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BC368: Biochemistry of the Cell II
Citric Acid Cycle
Chapter 16
March 12, 2015
3 stages of respiration
Production of acetyl-CoA
(e.g., during glycolysis and
the bridging reaction)
Oxidation of acetyl-CoA via
the citric acid cycle
Electon transport and
oxidative phosphorylation to
produce lots of ATP
Fig 16-1
Mitochondrial Architecture
Glycolysis takes place in
the cytosol
The citric acid cycle takes
place in the mitochondrial
matrix
The Bridging Reaction
H+ +
The Bridging Reaction
Fig 16-2
The Bridging Reaction
E1: orange
E2: green
E3: yellow
Pyruvate dehydrogenase complex
1. Decarboxylation
3. Acetyl group to CoA
2. Oxidation
4. Restore enzyme
Fig 16-6
Fig 16-6
Pyruvate dehydrogenase complex
Step 1. Decarboxylation
Fig 16-6
Fig 16-6
Step 1: Decarboxylation
TPP is derived from
vitamin B1
Common for
decarboxylation
reactions
Carries carbon
groups transiently
Fig 14-15
Pyruvate dehydrogenase complex
Step 2. Oxidation,
with reduction of E2
Fig 16-6
Fig 16-6
Step 2: Oxidation
Hydroxyethyl group is oxidized to acetyl group,
transferred to lipoamide of E2, which is reduced.
Lipoic Acid “Swinging Arm”
Swinging arm
acyl group
carrier
Transfers
intermediates
between
different enzyme
sites
of interest
here
The Marsh Test
Pyruvate dehydrogenase complex
Step 3. Transfer to CoA
Fig 16-6
Fig 16-6
Step 3: Transfer to CoA
Acetyl group is transferred to coenzyme A by E2.
Coenzyme A
Derived from Vitamin B5 (pantothenic acid)
“Activates” the acetyl group
Fig 16-3
Pyruvate dehydrogenase complex
Step 4. Restoring the
enzyme
Fig 16-6
Fig 16-6
Step 4: Restoring the enzyme
FAD of E3 reoxidizes dihydrolipoamide.
NAD+ reoxidizes FADH2.
Fig 16-6
FAD/FADH2
Derived from
Vitamin B2
(riboflavin)
1 or 2 electron
acceptor
NAD+/NADH
Derived from
Vitamin B3
(niacin)
2 electron
acceptor
Pyruvate dehydrogenase complex
Fig 16-6
Fig 16-6
Coenzyme A
Acetyl group is activated in two ways:
Carbonyl carbon is activated for attack by nucleophiles
Methyl carbon is more acidic
Fig 16-3
The Citric Acid Cycle
Reaction 1: Condensation
Citrate synthase
mechanism
1. deprotonation of
methyl group of
acetyl-CoA
Fig 16-9
Citrate synthase
mechanism
2. enolate attacks
carbonyl of OA,
forming citroyl-CoA
Fig 16-9
Citrate synthase
mechanism
3. hydrolysis of thioester releases citrate and
CoA
Fig 16-9
Reaction 2: Isomerization
A symmetric molecule that acts
asymmetric!
Chemically, these
carbons are
identical!
A symmetric molecule that acts
asymmetric!
Chemically, these
carbons are
identical!
So both these
products
should be
formed
A symmetric molecule that acts
asymmetric!
Chemically, these
carbons are
identical!
So both these
products
should be
formed
Prochiral
molecules can act
chiral!
Reaction 3: Oxidative Decarboxylation
Reaction 4: Oxidative Decarboxylation
Reaction 5: Substrate-level phosphorylation
Succinyl-CoA synthetase reaction
Hydrolysis of CoA-SH
drives phosphorylation of
succinate within the
enzyme-substrate complex
Succinate transfers its
phosphate group to the
enzyme
Enzyme phosphorylates
GDP
Reactions 6, 7, and 8
Oxidation
Hydration
Oxidation
Summary of TCA
Fig 16-14
Regulation
Irreversible reactions
are regulated
In general, energy
charge is key:
AMP/NAD+ activate
ATP/NADH inhibit
Product inhibition
Fig 16-19
Anaplerotic Reactions
Fig 16-16
Anaplerotic Reactions
Example: pyruvate carboxylase, which uses a
biotin (vitamin B7) cofactor to carry CO2
Case Study
Daniel plans to enter the Mr. Colby
contest and wants to get jacked.
He has begun adding raw eggs to
his diet and is up to a dozen a day.
Unfortunately, he has been
experiencing lactic acidosis during
his weight training and
hypoglycemia between meals.
What’s up with Daniel?
Case Study
KD ≈ 10-15 M
Pyruvate carboxylase
Carboxyl group of
bicarbonate is
“activated” by
phosphorylation
Pyruvate carboxylase
“Activated” CO2 is
passed to biotin
cofactor with loss
of Pi
Pyruvate carboxylase
CO2 is passed to second active site for rxn with pyruvate
Pyruvate carboxylase
CO2 is released for reaction with pyruvate
to form OA.
Glyoxylate
cycle
Plants and some
microorganisms can
convert acetyl-CoA to
oxaloacetate for net
gain of carbon and net
synthesis of TCA
intermediates
Fig 16-22
Intersection with
TCA
Glyxoylate pathway
runs simultaneously
with TCA but in a
different compartment.
Fig 16-24
Coordinated
regulation
Isocitrate is a branch
point; its fate depends
on relative activities of
isocitrate
dehydrogenase (TCA)
and isocitrate lyase
(glyoxylate cycle).
Fig 16-25
Case Study
Vania can’t believe that she feels so lousy.
Even though it is St. Patrick’s Day
weekend and she’s been up all night
partying, she’s never felt this bad before.
Her head is pounding, and she feels tired,
weak, dizzy, and sick to her stomach. She
would drink some water, but she lost her
Nalgene bottle last week somewhere, and
the walk to the dining hall is just way too
far.
1. What is wrong with Vania?
2. What are the consequences of dehydration on metabolism?
3. What are the metabolic breakdown products of ethanol?
4. What role do these metabolic products play in the citric acid cycle?
5. What would you recommend to Vania?