Thank you

1. Mamatha G
MSC MLT (MEDICAL BIOCHEMISTRY)
ST.JOHNS MEDICAL COLLEGE
NIP/NIAHS

2. CITRIC ACID CYCLE
• Synonyms: TCA cycle (tricarboxylic acid cycle),
Krebs’ cycle, Krebs’ citric acid cycle
• About 65-70% of the ATP is synthesized in
Krebs cycle
• Citric acid cycle essentially involves the
oxidation of acetyl CoA to CO2 and H2O.
• connecting almost all the individual metabolic
pathways

4. REACTIONS OF CITRIC ACID CYCLE
Reactions of citric acid cycle are arbitrarily divided into four stages for
discussion:
• An irreversible reaction and an
exergonic reactiongives out 7.8
Kcal.
• • Acetyl group of acetyl-CoA is
transferred to OAA, no oxidation
or decarboxylation is involved.
• • A molecule of H2O is required
to hydrolyse the “high energy”
bond linkage between the acetyl
group and CoA, the energy
released is used for citrate
condensation. No ATP is required.
• COA-SH released is reutilised
for oxidative decarboxylation of
PA

5. 2.Formation of cis-aconitic acid and
isocitric acid from citric acid
• Citric acid is converted to
isocitric acid by the enzyme
aconitase. This conversion
takes place in two steps:
• Formation of cis-aconitic
acid from citric acid as a
result of asymmetric
dehydration, and
• Formation of isocitric acid
from cis-aconitic acid as a
result of stereospecific
rehydration
Both processes are catalysed by the
same enzyme Aconitase which
requires Fe++

6. Energetics: No ATP formation at this
stage
• Stage II: The six-carbon isocitric acid is
converted to a derivative of the four carbon
succinyl-CoA. The isocitric acid undergoes
oxidation followed by decarboxylation to give
α-oxoglutarate (5 C) (α-ketoglutarate)

7. 1. Formation of oxalosuccinic acid and
α-oxo-glutarate from isocitric acid
• Since it is not possible to
separate the
dehydrogenase from the
decarboxylase activity, it
is concluded that these
two reactions are
catalyzed by a single
enzyme.
• It is believed that oxalo-
succinate is not a free
intermediate but rather
exists bound to the
enzyme

8. • Isocitrate dehydrogenase (ICD) enzyme: Three
types described: • One NADP dependant ICD
found in cytosol. • Another NADP dependant
ICD exists in mitochondria, greater activity and
more widely distributed. • One NAD+
dependant ICD, found only in mitochondria.
• Respiratory chain-linked oxidation of isocitrate
proceeds almost completely through the
NAD+ dependant ICD in mitochondrion.

9. 2. Oxidative decarboxylation of α-
oxoglutarate to succinyl-CoA:
• : This reaction is analogous to
oxidative decarboxylation of
Pyruvic acid to acetyl-CoA.
Enzyme is α-Ketoglutarate
dehydrogenase complex,
• It requires identical coenzymes
and cofactors: TPP, Lipoic acid,
CoASH, FAD, NAD+ and Mg++.
Reaction steps are similar to
PDH reaction. The reaction is
irreversible

10. Stage III
• Stage III The product of
preceding stage succinyl-CoA
is converted to succinic acid to
continue the cycle. Enzyme
catalysing this reaction is
succinate thiokinase (also
called as succinyl-CoA
synthase) Reaction requires
GDP or IDP, which is converted
in presence of Pi to either GTP
or ITP

11. • • The release of free energy from
oxidative decarboxylation of α-
oxoglutarate is sufficient to
generate a high energy bond in
addition to the formation of
NADH. • In presence of enzyme
nucleoside diphosphate kinase,
ATP is produced either from GTP
or ITP
• Thus, ATP is produced at
substrate level without
participation of electron
transport chain. This is the only
example of substrate level
Phosphorylation in TCA cycle

12. Stage IV
• Stage IV This involves three successive reactions in
which succinic acid is oxidised to oxaloacetate (OAA).
• 1. Oxidation of succinic acid to fumaric acid: It is a
dehydrogenation reaction catalysed by the enzyme
succinate dehydrogenase, hydrogen acceptor is FAD.
The enzyme is Ferri-flavoprotein, mol. wt = 200,000
containing FAD and Iron-sulphur (Fe: S), contains 4
atoms of non-haem Fe and one FAD per mol. of
enzyme.
• In contrast to other enzymes of TCA cycle, this enzyme
is bound to the inner surface of the inner
mitochondrial membrane.

13. • This is the only dehydrogenation in citric acid
cycle which involves direct transfer of H from
substrate to a flavoprotein without the
participation of NAD+.

14. 2. Formation of Malic Acid from
Fumaric Acid
• In addition to being
specific for the L-isomer
of malonate, fumarase
catalyses the addition
of the elements of
water to the double
bond of fumarate in the
‘trans’ configuration

15. 3. Oxidation of malic acid to
oxaloacetate (OAA):
• reaction is catalyzed by the
enzyme Malate dehydrogenase
which requires NAD+ as H-
acceptor.
• OAA produced acts ‘Catalytically’,
combines with a fresh molecule
of acetyl-CoA and the whole
process is repeated. Note:
Although the equilibrium of this
reaction strongly favours L-malate
the net reaction is toward the
formation of OAA as this
compound together with the
other product of reaction like
NADH and FADH2 are removed
continuously

16. TCA CYCLE IS CALLED AMPHIBOLIC IN
NATURE—WHY?
• TCA cycle has dual role
• • catabolic, and
• • anabolic.
• (a) Catabolic role: The two carbon compound
acetyl-CoA produced from metabolism of
carbohydrates, Lipids and Proteins are oxidised in
this cycle to produce CO2, H2O and energy as
ATP. (b) Anabolic or synthetic role:Intermediates
of TCA cycle are utilised for synthesis of various
compounds

17. • Examples 1. Transamination: Synthesis of non-essential amino acids:
Transaminase (aminotransferase) reactions produce Ketoacids, PA, OAA
and α-ketoglutarate, from alanine, asparate and glutamate respectively.
Because these reactions are reversible, TCA cycle serves as a source of C-
skeletons for the synthesis of nonessential amino acids. 2. Formation of
glucose: (Gluconeogenesis): Other amino acids contribute to
gluconeogenesis because all or part of their C-Skeletons enter TCA cycle
after deamination or transamination. • Pyruvate forming amino acids:
Glycine, alanine, serine, cysteine/cystine, threonine, Hydroxy-Proline and
tryptophan. • α-Ketoglutarate forming amino acids: Arginine, histidine,
glutamine and proline. • Fumarate forming amino acids: Phenylalanine
and tyrosine • Succinyl-CoA forming amino acids: Valine, Methionine and
Isoleucine. 3. Fatty acid synthesis: Acetyl-CoA formed from PA by the
action of PDH complex is the starting material for long chain FA synthesis
(palmitic acid). But this synthesis is extramitochondrial, whereas acetyl-
CoA

18. • 4. Synthesis of cholesterol and steroids: Acetyl-
CoA is used for synthesis of cholesterol, which in
turn is required for synthesis of steroids. 5. Haem
synthesis: Succinyl-CoA produced in TCA cycle
takes part in heme synthesis. 6. Formation of
acetoacetyl-CoA: Succinyl-CoA is utilised for
formation of ‘acetoacetyl-CoA’ from acetoacetate
in extrahepatic tissues (Refer ketolysis).
Formation and fate of succinyl-CoA (Active
succinate) is shown schematically in

19. Regulation of TCA Cycle
• 1. As the primary function of TCA cycle is to
provide energy, respiratory control via the ETC
and oxidative phosphorylation exerts the main
control. 2. In addition to this overall and coarse
control, several enzymes of TCA cycle are also
important in the regulation. Three Key enzymes
are: • Citrate synthase • Isocitrate dehydrogenase
(ICD) • α-oxoglutarate dehydrogenase These
enzymes are responsive to the energy status as
expressed by the ATP/ADP ratio and NADH/NAD+
ratio

20. • • Citrate synthase enzyme is allosterically inhibited by ATP and long-chain acyl-
CoA. • NAD+-dependant mitochondrial isocitrate dehydrogenase (ICD) is activated
allosterically by ADP and is inhibited by ATP and NADH. • α-oxoglutarate
dehydrogenase regulation is analogous to PDH complex. 3. In addition to above,
succinate dehydrogenase enzyme is inhibited by OAA and the availability of OAA is
controlled by malate dehydrogenase, which depends on NADH/NAD+ ratio.
• Bioenergetics: Overall energy production in glycolysis cum TCA cycle in presence of
O2 is summarised in box an next page.
• Efficiency 1. Complete oxidation of glucose to CO2 and H2O in a ‘Bomb
calorimeter’ yields 686,000 calories which is liberated as heat. 2. When oxidation
occurs in tissues, some of this energy is not lost immediately as ‘heat’ but
captured as “high energy PO4 bonds”. At least 38 high energy PO4 bonds are
generated per molecule of glucose oxidised to CO2 and H2O.
• 4. Most of ATP is formed as a result of oxidative phosphorylation resulting from re-
oxidation of reduced coenzymes, viz., NADH and FADH2 by the respiratory chain.
The remainder is generated by Phosphorylation at substrate level.

24. • Reactions of citric acid cycle Oxidative
decarboxylation of pyruvate to acetyl CoA by
pyruvate dehydrogenase complex is discussed
above. This step is a connecting link between
glycolysis and TCA cycle. A few authors,
however, describe the conversion of pyruvate
to acetyl CoA along with citric acid cycle.

25. • 1. Formation of citrate : Krebs cycle proper
starts with the condensation of acetyl CoA and
oxaloacetate, catalysed by the enzyme citrate
synthase.

26. • 2. and 3. Citrate is isomerized to isocitrate by the enzyme
aconitase. This is achieved in a two stage reaction of
dehydration followed by hydration through the formation
of an intermediate—cis-aconitate. 4. and 5. Formation of D-
ketoglutarate : The enzyme isocitrate dehydrogenase (ICD)
catalyses the conversion (oxidative decarboxylation) of
isocitrate to oxalosuccinate and then to D-ketoglutarate.
The formation of NADH and the liberation of CO2 occur at
this stage. 6. Conversion of D-ketoglutarate to succinyl CoA
occurs through oxidative decarboxylation, catalysed by D-
ketoglutarate dehydrogenase complex. This enzyme is
dependent on five cofactors—TPP, lipoamide, NAD+, FAD
and CoA. The mechanism of the reaction is analogous to
the conversion of pyruvate to acetyl CoA

27. • 7. Formation of succinate : Succinyl CoA is converted to succinate by
succinate thiokinase. This reaction is coupled with the
phosphorylation of GDP to GTP. This is a substrate level
phosphorylation. GTP is converted to ATP by the enzyme nucleoside
diphosphate kinase. GTP + ADP l ATP + GDP 8. Conversion of
succinate to fumarate : Succinate is oxidized by succinate
dehydrogenase to fumarate. This reaction results in the production
of FADH2 and not NADH. 9. Formation of malate : The enzyme
fumarase catalyses the conversion of fumarate to malate with the
addition of H2O. 10. Conversion of malate to oxaloacetate : Malate
is then oxidized to oxaloacetate by malate dehydrogenase. The third
and final synthesis of NADH occurs at this stage. The oxaloacetate is
regenerated which can combine with another molecule of acetyl
CoA, and continue the cycl

28. • Energetics of citric acid cycle During the process
of oxidation of acetyl CoA via citric acid cycle, 4
reducing equivalents (3 as NADH and one as
FADH2) are produced. Oxidation of 3 NADH by
electron transport chain coupled with oxidative
phosphorylation results in the synthesis of 9 ATP,
whereas FADH2 leads to the formation of 2 ATP.
Besides, there is one substrate level
phosphorylation. Thus, a total of twelve ATP (10
as per recent evidence) are produced from one
acetyl CoA.

29. • Role of vitamins in TCA cycle Four B-complex
vitamins are essential for Krebs cycle, and thus
energy generation 1. Thiamine (as TPP) as a
coenzyme for D-ketoglutarate dehydrogenase. 2.
Riboflavin (as FAD) as a coenzyme for succinate
dehydrogenase. 3. Niacin (as NAD+) as electron
acceptor for isocitrate dehydrogenase, D-
ketoglutarate dehydrogenase and malate
dehydrogenase. 4. Pantothenic acid (as coenzyme
A) attached to active carboxylic acid residues i.e.
acetyl CoA, succinyl CoA.

30. Inhibitors of Krebs cycle The important
enzymes of TCA cycle inhibited by the
respective inhibitors are listed

31. • Fluoroacetate – a suicide substrate : The
inhibitor fluoroacetate is first activated to
fluoroacetyl CoA which then condenses with
oxaloacetate to form fluorocitrate.

32. • Regulation of citric acid cycle The cellular demands of ATP are
crucial in controlling the rate of citric acid cycle. The regulation is
brought about either by enzymes or the levels of ADP. Three
enzymes—namely citrate synthase, isocitrate dehydrogenase and
D-ketoglutarate dehydrogenase—regulate citric acid cycle. 1. Citrate
synthase is inhibited by ATP, NADH, acetyl CoA and succinyl CoA. 2.
Isocitrate dehydrogenase is activated by ADP, and inhibited by ATP
and NADH. 3. D-Ketoglutarate dehydrogenase is inhibited by
succinyl CoA and NADH. 4. Availability of ADP is very important for
the citric acid cycle to proceed. This is due to the fact that unless
sufficient levels of ADP are available, oxidation (coupled with
phosphorylation of ADP to ATP) of NADH and FADH2 through
electron transport chain stops. The accumulation of NADH and
FADH2 will lead to inhibition of the enzymes (as stated above) and
also limits the supply of NAD+ and FAD which are essential for TCA
cycle to proceed.