The Citric Acid Cycle

Tricarboxylic Acid cycle(TCA)

Krebs Cycle

1 Dr. Suheir Ereqat 2024

 In Cytosol

In Mitochondria

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Pyruvate dehydrogenase
complex

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 Pyruvate Dehydrogenase

Pyruvate Dihydrolipoyl
Dihydrolipoyl
dehydrogenase dehydrogenase
transacetylase

•irreversible;
• in mitochodria.

5 Oxidative –decarboxylation of Pyruvate

 Pyruvate dehydrogenase complex:

E1 pyruvate dehydrogenase
3 Es E2 dihydrolipoyl transacetylase
E3 dihydrolipoyl dehydrogenase

Thiamine pyrophosphate, TPP (VB1)

5 Coenzymes Lipoic Acid
(Vitamins) CoA (pantothenic acid, VB5)
FAD (VB2)
NAD+ (VB3)

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 thioester

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Oxidative decarboxylation of pyruvate to acetyl-CoA
by the PDH complex.
Step 1 pyruvate reacts with the bound thiamine pyrophosphate (TPP) of
E1 undergoing decarboxylation to the hydroxyethyl derivative

Pyruvate dehydrogenase also carries out step 2 , the transfer of two electrons and the
acetyl group from TPP to the oxidized form of the lipoyllysyl group of the core enzyme
E2 to form the acetyl thioester of the reduced lipoyl group.

Step 3 is a transesterification in which the -SH group of CoA replaces the -SH group of
E2 to yield acetyl-CoA and the fully reduced (dithiol) form of the lipoyl group.

In step 4 E3 promotes transfer of two hydrogen atoms from the reduced lipoyl groups of
E2 to the FAD prosthetic group of E3, restoring the oxidized form of the lipoyllysyl group
of E2.

In step 5 the reduced FADH2 of E3 transfers a hydride ion to NAD,

forming NADH. The enzyme complex is now ready for another catalytic
cycle.

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Pyruvate dehydrogenase complex

---

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Regulation of Pyruvate Dehydrogenase Complex:
Product inhibition (feedback inhibition) by NADH &
acetyl CoA:
 NADH competes with NAD+ for binding to E3.
 Acetyl CoA competes with CoASH for binding to E2.

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During starvation:
 Pyruvate Dehydrogenase Kinase increases in amount
in most tissues, including skeletal muscle, via increased
gene transcription.
 Under the same conditions, the amount of Pyruvate
Dehydrogenase Phosphatase decreases.
The resulting inhibition of Pyruvate Dehydrogenase
prevents muscle and other tissues from catabolizing glucose
& gluconeogenesis precursors.
 Metabolism shifts toward fat utilization.
 Muscle protein breakdown to supply gluconeogenesis
precursors is increased.
 Available glucose is spared for use by the brain.

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 Pyruvate dehydrogenase deficiency

Pyruvate Dehydrogenase Deficiency (PDH) is a genetic disease that

involves human metabolism. Some of the genetic causes are extremely
rare in the order of 1 in millions. It affects a gene which codes for a critical
enzyme complex. PDH deficiency is most commonly linked to the alpha
unit of E1, which is X-linked, but autosomal recessive variants also exist.

PDH deficiency causes elevated serum lactate, pyruvate and alanine

(lactic acidosis). Symptoms are varied, and include developmental and
neurological defects which generally result in death .

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 Treatment
• Use of a ketogenic diet:
The ketogenic diet is a high-fat, adequate-protein, low-carbohydrate
diet. The diet mimics aspects of starvation by forcing the body to
burn fats rather than carbohydrates.

• Current research is being conducted on the viability

of Dichloroacetic acid to treat the lactic acidosis commonly
accompanied by this disorder.

• Salts of DCA have been studied as potential drugs because they

stimulates the activity of the enzyme pyruvate dehydrogenase by
inhibiting the enzyme pyruvate dehydrogenase kinase. Thus, it
decreases lactate production by shifting the metabolism of pyruvate
from glycolysis towards oxidation in the mitochondria.

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 Thiamin (Vitamin B1) deficiency causes
Beriberi
Thiamine pyrophosphate (TPP) is an important coenzyme of pyruvate
dehydrogenase complex, or PDC a critical enzyme in glucose metabolism.
Thiamine is neither synthesized nor stored in good amounts by most
vertebrates.
It is required in the diets of most vertebrates. Thiamine deficiency
ultimately causes a fatal disease called Beriberi characterized by
neurological disturbances, paralysis, atrophy of limbs and cardiac failure.
Note that brain exclusively uses aerobic glucose catabolism for energy and
PDC is very critical for aerobic catabolism. Therefore thiamine deficiency
causes severe neurological symptoms.

Prevalence
Beriberi is rare in developed countries because most foods are now vitamin-
enriched. one can get enough thiamine by eating a normal, healthy diet.
Today, beriberi occurs mostly in patients who abuse alcohol. Drinking
heavily can lead to poor nutrition, and excess alcohol makes it harder for
the body to absorb thiamine.

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TCA cycle •

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Reactions of TCA cycle ( 8 reactions)

Citrate synthase
Aconitase
Iso-citrate dehydrogenase
a ketoglutarate dehydrogenase
Succinyl-CoA synthetase
(=succinate thiokinase)
Succinate dehydrogenase
Fumerase
Malate dehydrogenase
 induced fit mechanism
Citrate synthase

Binding of OAA to the enzyme

results in conformational
change which facilitates the open form
binding of the next substrate,
the acetyl Coenzyme A. There
is a further conformational
change which leads to
formation of products.
Acetyl coA
Closed form

OAA

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 Products of one turn of the TCA cycle
Conservation of energy in
the TCA: The two carbon
acetyl group generated in
PDH reaction enter the
TCA, and two molecules
of CO2 are released in on
cycle. Thus there is
complete oxidation of
two carbons during one
cycle. Although the two
carbons which enter the
cycle become the part of
oxaloacetate, and are
released as CO2 only in
the third round of the
cycle, the energy
released due to this
oxidation is conserved in
the reduction of 3 NAD+,
1 FAD molecule and
synthesis of 1GTP
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molecule which is
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 To Summarize…

• 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 NADH
and FADH2.

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stereospecific enzyme :
Does not work on maleate or D malate.

Inhibited by malonate

Bound to IMM

NAD/NADP
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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.

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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 dyhdogenase 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 synthatse: 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..

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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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 TCA cycle is a source of Biosynthetic Intermediates
Amphibolic nature of the TCA cycle
serves in both catabolic and anabolic processes.

If the TCA intermediates are used for synthetic reactions (amino acids, Fatty
acids, Glucose), they are replenished by anaplerotic reactions in the cells
(indicated by red colours).
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 Filling up Reactions

and nervous tissues

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The most important anaplerotic reaction:

pyruvate carboxylase

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 TCA Cycle is carefully Regulated

Rate controlling enzymes: Key regulatory

Citrate synthase
Isocitrate dehydrogenase
a-keoglutarate dehydrogenase

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 Regulation of the TCA cycle
= by availability of substrates and consumption of produts

regulatory activation inhibition

enzyme
citrate synthase ADP   NADH / NAD+,ATP
 succinyl-CoA
 long chain F.A, citrate
isocitrate   ATP / ADP   NADH / NAD+
dehydrogenase  Ca2+   ATP / ADP

a-ketoglutarate  Ca2+   NADH / NAD+

dehydrogenase   ATP / ADP
 GTP
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 Summary: Regulation of metabolite flow from the PDH
complex through the citric acid cycle

• 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, as shown here, stimulates energy-
yielding metabolism to replace the ATP
consumed by contraction.
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