Hope it shall help in better understanding of citric acid cycle or TCA cycle.

1. 1
Berg • Tymoczko • Stryer
Biochemistry
Sixth Edition
Chapter 17:
Th Cit i A id C lThe Citric Acid Cycle
Copyright © 2007 by W. H. Freeman and Company
The Citric Acid Cycle
• Glycolysis produces just
2 ATP molecules
• Aerobic metabolism of
glucose → CO2 gives
more ATP
• Main part called the citric
acid cycle
– Tricarboxylic acid (TCA)
cyclecycle
– Krebs cycle
–

2. 2
The Citric Acid Cycle
• Pyruvate must be converted to acetyl-CoA
• Pyruvate + coenzyme A + NAD+ → acetyl-CoA +
CO + NADHCO2 + NADH
• Acetyl-CoA then enters the citric acid cycle,
which occurs inside mitochondria
The Citric Acid Cycle
• Citric acid cycle the
“metabolic hub” of the
cell
6 carbon
tricarboxylic
acid
cell
– Fuels aerobically
oxidized
– A source of
precursors for amino
acids nucleotideacids, nucleotide
bases, porphyrin
–

3. 3
The Citric Acid Cycle
• How does the citric acid cycle connect to other
metabolic pathways?
The Citric Acid Cycle
• Pyruvate + coenzyme A + NAD+ → acetyl-CoA +
CO2 + NADH
– Pyruvate transported through membrane protein– Pyruvate transported through membrane protein
into mitochondria
– Pyruvate dehydrogenase complex catalyzes this
irreversible reaction
• Complex of 3 enzymes
• Member of a large family with masses from 4 million toMember of a large family, with masses from 4 million to
10 million daltons
•

4. 4
The Citric Acid Cycle
The Citric Acid Cycle

5. 5
Mechanism of Pyruvate → Acetyl CoA
• Pyruvate + CoA + NAD+ → Acetyl CoA + CO2 + NADH + H+
– Requires 5 coenzymes
• Catalytic cofactors: thiamine pyrophosphate (TPP), lipoic acid, and
FADFAD
• Stoichiometric cofactors: CoA and NAD+
–
Mechanism of Pyruvate → Acetyl CoA

6. 6
Mechanism of Pyruvate → Acetyl CoA
• Decarboxylation
– Catalyzed by E1 of pyruvate dehydrogenase
complexcomplex
Mechanism of Pyruvate → Acetyl CoA
• Oxidation
– Catalyzed by the pyruvate
dehydrogenase component of thedehydrogenase component of the
complex (E1)
–

7. 7
Mechanism of Pyruvate → Acetyl CoA
• Transfer to CoA
– Catalyzed by dihydrolipoyl transacetylase (E2)
Thioester bond remains in product– Thioester bond remains in product
–
Mechanism of Pyruvate → Acetyl CoA
• Step 4: Dihydrolipoamide oxidized to lipoamide
– Catalyzed by dihydrolipoyl dehydrogenase (E3)
2 e transferred to FAD then to NAD+– 2 e- transferred to FAD then to NAD+
–
–

8. 8
Mechanism of Pyruvate → Acetyl CoA
• Complex structure of the complex
12 E3 (αβ) N-terminus
24 E1 (α2β2)
8 E2 (α3)
Mechanism of Pyruvate → Acetyl CoA
• Advantages of a compact multienzyme complex
– Reactions more efficient because reactants and
enzymes so close to each other increases overallenzymes so close to each other, increases overall
rate and minimizes side reactions
• Lipoamide swings to pyruvate dehydrogenase to
accept acetyl group
• Swings to transacetylase to transfer it to CoA-SH
• Swings to dihydrolipoyl dehydrogenase to regenerate
sulfhydryl groups
–

9. 9
Reactions of the Citric Acid Cycle
• 1, Formation of citrate, a condensation reaction
– Catalyzed by citrate synthase
–
Reactions of the Citric Acid Cycle
• Mechanism of citrate synthase, how does it
prevent hydrolysis of acetyl CoA?
– Large conformational changes during catalysis– Large conformational changes during catalysis
–
Oxaloacetat
e
Acetyl CoA CoA Citrate
Enzym
e
Enzyme
Condensation
–
Reaction

10. 10
Reactions of the Citric Acid Cycle
Acetyl CoA transformed
to enol intermediate
Citryl CoA causes
conformational
changes that close
active siteactive site
Reactions of the Citric Acid Cycle
• 2, Isomerization of citrate to isocitrate
–
–

11. 11
Reactions of the Citric Acid Cycle
• Aconitase in a class called iron-sulfur proteins
–
– 4 Fe atoms complexed to 4 sulfides and 3
cysteine S, one Fe binds to citrate through COO-
& OH groups
Reactions of the Citric Acid Cycle
• Fluoracetatyl-CoA also a substrate for citrate
synthase
– Fluoracetate found in leaves of some poisonous– Fluoracetate found in leaves of some poisonous
plants
– Fluorocitrate inhibits aconitase (enzyme in next
rxn of citric acid cycle)

12. 12
Reactions of the Citric Acid Cycle
• 3, 1st oxidation, formation of α-ketoglutarate and
CO2
––
–
Reactions of the Citric Acid Cycle
• 4, 2nd oxidation, formation of succinyl-CoA and
CO2
– Another oxidative decarboxylation catalyzed by– Another oxidative decarboxylation, catalyzed by
the α-ketoglutarate dehydrogenase complex
–

13. 13
Reactions of the Citric Acid Cycle
• 5, Formation of succinate
– Catalyzed by succinyl CoA synthetase
–
Reactions of the Citric Acid Cycle
• 6, Formation of fumarate, an FAD-linked
oxidation
– Catalyzed by succinate dehydrogenase an– Catalyzed by succinate dehydrogenase, an
integral protein of the mitochondrial membrane,
also is directly associated with the electron-
transport chain
– Because FAD covalently bound, transfer e- to Fe-
S clusters of the protein, then to electron transportE ES clusters of the protein, then to electron transport
chain
–
E- E-

14. 14
Reactions of the Citric Acid Cycle
• 7, Formation of L-malate by hydration
–
Reactions of the Citric Acid Cycle
• 8, The final oxidation, regeneration of
oxaloacetate
– Catalyzed by malate dehydrogenase– Catalyzed by malate dehydrogenase
– 2.5 ATP for each NADH
–
ΔGo’ = + 29.7 kJ/mol

15. 15
Reactions of the Citric Acid Cycle
• Net of steps 6-8
–
Summary of Reactions
• Pyruvate dehydrogenase complex in conjunction
with the citric acid cycle yields
––
–
–
–
• Pyruvate dehydrogenase complex:
– Pyruvate + CoA-SH + NAD+ → Acetyl-CoA +
NADH + CO2 + H+

16. 16
Summary of Reactions
• Citric acid cycle
– Acetyl-CoA + 3 NAD+ + FAD + GDP + Pi + 2 H2O
→ 2 CO2 + COA-SH + 3 NADH + 3 H+ + FADH2 +→ 2 CO2 + COA SH + 3 NADH + 3 H + FADH2 +
GTP
• Overall reaction
– Pyruvate + 4 NAD+ + FAD + GDP + Pi + 2 H2O →
3 CO2 + 4 NADH + FADH2 + GTP + 4 H+
E t l ATP d ti t• Eventual ATP production per pyruvate:
– 4 NADH → 10 ATP
– 1 FADH2 → 1.5 ATP
– 1 GTP → 1 ATP
– Sum: 12.5 ATP per pyruvate (25 per glucose)
Summary of Reactions
• Interesting points
– Enzymes of the citric acid cycle may be physically
associated with each other leading products toassociated with each other, leading products to
pass directly from one to the other in a process
called “substrate channeling”
– Citric acid cycle strictly aerobic because O2
required to regenerate NAD+ and FAD in the
mitochondriontoc o d o
– Net pyruvate → acetyl CoA has ΔGo’ = -33.4
kJ/mol
–

17. 17
Metabolic Control

18. 18
Metabolic Control
• Entry into cycle & rate of cycle
tightly controlled
• Pyruvate → acetyl CoA• Pyruvate → acetyl CoA
irreversible in animals
– C oxidized to CO2 by TCA cycle
– Incorporated into lipids
• Pyruvate dehydrogenase (PDH)
complex inhibited by products
–
–
Metabolic Control
• PDH complex activated by ADP and pyruvate
–
ADP & pyruvate inhibit the kinase that turns off– ADP & pyruvate inhibit the kinase that turns off
PDH
– Both the kinase and phosphatase are associated
with the PDH complex

19. 19
Metabolic Control
• How is the phosphatase activated?
– Recall the β-adrenergic receptor is stimulated by
epinephrine leads to release of Ca2+ intoepinephrine, leads to release of Ca into
cytoplasm and stimulates muscle contraction
–
Metabolic Control
• Citric acid cycle controlled at 3 points, rxns of
– Citrate synthase, isocitrate dehydrogenase, & the
α-ketoglutarate dehydrogenase complexg y g p
• Citrate synthase
– Inhibited by
– Activated by
• Isocitrate dehydrogenase
– Activated byActivated by
– Inhibited by
• The α-ketoglutarate dehydrogenase complex
– Inhibited by

20. 20
Metabolic Control
• Cells in a resting • Cells in a highly active
Relationship between metabolic state of a cell and the ATP/ADP
and NADH/NAD+ ratios
Cells in a resting
metabolic state
– Need and use little
energy
– High ATP, low ADP
l l i l hi h
Cells in a highly active
metabolic state
– Need and use more
energy than resting cells
– Low ATP, high ADP
levels imply low
ATP/ADP ratiolevels imply high
ATP/ADP ratio
– High NADH, low NAD+
levels imply high
NADH/NAD+ ratio
ATP/ADP ratio
– Low NADH, high NAD+
levels imply low
NADH/NAD+ ratio
Metabolic Control
• Inhibition of isocitrate dehydrogenase leads to
buildup of citrate
– Citrate signals glycolysis to stop– Citrate signals glycolysis to stop
– Can be a source of acetyl CoA for fatty acid
synthesis
• Inhibition of α-ketoglutarate dehydrogenase
leads to buildup of α-ketoglutarate
– Used as precursor for synthesis of many amino
acids and purine baes

21. 21
Metabolic Control
TCA Cycle & Anabolism
• Supply of cycle components need to be
replenished to keep cycle operating as they are
used for synthesisused for synthesis
– Anaplerotic reaction – reaction that replenishes a
citric acid cycle intermediate
– [Oxaloacetate] must allow acetyl-CoA to enter
cycle
In mammals Pyruvate + CO + ATP + H O →– In mammals, Pyruvate + CO2 + ATP + H2O →
oxaloacetate + ADP + Pi + 2 H+
–

22. 22
TCA Cycle & Anabolism
Beriberi
• Beriberi – a disorder caused by a lack of
thiamine (vitamin B1) in the diet, results in weight
loss, pain, emotional disturbance, weakness,loss, pain, emotional disturbance, weakness,
irregular heart rate
• Rare except in the Far East where rice is major
food
– Rice has a low content of thiamine
O i ll l h li ill b l i h d– Occasionally alcoholics will be malnourished
enough to suffer beriberi
• What is the biochemistry of this?
– Thiamine is precursor to thiamine pyrophosphate
(TPP), a coenzyme of pyruvate dehydrogenase,

23. 23
Beriberi
Beriberi

24. 24
Glyoxylate Cycle
• Plants and bacteria
can synthesize
carbohydrates fromcarbohydrates from
acetyl-CoA
– Similar to TCA
cycle, but
decarboxylations
bypassed & 2
Unique
reactions of
glyoxylate
cycle
bypassed & 2
acetyl-CoA
molecules enter per
cycle
– Lets them grow on
acetate
Carbohydrate
s
Summary
• Pyruvate dehydrogenase complex links
glycolysis to the citric acid cycle
• TCA cycle starts with condensation of 4C + 2C• TCA cycle starts with condensation of 4C + 2C
molecule, 4C molecule regenerated
• 12.5 ATP/pyruvate from TCA cycle & PDH
reaction
• TCA cycle tightly controlled
– Control closely tied to energy status of cell
• TCA cycle gives provide synthetic precursors
• Glyoxylate cycle lets plants & bacteria
synthesize carbohydrates from acetyl-CoA