CITRIC ACID

CYCLE
Floro B. Madarcos, MD
Dept. of Biochemistry
UERMMMCI
 CITRIC ACID CYCLE
Glucose
CO2 Biological
Glycolysis + Oxidation
H2O

Anaerobic Pyruvate Acetyl CoA

& Aerobic
Cytoplasmic Pyruvate &
Lactate Mitochondrial
CITRIC ACID CYCLE
I. Other Names
n Krebs Cycle
n Tricarboxylic Acid

Cycle
CITRIC ACID CYCLE
II. Site
Mitochondrion (matrix)
CITRIC ACID CYCLE
III. Major Function
The final common pathway for oxidation
of carbohydrates, lipids and proteins
via the formation of a common
metabolite Acetyl CoA.
ACETYL CoA AS A COMMON METABOLITE OF OXIDATION OF FUEL NUTRIENTS
Glucose Fatty acids Ketone
β-oxidation bodies
Pyruvate

Acetate ACETYL CoA (C2) Amino acids

Citrate synthase

(C4) OXALOACETATE
CITRATE (C6)
Malate
NADH + H+ H 2O
dehydrogenase Aconitase
NAD
(C4) MALATE ISOCITRATE (C6)
NAD
Fumarase Isocitrate
H 2O TCA NADH + H+ dehydrogenase

(C4) FUMARATE
CYCLE CO2
α - KETOGLUTARATE (C5)
FADH2 NAD
Succinate
α - ketoglutarate
dehydrogenase NADH + H+
FAD dehydrogenase
CO2
(ATP) complex
(C4) SUCCINATE
GTP ADP + Pi SUCCINYL-CoA (C4)
Mg+2
CoA Succinyl CoA thiokinase
CITRIC ACID CYCLE

IV. End Products

CO2
3 NADH/FADH2
ATP
 V. PATHWAYS OF CITRIC
ACID CYCLE
Oxidative Decarboxylation of Pyruvate
Cytosol

Mitochondria
Pyruvate Dehydrogenase Complex
Pyruvate
TPP Pyruvate dehydrogenase (E1)
translocase
S~CoA
Lipoate Dihydrolipoyl transacetylase(E2)
C1OOH FAD Dihydrolipoyl dehydrogenase(E3)
| |
Pyruvate C2=O C1=O
(C3) |
H+|
C3H3 C2H3
CO2
Pyruvate NAD+ NADH
Glycolysis CoASH Acetyl CoA
(C3)
ΔG0 = - 8.0 kcal/mole(C2; monocarboxylic)
E1-TPP E2-lipoate E3-FAD
def
Congenital lactic acidosis
 CATALYTIC MECHANISM OF PYRUVATE
DEHYDROGENASE COMPLEX
H
II -
H3C - COO
H 3C R1 H 3C R1
Pyruvate
(1)
+ +
R-N S R-N S
C Pyruvate C
dehydrogenase
H H3C – CH - OH
TPP
Hydroxyethylthiamine
H+ CO2 pyrophosphate
(HETPP)
1. FIRST STEP
a. Pyruvate reacts with TPP, the prosthetic group of E1 via pyruvate decar-
boxylase.
b. CO2 is released and hydroxyethylthiamine pyrophosphate (HETPP)
intermediate is formed.
 CATALYTIC MECHANISM OF PYRUVATE
DEHYDROGENASE COMPLEX
H3C – CH - OH
H 3C R1 H 3C R1 H 3C R1
(2)
+ +
R-N S R-N S R-N S
C C C
+
H3C – CH - OH H 3C – C = O
TPP Carbanion
Hydroxyethylthiamine Acetyl-TPP
pyrophosphate H+ O +
(HETPP) II
+ H 3C - C
+
HS SH
S E2 E2
S HS S E2

Lipoamide Dihydrolipoamide Acetyllipoamide

 CATALTIC MECHANISM OF PYRUVATE
DEHYDROGENASE COMPLEX
O
II
H 3C - C O
(3) SH + HS - CoA HS II
E2 E2 + H3C – C – S - CoA
S HS

Acetyllipoamide Dihydro- Acetyl CoA

lipoamide

(4) HS S
E2 E2
HS + E3 - FAD
S + E3 – FADH2

Dihydrolipoamide Lipoamide

(5) E3-FADH2 + NAD+ E3-FAD + NADH + H+

V. PATHWAYS OF CITRIC ACID CYCLE
1. Synthesis of Citrate from Acetyl CoA and Oxaloacetate
C1OO-
| carbonyl carbon of
C2= O oxaloacetate
| -
methyl carbon C3H2 C 1OO
of acetyl CoA | |
C4OO- C2H2
Oxaloacetate (C4) H2O HS-CoA |
S CoA~ (dicarboxylic acid) HO – C3 – C4OO-
| |
C1 = O C5H2
| (rate-limiting)
|
C2H3 Citrate synthase C6OO-
Acetyl CoA (C2) Citrate (C6)
(monocarboxylic acid) ΔG0 = - 7.7 kcal (tricarboxylic acid)
 V. PATHWAYS OF CITRIC
ACID CYCLE
2. Isomerization of Citrate to Isocitrate
Fluorocitrate
C1OO- -
C1OO- Aconitase C1OO-
| | (or aconitase |
C2H2 Aconitase hydratase)
(or aconitase C2H2 C2H2
| hydratase) H2O| |
HO – C3 – C4OO- C3 – C4OO- H – C3 – C4OO-
| || |
C5H2 C5H HO – C5 – H Carboxyl
| | | group of
H2O
C6OO- C6OO - C6OO- isocitrate
Citrate (C6) Cis-Aconitate
(tricarboxylic acid)
Isocitrate (C6)
ΔG0 = + 1.5 kcal (tricarboxylic acid)
 V. PATHWAYS OF CITRIC
ACID CYCLE
3. Oxidation and Decarboxylation of Isocitrate
Key or major β-carboxylate
regulatory of oxalosuccinate C1OO-
C1OO- C1OO-
enzyme |
| |
C2H2
C2H2 C2H2
|
| |
Isocitrate CO2 C3H2
H – C3 – C4OO- H – C3 – C4OO-
dehydrogenase |
| |
C4 = O
HO – C5 – H C5 = O
|
| Mg+2 |
H+ C5OO-
C6OO- Mn+2 C6OO-
α-keto-
Isocitrate (C6) NAD+ NADH Oxalosuccinate (C6)
+ H+ (unstable keto acid) glutarate (C5)
(alcohol) (ketone)
ΔG0 = - 5.3 kcal
V. PATHWAYS OF CITRIC ACID CYCLE
4. Oxidative Decarboxylation of α-ketoglutarate
α-ketoglutarate dehydrogenase complex: α-ketoglutarate
dehydrogenase, Dihydrolipoamide succinyl transferase,
Dihydrolipoamide dehydrogenase -

Coenzymes:
C1OOH 1.  TPP C1OO- Arsenite
| 2.  Lipoic Acid |
C2H2 3.  FAD C2H2
4.  NAD+
| 5.  CoA (CoASH) |
C3H2 C3H2
| |
C4 = O C4 = O
| CoASH CO2 |
C5OO-
NAD+
NADH S ~ CoA
+ H+
α-Ketoglutarate (C5) Succinyl CoA (C4)
ΔG0 = - 8.0 kcal
 V. PATHWAYS OF CITRIC
ACID CYCLE
5. Cleavage of Succinyl CoA
C1OO-
Succinate thiokinase
|
C2H2 or C1OO-
Succinyl CoA synthetase |
|
C2H2
C3H2
|
|
C3H2
C4 = O
Mg+2 |
|
C4OO-
S ~ CoA
HS-CoA
GDP GTP
+ (ATP)
Succinate (C4)
Succinyl CoA (C4) Pi (dicarboxylic acid)
ΔG0 = - 0.7 kcal
 COO- CATALYTIC MECHANISM OF
I
CH2 SUCCINATE THIOKINASE
I 1. Phosphate displaces CoA from
CH2 Succinyl from a bound succinyl CoA
I CoA molecule, forming the mixed
anhydride succinyl PO4 as an
C=O
O- COO- intermediate.
I
I 2. The phosphoryl group is then
I S - CoA NH transferred from succinyl PO4 to a
-O – P = O :N CH 2
histidine residue of the enzyme
I I
to form a relatively stable covalent
OH His CH2 phosphoenzyme intermediate.
HS- HS-CoA I 3. Succinate is released, and the
CoA C phosphoenzyme intermediate
O transfers its phosphoryl group to
O
Succinate GDP, forming GTP.
COO-
I H+
CH2 + H+
Succinyl +
I
phosphate GDP
CH2 GTP
intermediate
I H+ O
C=O II
I -O – P
N NH :N NH
O I
I :N NH -O
-O – P = O His His
GDP
I His +
-O H+ GTP
 V. PATHWAYS OF CITRIC ACID CYCLE
6. Oxidation of Succinate to Fumarate
COO-
I
CH2
I
COO-
Malonate
- (dicarboxylic acid)
C1OO-
Succinate dehydrogenase C1OO-
|
or |
C2H2
Complex II H – C2
|
C3H2 ||
| C3 – H
C4OO- |
C4OO-
Succinate (C4) FAD FADH2
Fumarate (C4)
(dicarboxylic acid) (dicarboxylic acid)
ΔG0 = 0 kcal
 V. PATHWAYS OF CITRIC
ACID CYCLE
7. Hydration of Fumarate

C1OO- H2O C1OO-

Fumarase
| |
(or fumarate hydratase
H – C2 HO – C2 – H
|| |
C3– H H – C3 – H
| H2O |
C4OO- C4OO-
ΔG0 = 0 kcal L-Malate (C4)
Fumarate (C4)
(alkene) (alcohol)
 V. PATHWAYS OF CITRIC
ACID CYCLE
8. Oxidation of Malate to Oxaloacetate
NADH
NAD+ + H+

C1OO- C1OO-
| |
HO – C2 – H Malate dehydrogenase C2 = O
| |
C3 – H2 C3 – H2
| |
NADH
C4OO- NAD+ + H+ C4OO-

L- Malate (C4) Oxaloacetate (C4)

(alcohol) ΔG0 = + 7.1 kcal (ketone)
VI. ENERGETICS OF THE TCA
ACETYL-CoA
- 7.7 kcal

OXALOACETATE CITRATE
+7.1 kcal NAD +1.5 kcal

NADH+ + H+
MALATE ISOCITRATE
NAD
0 kcal
NADH+ + H+ - 5.3 kcal
ΔG0= -13.1 kcal CO2
FUMARATE
α - KETOGLUTARATE
FADH2 NAD

0 kcal FAD NADH+ + H+

- 8 kcal
CO2
GTP
SUCCINATE
(ATP) ADP + Pi SUCCINYL-CoA
Mg+
CoA
- 0.7 kcal
 PYRUVATE (C3)
VII. SUMMARY OF NAD KREBS CYCLE
TPP
Pyruvate dehydrogenase complex NADH + H+

ACETYL-CoA CO2
(C2)
Citrate synthase Fluorocitrate

(C4) OXALOACETATE
H 2O CITRATE (C6)
Malate -
NADH + H+
dehydrogenase Aconitase
NAD
(C4) MALATE ISOCITRATE (C6)
NAD
Fumarase TPP Isocitrate
H 2O E NADH + H+ dehydrogenase
ATP T CO2
(C4) FUMARATE Oxid. C
Phospho α - KETOGLUTARATE (C5)
FADH2 NAD
Succinate
TPP α - ketoglutarate
dehydrogenase
FAD H2O O2 NADH + H+ dehydrogenase
- CO2
(ATP) complex
(C4) SUCCINATE
GTP ADP + Pi SUCCINYL-CoA (C4) -
Mg+2
Malonate Succinyl CoA thiokinase Arsenite
CoA
VIII. OTHER ASPECTS OF THE
KREBS CYCLE
IX. CATABOLISM OF THE MAJOR NUTRIENTS
CONVERGE ON THE TCA CYCLE
 X. ROLE OF VITAMINS IN KREBS CYCLE
Vitamin Active Form Function

Coenzyme for decarboxylation in

Thiamine (B1) Thiamine the pyruvate dehydrogenase
pyrophosphate complex, isocitrate dehydrogenase
(TPP) & α-ketoglutarate dehydrogenase
complex
Flavin adenine Cofactor of succinate
Riboflavin (B2) dinucleotide dehydrogenase
(FAD)
Electron acceptor for isocitrate
Nicotinamide dehydrogenase, α-ketoglutarate
Niacin (B3) adenine dehydrogenase complex & malate
dinucleotide dehydrogenase

Component of Cofactor attached to “active”

Pantothenic Coenzyme A carboxylic acid residues, such as
acid Acetyl CoA and Succinyl CoA
 XI. AMPHIBOLIC NATURE OF CAC
Acetyl CoA
PYRUVATE ACETYL-CoA
Alanine
Amino acids Fatty acid synthesis
OXALOACETATE CITRATE Steroid synthesis
Aspartate à
Asparagine
Urea, Purine MALATE ISOCITRATE
Pyrimidines:
Cytosine Glutamate
Uracil AA synthesis
Thymine FUMARATE
α - KETOGLUTARATE

Gluconeogenesis Neurotransmitter
(brain)
SUCCINATE
SUCCINYL-CoA
Heme synthesis
Cytochromes
Other heme proteins
 XII. ANAPLEROTIC REACTIONS OF TCA
AAs PYRUVATE ACETYL-CoA
CO2 CHOs
Pyruvate FAs
ATP carboxylase AAs
a
ADP
OXALOACETATE CITRATE
e
Aspartate
Alanine
Serine MALATE ISOCITRATE

Amino acids
b
FUMARATE TA
Glutamate α - KETOGLUTARATE
d
Amino acids GDH

SUCCINATE
SUCCINYL-CoA
c
Valine
Isoleucine Propionyl CoA Odd chain FAs
 XIII. REGULATION OF KREBS CYCLE
PYRUVATE

Pyruvate dehydrogenase
--
ACETYL-CoA
ATP, NADH, Acetyl CoA
+ Ca+2

Citrate synthase
- ATP, NADH, Succinyl CoA
Long-chain Fatty Acyl CoA, Citrate

OXALOACETATE

-
CITRATE
ATP,
NADH Malate dehydrogenase

-
MALATE ISOCITRATE
ATP, NADH

+
Isocitrate dehydrogenase
ADP, NAD+, Ca+2
FUMARATE
α - KETOGLUTARATE
α–Ketoglutarate
dehydrogenase - ATP, GTP, NADH,
Succinyl CoA

SUCCINATE +
SUCCINYL-CoA
Ca+2
 XIV. CLINICAL CORRELATION
BIOCHEMICAL BASIS OF BERI-BERI
1. Common among population with rice as the major food
(Far East).
2. Aggravated by eating polished rice whose outer layer rich
in thiamine is removed.
3. Thiamine pyrophosphate (the active form of thiamine) is a
coenzyme of pyruvate dehydrogenase and α-ketoglutarate
dehydrogenase.
4. Deficiency of thiamine à ↓ TPP → impaired glucose
oxidation esp. in the nervous system à ↓ ATP: muscle
weakness and atrophy, pain in the limbs, fatigue, cardiac
enlargement, ↓ CO and edema due to ↓ venous return.
5. Hence, neurologic and cardiovascular disorders due to
dietary deficiency of thiamine (Vit. B1).
XV. ATP YIELD PER MOLECULE OF ACETYL
CoA OXIDIZED THROUGH THE TCA
1 NADH = 2.5 ATPs 1 FADH = 1.5 ATPs
Reaction Mechanism ATP Yield
Isocitrate NADH, Oxid. 5
dehydrogenase Phospho.
α-ketoglutarate NADH, Oxid. 5
dehydrogenase Phospho.
Succinyl CoA Substrate-level 2
synthase Phospho.
Succinate FADH2, Oxid. 3
dehydrogenase Phospho.
Malate NADH, Oxid. 5
dehydrogenase Phospho.
TOTAL - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 20
Pyruvate NADH, Oxid. Phospho. 5
2 Pyruvates From Glycolysis Oxidized Through The TCA = 32 ATPs
XVI. ATP YIELD FOR COMPLETE OXIDATION OF GLUCOSE
Reaction Mechanism Mole ATP /
Mole
Glucose

A. Glycolysis
Hexokinase-Glucokinase Phosphorylation -1
PFK-1 Phosphorylation -1
G-3 - PDH NADH, Oxid. Phospho. +2.5 (1.5)
Phosphoglycerate kinase Substrate-level Phospho. +2
Pyruvate kinase Substrate-level Phospho. +2

1 NADH = 2.5 ATPs 1 FADH = 1.5ATPs

B. CAC
Pyruvate dehydrogenase +5
NADH, Oxid. Phospho.
Isocitrate dehydrogenase +5
NADH, Oxid. Phospho.
α-ketoglutarate dehydro- +5
NADH, Oxid. Phospho.
genase
Succinyl CoA synthetase +2
Substrate-level Phospho.
Succinate dehydrogenase +3
FADH2, Oxid. Phospho.
Malate dehydrogenase +5
NADH Oxid. Phospho.
TOTAL 30-32
 XVII. SHUTTLE MECHANISMS FOR
CYTOPLASMIC REDUCING EQUIVALENTS

The ATP yield depends on the route of

transport of redox equivalents to the
mitochondria:
a. Malate-Aspartate Shuttle = 2.5 ATP/
NADH molecule (2 NADH x 2.5 = 5 ATP)
b. Glycerol-Phosphate Shuttle = 1.5
ATP/NADH2 molecule (2 NADH2 X 1.5 =
3 ATP)
 XVIII. SUMMARY OF CITRIC ACID
CYCLE REACTIONS

Reactants Acetyl CoA + 3 NAD+ + 1 GDP + Pi + 2 H2O

Products CoA-SH + 3 NADH + 3 H+ 1 GTP + 2 CO2

 XIX. QUESTION 1

Starting with one pyruvate to

make acetyl CoA, how
many ATPs are
produced?
 ANSWER 1
Pyruvate dehydrogenase
Pyruvate Acetyl CoA

NAD+ NADH+
+ H+
ETC

2.5 ATPs
 QUESTION 2
Starting with one pyruvate to make
acetyl CoA and entering into
the CAC, how many ATPs
are produced?
 ANSWER 2
Pyruvate
dehydrogenase
Pyruvate Acetyl CoA 2 NADH 5 ATP

Isocitrate 2 NADH 5 ATP

dehydrogenase

α-ketoglutarate 2 NADH 5 ATP

dehydrogenase

Succinyl CoA 1 NADH 2 ATP

thiokinase

Succinate 3 ATP
dehydrogenase 2 FADH

Malate
2 NADH 5 ATP
dehydrogenase
TOTAL 25 ATP
 QUESTION 3

Starting with one acetyl

CoA, how many ATPs
are produced?
 ANSWER 3
Isocitrate
2 NADH 5 ATP
dehydrogenase

α-ketoglutarate 2 NADH 5 ATP

dehydrogenase

Succinyl CoA 2 ATP

1 NADH
thiokinase

Succinate 3 ATP
dehydrogenase 2 FADH

Malate
2 NADH 5 ATP
dehydrogenase
TOTAL 20 ATP
 QUESTION 4
Starting with glycogen producing
glucose 6-phosphate, what
is the end product of
aerobic glycolysis?
ANSWER 4

2 Pyruvate
 QUESTION 5
Starting with pyruvate, how many
CO2 molecules are produced
upon complete metabolism
in the CAC?
 ANSWER 5
Pyruvate
dehydrogenase 1 CO2

Isocitrate
dehydrogenase 1 CO2

α-ketoglutarate
dehydrogenase 1 CO2

TOTAL 3 CO2