Connections
16 Lecture | The Citric Acid Cycle
2009 W. H. Freeman and Company
Glucose Song
Only a Small Amount of Energy
Available in Glucose is Captured in
Glycolysis
Glycolysis
G = -146 kJ/mol
2
GLUCOSE
Full oxidation (+ 6 O2)
G = -2,840 kJ/mol
6 CO2 + 6 H2O
Glycolysis ends with Pyruvate
How do we get from Pyruvate into the
Citric Acid Cycle.
Convert it to Acetyl CoA?
Why?
In Eukaryotes, Citric Acid Cycle
Occurs in Mitochondria
Glycolysis occurs in the cytoplasm
Citric acid cycle occurs in the mitochondrial matrix
Oxidative phosphorylation occurs in the inner membrane
Except succinate dehydrogenase, which is located in the inner membrane
Conversion of Pyruvate to Acetyl-CoA
Oxidative decarboxylation of pyruvate
Forms 2 C substrate
acetyl-CoA can enter the citric acid cycle and
yield energy
acetyl-CoA can be used to synthesize storage
lipids
Pyruvate Dehydrogenase (PDH)
24 60 copies of
E2 depending on
Species.
5 cofactors or
Prosthetic groups
5 times Larger than
Ribosome
Why such a complex reaction for
a simple substrate?
Pyruvate Dehydrogenase
Complex (PDC)
PDC is a large (Mr = 7.8 106 Da) multienzyme complex
- pyruvate dehydrogenase (E1)
- dihydrolipoyl transacetylase (E2)
- dihydrolipoyl dehydrogenase (E3)
short distance between catalytic sites allows channeling
of substrates from one catalytic site to another
channeling minimizes side reactions
activity of the complex is subject to regulation (ATP)
5-Step Reaction Mechanim
for Pyruvate Decarboxylation
Step 1: Decarboxylation of pyruvate to an aldehyde
Step 2: Oxidation of aldehyde to a carboxylic acid
Step 3: Formation of acetyl CoA
Step 4: Reoxidation of the lipoamide cofactor
Step 5: Regeneration of the oxidized FAD cofactor
Chemistry of Oxidative
Decarboxylation of Pyruvate
NAD+ and CoA-SH are co-substrates
TPP, lipoyllysine and FAD are prosthetic groups
Pyruvate decarboxylase
In ethanol production
Structure of Lipoyllysine
prosthetic groups - strongly bound to protein.
lipoic acid is covalently linked to the enzyme
via a lysine residue.
Structure of CoA
coenzymes or co-substrates are not a permanent
part of the enzymes structure.
they associate, fulfill a function, and dissociate
The function of CoA is to accept and carry acetyl
groups
Why such a large complex?
Regulation
Channeling substrate
Preventing inadvertent oxidation of
intermediates.
The Citric Acid Cycle
Goals today:
Review TCA Cycle.
Focus on relevant enzyme mechanisms
Reduced products
Energetic products
Other interesting reasons to study TCA
Cycle.
Sequence of Events
in the Citric Acid Cycle
Step 1: C-C bond formation to make citrate
Step 2: Isomerization via dehydration, followed by
hydration
Steps 3-4: Oxidative decarboxylations to give 2
NADH
Step 5: Substrate-level phosphorylation to give GTP
Step 6: Dehydrogenation to give reduced FADH2
Step 7: Hydration
Step 8: Dehydrogenation to give NADH
The Citrate Synthase Reaction
The only cycle reaction with C-C bond
formation
Essentially irreversible Fit process
Induced in the Citrate
Synthase
Conformational change occurs
upon binding oxaloacetate
(a) Open conformation:
free enzyme does not have a binding site for acetyl CoA
(b) Closed conformation:
binding of oxaloacetate creates site for binding of acetyl
CoA
Reactive carbanion is protected in the closed conformation.
Why make an isomer of citrate?
I will present an alternate hypothesis at
the end of the lecture.
Isomerization of Citrate by
Aconitase
Citrate, a tertiary alcohol, is a poor substrate for
oxidation
Elimination of H2O from citrate gives a cis C=C
bond
Addition of H2O to cis-aconitate is stereospecific
Isocitrate, a secondary alcohol, is a good
substrate for oxidation
Oxidation of -ketoglutarate
Enzyme: -ketoglutarate dehydrogenase
complex
Similar to pyruvate dehydrogenase complex
Same coenzymes, identical mechanisms
E1 had differences
Changes allow
Different substrate
specificity
Succinate Dehydrogenase
Covalently bound FAD is reduced to FADH2
FADH2 passes electrons to coenzyme Q
Reduced coenzyme (QH2) can be used to make
ATP
Electron transfer by FeS cofactors.
Hydration of Fumarate to Malate
Fumarase is highly stereospecific
OH- adds to fumarate
then H+ adds to the carbanion
Net effect: trans addition of water
Reversible reaction
Oxidation of Malate to
Oxaloacetate
Thermodynamically unfavorable reaction
Oxidation occurs because oxaloacetate
concentration is very low as it is continuously used
to make citrate
Products from One Turn of the
Cycle
Net Effect of the Citric Acid Cycle
Acetyl-CoA + 3NAD+ + FAD + GDP + Pi + 2 H2O
2CO2 +3NADH + FADH2 + GTP + CoA + 3H+
intermediates in the cycle are not depleted
Regenerate oxaloacetate molecule with a handle
for enzyme reactions.
Only one GTP/ATP produced
1 glucose = 976 kJ/mol. ~ 65% efficient
Citric Acid Summary
Know accounting of ATP/GTP and NADH FADH2
produced.
Particular focus on Pyruvate Dehydrogenase/ketogluterate cofactors and coenzymes and detailed
mechanism of reaction substrate channeling.
Aconitase cofactors and reaction.
Role of the Citric Acid Cycle in
Anabolism
Anaerobic organisms
Anaerobic organisms possess many of
the citric acid cycle enzymes for
anabolic reactions
Anaplerotic Reactions
Anaplerosis is the act of replenishing
these reactions replenish metabolites for the cycle
four carbon intermediates are formed by
carboxylation of three-carbon precursors
Regulation of the Citric Acid
Cycle
How would you regulate a cycle?
TCA Cycle Old and Boring?
How can we make it exciting?
Bi-functional Enzymes
Aconitase
FeS cluster
Citrate to Isocitrate
FeS cluster labile
Removal of cluster
Produces IRP-1
3Fe4S cluster in intermediate in removal of cluster.
Succinate Dehydrogenase had IRE Why?
Transferrin Cycle
Ferritin Iron Release
Reducing agents
Sulfur-containing compounds
Cytosolic Isocitrate
Dehydrogenase
Uses NADP+ instead of NAD+
Why?
NADPH vs NADH
Glutathione
Glutathione Reductase
Glutathione Peroxidase
Pentose Phosphate pathway