TCA cycle overview Glycolysis converts glucose to pyruvate Produces 2 molecules of 2ATP per glucose Large amounts of potential energy from glucose remains unused Aerobic oxidation of pyruvate ensures that this energy is not lost The TCA cycle is the final common pathway for the oxidation of fuel molecules such as amino acids, fatty acids and carbohydrates The cycle is also an important source of precursors, not only for the storage forms of fuel, but also for the building blocks of many other molecules such as amino acids, nucleotides bases and sterols

1. Prof. Riaan den Haan
Hans Adolf Krebs. Biochemist;
born in Germany. Worked in
Britain. His discovery in 1937 of
the ‘Krebs cycle’ of chemical
reactions was critical to the
understanding of cell
metabolism and earned him
the 1953 Nobel Prize for
Physiology or Medicine.

2.  Aerobic cells use a metabolic “wheel”, the citric acid-/Krebs-
/tricarboxylic acid cycle to generate energy from pyruvate
 In eukaryotes the reactions of the citric acid cycle take place
inside mitochondria

3.  Glycolysis converts glucose to pyruvate
 Produces 2 molecules of ATP per glucose
 Large amounts of potential energy from glucose remains
unused
 Aerobic oxidation of pyruvate ensures that this energy is
not lost
 The TCA cycle is the final common pathway for the
oxidation of fuel molecules such as amino acids, fatty acids
and carbohydrates
 The cycle is also an important source of precursors, not
only for the storage forms of fuel, but also for the building
blocks of many other molecules such as amino acids,
nucleotide bases and sterols

4.  The TCA cycle consists of a series of
oxidation-reduction reactions in the
mitochondria
 Under aerobic conditions pyruvate from
glycolysis is oxidatively decarboxylated to
acetyl-coenzyme A (acetyl-CoA)
 Acetyl-CoA is oxidized to two molecules of
carbon dioxide
 This involves a 24-electron oxidation process
 This oxidation process results in the
reduction of coenzymes NAD+  NADH and
FAD  FADH2, some ATP (or equivalent) is
also produced
 The coenzymes are transported to the
electron transport pathway, where they are
oxidized to drive the synthesis of ATP
The function of the TCA cycle is the harvesting
of high-energy electrons from carbon fuels

6.  The TCA cycle seems like a complicated way to oxidize acetate units to
CO2 – why?
 The oxidation of acetate to CO2 is used to reduce NAD+ to NADH
 But, this cannot happen in a single step because acetate can’t be
directly oxidized into CO2
 Oxidation of an acetyl group to CO2 requires a C-C cleavage
 This has to be done in an energetically favorable way under cellular
conditions

7.  C-C cleavage reactions in biological systems can only occur in one of
two ways:
◦ Between carbons Cs α and β to a carbonyl group
◦ α -cleavage of an α -hydroxyketone (between COO- and C-OH)

8.  Neither of these strategies suitable for acetate (or Acetyl-CoA)
◦ No β-carbon
◦ No hydroxyl (OH) group
 Instead: acetate is condensed with oxaloacetate to form citrate
 Citrate contains a β-carbon and can undergo β-cleavage
 Therefore
◦ During the TCA cycle, 2 carbons flow into the cycle via acetyl-CoA
◦ Acetyl-CoA cannot be oxidized directly
◦ These 2 carbons condense with the final substrate of TCA cycle (oxaloacetate)
to form a 6-carbon structure citrate
◦ Citrate can undergo oxidation
◦ This oxidation leads to the generation of 2 carbon dioxide molecules
TCA cycle combines β-cleavage reaction with oxidation to form CO2,
regenerate oxaloacetate and capture liberated energy in NAD and ATP

9.  Sources: glucose (through pyruvate), fatty acids, amino acids, ketone
bodies
 Central role in citric acid cycle, oxidative phosphorylation, fatty acid
metabolism, cholesterol biosynthesis

10.  Glycolysis occurs in the cytoplasm and the TCA cycle and oxidative
phosphorylation in the mitochondria
 Pyruvate must enter the mitochondria to enter the TCA cycle
 Pyruvate is then converted to acetyl-CoA by the process of oxidative
decarboxylation via the pyruvate dehydrogenase enzyme complex

11.  Multi-enzyme complex (series of enzymes linked
together), noncovalent assembly of three enzymes in
this case – uses 5 co-enzymes
 Catalyzes oxidative decarboxylation of pyruvate to
acetyl-CoA
◦ Combines a redox reaction (pyruvate donates electron, NAD+
receives electron) and decarboxylation (loss of carbons in
form of carbon dioxide)

12.  Large multi-enzyme complex
 Multiple copies of three enzymes: E1, E2, E3
 E1: pyruvate dehydrogenase
 E2: dihydrolipoamide acetyltransferase
 E3: dihydrolipoamide dehydrogenase
 Inner core
◦ Icosahedral structure
◦ 60 copies of E2
◦ 12 copies of E3 binding protein (E3BP) that links E3 to E2
 Periphery:
◦ 30 copies of E1 (tetramer of subunits a2b2)
◦ 12 copies of E3 (homodimer)

13.  Eukaryotic PDH is one of the largest multi-enzyme
complexes known, ~50nM and ~9.5 mega-Daltons

14.  Coenzyme = Cofactor which is loosely bound to the
enzyme
 Prosthetic group = Cofactor which is tightly bound to
the enzyme
Enzyme Abbreviated Prosthetic group
Pyruvate dehydrogenase E1 Thiamine pyrophosphate (TPP)
Dihydrolipoyl transacetylase E2 Lipoic Acid (Lipoamide)
Dihydrolipoyl dehydrogenase E3 FAD
Other Coenzymes
Coenzyme A
NADH and NADPH

15.  Thiamine pyrophosphate (TPP)
◦ Prosthetic group on E1
◦ Synthesized from Thiamine (Vitamin B1)
◦ Assists in the decarboxylation process
 The flavin coenzyme (FAD)
◦ Prosthetic group on E3
◦ Synthesized from riboflavin (Vitamin B2)
◦ Involved in one or two electron transfer reactions
 The nicotinamine coenzyme (NAD)
◦ Synthesized from nicotinamide (Vitamin B3)
◦ NAD+/NADH carry out hydride transfer reactions
 Lipoic acid (Lipoamide)
◦ Prosthetic group on E2
◦ Contains a disulfide and acts as an arm to transfer acetyl group
 Coenzyme A
◦ Synthesized from cysteamine, panthothenate and ATP
◦ In TCA cycle it carries the acetyl group in acetyl-CoA

16. 1. Pyruvate reacts with TPP and is decarboxylated (E1)
• Carbon dioxide is released
• Hydroxyethyl-TPP is formed
2. Hydroxyethyl-TPP reacts with lipoamide (E1/E2)
• Hydroxyethyl group is transferred to lipoic acid (lipoamide)
• Oxidized to form acetyl dihydrolipoate (-lipoamide)
3. Acetyl group is transferred to CoA to form acetyl-CoA and lipoamide-
dithiol (reduced disulfide) (E2)
4. The reduced lipoamide is reoxidized by FAD to form FADH2 (E3)
5. The FADH2 is reoxidized by NAD+ to form NADH

17.  Thus the final products of the PDH complex reaction are
◦ Acetyl-CoA
◦ NADH
◦ CO2
 All other reactants are regenerated back to their original form

19.  Glycolysis converts glucose to pyruvate
 Pyruvate is fed into TCA cycle under aerobic conditions
 Pyruvate is converted to acetyl-CoA via the multi-
enzyme pyruvate dehydrogenase
 Pyruvate dehydrogenase consists of 3 enzymes and
needs 5 different coenzymes
 End products are CO2, acetyl-CoA, and NADH
https://www.youtube.com/watch?v=_cXVleFtzeE

20.  First 4 reactions in TCA cycle ends in the
production of succinyl-CoA and 2
molecules of CO2
Substrate Product Enzyme Other
Oxaloacetate +
Acetyl-CoA
Citrate Citrate synthase
Citrate Isocitrate Aconitase
Isocitrate α-ketoglutarate Isocitrate
dehydrogenase
CO2
α-ketoglutarate Succinyl-CoA α-ketoglutarate
dehydrogenase
CO2

21.  First reaction in TCA cycle is a synthase reaction - a new molecule is
made but ATP is not used
 Oxaloacetate + Acetyl-CoA + H2O  Citrate + CoA
 Initiates TCA cycle
 Acetyl-CoA condenses with oxaloacetate to form citrate
 Citrate synthase is a dimer – each subunit binds oxaloacetate and
acetyl-CoA
◦ Binding of oxaloacetate induces a conformational change which
facilitates the binding of acetyl-CoA

22.  Reaction is irreversible due to large energy of forward
reaction (large negative ∆G)
 NADH is an allosteric inhibitor
◦ NADH is a product of the TCA cycle
◦ TCA cycle will slow down if too much NADH
 Succinyl-CoA is an allosteric inhibitor
◦ Later intermediate in cycle
◦ Analog of acetyl-CoA
◦ Can bind citrate synthase but not react with oxaloacetate =
inhibition of reaction

23.  Citrate is a poor substrate for oxidation
 Aconitase isomerizes citrate to yield isocitrate which has a secondary -OH, which
can be readily oxidized
 Isomerization of citrate (tertiary alcohol) to isocitrate (secondary alcohol)
Citrate [H2O + cis-Aconitate ]Isocitrate
 Aconitase contains an iron-sulfur center as a prosthetic group which facilitates the
rearrangement reaction
 Dehydration reaction followed by a hydration with an aconitate intermediate
◦ Water is abstracted from citrate to yield aconitate (dehydration)
◦ H and OH are added back in opposite positions to produce isocitrate
 Citrate needs to become isocitrate so that in next step decarboxylation is possible

24.  Oxidative decarboxylation of isocitrate yields α-ketoglutarate
Isocitrate  α-ketoglutarate
 Oxidation of isocitrate linked to reduction of NAD+  NADH
 Isocitrate is oxidatively decarboxylated (carbon removed to
yield CO2)
 Isocitrate dehydrogenase is a link to the electron transport
pathway through the production of NADH

25.  Reaction is irreversible due to large energy of forward
reaction (large negative ∆G)
 NADH and ATP are allosteric inhibitors (high energy)
 ADP is allosteric activator (low energy)

26.  A second oxidative decarboxylation
α-ketoglutarate  succinyl-CoA
 Oxidative decarboxylation (oxidative removal of a carbon to yield
CO2 and NADH)
 Multi-enzyme complex similar to pyruvate dehydrogenase
◦ α-ketoglutarate dehydrogenase
◦ Dihydrolipoyl transsuccinylase
◦ Dihydrolipoyl dehydrogenase

27.  Reaction is identical to pyruvate dehydrogenase - structurally and
mechanistically
 Instead of acetyl-CoA, succinyl-CoA (an analog) is formed
 Five coenzymes used - TPP, CoASH, lipoic acid, NAD+, and FAD

28.  Last 4 reactions in TCA cycle produces GTP,
FADH2 and NADH
 Oxaloacetate is regenerated and cycle begins
again
Substrate Product Enzyme Other
Succinyl-CoA Succinate Succinyl-CoA
synthetase
GDP  GTP
Succinate Fumarate Succinate
dehydrogenase
FAD  FADH2
Fumarate Malate Fumarase
Malate Oxaloacetate Malate
dehydrogenase
NAD+  NADH

29.  A nucleoside triphosphate is made
 This is possible because succinyl-CoA is a high-energy intermediate
 The reaction removes the CoA group and yields succinate – energy
used to drive the phosphorylation of GDP to GTP
 Substrate level phosphorylation – substrate rather than electron-
transport chain provides the energy for phosphorylation
 GTP produced by mammals in this reaction is converted to ATP via
nucleoside diphosphate kinase
 GTP + ADP  ATP + GDP

30.  An FAD-dependent oxidation of a single bond to a double bond
 Succinate dehydrogenase catalyses the conversion of succinate to
fumarate and reduction of FAD to FADH2
 Dehydrogenase – thus removal of hydrogen atoms
 This enzyme is bound to the inner mitochondrial membrane
 Is part of BOTH TCA cycle AND electron transport pathway
 The electrons transferred from succinate to FAD (to form FADH2) are
passed directly to ubiquinone (UQ) in the electron transport pathway
 Succinate dehydrogenase is a dimeric protein = two subunits, one large
one small
 FAD covalently bound to larger subunit

31.  Succinate dehydrogenase contains 3 different iron-sulfur clusters:
 These iron-sulfur clusters receive the electrons captured by FAD
and pass them onto the electron transport chain

32.  Hydration of fumarate to malate (addition of water)
 Hydration involves trans-addition of the elements of water
across the double bond
 Prepares structure so it can donate electrons to NAD+ in next
reaction

33.  Malate Dehydrogenase completes the cycle by oxidizing malate to
oxaloacetate
 Hydrogen donated by malate to reduce NADH
 The carbon that gets oxidized is the one that received the -OH in the
previous reaction
 This reaction is energetically expensive:
∆Go' = +30 kJ/mol
 Thus the concentration of oxaloacetate in the mitochondrial matrix is
quite low (though ∆G is close to 0 in cellular conditions)
 However, the malate dehydrogenase reaction is pulled forward by the
favorable citrate synthase reaction

34.  TCA cycle involves a flow of carbon
molecules in and out of the cycle.
 TCA cycle starts with the addition of 2-
carbon molecules from acetyl-CoA to the
TCA cycle intermediate oxaloacetate,
which contains 4-carbons, to make a 6-
carbon molecule citrate.
 6-carbon citrate is rearranged (isocitrate)
 6-carbon isocitrate is decarboxylated (loses a carbon in form of
carbon dioxide) to 5-carbon molecule (α-ketoglutarate) and then
again to a 4-carbon molecule (succinyl-CoA)
 Next 4 molecules contain 4-carbons
 And then cycle starts again

35.  One acetate through the cycle produces two CO2, one ATP, four reduced
coenzymes
 Energy released through oxidation of acetyl-CoA is conserved in the
reduction of NAD+, FAD+ and the synthesis of GTP which can be converted
to ATP
 The TCA cycle is exergonic, with a net ΔGº' for one pass around the cycle
of -40 kJ/mol
 The combination of glycolysis and TCA produce 12 reduced coenzymes,
which can eventually produce over 32 molecules of ATP
 Two carbon molecules enter the cycle as acetyl-CoA and leave as two
carbon dioxide molecules
 The carbonyl C of acetyl-CoA becomes CO2 only in
the second turn of the cycle (following entry of
acetyl-CoA )
 The methyl C of acetyl-CoA survives two cycles
completely, but half of what's left exits the cycle
on each turn after that

36. 1. Citrate synthase: catalyzes the condensation of acetyl-CoA and
oxaloacetate to yield citrate.
2. Aconitase: isomerizes citrate to the easily oxidized isocitrate.
3. Isocitrate dehydrogenase: oxidizes & decarboxylates isocitrate to form
-ketoglutarate. (1st NADH and CO2).
4. -ketoglutarate dehydrogenase: oxidatively decarboxylates -
ketoglutarate to succinyl-CoA. (2nd NADH and CO2).
5. Succinyl-CoA synthetase converts succinyl-CoA to succinate. Forms
GTP.
6. Succinate dehydrogenase: catalyzes the oxidation of central single bond
of succinate to a trans double bond, yielding fumarate and FADH2.
7. Fumarase: catalyzes the hydration of the double bond to produce
malate.
8. Malate dehydrogenase: reforms oxaloacetate by oxidizing secondary
OH group to ketone (3rd NADH).
The function of the TCA cycle is the harvesting of high-energy electrons from
carbon fuels
Final products of citric acid cycle: 2 CO2 molecules. 3 NADH, 1 FADH2, and 1
GTP

37. - You need to be able to
draw a flow diagram of
the citric acid cycle
showing substrates,
enzymes, coenzymes and
products
- You don’t need to know
the structures or
mechanisms
- You need to be able to
write a short sentence
describing each reaction
https://www.youtube.co
m/watch?v=_cXVleFtzeE

38.  TCA cycle not only functions to convert carbon to electrons and energy,
but also provides many intermediates for other biosynthetic pathways
 α-Ketoglutarate transaminated to make glutamate, which is used to make
nucleotides, as well as arginine and proline
 Succinyl-CoA is used to make porphyrins (e.g. of a porphyrine is heme,
found in hemoglobin)
 Fumarate is a precursor in production of aspartate which is used to make
nucleotides, as well as other amino acids threonine, methionine,
isoleucine and lysine

39.  Oxaloacetate also precursor in aspartate production, can also be
decarboxylated to form PEP (fed back into glycolysis) as well as aromatic
amino acids
 Citrate exported from mitochondria and broken down to oxaloacetate
and acetyl-CoA
◦ Acetyl-CoA functions in cytoplasm as precursor in fatty acid synthesis
◦ Oxaloacetate is reduced to malate, which is either transported back to
mitochondria or decarboxylated to pyruvate

40.  All 20 common amino acids can be made from metabolites
derived from glycolysis & the TCA cycle (highlighted in orange).

42.  TCA cycle lies between glycolysis and the electron transport
chain
 Must be tightly controlled to prevent wasting metabolic
energy in production of unnecessary ATP or to prevent
energy shortage in cell
 TCA cycle also important producer of precursors for other
pathways, which would also be effected if TCA cycle went
uncontrolled
 Regulation occurs at 4 important points:
◦ Pyruvate converted to acetyl-CoA (not part of TCA cycle but input of
acetyl-CoA is needed for cycle to occur)
◦ 3 enzymatic steps in the TCA cycle: citrate synthase, isocitrate
dehydrogenase, α-ketoglutarate dehydrogenase

43.  Changes in free energy of the reactions of TCA cycle indicates 3
irreversible steps – the key regulatory sites
◦ A step is irreversible when the free energy for the forward reaction
is so large that it occurs spontaneously and is not in equilibrium with
the reverse reaction = large negative free energy
 Citrate synthase
 Isocitrate dehydrogenase
 α-ketoglutatrate dehydrogenase complex

44.  Pyruvate is an important metabolite
 Under aerobic conditions, pyruvate is converted to acetyl-CoA
by pyruvate dehydrogenase
 This is an irreversible step, and must be tightly regulated
 Pyruvate dehydrogenase is allosterically regulated
◦ High concentrations of ATP, NADH, acetyl-CoA inhibit
◦ High concentrations of NAD+, CoA activate
 High concentrations of acetyl-CoA inhibits the transacetylase
component of E2
 High concentrations of NADH inhibits the dihydrolipoyl
dehydrogenase component of E3

45.  Pyruvate dehydrogenase kinase is a
regulatory enzyme that is part of the
pyruvate dehydrogenase complex in
mammals
◦ Dehydrogenase kinase is allosterically
activated by NADH and acetyl-CoA
◦ Pyruvate dehydrogenase is
phosphorylated on E1
◦ This blocks the first step of catalysis,
the decarboxylation of pyruvate
 Pyruvate dehydrogenase phosphatase
is associated with the dehydrogenase
complex when NADH and acetyl-CoA
levels are low
◦ Keeps E1 dephosphorylated and thus
active
◦ Is inactivated by high NADH or acetyl-
CoA

46.  TCA cycle is regulated by
feedback inhibition
 Feedback inhibition = products
from the system inhibit enzymes
in the same system
 The principle signals are acetyl-
CoA, succinyl-CoA, ATP, ADP,
AMP, NAD+ and NADH.
Enzyme Activators Inhibitors
Pyruvate
dehydrogenase
NAD+, CoA Acetyl CoA,
NADH, ATP
Citrate synthase ATP, NADH,
succinyl-CoA
Isocitrate
dehydrogenase
NAD+, ADP ATP, NADH
α-ketoglutarate
dehydrogenase
AMP NADH,
succinyl-CoA