Pyruvate oxidation and the

citric acid cycle

Dr. Namrata Chhabra
Professor, Department of Biochemistry, Cell Biology and
Genetics
[email protected]

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List of contents
1. Goals and objectives
2. Pretest
3. Sources and the fate of pyruvate
4. PDH complex-Composition, reaction catalyzed, energetics and regulation
5. PDH complex- Clinical significance
6. TCA cycle-reactions, energetics, inhibitors and regulation
7. Amphibolic role of the TCA cycle
8. Role of coenzymes
9. Anaplerotic reactions
10. Key concepts
11. Posttest
12. Feedback

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 1. Goals and objectives

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GI1.4. To demonstrate knowledge of the importance of pyruvate
oxidation and the central metabolic role of the citric acid cycle,
and their clinical significance.
Given a clinical vignette, scenario, graph, or table, students should be able
to:
GI1.4.1. Describe the PDH complex with regards to its location, reaction
catalyzed, significance, and its regulation.
FO1.11.3. Analyze the effect of enzyme concentration, temperature, pH, and
cofactors on the initial (Vo) and maximum velocity (Vmax) of a reaction.
GI1.4.2. Describe the purpose of the TCA cycle, its location, and reactions.
cycle, its location, and reactions.
FO1.9.5. Categorize the 20 naturally occurring amino acids based on side
chains (R groups), polarity, metabolic fate, and nutritional requirements.
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GI1.4. To demonstrate knowledge of the importance of
pyruvate oxidation and the central metabolic role of the
citric acid cycle, and their clinical significance.
GI1.4.3. Explain the regulation of the TCA cycle and the significance of
mitochondrial NAD+: NADH ratio, ADP: ATP ratio, and succinyl CoA
concentration.
GI1.4.4. Differentiate the effects of inhibitors—arsenate and fluoroacetate—
on the citric acid cycle.
FO1.11.5. Differentiate competitive and non-competitive inhibition
GI1.4.5. Compare the energetics of oxidation of glucose under aerobic and
anaerobic conditions.
GI1.4.6. Correlate the central role of the TCA cycle with glycolysis,
gluconeogenesis, oxidative phosphorylation, fatty acid metabolism, and
amino acid metabolism.
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2.Pretest

Please click the above

link
or
scan the QR code

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3.Sources and fates of pyruvate

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 Enzymes
Glucose 2x Glyceraldehyde-3-P
ATP 1.Hexokinase/Glucokinase Pi 2NAD+
1 ADP 2.Phosphohexose isomerase 6 2NADH+2H+
Glucose-6-P 3. Phosphofructo kinase-1 2X 1,3 Bisphosphoglycerate
4. Aldolase 2ADP
2 7
5. Phosphotriose isomerase 2ATP
Fructose-6-P 6. Glyceraldehyde-3-P 2x 3, Phosphoglycerate
dehydrogenase
A quick 3
ATP
7.Phosphoglycerate Kinase 8
ADP
recap of Fructose-1,6
8. Phosphoglycerate mutase 2x 2, Phosphoglycerate
9. Enolase
glycolysis Bisphosphate 10. Pyruvate kinase 9 2H2O

Glyceraldehyde-3-P 2xPhosphoenolpyruvate
4
2ADP
5 10 2ATP
Reactions of
Dihydroxyacetone-P 2x Pyruvate
Glycolysis
Phase of energy Investment Phase of energy evolution

Energy expenditure= 2 ATP Energy evolution= 2 /7 ATP

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ATP, NADH and Pyruvate are the products of glycolysis
Sources and fate of Pyruvate
Sources Fate

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 4.PDH complex-composition, reaction catalyzed,
energetics and regulation

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Pyruvate to Acetyl Co A conversion
• Under aerobic conditions, pyruvate is transported into mitochondria
by a proton symporter.
• In the mitochondrial matrix, pyruvate is oxidatively decarboxylated by
the pyruvate dehydrogenase complex to form acetyl CoA.
• This irreversible reaction is the link between glycolysis and the citric
acid cycle.

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 Glucose

Cytoplasm
Glycolysis
Symport

Pyruvate H+
Mitochondrial
membrane
H+
Pyruvate
PDH Complex
Mitochondrial
matrix Acetyl Co A

Pyruvate transport
TCA into
Cycle
mitochondrion
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Reaction catalyzed by PDH complex

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Components of Pyruvate dehydrogenase complex

1) Enzymes- The pyruvate dehydrogenase complex is a large, highly

integrated complex of 2 types of enzymes-
A)- Catalytic enzymes
a) Pyruvate dehydrogenase (E1)
b) Dihydrolipoyl transacetylase (E2)
c) Dihydrolipoyl dehydrogenase (E3)
B)- Regulatory Enzymes
a) PDH Kinase
b) PDH Phosphatase

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Components of Pyruvate dehydrogenase complex

2) Coenzymes of PDH complex

Five coenzymes:
•Thiamine pyrophosphate (TPP),
•Lipoic acid,
•CoASH,
•FAD and
•NAD+

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Reaction catalyzed by PDH Complex

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Regulation of PDH complex
• PDH complex is highly regulated by covalent modification and a
variety of allosteric effectors.
• Covalent modification- It is regulated by phosphorylation of the
pyruvate dehydrogenase component of the multienzyme complex.
• Allosteric regulation- Pyruvate dehydrogenase is inhibited by its
products, Acetyl CoA and NADH.

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Regulation of
PDH complex
NADH and Acetyl-CoA are
powerful positive effectors on
PDH kinase,
The kinase is activated by
increases in the [ATP]/[ADP],
[Acetyl-CoA]/[CoASH], and
[NADH]/[NAD+] ratios.
the enzyme thus inactivates PDH
by converting it to the
phosphorylated form

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Energetics of PDH complex
• Two pyruvate molecules are obtained from one glucose molecule
through glycolysis.
• Each of the pyruvate yields one NADH, thus there are two NADH
molecules to be oxidized through the electron transport chain.
• Each of NADH yields 2.5 ATP molecules, thus a total of 5 ATP
molecules are produced at the level of PDH complex.

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 5.PDH complex- Clinical significance

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Case study
• A full-term female infant failed to gain weight and showed metabolic
acidosis in the neonatal period.
• A physical examination at 6 months showed failure to thrive,
hypotonia, small muscle mass, severe head lag, and a persistent
acidosis (pH 7.0 to 7.2).
• Blood lactate, pyruvate, and alanine were greatly elevated.
• What might be the probable diagnosis of this patient?

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Case discussion
• The child is most probably suffering from Pyruvate dehydrogenase
complex deficiency.
• Metabolic acidosis, elevation of blood pyruvate, lactate and alanine,
all signify the non conversion of pyruvate to Acetyl Co A.
• The persistent metabolic acidosis is due to accumulation of lactate in
blood.

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PDH Complex deficiency
• A rare disorder of carbohydrate metabolism caused by a deficiency of
one or more enzymes in the pyruvate dehydrogenase complex.
• The age of onset and severity of disease depends on the activity level
of the PDC enzymes.
• It is characterized by energy deficit and neurological deficit.

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Pathophysiology
1) Energy Deficit-A deficiency in this enzymatic complex limits the
production of Acetyl Co A and thus citrate.
• Because citrate is the first substrate in the citric acid cycle, the cycle
cannot proceed.
• The magnitude of the energy deficit depends on the residual activity
of the enzyme.

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Pathophysiology (contd.)
2) Neurological deficit
• Severe enzyme deficiencies may lead to congenital brain
malformation because of a lack of energy during neural development.
• Underlying neuropathology is not usually observed in individuals
whose onset of pyruvate dehydrogenase complex deficiency is in
childhood.

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Clinical Manifestations
The signs of poor neurological development or degenerative lesions
are:
• Poor acquisition or loss of motor milestones,
• poor muscle tone,
• new onset seizures, and
• periods of in-coordination (i.e.ataxia) abnormal eye movements,
• poor response to visual stimuli,
• mental delay, psychomotor delays and growth retardation.

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Laboratory diagnosis and treatment

• High blood lactate and high pyruvate levels with or without

lactic acidemia are diagnostic.
• Cofactor supplementation with thiamine, carnitine, and lipoic
acid is the standard of care.
• Ketogenic diets (with restricted carbohydrate intake) have
been used to control lactic acidosis with minimal success.

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 6. TCA cycle-reactions, energetics and regulation

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Introduction
• The citric acid cycle is the central metabolic hub of the cell.
• It is the final common pathway for the oxidation of fuel molecule
such as amino acids, fatty acids, and carbohydrates.
• In eukaryotes, the reactions of the citric acid cycle take place inside
mitochondria, in contrast with those of glycolysis, which take place in
the cytosol.

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Overview of the Citric Acid Cycle

Reduced
Acetyl co A CO2
Coenzymes

Electron Energy
transport chain ATP

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 The poison Fluoroacetate
Succinate (fluoro acetyl-CoA) condenses
dehydrogenase is the with oxaloacetate to form
only enzyme which is fluorocitrate, which inhibits
bound to the inner Aconitase, causing citrate to
surface of the inner accumulate.
mitochondrial
membrane.

Reactions
of
the Citric
This is the only example in
the citric acid cycle of
Acid Cycle
substrate level
phosphorylation.

Arsenite inhibits the α-

ketoglutarate dehydrogenase
enzyme.
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Role of oxaloacetate in citric acid cycle

• The four-carbon molecule, oxaloacetate that initiates the first

step in the citric acid cycle is regenerated at the end of one
passage through the cycle.
• The oxaloacetate acts catalytically: it participates in the oxidation
of the acetyl group but is itself regenerated.
• Thus, one molecule of oxaloacetate is capable of participating in
the oxidation of many acetyl molecules.

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Regulation of the TCA cycle
• The most likely sites for regulations are the nonequilibrium reactions
catalyzed citrate synthase, isocitrate dehydrogenase, and α-
ketoglutarate dehydrogenase.
• The dehydrogenases are activated by Ca2+, which increases in
concentration during muscular contraction and secretion, when there
is increased energy demand.

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Regulation of TCA cycle enzymes
a) Citrate synthase- There is allosteric inhibition of citrate synthase by
ATP and long-chain fatty acyl-CoA.
b) Isocitrate dehydrogenase- is allosterically stimulated by ADP, which
enhances the enzyme's affinity for substrates.
In contrast, NADH inhibits iso-citrate dehydrogenase by directly
displacing NAD+. ATP, too, is inhibitory.

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Regulation of TCA cycle enzymes
c) α-ketoglutarate dehydrogenase -α-
Ketoglutarate dehydrogenase is inhibited by
succinyl CoA and NADH.
In addition, α-ketoglutarate dehydrogenase is
inhibited by a high energy charge. Thus, the rate
of the cycle is reduced when the cell has a high
level of ATP.
d) Succinate dehydrogenase is inhibited by
oxaloacetate, and the availability of
oxaloacetate, as controlled by malate
dehydrogenase, depends on the [NADH]/[NAD+]
ratio.

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Inhibitors of TCA cycle- Arsenite poisoning
Arsenite (the trivalent form of Arsenic) forms a stable complex with the
- thiol group of lipoic acid, making that compound unavailable to serve
as a coenzyme. Arsenic poisoning is caused due to inhibition of the
enzymes that require lipoic acid as a coenzyme.
The enzymes requiring lipoic acid are:
1) Pyruvate dehydrogenase complex,
2) Alpha keto glutarate dehydrogenase complex and
3) Alpha ketoacid dehydrogenase complex.

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Arsenic poisoning
• In the presence of Arsenite, there is inhibition of TCA cycle affecting
brain, causing neurological manifestations and death.
• Not only lipoic acid but all enzymes containing -SH groups are
affected by Arsenic poisoning.
• Arsenate (pentavalent form of Arsenic) can interfere with glycolysis at
the step of Glyceraldeyde-3-P dehydrogenase thereby causing
decreased ATP and NADH production by glycolysis, without inhibiting
the pathway itself.

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Fluoroacetate- a rat poison
• Fluoroacetate is structurally similar to acetate, which has an important role
in cellular metabolism.
• Fluoroacetate disrupts the citric acid cycle by combining with coenzyme A
to form Fluoroacetyl CoA, which reacts with oxaloacetate in the presence
of citrate synthase (host enzyme) to produce fluorocitrate.
• Fluorocitrate binds very tightly to Aconitase, to inhibit its action, thereby
inhibiting the citric acid cycle as a whole. This is an example of suicidal
inhibition.

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Energy yield per Acetyl Co A per turn of cycle
• Three molecules of NADH and one of FADH2 are produced for each
molecule of acetyl-CoA catabolized in one turn of the cycle.
• These reducing equivalents are transferred to the respiratory chain, where
reoxidation of each NADH results in formation of 2.5, and 1.5 ATP of
FADH2.
• Consequently, 9 high-transfer-potential phosphoryl groups are generated
when the electron-transport chain oxidizes 3 molecules of NADH and 1
molecule of FADH2,
• In addition, 1 ATP (or GTP) is formed by substrate-level phosphorylation
catalyzed by succinate thiokinase.

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 Energy yield per Acetyl co A per
turn of cycle

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Energy yield when glucose is completely
oxidized to CO2 and H2O
Process Energy output
Glycolysis (aerobic) 7
PDH Complex (2 pyruvate) 2X2.5=5
TCA cycle ( 2 Acetyl CoA) 2X10=20
Total Energy output 32

Process Energy output

Glycolysis (anaerobic) 2
PDH Complex (2 pyruvate) 0
TCA cycle ( 2 Acetyl CoA) 0
Total Energy output 2
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 7. Amphibolic role of TCA cycle

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 Significance of TCA Cycle
• The citric acid cycle is not only a pathway for oxidation of two-
carbon units
• but is also a major pathway for gluconeogenesis, synthesis of
non-essential amino acids and fatty acid synthesis.
• Because it functions in both oxidative and synthetic processes,
it is amphibolic.

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 A) Catabolic role of TCA Cycle

• The citric acid cycle is the final common pathway for the
oxidation of carbohydrate, lipid, and protein because
glucose, fatty acids, and most amino acids are metabolized
to acetyl-CoA or intermediates of the cycle.

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 The function of the citric acid
cycle is the harvesting of high-
energy electrons from carbon
fuels.
1 acetate unit generates
approximately 10 molecules of
ATP per turn of the cycle.

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Sources of Acetyl Co A

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 Catabolic role of Odd chain fatty acids

TCA Cycle Methionine &Isoleucine

Side chain of cholesterol

Even chain fatty acids,

Pyruvate and ketone
bodies
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B) Anabolic role of TCA cycle
• As a major metabolic hub of the cell, the citric acid cycle also provides
intermediates for biosynthesis of various compounds.
• i) Role in Gluconeogenesis- All the intermediates of the cycle are
potentially glucogenic, since they can give rise to oxaloacetate, and
hence net production of glucose (in the liver and kidney, the organs
that carry out gluconeogenesis.

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B) Role of TCA cycle in Glucose

Gluconeogenesis Glycolysis

Role in gluconeogenesis 3-Phosphoglycerate

Pyruvate Lactate

2-Phosphoglycerate
Acetyl Co A

TCA cycle Oxaloacetate Co ASH

Phosphoenol
intermediates Pyruvate
Citrate
beyond Alpha- Malate
ketoglutarate are TCA
Isocitrate
potentially Fumarate Cycle
glucogenic.
Succinate Alpha keto glutarate

Acetyl Co A is not glucogenic.

Succinyl Co A
Why ??
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ii) Role in synthesis of nonessential amino acids
Since the transamination reactions are reversible, the cycle also
serves as a source of carbon skeletons for the synthesis of some
amino acids like Alanine, aspartate, Asparagine Glutamate , glutamine
etc.
Glutamine Purine nucleotides

Transamination Proline
Alpha-keto glutarate Glutamic acid
Arginine
Transamination
Pyruvate Alanine

Transamination
Oxaloacetate Aspartate Asparagine
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Role of TCA cycle in fatty acid synthesis
iii) Role in fatty acid synthesis- Acetyl-
CoA, formed from pyruvate by the action
of pyruvate dehydrogenase, is the major
substrate for long-chain fatty acid
synthesis .
Acetyl Co A can also be used for the
synthesis of cholesterol, steroids etc.

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v) Role in purine and pyrimidine synthesis

Acetyl Co A is utilized for the

synthesis of fatty acids, steroids,
cholesterol, ketone bodies as well as
Acetyl Choline.

Succinyl Co A is utilized for

Heme synthesis as well as
for utilization of ketone
bodies. Glutamic acid is a precursor
of GABA, an inhibitory
neurotransmitter
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 Role of vitamins (coenzymes)
• Riboflavin, in the form of flavin adenine dinucleotide (FAD), a cofactor
for succinate dehydrogenase
• Niacin, in the form of nicotinamide adenine dinucleotide (NAD), the
electron acceptor for isocitrate dehydrogenase, α-ketoglutarate
dehydrogenase, and malate dehydrogenase;
• Thiamine(vitamin B1) , as thiamine pyro phosphate, the coenzyme
for decarboxylation in the α -ketoglutarate dehydrogenase reaction;
• Pantothenic acid, as part of coenzyme A such as acetyl-CoA and
succinyl-CoA and
• Biotin- in CO2 fixation reaction to compensate oxaloacetate
concentration.

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Anaplerotic reactions
• Anaplerosis is the act of replenishing TCA cycle intermediates that
have been extracted for biosynthesis (in what are called
cataplerotic reactions).
• The TCA Cycle is a hub of metabolism, with central importance in
both energy production and biosynthesis.
• Therefore, it is crucial for the cell to regulate concentrations of
TCA Cycle metabolites in the mitochondria.

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Anaplerotic
reactions

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Key concepts
• Under aerobic conditions pyruvate is oxidatively decarboxylated by
the pyruvate dehydrogenase complex to form acetyl CoA.
• This irreversible reaction is the link between glycolysis and the citric
acid cycle.
• PDH complex is highly regulated by a variety of allosteric effectors and
by covalent modification.
• A total of 5 ATP molecules are produced at the level of PDH complex
• A deficiency of PDH complex is characterized by energy deficit and
neurological deficit

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Key concepts
• Citric acid cycle is the final common pathway for the oxidation of fuel
molecule such as amino acids, fatty acids, and carbohydrates.
• The oxaloacetate acts catalytically: it participates in the oxidation of the
acetyl group but is itself regenerated.
• 1 acetate unit generates approximately 10 molecules of ATP.
• The activity of Citric acid cycle is immediately dependent on the supply of
NAD+, which in turn, because of the tight coupling between oxidation and
phosphorylation, is dependent on the availability of ADP.
• TCA cycle functions in both oxidative and synthetic processes, thus, it is
amphibolic.

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Posttest

Please click the above link

or
scan the QR code

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Feedback

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or
scan the QR code to
provide your feedback

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 Thank you

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Contact details

Professor (Dr.) Namrata Chhabra

Department of Biochemistry, Cell Biology and Genetics
Room GC6
[email protected]

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