tyrosine and phenylalanine yield fumarate
• CAC intermediates from amino acids must undergo a different pathway to prevent an
increase in amount of oxaloacetate.
 • Oxaloacetate is catalyzed by phosphoenolpyruvate carboxykinase ,using up
GTP, to form phosphoenolpyruvate.
• Phosphoenolpyruvate catalyzed by pyruvate kinase to form pyruvate
• Pyruvate catalyzed by pyruvate dehydrogenase to form acetyl CoA

Slide 10

CORNELLUNIVERSITY

Pyruvate dehydrogenase is a mitochondrial matrix enzyme that

catalyzes pyruvate to form acetyl CoA. It is a multi-enzyme complex
similar to α-ketoglutarate dehydrogenase that has several co-factors:
thiamin diphosphate, lipoate, NAD+, FAD and Co-A
Slide 11

Acetyl CoA is also a major substrate for long chain fatty acid synthesis. Fatty acid
synthesis occurs in the cytosol. The mitochondrial membrane is impermeable to
acetyl CoA. We need a transport shuttle again. Citrate is transported out to the
cytosol then catalyzed by citrate lyase to form oxaloacetate and acetyl CoA. As
discussed in the previous slides, citrate is only transported out when there is
excess of isocitrate which inhibits aconitase.
The oxaloacetate can not enter the mitochondrion so is reduced to malate by
malate dehydrogenase at the expense of NADH. Malate is catalyzed by malic
enzyme to form pyruvate reducing NADP+ to NADPH. This reaction of malate to
pyruvate is the source of half of the NADPH required for fatty acid synthesis.
Slide 12

REGULATIONOFTHECAC

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CAC - energy yielding metabolism. CAC activity is dependent on the supply of NAD
which is tightly coupled to the ETC and phosphorylation which is dependent on
ADP.

Main sites for regulation and are non-equilibrium

- Pyruvate dehydrogenase (catalyzes pyruvate to acetyl CoA)
- Citrate synthase (catalyzes acetyl CoA and oxaloacetate to citrate)
- Isocitrate dehydrogenase (catalyzes isocitrate to α-ketoglutarate )
- α-ketoglutarate dehydrogenase (catalyzes α-ketoglutarate to succinyl CoA)

Dehyrogenases are activated by Ca2+ which increases in concentration during

contraction of muscle and during secretion by other tissues when there is increase
in energy demand.
Slide 13

SlideShare

Inhibitors of the CAC

- Fluroacetate : condenses w oxaloacetate to form fluorocitrate which inhibits
aconitase
- Ammonia : high levels inhibits α-ketoglutarate dehydrogenase
- Arsenite : inhibitor of α-ketoglutarate dehydrogenase
- Malonate : inhibitor of succinate dehydrogenase (Complex 2 in the ETC