Lec 6: TCA cycle
TCA cycle reactions, regulations, and glyoxylate shunt
Reference material
Biochemistry 4th edition, Mathews, Van Holde, Appling, Anthony‐Cahill. Pearson ISBN:978‐0‐13‐800464‐4
Lehninger Principles of Biochemistry 4th edition, David L. Nelson, Michael M. Cox. W. H. Freeman ISBN:978‐0716743392
TCA cycle
•
TCA cycle (tricarboxylic acid cycle)
= Citric acid cycle (citrate is tricarboxylic acid..)
= Krebs cycle (discovered by Hans Krebs in 1937)
•
Functions of the TCA cycle
• the central pathway for recovering energy from several metabolic fuels
• providing intermediate for several
anabolic pathways
• passing electrons to O2 in the subsequent oxidation phosphorylation processes
•
Oxidants other than O2 in various organisms
• NO3–
• SO42–
• Fe3+
Citrus fruits
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The oxidation of acetyl‐CoA to CO2 in the TCA cycle generates energy currencies
O
CoA
O
-O
OO
OH
-O
O
O
-O
O
OH
O
-O
OO
OO
O-
-O
OH
O
-O
O
O
O
-O
O
O-
O-
-O
O
O
O
-O
O
O
O-
O
-O
CoA
O
(1) Citrate synthase catalyzes C‐C bond formation between acetate and oxaloacetate
Driven mainly by thioester hydrolysis
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(1) Citrate synthase changes conformation in response to substrate binding (induced fit)
Substrates bind sequentially: oxaloacetate, then acetyl-CoA
(1) In the rate‐limiting step, acetyl‐CoA is deprotonated to form an enolate
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(1) The nucleophilic enolate attacks the carbonyl of oxaloacetate to yield citryl‐CoA
(1) Hydrolysis of the “high‐energy” thioester
citryl‐CoA makes the reaction irreversible
A second conformational change allows hydrolysis at this step
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(2) Aconitase catalyzes the stereospecific conversion of citrate to isocitrate
(2) Starting with a radio‐labeled acetyl group yields label in only one position
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(2) Aconitase can distinguish between the pro‐R and pro‐S substituents of citrate
pro‐S
pro‐R
OH is always added here
(3) Isocitrate dehydrogenase catalyzes the oxidative decarboxylation of isocitrate
ΔG'°= ‐21 kJ/mol
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(3) In the first step, isocitrate is oxidized to oxalosuccinate, an unstable intermediate
(3) In the second step, oxalosuccinate is decarboxylated
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(3) In the final step, the enolate rearranges and is protonated to form the keto product
H+
(4) α‐KG DH complex couples an oxidative decarboxylation with thioester formation
‘high‐energy’
intermediate
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(4) α‐Ketoglutarate DH complex is similar to pyruvate DH complex
Enzyme
PDH complex
α‐KGDH complex
Cofactors
E1
Pyruvate DH
α‐Ketoglutarate DH
TPP
E2
Dihydrolipoyl
transacetylase
Dihydrolipoyl
transsuccinylase
Lipoic acid,
Coenzyme A
E3
Dihydrolipoyl DH
Dihydrolipoyl DH
FAD, NAD
How do E1, E2, and E3 differ between PDH complex and a‐KG complex?
(5) Succinyl‐CoA synthetase couples thioester hydrolysis with NTP synthesis
ATP
equivalent
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(5) In the first step, the “high‐energy” thio‐
ester becomes a “high‐energy” anhydride
‘high‐energy’
intermediate
(5) In the second step, the enzyme becomes the “high‐energy” compound
‘high‐energy’
intermediate
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(5) In the third step, the phosphoryl group is transferred to form the “high‐energy” NTP
ATP
equivalent
(6) The integral membrane protein succinate DH catalyzes succinate oxidation
(tightly bound in enzyme)
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(7) Fumarase catalyses the stereospecific conversion of fumarate to L‐malate
fumarase
fumarase
(8) Malate dehydrogenase catalyzes the final step in regenerating oxaloacetate
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The slowest steps of the citric acid cycle have negative ΔG’s, and are regulated
Which enzymes should be regulated?
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Compounds reflecting energy status and energy use are regulators of the TCA cycle
• NADH
• Product inhibitor of NAD+‐using
dehydrogenases
• Inhibitor of citrate synthase
• Pathway intermediates
• Citrate and succinyl‐CoA act via
product inhibition or competitive
feedback inhibition
• Levels of substrates OAA and
acetyl‐CoA determine activity of
citrate synthase
• Adenylates
• Allosteric inhibitors (ATP) or
activators (ADP) of isocitrate DH
• Ca2+ (muscle contraction)
• Allosteric activator of the
dehydrogenases
TCA cycle intermediates are made and used in additional metabolic pathways
• Cataplerotic reactions
use cycle intermediates
to make:
•
•
•
•
Glucose
Amino acids
Lipids
Cofactors
• Anaplerotic reactions
generate cycle
intermediates from:
• Pyruvate
• Amino acids
• Odd‐chain fatty acids
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Major Anaplerotic reactions: from pyruvate or PEP
HCO3‐
OPO 32O-
O
-O
PEP carboxylase
O
OO
O
Oxaloacetate
PEP
OPO 32OO
O
CO2
GDP
GTP
OO
PEP carboxykinase
PEP
-O
O
Oxaloacetate
O
O
O-
HCO3‐ ATP
ADP
-O
OO
O
Pyruvate
O
Pyruvate carboxylase
Oxaloacetate
HCO3‐ NADPH NADP+
-O
O
O
O
-
OO
OH
O
Pyruvate
Malic enzyme
Malate
Amino acids and TCA cycle intermediates are readily inter‐
converted
Reductive
amination:
Transamination:
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Production of pyruvate increases flux through TCA cycle by increasing [substrate]
fatty acid oxidation
pyruvate
carboxylase
(with biotin)
PDHC
aminotransferase
• Action of PDH complex
increases [acetyl‐CoA] (as
does FA oxidation), but
[OAA] can limit flux
• Pyruvate carboxylase is
activated by acetyl‐CoA,
and can generate more
OAA to enhance flux
• Pyruvate can also act in
transamination rxns,
yielding α‐KG (from Glu)
or OAA (from Asp)
The oxidation of acetyl‐CoA to CO2 in the TCA cycle generates energy currencies
O
CoA
O
-O
OO
OH
-O
O
O
-O
O
OO
OH
O
-O
OO
O-
-O
O
OH
-O
O
-O
O
O-
O-
-O
O
O
O
-O
O
O
O
O
O-
O
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-O
CoA
O
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Carbon Labelling experiment
O
CoA
O
-O
OO
OH
-O
O
O
-O
O
O
OH
O
-O
OO
O-
O-
-O
OH
O
-O
O
O
O
-O
O
O-
O-
-O
O
O
O
-O
O
O
O-
O
-O
CoA
O
*note: because succinate is
symmetrical, C2 and C3 cannot be distinguished
Fate of Acetyl‐CoA in TCA cycle
How Acetyl‐CoA is incorporated into TCA cycle?
Where do the carbons of acetyl‐CoA end up?
How many times the TCA cycle has to operate to oxidize a particular labeled acetyl‐CoA to CO2?
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First round
Unlike citrate, succinate does not differentiate between the two carboxylates for subsequent reaction to fumarate and to malate…
O
-O
O
O-
-O
OO
O
For fumarase & malate dehydrogenase
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At the end of one round of TCA cycle, the labeled carbon of acetyl‐CoA end up at either C1 & C2 OR C3 & C4 of Oxaloacetate
At the end of first round
If we look at the labeled carbon at C1 & C2 of Oxaloacetate…
NOTE: one of the labeled carbon
is kicked out in the Second round…
Second round, a new acetyl‐CoA comes in
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NOTE: one of the labeled carbon
is kicked out in the Second round…
At the end of second cycle
Third Round 國立交通大學生物科技學系 蘭宜錚老師
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At the end of third round, the labeled carbon is still there
At the end of third round
The labeled carbon now on C4 of oxaloacetate… eventually gets decarboxylated at isocitrate
dehydrogenase
Fourth Cycle
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Glyoxylate shunt: A bypass in TCA to avoid CO2 loss
O
O-
In plants, bacteria, and fungi, glyoxylate shunt bypasses the decarboxylation steps in TCA cycle and conserves/utilizes carbon for gluconeogenesis…
O
CoA
O
O-
O
-O
What is the net reaction
From Acetyl‐CoA to Oxaloacetate???
OH
-O
OO
O
O
-O
O-
O
O
O
H
OO
O
-O
OO
OH
OH
O-
-O
O
-O
O
O
O
-O
OO
O
FADH2
-O
No carbon lost to CO2 from Isocitrate to succinate
OO
Two enzymes for Glyoxylate shunt
OH
O-
-O
O
-O
O
Isocitrate lyase
O
+ H2O
isocitrate
O
O
-O
O
-
O
O-
H
+
O
succinate
glyoxylate
O
O
CoA
+
O-
H
Malate synthase
O
-O
O-
O
O
Acetyl‐CoA
glyoxylate
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Carbon‐carbon bond formation!
OH
malate
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