TRICARBOXYLIC ACID CYCLE

and
HEXOSE MONOPHOSPHATE SHUNT
Prof. Goncagül Haklar, M.D.
Dept. of Biochemistry, School of Medicine, Marmara University
Marmara University Pendik E&R Hospital Biochemistry Laboratory

Year 1; December 16, 2019

2
 AIM

to be able to describe the contents of TCA cycle

to be able to review the metabolic control of TCA

cycle
to be able to summarize the function of the HMS in
production of NADPH and ribose precursors for
nucleic acid synthesis.
to be able to relate defects in the HMS to
disease conditions.
THE CITRIC ACID CYCLE

=Tricarboxylic acid (TCA) cycle due to tricarboxylate

intermediates,

=Krebs cycle (for biochemist Hans Krebs)

The wheel is turnin’ and

the sugars are burnin’

4
 Acetyl-CoA was
? discovered only in 1951

acetyl phosphate

The original
citric acid cycle

Krebs and Johnson (1937) “The role of citric acid in the intermediate
metabolism in animal tissues. Enzymologica 4:148-156.
This paper was submitted to Nature but was rejected!!! 5
 The Nobel Prize in Physiology or Medicine 1953
"for his discovery of co-enzyme A
"for his discovery
and its importance for intermediary
of the citric acid cycle"
metabolism"

Krebs discovered
the urea cycle in
1932 before he
elucidated
the citric acid
cycle!

Hans Adolf Krebs Fritz Albert Lipmann

(1900-1981) (1899-1986)
United Kingdom USA
6
 Chemical Structure of ATP

Adenine Base

3 Phosphates Ribose Sugar

7
 All Types of Molecules can be used
to form ATP by Cell Respiration:
Proteins, Carbohydrates, and Lipids must first be broken
down into their monomers and absorbed in the small
intestine.

Monomers may be further broken down into intermediate

molecules before entering different parts of Cell respiration
to ultimately form ATP.

8
9
 Cell Respiration can be divided into 4 Parts:
1) Glycolysis
2) Oxidation of Pyruvate / Transition Reaction
3) The Krebs Cycle
4) The Electron Transport Chain and Chemiosmotic Phosphorylation

10
 Where do the 4 parts of Cellular
Respiration take place?
Glycolysis:
– Cytosol
Oxidation of Pyruvate:
– Matrix
The Krebs Cycled:
– Matrix
Electron Transport Chain
and Oxidative
Phosphorylation:
– Cristae
11
12
13
In aerobic organisms, pyruvate
(formed through glycolysis)
oxidized to CO2 and acetyl-CoA
using coenzyme A

Subsequent oxidation of acetyl

group carried out using the citric
acid cycle

14
Reactions occur in mitochondrial matrix
Common final degradative pathway for breakdown of
monomers of CHO, fat and protein to CO2 and H20
– Electrons removed from acetyl groups and
attached to NAD+ and FAD
– Small amount of ATP produced from substrate
level phosphorylation
TCA cycle also provides intermediates for anabolic
functions (eg gluconeogenesis)

15
 Citric Acid Cycle is Amphibolic
✓ Catabolic and anabolic
✓ Aerobic catabolism of carbohydrates, lipids, and amino
acids merge at citric acid cycle
▪ Oxidized acetyl-CoA formed from metabolism of all
three
✓ Intermediates of citric acid cycle are starting points for
many biosynthetic pathways

16
The Amphibolic Citric Acid Cycle

17
 Citric Acid Cycle, cont.
Controlled metabolic oxidation
– Compounds oxidized in discrete steps
– Energy released largely conserved when NAD+ is
reduced to NADH+H+
– Re-oxidation of reduced coenzymes produce more
energy (ATP) through electron transport and
oxidative phosphorylation

18
NETS: 3NADH, 1ATP, 1FADH2, & 2CO2
19
 Net reaction-I
Circular pathway
– Regenerate oxaloacetate starting material in last step
– Cycle is a multi-step enzyme, can catalyze unlimited acetyl
groups
Carbon flow:
– 2 carbons that enter the cycle are
not the same carbons lost as CO2
Series of 8 enzyme-catalyzed reactions
Transfer of 4 pairs of electrons

20 20
 Net reaction-II
Production of energy-rich molecules:
– Most of energy released is conserved in the form of
reduced coenzymes NADH and FADH2
– Oxidation of reduced coenzymes through ETC and
production of ATP
– Step 5 is substrate-level phosphorylation
Net reaction:
Acetyl-CoA + 3 NAD+ + FAD + GDP + Pi + 2 H2O →
2 CO2 + 3 NADH + FADH2 + CoA + GTP + 3 H+

21
22
23
 Synthesis of Acetyl-CoA
Not part of cycle, but must occur first
First step: entry of pyruvate into the mitochondria
– In aerobic cells, all enzymes of the citric acid cycle are
located within the mito.
– Pyruvate passes through outer membrane via aqueous
channels formed by transmembrane proteins (porins)
– Pyruvate translocase is a protein embedded in the inner
mitochondrial membrane which transports pyruvate from the
intermembrane space to the mitochondrial matrix (interior
space of the mitochondrion)

24
 Generation of Acetyl-CoA
• Coenzyme A receives 2
carbons from pyruvate
• Thioester linkage
– ∆G°’=-31kJ mol-1
• Multi-enzyme process
– Pyruvate + CoA + NAD+
→ acetyl CoA + CO2 +
NADH

25
 Conversion of Pyruvate to Acetyl-CoA
Oxidative decarboxylation
– Series of 5 reactions, irreversible, mechanism is highly complicated
Catalyzed by complex of enzymes and cofactors
– Pyruvate dehydrogenase complex
– Multi-enzyme structure located in mito. matrix
– Contains multiple copies of three non-covalently associated enzymes
and five cofactors
– E1 and E3 surround core of 24-60 E2 chains
Overall reaction:
CH3C(O)CO2- + NAD+ + CoASH → CH3C(O)-SCoA + NADH + CO2

26
 Pyruvate Dehydrogenase Complex (PDC)

It is a multi-enzyme complex containing three enzymes

associated together non-covalently:

E-1 : Pyruvate dehydrogenase, uses Thiamine pyrophosphate

as cofactor bound to E1

E-2 : Dihydrolipoyl transacetylase, Lipoic acid bound, CoA as

substrate

E-3 : Dihydrolipoyl dehydrogenase, FAD bound, NAD+ as

substrate

27
Advantages of multienzyme complex:
1. Higher rate of reaction: Because product of one enzyme
acts as a substrate of other, and is available for the active
site of next enzyme without much diffusion.

2. Minimum side reaction.

3. Coordinated control.

28
Pyruvate Dehydrogenase Multienzyme Complex
Pyruvate dehydrogenase - E1
– Pyruvate + TPP → Hydroxyethyl TPP + CO2
Dihydrolipoyl transacetyase - E2
– HOEt-TPP + lipoamide→ Acetyl-dihydrolipoamide + TPP
– Acetyl-dihydrolipoamide + CoA-SH → acetyl CoA +
dihydrolipoamide
Dihydrolipoyl dehydrogenase - E3
– NAD+ + dihydrolipoamide → NADH + H+ + lipoamide

29
TPP Cofactor
(Coenzyme)
TPP is derived from
vitamin B1

Common for
decarboxylation
reactions

Carries carbon groups

transiently

30
Coenzyme A
Derived from Vitamin B5 (pantothenic acid)

“Activates” the acetyl group

31
FAD/FADH2

Derived from
Vitamin B2
(riboflavin)

1 or 2 electron
acceptor

32
 NAD+/NADH

Derived from
Vitamin B3 (niacin)

2 electron
acceptor

33
34
35
 Pyruvate dehydrogenase

• Thiamine Pyrophosphate (TPP)

• Attacks carbonyl, releases CO2
• Hydroxyethyl TPP remains bound
36
 Lipoamide flexible redox arm

Covalent E2 cofactor
S-S can be reduced to SH, SH
Lipoamide is a target for arsenic toxicity
37
 Dihydrolipoyl transacetyase

Lipolysyl side chain extends to E1

Transfers hydroxyethyl from TPP to Dihydrolipoamide
Transfers Acetyl group to CoA
38
Thiamin (Vitamine B1) deficiency causes Beriberi:
Thiamine pyrophosphate (TPP) is an important cofactor of
PDH. Thiamine is neither synthesized nor stored in good
amounts by most vertebrates. It is required in the diets of
most vertebrates. Thiamine deficiency ultimately causes a
fatal disease called Beriberi characterized by neurological
disturbances, paralysis, atrophy of limbs and cardiac
failure. Note that brain exclusively uses aerobic glucose
catabolism for energy and PDH is very critical for aerobic
catabolism. Therefore thiamine deficiency causes severe
neurological symptoms.

39
Arsenic Poisoning: Arsenic compounds are poisonous
because they covalently bind to sulfhydryl compounds
(SH- groups of proteins and cofactors).
Dihydrolipoamide is a critical cofactor of PDH, and it
has two-SH groups, which are important for the
reaction. These –SH groups are covalently inactivated
by arsenic compounds

40
 Arsenic Compound poisoning: Inactivation of E-2 of PDH, and other proteins.

Organic Arsenical were used as

antibiotics for the treatment of
syphilis and trypanosomiasis.
Micro-organisms are more
sensitive to organic arsenicals
than humans.
But these compounds had severe
side effects and As-poisoning.
Fowler’s solution, the famous 19th
century tonic contained 10mg/ml
As. Charles Darwin died of As
poisoning by taking this tonic.
Napoleon Bonaparte’s death was
also suspected to be due to As
poisoning.

41
 Dihydrolipoyl dehydrogenase

Exchange of enzyme disulfide for lipoamide

– E3 S-S + dihydrolipoamide → E3–SH, SH + lipoamide
Bound FAD cofactor oxidizes E3 to disulfide
– E3– SH, SH + FAD → E3 S-S +
NAD+ oxidizes FADH2 to regenerate FAD
– E3FADH2 + NAD+ → E3FAD + NADH+ + H+

42
43
 1. Formation of citrate
Oxaloacetate reacts with acetyl-CoA to form citrate and
coenzyme A
Aldol condensation
– Only C-C bond-forming reaction in cycle
Irreversible
Enzyme = citrate synthase

44
 Citrate synthase
COO-
CoA
COO- H2O CH2
S
C=O HO-C–COO-
C=O
CH2 CH2
H3C CoASH
COO- COO-

✓ Oxaloacetate binds - cleft closure

✓ Acetyl-CoA binds to closed form only
✓ Acid base catalysis forms Enolate of Acetyl-CoA
✓ Nucleophilic attack on OAA C=O forms Citroyl CoA
✓ Citrate and CoA-SH are released

45
1. Citrate synthase: Formation of Citroyl CoA intermediate.
2. Binding of Oxaloacetate to the enzyme results in
conformational change which facilitates the binding of
the next substrate, the acetyl Coenzyme A. There is a
further conformational change which leads to formation
of products. This mechanism of reaction is referred as
induced fit model.

46
 Citrate synthase
Dimer of two identical subunits
Changes in conformation
– Binding of oxaloacetate
– Domains move closer to form binding site for
acetyl-CoA
– Formation of intermediate
– Enzyme closes around intermediate
• Prevent side reactions by shielding thiol
ester linkage of acetyl-CoA from
hydrolysis by solvent
• Intermediate hydrolyzed by bound water
molecule
– Enzyme opens and products leave active site
47
 2. Isomerization of citrate to isocitrate
Citrate is a 3° alcohol
– Cannot be oxidized to keto acid
Isocitrate is a 2° alcohol
– Easily oxidized
Enzyme = aconitase (aconitate hydratase)

48
 2. Isomerization of citrate to isocitrate
Mechanism:
– First step: elimination of H2O to from alkene
intermediate (cis-aconitate)
– Second step: stereospecific addition of water to form
(2R, 3S)-isocitrate
– Reaction near equilibrium

49
 Aconitase
COO- COO- COO-
CH2 CH2 H2O CH
2
HO-C–COO- C–COO- C–COO-
CH2 CH2 HO-CH
COO- COO- COO-
H2O

✓ Transfers C3-OH of citrate to C2-OH of isocitrate

✓ Cis Aconitate (C2-C3 double bond) intermediate
✓ Iron sulfur cluster [4Fe - 4S]
✓ Fluoroacetate is converted to Fluorocitrate
inhibits aconitase

50
3. Oxidative decarboxylation of isocitrate to form
a-ketoglutarate

First of four oxidation-reduction reactions

– NAD+ is oxidizing agent
Enzyme = isocitrate dehydrogenase
Irreversible
One of rate-limiting steps in cycle

51
 3. Oxidative decarboxylation of isocitrate to form
a-ketoglutarate (Mechanism)
– First step: alcohol oxidized by transfer of H:- from C2 to NAD+
• Intermediate = oxalosuccinate, an unstable b-keto acid
• First molecule of NADH formed
– Second step: intermediate undergoes b-decarboxylation to form
an a-keto acid, which is released from enzyme
– First molecule of CO2 produced

52
 Isocitrate dehydrogenase

✓ Isocitrate + NAD+ → a ketoglutarate + CO2 + NADH + H+

✓ Initial oxidation of isocitrate to oxalosuccinate
✓ Decarboxylation to give CO2 and a-ketoglutarate
53
4. Oxidative decarboxylation of a-ketoglutarate
to form succinyl-CoA
Catalyzed by multi-enzyme a-ketoglutarate dehydrogenase
complex
 a-ketoglutarate dehydrogenase (E1)
– Dihydrolipoamide succinyltransferase (E2)
– Dihydrolipoamide dehydrogenase (E3)
– Analogous to pyruvate-to-acetyl-CoA reaction catalyzed by
pyruvate dehydrogenase complex
• Same coenzymes
• Similar complicated mechanism

54
4. Oxidative decarboxylation of a-ketoglutarate
to form succinyl-CoA
Product is high-energy thioester
Key regulatory step of citric acid cycle
Second molecule of NADH produced
Second molecule of CO2 produced

55
 a-Ketoglutarate Dehydrogenase
COO- COO- CO2
CH2 CH2
CoASH
CH2 CH2 NADH
C=O C=O
NAD
COO- S–CoA H+

Similar to Pyruvate Dehydrogenase

– Product is Succinyl CoA instead of Acetyl CoA
Multienzyme complex
– E1- dehydrogenase, E2- dihydrolipoyl transsuccinylase,
E3 identical to PD-E3
56
4. a-Ketoglutarate dehydrogenase: This is a complex of different
enzymatic activities similar to the pyruvate dehydrogenase
complex. It has the same mechanism of reaction with E1, E2 and
E3 enzyme units. NAD+ is an electron acceptor.

57
 Halfway through the cycle…

So far…
– Net oxidation of two carbon atoms to produce two
molecules CO2

In the next four reactions…

– Four-carbon succinyl group of succinyl CoA
converted back to oxaloacetate
– As oxaloacetate is regenerated, additional acetyl-
CoA enters the citric acid cycle to be oxidized

58
5. Conversion of succinyl-CoA to succinate
Substrate-level phosphorylation
– Cleavage of high-energy thioester bond
– Free energy conserved through the synthesis of nucleoside
triphosphate
• GTP in mammals, ATP in plants and bacteria
– GDP regenerated and ATP produced from the reaction of
GTP with ADP
• GTP + ADP GDP + ATP
• Nucleoside diphosphate kinase
Enzyme= succinyl-CoA synthetase (=aka succinate
thiokinase)
59
5. Conversion of succinyl-CoA to succinate
Mechanism:
– Phosphate displace CoA from bound succinyl-CoA molecule
– Phosphoryl group transfers to His residue of enzyme
– Succinate released
– Phosphoryl group transferred to GDP (or ADP)

60
Succinyl-CoA synthetase mechanism

61
 Succinyl-CoA Synthetase
COO- COO-
CH2 CH2 GTP
GDP
CH2 CH2
C=O COO- CoASH
Pi
S–CoA

Products Succinate, GTP and CoASH

– ATP instead of GTP in plants and bacteria
Thioester free energy almost entirely conserved in
phospho anhydride
Phosphoryl enzyme intermediate

62
5. Succinyl CoA synthetase: Succinyl CoA, like Acetyl CoA has
a thioester bond with very negative free energy of hydrolysis.
In this reaction, the hydrolysis of the thioester bond leads to
the formation of phosphoester bond with inorganic phosphate.
This phosphate is transferred to Histidine residue of the
enzyme and this high energy, unstable phosphate is finally
transferred to GDP resulting in the generation of GTP.

63
 6. Oxidation of succinate to fumarate
Dehydrogenation (loss of H2; oxidation)
– Stereospecific to form trans double bond only
Catalyzed by succinate dehydrogenase complex
– Embedded in inner mitochondrial membrane, rather
than in mitochondrial matrix.

64
6. Oxidation of succinate to fumarate

Oxidation of alkane requires stronger oxidizing

agent than NAD+ (hence FAD)
– FADH2 produced is re-oxidized by
coenzyme ubiquinone (Q) to reform
FAD and ubiquinol (QH2)

65
 6. Oxidation of succinate to fumarate
Competitive inhibitor = malonate
– -O2C-CH2-CO2-
– Binds to active site through carboxylate groups
– Cannot undergo dehydrogenation
– Inhibition reactions used by Krebs to determine citric
acid cycle reaction sequence
Symmetrical molecule evenly distributes carbons in
remainder of products throughout the cycle

66
 Succinate Dehydrogenase
COO- H COO-
CH2 C
CH2 E-FAD C E-FADH2
-OOC H
COO-

Electrons accepted by covalently bound FAD

Embedded in mitochondrial membrane

– FADH2 reoxidized by electron transport chain

67
7. Hydration of fumarate to form L-malate
Reversible reaction, near equilibrium
Stereospecificity
1. trans addition of water to double bond of fumarate
2. Only trans double bond will react
Enzyme = fumarase

68
8. Oxidation of L-malate to regenerate OAA
Formation of third molecule of NADH

Reaction is endergonic, and concentration of product is low

at equilibrium

– Next reaction in cycle (1) is highly exergonic

– Product used immediately

Enzyme = malate dehydrogenase

69
 Malate Dehydrogenase
✓ Hydride transfer from malate to NAD regenerates
oxaloacetate for another cycle

✓ Reaction pulled by citrate synthase consumption of OAA

70
REGULATION

71
 Regulation of the Citric Acid Cycle-I
Recall that build-up of citrate slows
glycolysis/production of pyruvate
Regulation also depends on continuous supply of
acetyl-CoA (from pyruvate), NAD+, FAD, and ADP

72
 Regulation of the Citric Acid Cycle-II

Regulated enzymes in the citric acid cycle:

– Citrate synthase (reaction 1)
• Allosterically inhibited by high concentrations
of citrate, succinyl-CoA, NADH, ATP
– Isocitrate dehydrogenase (reaction 3)
• Activity stimulated by ADP, NAD+, and Ca2+,
isocitrate, Mg2+, (links with muscle contraction)
• Inhibited by ATP and NADH
• Inactivated by phosphorylation

73
 Regulation of the Citric Acid Cycle-III

Regulated enzymes in the citric acid cycle:

– a-Ketoglutarate dehydrogenase (reaction 4)
• Stimulated by Ca2+,
• Inhibited by ATP, GTP, NADH and succinyl-
CoA
– All three of these enzymes catalyze reactions that
represent important metabolic branch points

74
 Regulation of Pyruvate → Acetyl CoA
PDH reaction regulated to spare pyruvate from being irreversibly lost
– Glucose important for brain and once converted to Acetyl CoA
cannot be used for glucose synthesis
PDH regulated by phosphorylation and allosteric control
– Dephosphorylation activates PDH
• Phosphatase enzyme activated by high Ca2+
– Phosphorylation inactivates PDH
• Kinase activated by acetyl CoA and NADH
– PDH allosterically inhibited by: allosterically act. by: AMP
• ATP
• Acetyl CoA
• NADH

75
 Control of Pyruvate Dehydrogenase
• Product Inhibition by NADH and acetyl-CoA
– Competitive inhibition of substrate binding
– E2 and E3 are reversible
• Phosphorylation inactivates mammalian E1
– Pyruvate dehydrogenase kinase
• Stimulated by NADH and acetyl-CoA
– Pyruvate dehydrogenase phosphatase - activates E1
• Activated by Ca2+ as part of Insulin response
• Provides Acetyl CoA for fatty acid synthesis

76
77
 Anabolic Uses of Cycle Intermediates
• Gluconeogenesis
– Malate → OAA → → Glucose
• Lipid Biosynthesis
– Citrate → OAA + Acetyl CoA → → lipids
• Amino Acid Biosynthesis
– OAA and a-ketoglutarate
• Porphyrin Biosynthesis
– Succinyl CoA

78
79
Catabolic Sources of Cycle Intermediates

• Fatty acid oxidation

– Succinyl CoA
• Amino Acid degradation
– Succinyl CoA
• Transamination of Amino Acids
– OAA and a-ketoglutarate

80
Participation of TCA cycle in fatty acid
synthesis from glucose

81
Vitamins play key roles of the Citric Acid Cycle

⚫ Four of the B vitamins are essential in the

citric acid cycle :
1. Ribovlavin FAD
2. Niacin NAD
3. Thiamin TPP
4. Pantothenic acid CoA

82
 Why use ATP energy and not
energy from glucose?
Breaking down glucose yields too much energy for
cellular reactions and most of the energy would be
wasted as heat.

1 Glucose = 686 kcal

1 ATP = 7.3 kcal
1 Glucose → 36 ATP

How efficient are cells at converting glucose into ATP?

– 38% of the energy from glucose yields ATP, therefore
62% wasted as heat.
83
Anaerobic Respiration (no oxygen required, cytoplasm)
1. Glycolysis Glucose → 2 Pyruvate
(substrate level) 2 ATP 4 ATP (Net 2 ATP)
2 NADH

Aerobic Respiration (oxygen required, mitochondria)

2. Oxidation 2 Pyruvate → 2 CO2
of 2 NADH
Pyruvate 2 Acetyl CoA

3. Krebs Cycle 2 Acetyl CoA → 4 CO2

(substrate level) 2 ATP
6 NADH
2 FADH2
4. Electron
10 NADH → 32-34 ATP
Transport
2 FADH2 H 2O
Chain
Oxygen
(chemiosmotic) Total: 36-38 ATP produced
84
 ATP is made in two ways:
1) Substrate Level
Phosphorylation (glycolysis
& Krebs cycle)
2) Chemiosmotic
Phosphorylation (electron
transport chain)

Substrate-Level
Phosphorylation:
Energy and phosphate are
transferred to ADP using an
enzyme, to form ATP.
Phosphate comes from one
of the intermediate
molecules produced from
the breakdown of glucose.
85
 Glyoxylate cycle
Net conversion of fatty acids to glucose can occur in
germinating seeds, some invertebrates and some bacteria
via the glyoxylate cycle, which shares three steps with the
citric acid cycle but bypasses the two decarboxylation
steps, converting two molecules of acetyl-CoA to one
succinate.

87
Glyoxalate Cycle

88
 Partial overlapping
of the glyoxylate and
citric acid cycles

89
Conversion of fatty acids to glucose
(in germinating seeds) occurs in
three intracellular compartments
Partition of isocitrate
between the
glyoxylate and citric
acid cycles are
regulated by the
opposite effect of
common allosteric
effectors on the
isocitrate lyase and
the isocitrate
dehydrogenase.
91
Biosynthetic roles of the citric acid cycle

92
The amphibolic nature of Citric acid cycle: This pathway is utilized for the
both catabolic reactions to generate energy as well as for anabolic reactions to
generate metabolic intermediates for biosynthesis.

If the CAC intermediate are used for synthetic reactions, they are replenished by
anaplerotic reactions in the cells (indicated by red colours).

93
 Pentose Phosphate Pathway
(Hexose Monophosphate Shunt)

95
The pentose phosphate pathway is an alternate route for the
oxidation of glucose.

96
The Pentose Phosphate Pathway Has Two
Main Functions
Generation of NADPH
- mainly used for reductive synthesis of fatty acids,
steroids, amino acids via glutamate dehydrogenase; and
production of reduced glutathione in erythrocytes and other
cells.
- active in liver, adipose tissue, adrenal cortex, thyroid,
erythrocytes, testes, and lactating mammary gland
- not active in non-lactating mammary gland and has low
activity in skeletal muscle.
Production of ribose residues for nucleotide and nucleic
acid synthesis.

97
 Reactions Of The Pentose Phosphate
Pathway Occur In The Cytosol In Two Phases
Oxidative non-reversible phase
Non-oxidative reversible phase
NADP+, not NAD +, is used as hydrogen acceptor
1st phase
- Glucose 6-phosphate undergoes dehydrogenation and
decarboxylation to give a pentose, ribulose 5-phosphate,
which is converted to its isomer, D-ribose 5-phosphate.
- Overall equation of 1st phase:
Glucose 6-phosphate + 2 NADP++ H2O →
ribose 5-phosphate + CO2 + 2 NADPH + 2 H+
98
99
 Glucose-6-phosphate 6-Phospho- O O−
Dehydrogenase glucono- 1C
6CH OPO 2− 6 CH OPO 2− lactonase
2 3 2 3
NADPH + H+ HC OH
H
5 O OH NADP
+
H
5 O H2 O H+ 2
H H HO 3CH
4 OH H 1 4
OH H O
1 HC OH
4
OH H OH
3 2 3 2 HC OH
5
H OH H OH CH2OPO32−
6
glucose-6-phosphate 6-phoshogluconolactone 6-phosphogluconate

Glucose-6-phosphate Dehydrogenase catalyzes oxidation of

the aldehyde (hemiacetal), at C1 of glucose-6-phosphate, to a
carboxylic acid, in ester linkage (lactone).
NADP+ serves as electron acceptor.

100
 Glucose-6-phosphate 6-Phospho- O O−
Dehydrogenase glucono- 1C
6CH OPO 2− 6 CH OPO 2− lactonase
2 3 2 3
NADPH + H+ HC OH
H
5 O OH NADP
+
H
5 O H2 O H+ 2
H H HO 3CH
4 OH H 1 4
OH H O
1 HC OH
4
OH H OH
3 2 3 2 HC OH
5
H OH H OH CH2OPO32−
6
glucose-6-phosphate 6-phoshogluconolactone 6-phosphogluconate

6-Phosphogluconolactonase catalyzes hydrolysis of the ester

linkage, resulting in ring opening.
The product is 6-phosphogluconate.
Although ring opening occurs in the absence of a catalyst, 6-
Phosphogluconolactonase speeds up the reaction, decreasing
the lifetime of the highly reactive, and thus potentially toxic, 6-
phosphogluconolactone.
101
 O O− Phosphogluconate
1C Dehydrogenase
HC OH 1CH2OH
2 NADP + NADPH + H+
HO 3CH C O
2
HC OH HC OH
4 3
HC OH CO2 HC OH
5 4
CH2OPO32− CH2OPO32−
6 5
6-phosphogluconate ribulose-5-phosphate

Phosphogluconate Dehydrogenase catalyzes oxidative

decarboxylation of 6-phosphogluconate, to yield the 5-C
ketose ribulose-5-phosphate.
The OH at C3 (C2 of product) is oxidized to a ketone.
This promotes loss of the carboxyl at C1 as CO2.
NADP+ serves as oxidant.
102
 H O O
H H
C C
Reduction of NADP+ (as NH2 NH2
with NAD+) involves
transfer of 2 e− and 1 H+ to +
N N
2e− + H
+
the nicotinamide moiety.
R R
NADP+ NADPH

 NADPH, a product of the Pentose Phosphate Pathway,

functions as a reductant in anabolic (synthetic) pathways,
e.g., fatty acid synthesis.
 NAD+ serves as electron acceptor in catabolic pathways, in
which metabolites are oxidized.
The resultant NADH is reoxidized by the respiratory chain,
producing ATP.
103
NAD+ & NADP+ differ only Nicotinamide H O

in the presence of an extra Adenine C

Dinucleotide NH2
phosphate on the
O
adenosine ribose of NADP+. +
N
−
O P O CH2
O
nicotinamide
This difference has little to
H H
do with redox activity, but H H
is recognized by substrate- OH OH
binding sites of enzymes. O NH2

It is a mechanism for N
N

separation of catabolic and

synthetic pathways. −
O P O CH2
N N adenine
O
O H H
H H esterified to
OH OH Pi in NADP+

104
Regulation of Glucose-6-phosphate Dehydrogenase:
 Glucose-6-phosphate Dehydrogenase is the committed
step of the Pentose Phosphate Pathway.
This enzyme is regulated by availability of the substrate
NADP+.
 As NADPH is utilized in reductive synthetic pathways, the
increasing concentration of NADP+ stimulates the
Pentose Phosphate Pathway, to replenish NADPH.

105
The rest of the pathway converts ribulose-5-P to the 5-C
product ribose-5-P, or to 3-C glyceraldehyde-3-P & 6-C
fructose-6-P.
Additional enzymes include an Isomerase, Epimerase,
Transketolase, and Transaldolase.

106
Epimerase inter-converts CH2OH
stereoisomers ribulose-5-P C O
and xylulose-5-P. Epimerase
HO C H
Isomerase converts the
H C OH
ketose ribulose-5-P to the CH2OH
aldose ribose-5-P. C O CH2OPO32−
xylulose-5-
Both reactions involve H C OH phosphate
deprotonation to an
H C OH HC O
endiolate intermediate
followed by specific CH2OPO32− H C OH
reprotonation to yield the ribulose-5- H C OH
product. phosphate Isomerase
H C OH
Both reactions are
CH2OPO32−
reversible. ribose-5-
phosphate
107
Transketolase & Transaldolase catalyze transfer of 2-C or 3-
C molecular fragments respectively, in each case from a
ketose donor to an aldose acceptor.

108
 CH2OH
Transketolase
C O

CH2OH HC O HO C H

C O H C OH H C OH

HO C H H C OH HC O H C OH

H C OH
+ H C OH H C OH
+ H C OH

CH2OPO32− CH2OPO32− CH2OPO32− CH2OPO32−

xylulose- ribose- glyceraldehyde- sedoheptulose-

5-phosphate 5-phosphate 3-phosphate 7-phosphate
 Transketolase transfers a 2-C fragment from xylulose-5-P to
either ribose-5-P or erythrose-4-P.
 Transketolase utilizes as prosthetic group thiamine
pyrophosphate (TPP), a derivative of vitamin B1.
Pyruvate Dehydrogenase also utilizes TPP as prosthetic group.
109
 CH2OH
Transketolase
C O

CH2OH HC O HO C H

C O H C OH H C OH

HO C H H C OH HC O H C OH

H C OH
+ H C OH H C OH
+ H C OH

CH2OPO32− CH2OPO32− CH2OPO32− CH2OPO32−

xylulose- ribose- glyceraldehyde- sedoheptulose-

5-phosphate 5-phosphate 3-phosphate 7-phosphate

 Transfer of the 2-C fragment to the 5-C aldose ribose-5-

phosphate yields sedoheptulose-7-phosphate.
 Transfer of the 2-C fragment instead to the 4-C aldose
erythrose-4-phosphate yields fructose-6-phosphate.
110
 CH2OH
Transaldolase
C O H2C OH

HO CH C O

HC OH HC O HO CH

HC OH HC O HC OH HC OH

HC OH + HC OH HC OH + HC OH

H2C OPO32− H2C OPO32− H2C OPO32− H2C OPO32−

sedoheptulose- glyceraldehyde- erythrose- fructose-

7-phosphate 3-phosphate 4-phosphate 6-phosphate

Transaldolase catalyzes transfer of a 3-C dihydroxyacetone

moiety, from sedoheptulose-7-phosphate to glyceraldehyde-3-
phosphate.

111
The diagram at right (3) ribulose-5-P
summarizes flow of IS EP
15 C atoms through
Pentose Phosphate ribose-5-P (2) xylulose-5-P
Pathway reactions by TK
which 5-C sugars are
glyceraldehyde-3-P
converted to 3-C and
6-C sugars. sedoheptulose 7 P
IS = Isomerase
EP = Epimerase
fructose-6- P TA
TK = Transketolase
TA = Transaldolase erythrose-4-P

TK fructose-6-P
glyceraldehyde-3-P
112
In tissues requiring primarily NADPH rather than ribose 5-
phosphate, these pentose phosphates can be recycled into
glucose 6-phosphate. Overall, 6 five-carbon sugars are
converted to 5 six-carbon sugars

113
 Pentose Phosphate Pathway Protects Cells
Against Reactive Oxygen Species (ROS)
Molecular oxygen and partially reduced, reactive forms of oxygen.
Reduction of molecular O2 in a series of one-electron steps yields
superoxide, hydrogen peroxide, hydroxyl radical, and water. The
intermediate, activated forms of oxygen are known as reactive
oxygen species (ROS)

114
Role Of NADPH And Glutathione In
Protecting Cells Against ROS

115
 Glucose-6-phosphate Dehydrogenase
Deficiency Causes Hemolytic Anemia
Mutations present in some populations causes a deficiency
in glucose 6-phosphate dehydrogenase, with consequent
impairment of NADPH production.
Detoxification of H2O2 is inhibited, and cellular damage
results - lipid peroxidation leads to erythrocyte membrane
breakdown and hemolytic anemia.
Most G6PD-deficient individuals are asymptomatic - only in
combination with certain environmental factors (sulfa
antibiotics, herbicides, antimalarials, *divicine) do clinical
manifestations occur.
*toxic ingredient of fava beans

116
117
 Regulation of
Pentose Phosphate Pathway
The entry of glucose 6-phosphate into the pentose
phosphate pathway is controlled by the cellular
concentration of NADPH
NADPH is a strong inhibitor of glucose 6-phosphate
dehydrogenase
As NADPH is used in various pathways, inhibition is
relieved, and the enzyme is accelerated to produce more
NADPH
The synthesis of glucose 6-phosphate dehydrogenase is
induced by the increased insulin/glucagon ratio after a
high carbohydrate meal.

118
Depending on needs of a cell for ribose-5-phosphate, NADPH,
and ATP, the Pentose Phosphate Pathway can operate in various
modes, to maximize different products. There are three major
scenarios:

2 NADP+ 2 NADPH + CO2

glucose-6-P ribulose-5-P ribose-5-P

Pentose Phosphate Pathway producing

NADPH and ribose-5-phosphate

Ribulose-5-P may be converted to ribose-5-phosphate, a

substrate for synthesis of nucleotides and nucleic acids.
The pathway also produces some NADPH.
119
 2 NADP+ 2 NADPH + CO2
glucose-6-P ribulose-5-P ribose-5-P

fructose-6-P, &
glyceraldehyde-3-P
Pentose Phosphate Pathway producing
maximum NADPH

Glyceraldehyde-3-P and fructose-6-P may be

converted to glucose-6-P for reentry to the
linear portion of the Pentose Phosphate
Pathway, maximizing formation of NADPH.
120
 2 NADP+ 2 NADPH + CO2
glucose-6-P ribulose-5-P ribose-5-P

fructose-6-P, &
glyceraldehyde-3-P

to Glycolysis
for production of ATP
Pentose Phosphate Pathway producing
NADPH and ATP

Glyceraldehyde-3-P and fructose-6-P, formed from 5-C sugar

phosphates, may enter Glycolysis for ATP synthesis.
The pathway also produces some NADPH.
121
 2 NADP+ 2 NADPH + CO2
glucose-6-P ribulose-5-P ribose-5-P

fructose-6-P, &
glyceraldehyde-3-P

to Glycolysis
for production of ATP
Pentose Phosphate Pathway producing
NADPH and ATP
Ribose-1-phosphate generated during catabolism of nucleosides
also enters Glycolysis in this way, after first being converted to
ribose-5-phosphate.
Thus the Pentose Phosphate Pathway serves as an entry into
Glycolysis for both 5-carbon & 6-carbon sugars. 122
 Summary of the pentose phosphate pathway

3G6P + 6NADP+ + 3H2O

6NADPH + 6H+ + 3CO2 + 2F6P + GAP

Important intermediates

Ribose-5-phosphate (nucleic acids, histidine)

Erythrose-4-phosphate (aromatic amino acids)
123
124
125
126