The Citric Acid Cycle

Tricarboxylic Acid cycle

Krebs Cycle
Part II

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 Acetyl CoA

• Fate:
• 1. Complete oxidation of acetyl group in
TCA for energy
• 2. Conversion of the excess to ketone
bodies, acetoacetate and B-
hydroxybutyrate in liver
• 3. Transfer acetyl unit as citrate to
cytosol for fatty acid synthesis.
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Citric Acid cycle or Tricarboxylic Acid cycle or Krebs Cycle

•Pyruvate Dehydrogenase Complex (PDC) and its control

•Reactions of TCA cycle or CAC

•Amphibolic nature of TCA cycle

•Regulation of TCA cycle

•Reactions of Glycolysis are localized in Cytosol, and do not require any

oxygen.

whereas pyruvate dehydrogenase and TCA cycle reactions take place in

mitochondria where oxygen is utilized to generate ATP by oxydative
phosphorylation.

Consumption of oxygen (respiration) depends on the rate of PDC and

TCA reactions.
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In Cytosol

In Mitochondria

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Reactions of TCA cycle ( 8 reactions)

Citrate synthase
Aconitase
Iso-citrate dehydrogenase
a ketoglutarate dehydrogenase
Succinyl-Coenzyme A synthetase
Succinate dehydrogenase
Fumerase
Malate dehydrogenase

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 Products of one turn of the citric acid cycle

Four reactions of TCA cycle transfer

electron to either NAD+ or FAD

One turn- One GTP, 3 NADH, one FADH2=10 ATP

So one mole of glucose give??
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• To begin a turn of the cycle, acetyl-CoA donates its acetyl group to the
four-carbon compound oxaloacetate to form the six-carbon citrate.
• Citrate is then transformed into isocitrate, also a six-carbon molecule,
which is dehydrogenated with loss of CO2 to yield the five-carbon
compound ά-ketoglutarate (also called oxoglutarate).
• ά -Ketoglutarate undergoes loss of a second molecule of CO2 and
ultimately yields the four-carbon compound succinate.
• Succinate is then enzymatically converted in three steps into the four-
carbon oxaloacetate—which is then ready to react with another molecule
of acetyl-CoA.
• In each turn of the cycle, one acetyl group (two carbons) enters as acetyl-
CoA and two molecules of CO2 leave;
• One molecule of oxaloacetate is used to form citrate and one molecule of
oxaloacetate is regenerated. No net removal of oxaloacetate occurs;
one molecule of oxaloacetate can theoretically bring about oxidation of an
infinite
• Number of acetyl groups, and, in fact, oxaloacetate is present in cells in
very low concentrations.
• Four of the eight steps in this process are oxidations, in which the energy
of oxidation is very efficiently conserved in the form of the reduced
coenzymes
7 NADH and FADH2. 2/13/2021
Detailed Reactions of TCA cycle

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Reactions of Citric Acid Cycle
1. Citrate synthase:.
Binding of Oxaloacetate to the enzyme results in conformational change
which facilitates the binding of the next substrate, the acetyl Coenzyme
A. There is a further conformational change which leads to formation of
products. This mechanism of reaction is referred as induced fit model.

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2. Aconitase: This enzyme catalyses the isomerization reaction by
removing and then adding back the water ( H and OH ) to cis-
aconitate in at different positions. Isocitrate is consumed rapidly by
the next step thus deriving the reaction in forward direction.

Fluoroacetate is potent inhibitor of the cycle, where converted to flurocitrate

that inhibit aconitase.it is lethal in small doses and used as rat poison.the lethal
dose for 50% of animal consuming it, (LD50), is 0.2 mg per kilogram of body
weight. 2/13/2021
3. Isocitrate dehydrogenase: There are two isoforms of this enzyme,
one uses NAD+ and other uses NADP+ as electron acceptor.

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4. a-Ketoglutarate dehydrogenase: This is a complex of different
enzymatic activities similar to the pyruvate dehydogenase complex.
It has the same mechanism of reaction with E1, E2 and E3 enzyme
units. NAD+ is an electron acceptor.

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5. Succinyl CoA synthatase: Succinyl CoA, like Acetyl CoA has a
thioester bond with very negative free energy of hydrolysis. In
this reaction, the hydrolysis of the thioester bond leads to the
formation of phosphoester bond with inorganic phosphate. This
phosphate is transferred to Histidine residue of the enzyme and
this high energy, unstable phosphate is finally transferred to GDP
resulting in the generation of GTP.

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6. Succinate Dehydrogenase: Oxidation of succinate to fumarate.
This is the only citric acid cycle enzyme that is tightly bound to the
inner mitochondrial membrane. It is an FAD dependent enzyme.

Malonate has similar structure to Succinate, and it competitively

inhibits Succinate Dehydrogenase.

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7. Fumarase: Hydration of Fumarate to malate: It is a highly
stereospecific enzyme. Cis-Maleate > the cis form of fumarate is
not recognized by this enzyme.

Fumarase deficiency…………….

It catalyzes hydration of the trans double bond of fumarate but not the cis
double bond of maleate (the cis isomer of fumarate).

In the reverse direction (from L-malate to fumarate), fumarase is equally

stereospecific: D-malate is not a substrate.
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8. L-Malate dehydrogenase: Oxidation of malate to oxaloacetate: It
is an NAD+dependent enzyme.

Reaction is pulled in forward direction by the next reaction (citrate

synthase reaction) as the oxaloacetate is depleted at a very fast
rate.

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Efficiency of Biochemical engine in Living Systems:
Oxidation of one glucose yields 2840 kJ/mole energy
Energy obtained by biological engine: 32ATP X 30.5 kJ/Mol = 976
kJ/mol
Thus 34% efficiency is obtained if calculations are done using
standard conditions. But if concentrations in the cellular condition
are taken in account, the efficiency is close to 65%.

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Reactions of TCA cycle ( 8 reactions)

Citrate synthase
Aconitase
Iso-citrate dehydrogenase
a ketoglutarate dehydrogenase
Succinyl-Coenzyme A synthetase
Succinate dehydrogenase
Fumerase
Malate dehydrogenase

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 Products of one turn of the citric acid cycle

Four reactions of TCA cycle transfer

electron to either NAD+ or FAD+

One turn- One GTP, 3 NADH, one FADH2=10 ATP

So one mole of glucose give??
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 Anaerobic bacteria use incomplete
citric acid cycle for production of
biosynthetic precursors. They do
not contain a-ketoglutarate
dehydrogenase.
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. When the citric
acid cycle is deficient in
oxaloacetate or any other
intermediates, pyruvate is
carboxylated to produce more
21 oxaloacetate. 2/13/2021
Pyruvate carboxylase is a
regulatory enzyme and is
virtually inactive in the
absence of acetyl-CoA, its
positive allosteric
modulator.

Whenever acetyl-CoA, the

fuel for the citric acid
cycle, is present in excess,
it stimulates the pyruvate
carboxylase reaction to
produce more
oxaloacetate, enabling the
cycle to use more acetyl-
CoA in the citrate
synthase reaction.
22
The amphibolic nature of Citric acid cycle: This pathway is utilized for
the both catabolic reactions to generate energy as well as for anabolic
reactions to generate metabolic intermediates for biosynthesis.

If the TCA intermediate are used for synthetic reactions, they are
replenished by anaplerotic reactions in the cells.

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Transamination convert alpha ketoglutarate to glutamate,while OAA to aspartate
 Role of the citric acid cycle in anabolism
Intermediates of the
citric acid cycle are
drawn off as
fructose 1,6- bisphosphate precursors in many
biosynthetic
(+) pathways.

Shown in red are four

anaplerotic reactions
that replenish
depleted
cycle intermediates/
Regulated to keep the
level of intermediates
high enough to support
the activity of the citric
acid cycle.
Ex: Pyruvate
carboxylase, is activated
by the glycolytic
intermediate fructose
1,6- bisphosphate, which
accumulates when the
citric acid cycle operates
too slowly to process the
24 pyruvate generated
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glycolysis.
 anaplerotic reactions that replenish
depleted
cycle intermediates

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 Regulation of metabolite flow from the PDH
complex through the citric acid cycle

. Regulation by activation and inhibition of enzyme

activity
- In contrast to glycolysis which is regulated
primarily by PFK, the TCA cycle is controlled
by regulation of several enzymatic activities.

Rate controlling enzymes:

The 3 strongly exergonic steps
Citrate synthase
Isocitrate dehydrogenase
a-keoglutarate dehydrogenase
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 Regulation of metabolite flow from the PDH
complex through the citric acid cycle

Three factors govern the

rate of flux through the
cycle:
Substrate availability; The
availability of the substrates for
citrate synthase (acetyl-CoA and
oxaloacetate) varies with the
metabolic state of the cell and
sometimes limits the rate of citrate
formation.
Inhibition by accumulating products;
NADH, a product of isocitrate and
ketoglutarate oxidation, accumulates
under some conditions, and at high
[NADH]/[NAD] both dehydrogenase
reactions are severely inhibited.
Allosteric feedback inhibition of the
enzymes that catalyze early steps in
the cycle (succinyl-CoA, citrate, and
ATP).

Citrate and PFK1?

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• The PDH complex is allosterically inhibited when [ATP]/[ADP],
[NADH]/[NAD], and [acetyl-CoA]/[CoA] ratios are high,
indicating an energy-sufficient metabolic state.

• The rate of flow through the citric acid cycle can be limited
by the availability of the citrate synthase substrates,
oxaloacetate and acetyl-CoA, or of NAD, which is depleted
by its conversion to NADH, slowing the three NAD-
dependent oxidation steps.

• Feedback inhibition by succinyl-CoA, citrate, and ATP also

slows the cycle by inhibiting early steps.

• In muscle tissue, Ca2 signals contraction and, (isocitrate

and alphaketoglutarate) not as PDH that PKA stimulated, in
contrary, it activate phosphorylase b enzyme during
glycogenolysis, stimulates energy-yielding metabolism to
replace the ATP consumed by contraction. 2/13/2021
 Fumarase is stereospecific

Does not work on maleate (cis) or D malate.

trans

Inhibited by malonate

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 Each turn of the glyoxylate
cycle (occurs in plants and
invertebrates) consumes two
molecules of acetyl-CoA and
produces one molecule of
succinate, which is then
available for biosynthetic
purposes. The succinate may
be converted through
fumarate and malate into
oxaloacetate, which can then
be converted to
phosphoenolpyruvate by PEP
carboxykinase, and thus to
glucose by gluconeogenesis.

Vertebrates do not have

the enzymes specific to the
glyoxylate cycle (isocitrate
lyase and malate synthase)
and therefore cannot bring
31 about the net synthesis of
glucose from lipids.
 Mutation in citric acid cycle cause cancer

• Mutation rare. Tumor suppressor gene

• Fumarase mutation cause tumor in smooth muscle and
kidney.
• Succinate dehydrogenase cause tumor of adrenal gland.
• Accumulation induce hypoxia inducible transcription factor
HIF alpha.

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•
Fumarase Deficiency
Fumarase deficiency is caused by a mutation in the fumarate hydratase
gene which encodes the enzyme that converts fumarate to malate in
the mitochondria.
• FUMARASE DEFICIENCY is due to other mutant allele, MUTATIONS OF
FH GENE ON CHROMOSOME 1.

• FUMARASE DEFICIENCY IS INHERITED IN AN AUTOSOMAL

RECESSIVE PATTERN

• 17 DIFFERENT MUTATIONS ON FH GENE ARE CURRENTLY KNOWN TO

CAUSE FUMARASE DEFICIENCY

• RARE, 100 PEOPLE AFFECTED WORLDWIDE

• AFFECTED INDIVIDUALS EXPERIENCE SLOW/DELAYED DEVELOPMENT

(GROWTH RATE, WEIGHT GAIN, ETC).

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SYMPTOMS…

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