Takusagawa’s Note
1
Chapter-16
Chapter 16: Citric Acid Cycle
1. CITRIC ACID CYCLE OVERVIEW (all carried out in mitochondrial matrix)
- Citric acid cycle is also called Krebs cycle or tricarboxylic acid (TCA) cycle.
- Citric acid cycle is the process that the pyruvate produced in glycolysis is further oxidized to
3CO2 to produce 4NADH, FADH2 and GTP (ATP). The NADH and FADH2 are utilized to
produce the “energy currency” ATP in oxidative phosphorylation.
O
Pyruvate
H3C C COO
CoASH + NAD+
pyruvate
dehydrogenase
CO2 + NADH
O
H3C C S CoA Acetyl-CoA
NADH + H+
COO+
NAD
C O
-
COO
HO CH
L-malate
CH2
COO7
H2O
H2O
-
COO
CoASH
COO-
cis-Aconitate
CH
COO-
CO2
COOIsocitrate
C O
3
C O
COO-
3
CH2
4
CH2
NAD
+
C O
COO-
CO2
α-Ketoglutarate
2CO2 are not come from
the original acetate.
1
CH2
H C COO
HO C H
COO-
CH2
H C COO
CoASH
COO-
H2O
2
COO
CH2
S CoA
Succinyl-CoA
NADH
+ H+
•
COOCH2
C COO
-
CH2
GDP + Pi
H2O
2
7. fumarase
8. malate
dehydrogenase
Succinate CH2
CH2
5
Citrate
4. α-ketoglutarate
dehydrogenase
5. succinyl-CoA
synthetase
6. Succinate
dehydrogenase
6
GTP
CH2
HO C COO
CH2
COO-
3. isocitrate
dehydrogenase
COO-
COO-
1
CH2
COOOxaloacetate
1. citrate
synthase
2. aconitase
Fumarate CH
HC
FAD
CoA
8
COO-
FADH2
COO-
Oxalosuccinate
NAD+
NADH
+ H+
Takusagawa’s Note
2
Chapter-16
-
Pyruvate generated from glycolysis is converted to acetyl-CoA before entering the citric acid
cycle.
- At the initial reaction, acetyl group from acetyl-CoA and oxaloacetate react to form citrate.
- 3NADH, FADH2 and GTP are generated from one acetyl-CoA oxidation.
- 2CO2 are released from the portion of oxaloacetate.
- At the final reaction, oxaloacetate is regenerated.
- Overall reaction in the citric acid cycle is:
3NAD+ + FAD + GDP + Pi + acetyl-CoA → 3NADH + FADH2 + GTP + CoA + 2CO2
From glucose:
Glucose + 2NAD+ + 2ADP + 2Pi → 2pyruvate + 2NADH + 2ATP
2pyruvate + 2NAD+ + 2CoA → 2acetyl-CoA + 2NADH + 2CO2
2acetyl-CoA + 6NAD+ + 2FAD + 2GDP + 2Pi → 6NADH + 2FADH2 + 2GTP + 2CoA + 4CO2
2GTP + 2ADP → 2ATP + 2GDP
.
Glucose + 10NAD+ + 4ADP + 4Pi + 2FAD → 10NADH + 2FADH2 + 4ATP + 6CO2
→ 30ATP + 4ATP + 4ATP = 38ATP
2. METABOLIC SOURCES OF ACETYL-COENZYME A
- Pyruvate is converted to acetyl-CoA before entering the citric acid cycle.
- The function of coenzyme A is a carrier of acetyl and other acyl group.
- Acetyl-CoA is a “high-energy” compound since it has a “high energy” S~C bond which
releases ∆G°’ = -31.5 kJ/mol by hydrolysis.
Acetyl group
O
S C CH 3
β-mercaptoethylamine residue
High energy bond
CH2
CH2
NH
C O
Adenosine-3'phosphate
CH2
CH2
NH2
NH
Pantothenic
acid residue
C O
N
N
HO C H
H3C C CH3
H2C
O
O
N
N
O
P O P O CH2
O-
O-
O
O
OH
-
-
O P O
2
Acetyl-coenzyme A (acetyl-CoA)
O
Chapter-16
3
Takusagawa’s Note
A. Pyruvate dehydrogenase is a multienzyme complex
- Acetyl-CoA is formed from pyruvate through oxidative decarboxylation by a multienzyme
complex named pyruvate dehydrogenase.
Pyruvate + CoA + NAD+ → acetyl-CoA + CO2 + NADH
- Pyruvate dehydrogenase multienzyme complex consists of:
1. Pyruvate dehydrogenase (E1)
2. Dihydrolipoyl transacetylase (E2)
3. Dihydrolipoyl dehydrogenase (E3)
-
Multienzyme complexes have catalytic advantages:
1. Rates of a series of reactions are enhanced since short diffusion distance.
2. Side reactions are minimized.
3. Reactions may be coordinately controlled.
-
The following coenzymes and prosthetic groups are required in pyruvate dehydrogenase
multienzyme complex:
- Thiamine pyrophosphate (TPP, Fig. 16-27) decarboxylase

- Flavin adenine dinucleotide (FAD, Fig. 14-28) redox
 See next page.
+
- Nicotinamide adenine dinucleotide (NAD , Fig. 12-2) redox 
- Coenzyme A (see previous page) acetyl-carrier
- Lipoamide (prosthetic group) acetyl-carrier
3
Chapter-16
4
4
Takusagawa’s Note
Chapter-16
5
Takusagawa’s Note
Acetyl-CoA formation occurs in five reactions
1. Pyruvate dehydrogenase (E1), a TPP-requiring enzyme, decarboxylates a pyruvate with the
intermediate formation of hydroxyethyl-TPP. This is the same reaction catalyzed by yeast
pyruvate decarboxylase (pyruvate → acetylaldehyde + CO2).
5
Takusagawa’s Note
6
Chapter-16
2. Hydroxyethyl group is transferred to the next enzyme (dihydrolipoyl transacetylase (E2)).
The hydroxyethyl group carbanion attacks the lipoamide disulfide of E2 and eliminate the
TPP to form acetyl-dihydrolipoamide-E2.
+
H
B:
3. The acetyl group is transferred to CoA to yield acetyl-CoA and dihydrolipoamide-E2.
4. Dihydrolipoamide-E2 is oxidized by dihydrolipoyl dehydrogenase (E3)
+
+
5. Reduced E3 is reoxidized by NAD+. Initially the enzyme’s sulfhydryl groups (-SH) are
reoxidized by the enzyme-bound FAD, yielding FADH2, then FADH2 is reoxidized by
NAD+, producing NADH.
6
Takusagawa’s Note
7
Chapter-16
The lipoyllysyl arm transfers intermediates between enzyme subunits
- Lipoyllysyl arm is quite long (14 Å).
14 Å
O
O
HN
N
H
S
S
Lipollysyl arm
(fully extended)
Arsenic compounds are poisonous because they covalently bind to the vicinal (adjacent) dithiols
of dihydrolipoamide.
7
Chapter-16
8
Takusagawa’s Note
B. Control of pyruvate dehydrogenase
Product inhibition
- When the relative concentrations of NADH and acetyl-CoA are high, the reversible reactions
catalyzed by E2 and E3 are driven backwards. Therefore formation of acetyl-CoA is
inhibited.
- Thus the E2 and E3 activities are controlled by product inhibition (acetyl-CoA for E2 and
NADH for E3).
Covalent modification (Eukaryotic complex only)
- E1 is regulated by phosphorylation/dephosphorylation. When the Ser of E1 is
phosphorylated, the enzyme is inactivated.
Insulin activates
Activators of phosphatase: Mg2+, Ca2+
Activators of kinase: Acetyl-CoA, NADH
Inhibitors of kinase: Pyruvate, ADP, Ca2+, high Mg2+, K+
-
Remember: Insulin inhibits phosphorylation and activates dephosphorylation in order to
reduce the [glucose] in blood at the starting point of glycolysis.
Now, insulin also works to reduce the end product of glycolysis, i.e., activates
dephosphorylation of E1 to convert pyruvate to acetyl-CoA.
Acetyl-CoA is not only the fuel of citric acid cycle, but also the precursor of fatty acids.
8
Takusagawa’s Note
9
Chapter-16
3. Enzymes of the citric acid cycle
A. Citrate synthase
- catalyzes the condensation of acetyl-CoA and oxaloacetate.
O
O
+
H3C C
S CoA
Acetyl-CoA
H2O
-
O C COO
H2C
CoA-SH
-
H2C C O
-
HO C COO
-
H2C
COO
-
COO
Citrate
Oxaloacetate
∆G°’ = -32.2 kJ/mol
Reaction mechanism
1. Asp-375 acts as a base to remove a proton from the methyl group of acetyl-CoA. His-274
acts as an acid to protonate the enolate oxygen.
2. Citryl-CoA is formed in a second concerted acid-base catalysis. His-320 acts as acid, and
His-274 acts as base.
3. Citryl-CoA is hydrolyzed to citrate and CoA. This hydrolysis (∆G°’ = -31.5 kJ/mol) pulls
the reaction 1 and 2.
1
CoASH
3
9
H2O
Takusagawa’s Note
10
Chapter-16
B. Aconitase
- catalyzes the reversible isomerization of citrate and isocitrate.
-
H2C COO
Citrate
H 2O
-
H2C COO
-
H C COO
C COO
-
C
COO
H
-
H2C COO
-
HO C COO
H C
H 2O
-
COO
-
HO C
-
COO
H
H
cis-Aconitate
Isocitrate
∆G°’ = 13.3 kJ/mol
Reaction mechanism
- Aconitase contains a covalently bound [4Fe-4S] iron-sulfur cluster, which is required for
catalytic activity. The Fea is coordinated by the hydroxyl and the central carboxyl groups.
1. His-101 acts as an acid to eliminate -OH as water, and Ser-642 acts as a base to eliminate a
proton from C2.
2. cis-Aconitate intermediate is flipped by 180° so that C2 and C3 are exchanged their
positions.
3. The reversed acid-base catalysis is taken place to yield (2R,3S)-isocitrate.
10
Takusagawa’s Note
11
Chapter-16
Fluorocitrate inhibits aconitase
- Fluoroacetate, one of the most toxic small molecules (LD50 = 0.2 mg/kg), is converted to
(2R,3R)-fluorocitrate, which specifically inhibits aconitase since Ser-642 cannot remove the
proton at C2.
Less acidic
Less toxic
Very toxic
C. NAD+-dependent isocitrate dehydrogenase
- catalyzes the oxidative decarboxylation of isocitrate to α-ketoglutarate to produce CO2 and
NADH.
-
H2C COO
NAD
+
NADH + H
+
-
H2C COO
-
HO C
C
COO
CO2
-
COO
O
H
α-Ketoglutarate
Isocitrate
-
+
CH2
H C COO
∆G°’ = -20.9 kJ/mol
There are two isozymes in mammalian cells.
1. NAD+-dependent form is in mitochondria and requires an Mn2+ or Mg2+.
2. NADP+-dependent form is in both cytosol and mitochondria.
D. α-Ketoglutarate dehydrogenase
- catalyzes the oxidative decarboxylation of an α-keto acid, releasing CO2, forming succinylCoA and reducing NAD+ to NADH.
+
H2C COO CoA-SH NAD
NADH
CH2
C
-
H2C COO
CH2
-
COO
C
O
+
S-CoA
O
α-Ketoglutarate
Succinyl-CoA
∆G°’ = -33.5 kJ/mol
11
CO2
-
-
Takusagawa’s Note
12
Chapter-16
α-Ketoglutarate dehydrogenase is a multienzyme complex that consists of α-ketoglutarate
dehydrogenase (E1), dihydrolipoyl transsuccinylase (E2), and dihydrolipoyl dehydrogenase
(E3).
The overall reaction closely resembles that are catalyzed by the pyruvate dehydrogenase
multienzyme complex, i.e.,
1. Decarboxylation -----------------------E1
 E2
2. Succinyl group transfer

3. Succinyl-CoA formation.
 E3
4. Oxidation of E2.
+

5. Reduction of NAD .
E. Succinyl-CoA synthetase
- hydrolyzes the “high-energy” compound succinyl-CoA with the coupled synthesis of a “highenergy” nucleosidetriphosphate (GTP).
H2C
COO-
CH2
GDP + Pi
GTP
-
COO
CoA-SH
CH2
C S-CoA
CH2
-
O
COO
Succinate
Succinyl-CoA
∆G°’ = -2.9 kJ/mol
-
The succinyl~CoA thioester bond energy is preserved through the formation of a series of
“high-energy” phosphate (~Pi). The succinate formation is as follows:
Pi
Succinyl~CoA
1
GDP~Pi (GTP)
CoASH
Succinyl~Pi
2
3
E-His~Pi
E-His
GDP
-
Succinate
GTP is converted to ATP by nucleoside diphosphate kinase.
∆G°’ = 0 kJ/mol
GTP + ADP ↔ GDP + ATP
12
E-His
Takusagawa’s Note
13
Chapter-16
F. Succinate dehydrogenase
- catalyzes stereospecific dehydrogenation of succinate to fumarate and produces FADH2.
-
-
FADH2
FAD
COO
COO
C H
H C H
H C
H C H
-
-
COO
Fumarate
COO
Succinate
-
-
∆G°’ = 0 kJ/mol
The FAD in succinate dehydrogenase is covalently bound to the enzyme. Thus, FADH2
cannot be oxidized as a cofactor. FADH2 is oxidized by the electron transport chain reaction
(See Chapter-17).
For the reason, succinate dehydrogenase is the only membrane-bound citric acid cycle
enzyme. The others are dissolved in the mitochondrial matrix.
The enzyme is strongly inhibited by malonate (structural analog of succinate).
-
COO
-
COO
H C H
H C H
H C H
-
COO
-
COO
Malonate
Succinate
+
In general, FAD and NAD are involved in different oxidation-reduction reactions.
- For example,
FAD
FADH2
H C H
C H
H C H
H C
Alkane
Alkene
-
The oxidation of alkane to alkene produces ∆G°’ ≈ -42 kJ/mol, whereas the FAD to FADH2
reduction requires ~42 kJ/mol (FAD + 2H+ + 2e- → FADH2, ∆E°’ = -0.219 V = (∆G°’ = 42
kJ/mol)). Thus, the oxidation of alkane to alkene is just enough to reduce FAD to FADH2,
but not enough to reduce NAD+ to NADH + H+ (∆G°’ = 61 kJ/mol).
-
The oxidation of alcohol to aldehyde (or ketone) produces more energy than the above case.
NAD
H C H
H C OH
Alcohol
-
+
+
NADH + H
H C H
C O
Aldehyde or ketone
Alcohol → aldehyde (or ketone)
∆G°’ ≈ -61 kJ/mol
+
+
+
NAD + 2H + 2e → NADH + H
∆E°’ = -0.315 V (∆G°’ = 61 kJ/mol)
The oxidation of alcohol to aldehyde is sufficient to reduce NAD+ to NADH2.
13
Takusagawa’s Note
14
Chapter-16
G. Fumarase
- catalyzes the hydration of fumarate’s double bond to form L-malate.
-
COO
H2O
-
COO
C H
HO C H
H C
H C H
-
-
COO
COO
L-Malate
Fumarate
∆G°’ = -3.8 kJ/mol
H. Malate dehydrogenase
- catalyzes the oxidation of L-malate’s hydroxyl group to ketone in a NAD+-dependent
reaction, regenerating oxaloacetate.
-
COO
NAD
+
NADH + H
-
COO
HO C H
C O
H C H
H C H
-
COO
-
COO
L-Malate
-
+
Oxaloacetate
∆G°’ = 29.7 kJ/mol
This reaction is relatively high endergonic reaction (∆G > 0).
However, the following two reasons, this reaction occurs.
1. [Oxaloacetate] is very low at equilibrium, i.e., RTlnKeq becomes negative where
[oxaloacetate][ NADH] < 1, i.e., lnK < 0.
Keq =
eq
[ malate] NAD +
[
]
2. The subsequent reaction (formation of citrate from oxaloacetate and acetyl-CoA) that is
highly exergonic pulls this reaction since the hydrolysis of “high-energy” thioester bond
of acetyl-CoA releases ∆G°’ = -31.5 kJ/mol energy. This is a reason why acetyl-CoA
enters the citric acid cycle.
14
Chapter-16
15
Takusagawa’s Note
I. Integration of the citric acid cycle
- Citric acid cycle results in the following chemical transformations.
1. One acetyl group (-COCH3) → 2CO2 (4-electron pair process).
O
-
+
CoA S C CH3 + 3H2O
2CO2 + CoA SH + 8H + 8e
2. Reduction of three NAD+ to three NADH (3-electron pairs process) and equivalent to
9ATP generation, i.e., 3NAD+ + 6H+ + 6e- → 3NADH + 3H+
3. Reduction of one FAD to FADH2 (1-electron pairs process) and equivalent to 2ATP
generation, i.e., FAD + 2H+ + 2e- → FADH2
4. Generation of one GTP (ATP).
Four electron pairs generated by one acetyl group oxidation are carried by 3NADH and
FADH2 to the oxidative phosphorylation pathway to generate 11ATP.
Thus, citric acid cycle generates 12ATP from one acetyl group and sends 4-electron pairs (8
electrons) to electron-transport chain, where they reduce two molecules of O2 to 4H2O, i.e.,
2O2 + 8H+ + 8e- → 4H2O.
15
Chapter-16
16
Takusagawa’s Note
4. REGULATION OF THE CITRIC ACID CYCLE
- Citrate synthase, isocitrate dehydrogenase and α-ketoglutarate dehydrogenase are the
citric acid cycle’s rate-controlling enzymes because those ∆G are negative.
- The citric acid cycle reactions are carried out in mitochondria, but most of the cycle’s
metabolites are present in both mitochondria and cytosol. Therefore it is difficult to establish
the rate-determining steps.
- However, three of the eight steps have significantly negative physiological free energy
changes. The enzymes involved in those steps are likely to function far from equilibrium
under physiological conditions.
Standard (∆G°’) and physiological (∆G) free energy changes
Reaction
Enzyme
∆G°’ (kJ/mol)
1
Citrate synthase
-32.2
2
Aconitase
+13.3
3
Isocitrate dehydrogenase
-20.9
-33.5
4
α-Ketoglutarate
dehydrogenase
5
Succinyl-CoA synthetase
-2.9
6
Succinate dehydrogenase
0.0
7
Fumarase
-3.8
8
Malate dehydrogenase
+29.7
-
∆G (kJ/mol)
Negative
~0
Negative
Negative
~0
~0
~0
~0
Unlike enzymes in glycolysis and glycogen metabolism, the citric acid cycle is largely
regulated by
1. substrate availability (rate of diffusion of substrate into mitochondria)
2. product inhibition. (NADH, ATP, citrate)
3. competitive feedback inhibition by intermediates further along the cycle.
16
Chapter-16
17
Takusagawa’s Note
Products and NADH are involved in feedback inhibition.
- ADP and ATP are allosteric regulators of isocitrate dehydrogenase. High [ADP] activates
the enzyme whereas high [ATP] inhibits the enzyme.
- Ca2+ activates pyruvate dehydrogenase, isocitrate dehydrogenase and α-ketoglutarate
dehydrogenase.
17
Takusagawa’s Note
18
Chapter-16
5. THE AMPHIBOLIC NATURE OF THE CITRIC ACID CYCLE
- In the muscle, the citric acid cycle works mainly degradation of acetyl-CoA to produce
bioenergies (ATP).
- In the liver, the citric acid cycle is amphibolic.
Note: Amphibolic = both anabolic and catabolic processes.
Anabolism:
Amino acids
Sugars
Fatty acids, etc.
Catabolism:
Energy yielding
materials, such
as proteins
⇒
Proteins
Nucleic acids
Lipids, etc.
⇒
Energy poor end
products, such as
CO2, NH3, H2O
Intermediates of citric acid cycle are also various precursors
18
Chapter-16
-
19
Takusagawa’s Note
Intermediates of citric acid cycle are also precursors of:
- Glucose biosynthesis.
- Lipid biosynthesis including fatty acid and cholesterol.
Note: Lipid biosynthesis is taken place in cytosol, but the mitochondrial acetyl-CoA
(processor) cannot be transported across the inner mitochondrial membrane. Thus, acetylCoA is converted to citrate by ATP-citrate lyase since citrate can cross the membrane.
Why citrate synthase is not used? --- Because no ATP is produced.
ADP + Pi + oxaloacetate + acetyl-CoA ↔ ATP + citrate + CoA
- Amino acid biosynthesis
α-ketoglutarate + NAD(P)H + NH4+ ↔ Glu + NAD(P)+ + H2O
α-ketoglutarate + Ala ↔ Glu + pyruvate
Oxaloacetate + Ala ↔ Asp + pyruvate
- Porphyrin biosynthesis
- utilizes succinyl-CoA as a starting material.
When the citric acid cycle intermediates are transported too much as precursors, the
concentration of oxaloacetate is very low. In this case, it is necessary to replenish citric acid
cycle intermediates. The main reaction is:
- Pyruvate + CO2 + ATP + H2O ↔ oxaloacetate + ADP + Pi
The citric acid cycle is truly at the center of metabolism
- Reduced products: NADH and FADH2 are reoxidized to produce ATP.
- The citric acid intermediates are utilized in the biosynthesis of many vital cellular constituents.
19