School Year - 1

of Committee III
Medicine Cellular Basis of
Medicine

TCA Cycle
(Citric acid cycle) (Krebs cycle)
Aysel Özpınar
(aysel.ozpinar@acibadem.edu.tr)
 LEARNING OBJECTIVES
•After this lecture you should be able to:

•Outline the sequence of reactions in the

tricarboxylic acid (TCA) cycle and explain the
purpose of the cycle.
•Identify the four oxidative enzymes in the TCA
cycle and their products.

A.Özpınar 2
 LEARNING OBJECTIVES
•Identify the two intermediates required in
the first step of the TCA cycle and their
metabolic sources.
•Identify four major metabolic intermediates
synthesized from TCA cycle intermediates.
•Describe how the TCA cycle is regulated by
substrate supply, allosteric effectors, covalent
modification, and protein synthesis.

A.Özpınar 3
The Most Important Metabolic Pathways
 TCA Cycle
Citric Acid Cycle
Krebs Cycle
A common pathway for the final
oxidation of all metabolic fuels
 The TCA cycle has two major
functions:

energy production and biosynthesis

A.Özpınar 8
TCA CYCLE IS AMPHIBOLIC
• Involved
– Catabolic Processes
– Anabolic Processes
• Gluconeogenesis
• Lipogenesis
• Synthesis od amino acids
TCA cycle is a
part of the
central
pathways of
energy
metabolism

A.Özpınar 10
Functions of the tricarboxylic acid cycle

Acetyl-CoA
is a common
product of
many catabolic
pathways

A.Özpınar 11
the final destination Most fuel molecules enter the
for metabolism of pathway as acetyl CoA
fuel molecules

the carbon skeletons of amino acids may also

enter the TCA cycle at various points.
 TCA Cycle
Acetyl CoA is 8 steps
converted to
CO2
Electrons are
released to
NADH and
FADH2

PDH is
important.
In aerobic organisms,
the citric acid cycle
is an essential
metabolic pathway
(along
with glycolysis,
the pyruvate
dehydrogenase
reaction,
and
oxidative
phosphorylation)
A.Özpınar 16
 Functions of the tricarboxylic acid cycle
 Four oxidative steps provide free energy
for ATP synthesis

 A common endproduct of carbohydrate,

fatty acid and amino acid metabolism.

 Acetyl-CoA is oxidized in the TCA cycle

to produce reduced coenzymes
by four redox reactions per turn of the
cycle.
A.Özpınar 17
 TCA Cycle

Electrons derived from the carbon skeletons

Electrons are cuptured and transferred by
the electron transport chain to oxygen, driving
the generation of ATP.

The reactions of the TCA cycle occur entirely

within the mitochondrial matrix.

A.Özpınar 18
Functions of the tricarboxylic acid cycle
The TCA cycle provides a common
ground for interconversion of fuels and
metabolites
Biosynthetic reactions proceeding from
the TCA cycle require the input of
carbons from intermediates other than
acetyl-CoA.
Such reactions are known as anaplerotic
(building up) reactions
A.Özpınar 20
The TCA cycle provides energy and metabolites
for cellular metabolism.

A.Özpınar 21
 Why the TCA cycle is described
as Amphibolic?
Because
of the
catabolic
and
anabolic
nature
of
the TCA
cycle.
A.Özpınar 22
 Anaplerotic Reactions
• As intermediates of the citric acid cycle are
removed to serve as biosynthetic precursors.
• The intermediates of the citric acid cycle are
replanished by anaplerotic reactions
• Anaplerotic reactions convert
either pyruvate or PEP
to oxaloacetate or malate
• The most important
anaplerotic reaction in
mammalian liver and
kidney is
• the reversible
carboxylation of
pyruvate by CO2 to form
oxaloacetate, catalyzed
by pyruvate carboxylase
• Pyruvate is also
converted to Malate by
Malic enzyme
Pyruvate is at the crossroads of metabolism.

A.Özpınar 25
Pyruvate is readily
formed from lactate
or alanine.
Acetyl-CoA and
oxaloacetate are
derived from pyruvate
through the catalytic
action of pyruvate
dehydrogenase and
pyruvate carboxylase,
respectively.
A.Özpınar 26
 Pyruvate carboxylase

It catalyzes carboxylation of pyruvate to

oxaloacetate.
The coenzyme, biotin, is covalently bound to
pyruvate carboxylase, and transfers the carbon
originating from CO2 to pyruvate
A.Özpınar 27
• PEP is converted to OAA
• this reaction is catalyzed by PEP
carboxylase
• PEP- carboxylase is activated by
the glycolytic intermediate
fructose 1,6-biphosphate
Krebs cycle in biosynthesis
 TCA Cycle
• Location: Mitochondrial matrix
• Functions: involved both
– Anabolic reactions: The intermediates of the
TCA cycle are used precursors in the
biosynthesis of many compounds.
– Catabolic reactions: provides the degredation
of two-carbon acetyl residues
Provides much of the energy for respiration
 Metabolic defects
in the TCA cycle are rare

A.Özpınar 32
 Biosynthesis of Acetyl CoA
The main entry
point for the TCA
cycle is
through
generation of
acetyl CoA
by oxidative
decarboxylation
of pyruvate.
A.Özpınar 33
 Biosynthesis of Acetyl CoA

Pyruvate is transported from

cytoplasm into the mitochondrial matrix.

A specialized pyruvate transporter is

responsible for this step.

A.Özpınar 34
THE PYRUVATE DEHYDROGENASE COMPLEX (PDC)
PDC serves as a bridge between
carbohydrates and the TCA cycle .
Its irreversibility explains in part why
acetyl-CoA cannot yield a net synthesis of
glucose.
The complex functions as a unit consisting
of three principal enzymes:
pyruvate dehydrogenase
dihydrolipoyl transacetylase
dihydrolipoyl dehydrogenase.
A.Özpınar 38
 The PDH Complex
• Multi-enzyme complex
– Three enzymes
– 5 co-enzymes
– Allows for efficient direct transfer of product from one
enzyme to the next
 THE PYRUVATE DEHYDROGENASE (PDH)
ENZYME COMPLEX

• Function:
– PDH links glycolysis and TCA cycle.
– Oksidizes pyruvate to CO2, and acetyl CoA
(substrate for the TCA cycle.
• Location:
• Mitochondrial matrix
 The PDH Reaction
• E1: pyruvate dehydrogenase
PDH removesE-1
CO2 and transfers the remaining acetyl
group to the enzyme bound coenzyme thiamine
pyrophosphate
E2: dihydrolipoyl transacetylase.
Dihydrolipoyl transacetylase transfers the acetyl CoA
to its lipoic acids coenzyme with a reduction of the
lipoic acid.

A.Özpınar 43
 E3: dihydrolipoyl dehydrogenase
Transfers electrons from reduced lipoic acid
to produce NADH and regenerate the oxidized
form of lipoic acid.

A.Özpınar 44
 Deficiencies of
thiamine or niacin can
cause serious central
nervous system
problems.
This is because brain
cells are unable to
produce sufficient
ATP (via the TCA
cycle) for proper
function
if pyruvate
Lipoic acid in the pyruvate dehydrogenase is
dehydrogenase complex. A.Özpınar inactive.] 45
Regulation of PDH Reaction
highly regulated

activated by
pyruvate, ADP and
Ca ions.

inhibited by
increases in the
ratio (NADH/NAD+)
and by the product acetyl-
CoA.
 PDH regulation
• Product inhibition
– Both Acetyl CoA and NADH
• Availability of substrate
– Adequate conc of CoA and NAD+
• Covalent modification
– PDH exist in two forms:
-Inactive-phosphorylated
-Active, dephosphorylated
hormonal regulation
insulin can activate PDH in
adipose tissue
Catecholamines can activate
PDH in cardiac muscle.
Regulation of PDH
 Genetic defects in PDH
• A defect in any of the protein subunits of PDH
can result in decrease or complete loss of
activity.
• Semptoms
– Lactic acidosis
– Neurological disorders
• Symptoms are varied, developmental defects
(especially of the brain and nervous system),
muscular spasticity early death
Treatment
tiamine,
lipoic acid
ketogenic diet may
help
 Reactions of the TCA Cycle
Oxaloacetate is first condensed with an acetyl
group from acetyl coenzyme A (CoA), and then
Oxaloacetate is regenerated as the cycle is
completed.

A.Özpınar 53
1.Step: Acetyl CoA enters the TCA cycle
by condensing with OAA to form citrate

Stimulated by AMP
 2. Step

Citrate
rearranges to
isocitrate in a
reaction
catalysed by
acotinase.

A.Özpınar 55
3. Step
Isocitrate dehydrogenase converts isocitrate
to alfa-ketoglutarate.
This is a dual reaction
that
combines
decarboxylation
to release CO2
and oxidation,
With capture of the
electrons in NADH.

This enzyme is the major regulatory enzyme of

the TCA cycle.
A.Özpınar 57
4. Step: Conversion of alfa-ketoglutarate
to succinyl CoA, CO2, and NADH is catalyzed.
 This reaction is again a combained
oxidation and decarboxylation.
By analog to PDH
complex,
this enzyme complex
is made up of three
enzyme activities
with similar array of
activities and
coenzymes
requirements.
A.Özpınar 59
5. Step
Succinyl CoA is hyrolyzed to succinate and CoA.
This reaction catalyzed by Succinate thiokinase
This reaction involves
smiltaneous coupling of
GDP and Pi to form GTP.

This is substrate-level
phosphorylation
with energy
being conserved in the
form GTP.
A.Özpınar 60
6. Step
Succinate is converted to fumarate with the
transfer of electrons to FAD to form FADH2.

This reaction
catalyzed by
Succinate
dehydogenase
(SDH), tightly reduction
associated with inner
mitochondrial
membrane
A.Özpınar 61
 7. Step
Fumarate to malat
which is converted to
OAA, completing the
cycle.

This hydration
reaction is
catalyzed by fumarase

A.Özpınar 62
8. Step
Malate to oxaloacetate
Another NADH is
formed in the
sythesis of OAA from
malat.
OAA is then able to
react with another
acetyl CoA molecule
to begin the cycle
again.
A.Özpınar 63
 Energy Produced by the TCA Cycle

Two carbon atoms enter the cycle as

acetyl CoA and leave as 2 CO2.
Four pairs of electrons are transferred
during one turn of the cycle:
three pairs of electrons reducing three
NAD+ to NADH
one pair reducing FAD to FADH2.

A.Özpınar 64
A.Özpınar 65
 Stoichiometry
The net reaction of the TCA cycle is:

Acetyl-CoA + 3NAD+ + FAD + GDP + Pi + 2H2O

2CO2 + 3NADH + FADH2 + GTP + 2H+ + HSCoA

 7.5

1.5

10

Number of ATP molecules produced from the oxidation of one

molecule of acetyl CoA (using both substrate-level and
oxidative phosphorylation).
A.Özpınar 67
 Regulation of the TCA Cycle
the TCA cycle is controlled
by the regulation of several
enzyme activities
The most important of these
regulated enzymes are those
that catalyze reactions with
highly negative ΔG0:
citrate synthase,
isocitrate
dehydrogenase
( rate limiting step)
α-ketoglutarate
dehydrogenase
complex. A.Özpınar 69
Inhibitors of TCA cyle
Fluoroacetate- Aconitase

Arsenite-alfa ketoglutarate
Dehyrogenase

Malonate-Succinate
dehydrogenase
(competitive inhibitor)
 Four different B vitamins play
important roles in functioning of
TCA cyle
• Riboflavin: Vit B2 (It exists in FAD)
– FAD is the prosthetic group in alfa ketoglutarate
Dehyrogenase enzyme system
– FAD is the prosthetic group of succinate
dehydrogenase
 Four different B vitamins play
important roles in functioning of
TCA cyle
• Niacin: Vitamin B3 (nicotinamide) It exists in
NAD
– NAD is a coeznyme of the following alfa
ketoglutarate dehyrogenase enzyme system
– NAD is a coeznyme of isocitrate dehyrogenase
– NAD is a coeznyme of malate dehyrogenase
 Four different B vitamins play
important roles in functioning of
TCA cyle
• Thiamine: Vitamine B1
– Thiamine pyrophosphate (TPP) is a coenzyme of
alfa ketoglutarate dehyrogenase enzyme system
• Pantothenic acid
– It is component of Coenzme A(CoASH) and
CoASH is the cofactor of active carboxylic acid
residues, such as acetyl-CoA and succinyl-CoA