1. No category

Berg • Tymoczko • Stryer
Biochemistry
Sixth Edition
Chapter 17:
Th Cit
The
Citric
i A
Acid
id Cycle
C l
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)
cycle
– Krebs cycle
–
1
The Citric Acid Cycle
• Pyruvate must be converted to acetyl-CoA
• Pyruvate + coenzyme A + NAD+ → acetyl-CoA +
CO2 + 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
– Fuels aerobically
oxidized
– A source of
precursors for amino
acids nucleotide
acids,
bases, porphyrin
–
2
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
into mitochondria
– Pyruvate dehydrogenase complex catalyzes this
irreversible reaction
• Complex of 3 enzymes
family, with masses from 4 million to
• Member of a large family
10 million daltons
•
3
The Citric Acid Cycle
The Citric Acid Cycle
4
Mechanism of Pyruvate → Acetyl CoA
• Pyruvate + CoA + NAD+ → Acetyl CoA + CO2 + NADH + H+
– Requires 5 coenzymes
• Catalytic cofactors: thiamine pyrophosphate (TPP), lipoic acid, and
FAD
• Stoichiometric cofactors: CoA and NAD+
–
Mechanism of Pyruvate → Acetyl CoA
5
Mechanism of Pyruvate → Acetyl CoA
• Decarboxylation
– Catalyzed by E1 of pyruvate dehydrogenase
complex
Mechanism of Pyruvate → Acetyl CoA
• Oxidation
– Catalyzed by the pyruvate
dehydrogenase component of the
complex (E1)
–
6
Mechanism of Pyruvate → Acetyl CoA
• Transfer to CoA
– Catalyzed by dihydrolipoyl transacetylase (E2)
– Thioester bond remains in product
–
Mechanism of Pyruvate → Acetyl CoA
• Step 4: Dihydrolipoamide oxidized to lipoamide
– Catalyzed by dihydrolipoyl dehydrogenase (E3)
–2e
e- transferred to FAD then to NAD+
–
–
7
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
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
–
8
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
–
Oxaloacetat Acetyl CoA
e
CoA Citrate
Enzyme
Condensation
Reaction
Enzym
e
–
9
Reactions of the Citric Acid Cycle
Acetyl CoA transformed
to enol intermediate
Citryl CoA causes
conformational
changes that close
active site
Reactions of the Citric Acid Cycle
• 2, Isomerization of citrate to isocitrate
–
–
10
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
plants
– Fluorocitrate inhibits aconitase (enzyme in next
rxn of citric acid cycle)
11
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,
decarboxylation catalyzed by
the α-ketoglutarate dehydrogenase complex
–
12
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,
dehydrogenase an
integral protein of the mitochondrial membrane,
also is directly associated with the electrontransport chain
– Because FAD covalently bound, transfer e- to FeS clusters of the
protein,
then to electron transport
E
EE
Echain
–
13
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
– 2.5 ATP for each NADH
–
ΔGo’ = + 29.7 kJ/mol
14
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+
15
Summary of Reactions
• Citric acid cycle
– Acetyl-CoA + 3 NAD+ + FAD + GDP + Pi + 2 H2O
→ 2 CO2 + COA
COA-SH
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+
• Eventual
E
t l ATP production
d ti per pyruvate:
t
– 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,
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
mitochondrion
toc o d o
– Net pyruvate → acetyl CoA has ΔGo’ = -33.4
kJ/mol
–
16
Metabolic Control
17
Metabolic Control
• Entry into cycle & rate of cycle
tightly controlled
• 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
PDH
– Both the kinase and phosphatase are associated
with the PDH complex
18
Metabolic Control
• How is the phosphatase activated?
– Recall the β-adrenergic receptor is stimulated by
epinephrine leads to release of Ca2+ into
epinephrine,
cytoplasm and stimulates muscle contraction
–
Metabolic Control
• Citric acid cycle controlled at 3 points, rxns of
– Citrate synthase, isocitrate dehydrogenase, & the
α-ketoglutarate
g
dehydrogenase
y g
complex
p
• Citrate synthase
– Inhibited by
– Activated by
• Isocitrate dehydrogenase
– Activated by
– Inhibited by
• The α-ketoglutarate dehydrogenase complex
– Inhibited by
19
Metabolic Control
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
levels
l iimply
l hi
high
h
ATP/ADP ratio
– High NADH, low NAD+
levels imply high
NADH/NAD+ ratio
• Cells in a highly active
metabolic state
– Need and use more
energy than resting cells
– Low ATP, high ADP
levels imply low
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
– 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
20
Metabolic Control
TCA Cycle & Anabolism
• Supply of cycle components need to be
replenished to keep cycle operating as they are
used for synthesis
– Anaplerotic reaction – reaction that replenishes a
citric acid cycle intermediate
– [Oxaloacetate] must allow acetyl-CoA to enter
cycle
– In mammals
mammals, Pyruvate + CO2 + ATP + H2O →
oxaloacetate + ADP + Pi + 2 H+
–
21
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,
irregular heart rate
• Rare except in the Far East where rice is major
food
– Rice has a low content of thiamine
– Occasionally
O
i
ll alcoholics
l h li will
ill b
be malnourished
l
i h d
enough to suffer beriberi
• What is the biochemistry of this?
– Thiamine is precursor to thiamine pyrophosphate
(TPP), a coenzyme of pyruvate dehydrogenase,
22
Beriberi
Beriberi
23
Glyoxylate Cycle
• Plants and bacteria
can synthesize
carbohydrates from
acetyl-CoA
– Similar to TCA
cycle, but
decarboxylations
bypassed & 2
acetyl-CoA
molecules enter per
cycle
– Lets them grow on
acetate
Unique
reactions of
glyoxylate
cycle
Carbohydrate
s
Summary
• Pyruvate dehydrogenase complex links
glycolysis to the citric acid cycle
• 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
24