Download 09: TCA Cycle TCA CYCLE

09: TCA Cycle
Citrate Synthase
Energy Metabolism
The TCA cycle acts as a
multistep catalyst that can
oxidize an unlimited number
of acetyl groups.
Mitochondrial localization:
concentrate reactants in small
volume to increase kinetic
Interactions.
“Metabolon”: enzyme
complexes operating in close
proximity. Kinetic advantage.
TCA CYCLE
Pyruvate
Acetyl-CoA
• Citrate
• Isocitrate
• α-ketoglutarate
•
•
•
•
•
Succinyl-CoA
Succinate
Fumarate
Malate
Oxaloacetate
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1. Synthesis of ACOA
(acetyl Coenzyme-A)
• Pyruvate + CoA + NAD+  acetyl-CoA + CO2 + NADH
• ΔGo = -47.9 kJ mol-1 ; oxidative decarboxyation
• Pyruvate dehydrogenase: multienzyme complex
• E1: pyruvate dehydrogenase
• 60 proteins
• E2: dihydrolipoyl transacetylase
• 12 proteins
• E3: dihydrolipoyl dehydrogenase
• 60 proteins
• Kinases & phosphatases
PD Regulation
Model Structures
CO2
adenine
-S-
C2
pyruvate
acetyl-CoA
acetaldehyde
group
> model page <
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2. Formation of Citrate
• ACOA + OAA + H20  CoA-SH + Citrate
• ΔGo’ = -31.5 kJ mol-1; enol condensation
CH2=C-O-
CITRATE SYNTHASE
CS Active Site
HIS320
ARG375
HIS274
CS Catalytic Activity
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Model Structures
C1
+
ACOA
C6
C4
C2
OAA
Citrate
2. Conversion to Isocitrate
• Citrate  Isocitrate
• ΔGo’ = +5 kJ mol-1; isomerization
Aconitase
Aconitase
• Contains 4Fe-4S complex in active site
• Electron transfer coordinated by HIS residues
H100
C420
FeS
citrate
C423
C357
H166
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Aconitase Rx
3. Formation of α-Ketoglutarate
• IC + NAD+  aKG + NADH + CO2
• ΔGo’ = -21 kJ mol-1
• Oxidative decarboxylation
Isocitrate Dehydrogenase
isocitrate
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IDH Mechanism
hydrogenation
redox
decarboxylation
•
•
•
•
Reduction of NAD+
Decarboxylation of C4 carbonyl
Hydrogenation of C3-C5 double bond
Mn2+ does not undergo redox change
Model Structures
• C4 of isocitrate is removed as CO2
leaving a 5 carbon sugar, αKG
CO2
C1
C5
isocitrate
aKG
4. Formation of Succinyl-CoA
• αKG + NAD+ + CoA-SH

Succinyl-CoA + NADH + CO2
ΔG o’ = -33 kJ mol-1
Oxidative decarboxylation
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Model Structure
C1
The C atom in position #2 of pyruvate is in position
C1 of succinyl-CoA.
5. Formation of Succinate
• Succinyl-CoA + GDP + Pi  Succinate + GTP + CoA-SH
• ΔGo’ = -2.1 kJ mol-1
• Delicate energy balance:
– Cleavage of the sulfhydryl bond in S-CoA: ΔGo’ = -32.6 kJ mol-1
– Formation of the phospho ester bond in GTP: ΔG o’ = +30.5 kJ mol-1
Succinyl-CoA Synthetase
E*PO32- intermediate
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SCS Step 1
• Substrate level phosphorylation of succinate
• Energy of the sulfhydryl bond conserved in
the phospho ester bond.
SCS Step 2
• PO3 transferred to the enzyme
• Energy of phospho ester bond maintained
unique intermediate
SCS Step 3
• PO3 transferred to GDP
• Energy of phospho ester bond maintained
3 substrate-level phosphorylation events with only a small
net change in the free energy of the system (-2 kJ mol-1)
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Succinate
Symmetrical dicarboxylic acid; carbon atom positions
C1 and C4 are not distinguishable because the
following reaction (SDH) is an equilibrium reaction that
is not stereospecific.
6. Formation of Fumarate
Succinate
Dehydrogenase
• Suc + FAD  Fum + FADH2
• ΔGo’ = +6 kJ mol-1
• Simple Redox coupling rex
• FAD  FADH2
– E’ = ~0.0 V
Succinate Dehydrogenase
Bacterial SDH/FOx
heme
FeS
FAD
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FAD is covalently linked to E
FAD
FAD is an ETS component
• Mitochondrial Electron Transport System
• SDH is the only TCA enzyme that is
membrane bound; the others are all
diffusive within the matrix (more or less)
• FADH2 needs to be re-oxidized before SDH
can function in another cycle.
• FADH2 transports electrons to the ETS
within the mitochondrial membrane.
7. Formation of Malate
• Fumarate + H2O  Malate
• ΔGo’ = -3.4 kJ mol-1
• Hydration of alkene
• L-Malate: enantiomer
Fumarase
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FUMARASE
H+
OHfumarate
malate
mechanism for sequential addition
8. Formation of OAA
• MAL + NAD+  OAA + NADH
• ΔGo’ = +29.7 kJ mol-1
• Hydroxyl group oxidized
Malate Dehydrogenase
NAD+
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Model Structures
C1
C1
C4
C6
C4
Citrate
C1 ~ pyruvate C2
C4 ~ OAA C1
C6 ~ OAA C4
OAA
OAA formation
• Same reaction mechanism as:
– Lactate dehydrogenase
– Alcohol dehydrogenase
– High conservation in NAD binding sites
• Where does the energy come from ?
– ΔGo’ = +29.7 kJ mol-1
– No input of energy to drive reaction . . .
– Concentration of OAA is very small
– Large ΔG of next reaction (CS);
• ΔG o’ = -31.5 kJ mol-1 ; SH bond energy is not conserved
1. Synthesis of ACOA
(acetyl Coenzyme-A)
• Pyruvate + CoA + NAD+  acetyl-CoA + CO2 + NADH
• ΔGo = -47.9 kJ mol-1 ; oxidative decarboxyation
• Pyruvate dehydrogenase: multienzyme complex
• E1: pyruvate dehydrogenase
• 60 proteins
• E2: dihydrolipoyl transacetylase
• 12 proteins
• E3: dihydrolipoyl dehydrogenase
• 60 proteins
• Kinases & phosphatases
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Carboxylic Acid Energy Metabolism
• Why does decarboxylation result in such
large releases of chemical energy for
both chemical work (CO2 cleavage) and
redox cycling (reduction of NAD+) ???
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