The Citric Acid

Cycle
The Citric Acid Cycle
• Synthesizes citrate (a tri-carboxylic acid)
using oxaloacetate and acetyl-CoA

• Citric acid cycle also called Tri-

Carboxylic Acid Cycle or Krebs Cycle

• Pathway is key in Biochemistry as it

intersects carbohydrate, fat and protein
metabolism

• The pathway oxidizes acetyl-CoA to

CO2 and H2O and regenerates
oxaloacetate while producing NADH,
FADH2 and ATP (GTP).
1. Oxidation of fatty
acids, glucose and
some amino acids to
yield acetyl-CoA
2. Oxidation of acetyl
groups in the Krebs
cycle (electrons
passed to NAD+ and
FAD)
3. Electrons from NADH
and FADH2 passed to
O2 and driving the
production of ATP
Glycolysis and the TCA cycle are at the Centre of
Metabolism
Some 500 metabolic reactions of a
typical cell are shown
schematically with the reactions of
glycolysis and the citric acid cycle
in red.

Other reactions either lead into

these two central pathways—
delivering small molecules to be
catabolized with production of
energy—or they lead outward and
thereby supply carbon compounds
for the purpose of biosynthesis.
 Glycolysis

Each of the 10 steps shown is

catalyzed by a different enzyme.

Note that step 4 cleaves a six-

carbon sugar into two three-
carbon sugars, so that the
number of molecules at every
stage after this doubles.

Step 6 begins the energy

generation phase of glycolysis,
which causes the net synthesis of
ATP and NADH molecules
Glycolysis occurs in the cytosol, however the
enzymes of the Krebs cycle are located in the
matrix of the mitochondria
Pyruvate Must Enter the Mitochondrial
Matrix for Aerobic Respiration

Symporter
The Pyruvate Dehydrogenase Complex
(PDH)
• Pyruvate dehydrogenase (E1)
• Dihydrolipoyl transacetylase (E2)
• Dihydrolipoyl dehydrogenase (E3)

• Eukaryotic complexes also contains additional

proteins:
• a protein kinase and a phosphoprotein
phosphatase
The PDH Complex
The PDH complex isolated from
mammals is approximately 50 nm
is diameter and is large enough to
be visualized with an electron
microscope.

The complex isolated from bovine

mitochondria shows a central core
of 60 copies of E2 in a pentagonal
dodecahedron with a diameter of
approximately 25nm, 30 copies of
E1 and 12 of E3.
The E2 Subunit has Three Functional
Domains
The E2 subunit has three
functionally distinct
domains:

1.The amino terminal lipoyl

domain which contains the
lipoyl-lys residue

2. A central E3 binding
domain

3. The inner core acetyl

transferase active site.
The conversation of pyruvate into acetyl CoA
consists of three steps:
decarboxylation, oxidation and transfer of the
resultant acetyl group to CoA
Substrate Channeling
The regulation of the mammalian PDH
complex
The Citric Acid Cycle
1. Citrate Synthase
 Citrate Synthase Undergoes a Number of
Conformational Changes

First, oxaloacetate binding induces the two domains to move together by 18o and
form a binding site for acetyl CoA.

When acetyl CoA is bound and citryl CoA is formed, the enzyme closes completely.

Only in the closed conformation can the hydrolysis of citryl CoA by a bound water
molecule occur. After hydrolysis the enzyme opens, allowing the products to leave.
 2. Aconitase

The tertiary hydroxyl group is not properly located in the citrate molecule for the
oxidative decarboxylations that follow.

Thus, citrate is isomerized into isocitrate to enable the six-carbon unit to undergo
oxidative decarboxylation.

The isomerization of citrate is accomplished by a dehydration step followed by a

hydration step.
 Binding of citrate to the iron-sulphur
complex of aconitase

A 4Fe-4S iron- sulphur cluster is a component of the active site of

aconitase. It aids substrate binding and is required for
catalytic addition / removal of H2O.

One of the iron atoms of the cluster binds to the carboxylate and
hydroxyl groups of citrate
 3. Isocitrate Dehydrogenase

An Oxidative Decarboxylation
Firstly the alcohol group of isocitrate is oxidized by the transfer of the hydrogen on
C2 as a hydride ion to NAD+ forming the intermediate oxalosuccinate, an unstable b-
keto acid.

A Mn2+ atom in the active site of the enzyme interacts with the oxalosuccinate
which increases the electron withdrawing capacity of the carbonyl group.
Oxalosuccinate then undergoes b-decarboxylation to form a-ketoglutarate.
4. a-Ketoglutarate Dehydrogenase
Complex

The oxidative decarboxylation of

α-ketoglutarate closely resembles that of
pyruvate, also an α-keto acid.
5. Succinyl CoA Synthetase
6. Succinate Dehydrogenase
 7. Fumerase

Stereospecific trans addition

of water to the double bond
of fumarate to form L-malate
8. Malate Dehydrogenase
Fates of the carbon atoms from OAA
and acetyl CoA
 Energy Return Anaerobic

Reaction Sequence ATP per Glucose

Phosphorylation of glucose -1

Phosphorylation of F-6-P -1

Dephosphorylation of 2 molecules +2
of 1,3-BPG

Dephosphorylation of 2 molecules of PEP +2

Formation of 2 molecules NADH 0

Total per glucose +2

 Energy Return Aerobic
Reaction Sequence ATP per Glucose

Glycolysis +2
2 molecules of NADH from glycolysis +3 (+5)
2 molecules of NADH from PDH +5
6 molecules of NADH from TCA cycle +15
2 molecules FADH2 from TCA cycle +3
2 molecules of ATP/GTP from TCA cycle +2
Total per Glucose +30 (+32)

NADH 2.5 ATP

FADH2 1.5 ATP