1) Most molecules enter the citric acid cycle as acetyl-CoA. The cycle has three stages: acetyl-CoA production, acetyl-CoA oxidation, and electron transfer. 2) The cycle uses oxygen as the ultimate electron acceptor, completely oxidizes organic substrates to CO2 and H2O, and conserves energy as ATP. Reactions occur in the mitochondrial matrix. 3) Key steps include the condensation of acetyl-CoA and oxaloacetate to form citrate, and a series of oxidation and decarboxylation reactions that generate NADH and FADH2 and regenerate oxaloacetate, completing the cycle.

1. Berg • Tymoczko • Stryer
Biochemistry
Sixth Edition
Chapter 17:
The Citric Acid Cycle
Copyright © 2007 by W. H. Freeman and Company

8. The Citric Acid Cycle = Krebs Cycle = Three
Carboyxlic Acid Cycle
 Most mols enter the cycle as Acetyl-CoA
 There are three stages
– Acetyl-CoA production
– Acetyl-CoA oxidation
– Electron transfer
 Its distinguishing characteristics are:
– The use of oxygen as the ultimate electron acceptor
– The complete oxidation of organic substrates to CO2 and H2O
– The conservation of much of the free energy as ATP
 The reactions of the citric acid cycle occur in the mitochondrial
matrix, in contrast with glycolysis.
 An overview of the citric acid cycle
 Reactions of the citric acid cycle

9. The Oxidative Decarboxylation of Pyruvate
1. The condensation of Acetyl-CoA and oxaloacetate
to form citrate
2. Isomerization of citrate
3. Oxidation of isocitrate
4. Oxidation of -KG to succinyl-CoA
5. Conversion of succinyl-CoA to succinate
6. Oxidation of succinate to fumarate
7. Hydration of fumarate to malate
8. Oxidation of malate to oxalacetate

10. The citric acid cycle
 Citric acid cycle (also called the Krebs cycle, TCA )
oxidizes Acetyl CoA to CO2 + H2O
 Acetyl CoA
 Most mols enter the TCA cycle as Acetyl CoA. The cycle
provides intermediates for biosynthesis. So, catabolism
of proteins, fats and carbohydrates occurs in the 3
stages of cellular respiration.
Stage I  oxidation of f.a, Glc, some a.a yields Acetyl CoA
Stage II  oxidation of acetyl groups via the TCA cycle includes 4
steps in which electrons are abstracted.
Stage III  Electrons carried by NADH and FADH2 are funnelled
into a chain of mitochondrial electron carriers--
respiratory chain- ultimately reducing O2 to H2O. This
electron flow drives the synthesis of ATP, in the
process of oxidative phosphorylation.

14. Cycles distinguishing characteristics are:
• The use of oxygen as the ultimate electron
acceptor.
• The complete oxidation of organic substrates to
CO2 and H2O.
• The conservation of much of the free energy as
ATP.
The reactions of the citric acid cycle occur inside mitochondria,
in contrast with those of glycolysis, which take place in the
cytosol.

17. An overview of the citric acid cycle:
• An overview of TCA cycle
– A 4C compound (oxaloacetate) condenses with a acetyl
unit to yield a 6C tricarboxylic acid (citrate).
– An isomer of citrate is then oxidatively decarboxylated.
– The resulting 5C (a-ketoglutarate) is oxidatively
decarboxylated to yield a 4C compound (succinate).
– Oxaloacetate is then regenerated from succinate.
• Reactions of the TCA cycle

22. The oxidative decarboxylation of pyruvate.
• This is done by a multi-enzyme complex located in
the mitochondrial matrix.
Pyruvate dehydrogenase complex
• Pyruvate  Acetyl CoA
– a major fuel of the citric acid cycle
– irreversible reaction.
• That means we cannot make pyruvate from Acetyl
CoA and also explains why glucose can not be
formed from Acetyl CoA in gluconeogenesis.

30. PD complex
 5 cofactors are involved in PD complex... All of which are
coenzyme derived from vits.
 The regulation of this enzyme complex also shows how a
combination of covalent modification and allosteric
regulation results in precisely regulated flux through a
metabolic step.
 Finally, the pyruvate dehydrogenase complex is the
prototype for 2 other important enzyme complexes that
we’ll cover later.
– -ketoglutarate dehydrogenase  TCA cycle
– -ketoacid dehydrogenase  a.a degradation

31. Reactions of PD complex
Step 1. Pyruvate reacts with the bound TPP of E1, undergoing deCO2
to form the Ohethyl derivative.
Step 2. The transfer of 2e- and the acetyl group from TPP to E2 to
form acetyl thioester of the reduced lipoyl group.
Step 3. Transesterification -SH group of CoA replaces the SH group
of E2 to yield AcetylCoA.
Step 4. E3 promotes transfer of 2H atoms from E2 to the FAD of E3
restoring the oxidized form of the lypoyllysyl group of E2.
Step 5. The reduced FADH2 ON E3 TRANSFERS HYDRIDE ION
TO NAD.

32. Pyruvate
Dehydrogenase

36. OXIDATIVE DECARBOXYLATION OF PYRUVATE
PDC is regulated by 2 mechanism.
1. Product inhibition
– Inhibited by Acetyl CoA
– High concentrations of NADH
2. Covalent modification:
PDC exists in 2 forms:
1. Active  nonphosphorylated
2. Inactive  phosphorylated form.
Phosphorylated and nonphosphorylated PDC can be
interconverted by 2 separate enzymes.
1. A kinase
2. A phosphotase

38. 8 STEPS IN THE TCA CYCLE
1) The condensation of acetylCoA and oxaloacetate to
form citrate
– The reaction uses an intermediate of the TCA cycle OA and
produces another intermediate of the cycle (citrate). Thus, the
entry of acetylCoA into the Krebs cycle does not lead to the
net production or consumption of cycle intermediates.
A refresher on enzyme nomenclature
• Synthases: catalyze condensation reactions in which no ATP, GTP is
required as an energy source.
• Synthetases: also catalyze condensation reactions but this name implies
that ATP or GTP is used for the synthetic reaction.
Citrate syntase is inhibited by ATP, NADH, succinyl CoA derivatives of
fatty acids.

43. Citrate, in addition to being an intermediate in the TCA
cycle, has other functions:
 Provides a source of AcetylCoA for fa synthesis.
 Citrate inhibits PFK, the rate limiting step in
glycolysis, and activates Acetyl-CoA carboxylase,
the rate limiting enzyme for fa synthesis.

44. 2) ISOMERIZATION OF CITRATE:
Citrate is isomerized to isocitrate by a dehydration
step followed by a hydration step. Cis-aconitate
occurs as an enzyme-bound intermediate.

46. 3) OXIDATION OF ISOCITRATE:
Isocitrate dehydrogenase catalyzes the irreversible
oxidadite deCO2 of isocitrate yielding the first of 3
NADH mols produced by the cycle and CO2.
– Enzyme is activated by ADP. Elevated levels of
mitochondrial ADP signals a need for the generation of
more high-energy phosphate (ATP).
– The enzyme is inhibited by ATP and NADH, which are
increased when the cell has abundant energy.

48. 4) OXIDATION OF -KG TO SUCCINYLCOA
– Irreversible reaction.
– Enzyme: –KGDC. It is similar to PDC reaction.
– It also has 3 enzymes (analogous to E1, E2, E3) and 5 cofactors.
(TPP, lipoic acid, FAD, NAD, and CoA)
– Enzyme is inhibited by ATP, GTP, NADH, and succinylCoA
– Enzyme is not regulated by phosphorylation/dephosphorylation
reactions as described for PDC, 2nd CO2, and 2nd NADH are
produced.

50. 5) CONVERSION OF SUCCINYL COA TO SUCCINATE:
– This reaction is coupled to the phosphorylation of GDP to GTP.
The energy content of GTP is the same as that of ATP, because
2 nucleotides are interconvertible by the nucleoside diphosphate
kinase reaction.
– This is an example of substrate -level phosphorylation in which
the ATP production is coupled to the conversion of substrate to
product, rather than resulting from respiratory-chain.

52. 6) OXIDATION OF SUCCINATE TO FUMARATE:
– FAD rather than NAD is the e-acceptor, since the
reducing power of succinate is not sufficient to
reduce NAD+. Malonate, a dicarboxylic acid that is
a structural analog of succinate, competitively
inhibits succinate dehydrogenase.

55. 7) HYDRATION OF FUMARATE TO MALATE:

56. 8) OXIDATION OF MALATE TO OXALOACETATE:

58. STOICHIOMETRY OF THE CYCLE
Summary of the reactions:
1. Two carbon atoms enter the cycle as acetyl CoA and leave
as CO2.
2. The TCA cycle does not involve the net consumption or
production of OA or any other intermediate of the cycle.
3. Four pairs of e- are transferred during one turn of the cycle:
3 pairs of e- reducing NAD+ to NADH and one pair
reducing FAD to FADH2.
ATP FORMATION IN THE AEROBIC OXIDATION OF A
MOLECULE OF GLC VIA GLYCOLYSIS, THE PDC
REACTION AND THE TCA CYCLE:

59. CITRIC ACID CYCLE COMPONENTS ARE
IMPORTANT BIOSYNTHETIC INTERMEDIATES.
 The TCA cycle is an amphibolic pathway, meaning it serves in both
catabolic and anabolic processes.
– It also provides precursors for many biosynthetic pathways.
– But if this is the case , we have to replace the ones used for the
biosynthesis of some molecules.
– Those reactions which replenish TCA acid cycle intermediates are
called anaplerotic reactions.
– Under normal circumstances there is a dynamic balance between the
reactions by which the cycle intermediates are used and those by which
they are replenished by the anaplerotic reactions.
– So that the concentrations of the citric acid cycle intermediates remain
almost constant.
 Given the number of biosynthetic products synthesized from the TCA
cycle intermediates, this cycle serves a critical role apart from its role
in energy yielding metabolism.

60. ATP Formation in the Aerobic Oxidation of GLC via
Glycolysis, the PDC Reaction, and the TCA Cycle
#ATP #ATP
REACTION or coenz formed
GLC  G-G-P -ATP -1
F-G-P  F-1,6-biP -ATP -1
G-3-P  1, 3 bisP Glycerate 2 NADH 6
1, 3 bis.Gly  3 P Gly 2 ATP 2
PEP P 2 ATP 2
Pyru  AcetylCoA 2 NADH 6
Iso.  αKG 2 NADH 6
αKG  Succ. 2 NADH 6
Succinyl  Succinate 2 GTP 2
Succinate  Fumarate 2 FADH2 4
Malate OA 2NADH 6

64. ANAPLEROTIC REACTIONS
REACTION TISSUE, ORGANS
1)
2)
3)
4)
 They are all reversible. When TCA needs OA, pyruvate is carboxylated to
OA. Free energy is required to attach CO2 to pyruvate comes from ATP.
Carboxylation of pyruvate also requires, like in other carboxylation
reactions BIOTIN, which is a prosthetic group of pyruvate carboxylase.

66. THREE ENZYMES
OF THE TCA CYCLE ARE REGULATED
 The TCA cycle is under tight regulation.
3 factors are important for the rate of flux
through the cycle.
1. Substrate availability
2. Product inhibition
3. Allosteric feedback inhibition of early
enzymes by later intermediates in the cycle.

67.  There are 3 irreversible steps in the cycle,
therefore potential sites for control. Those are
catalyzed by :
– Citrate synthase
– Isocitrate dehydrogenase
– -KG dehydrogenase.
 Each can become a rate limiting step under
certain circumstances. When acetyl CoA and OA
are available or not , citrate formation increase or
decrease.
 NADH increases (a product of the oxidation of
isocitrate and -KG) , NADH/[NAD] increases,
those dehydrogenase reactions are severely
inhibited.

70. The TCA
cycle is a
source of
biosynthetic
precursors

71. Regulation of
Carbohydrate
Metabolism

72. The disruption of pyruvate metabolism is the cause
of beriberi and heavy metal poisoning
 TPP deficiency causes beriberi
 Hg, Ar, and Pb have high affinity for -SH
 Lipoic acid is one of the cofactors in PDC
 PDC becomes inactive when lipoic acid is
bound to heavy metals.
 CNS solely depends on Glc metabolism
therefore effected by heavy metal poisoning.

76. The glyoxylate cycle permits AcetylCoA
to be incorparated into carbohydrates:
 The glyoxylate cycle , a modification of the TCA
cycle, is a biosnthetic pathway that leads to the
formation of glucose from AcetylCoA.
 It occurs in :
– Plants
– Bacteria
– Yeast
– Not in Animals

77. glyoxylate cycle
 The glyoxylate cycle is especially active in oily
seed plants.
 The glyoxylate cycle can be regarded as a shunt
within the TCA cycle.
The 6C intermediate isocitrate, rather than
undergoing decarboxylation, is converted to the 4C
mol succinate and 2C mol glyoxylate in a reaction
catalyzed by isocitrate lyase, the first of the 2
enzymes in this cycle.

81. More about the glyoxylate cycle
 In plants, the enzymes of the glyoxylate cycle are in the
membrane bound organelles and called glyoxysomes.
 Glyoxylate enzymes are not present in animal cells, thus
animals can not sustain growth on acetylCoA or 2C mols,
such as acetate.
 Role of the glyoxylate cycle:
• 4C and 6C compounds are made from 2C compounds
• Glucose is made from acetate
• It is also essential reaction sequence for seedlings of fat storing in
plants.
 TCA and Glyoxylate cycles are coordinately regulated.