Learning outcomes

Outline the metabolic fate of glucose in blood

Describe the biological role of Glycolysis and
indicate the reactants, products and key
regulatory enzymes
Explain the fate of Pyruvate in Aerobic and
Anaerobic Glycolysis
8 reactions catalyzed by unique mitochondrial
enzymes in TCA cycle.
Generation of compounds from intermediates of
TCA cycle.
Regulation of glycolysis and TCA cycle.
 Glucose homeostasis
Glucose metabolism is critical to normal
functioning.
Glucose acts both as a source of energy and
as a source of starting material for nearly all
types of biosynthetic reactions.
The organs that control plasma glucose levels:
Pancreas, liver, kidneys, brain, heart, muscle,
adipose tissues and intestine
The normal plasma glucose concentration
varies between about 70 and 120 mg/dL (3.9-
6.7 mM).
 Metabolic Fate of Glucose
Ribose Glycogen synthesis

Pentose phosphate Glycogen

6-phosphogluconate
UDP-glucose

Glucose Glucose 6-PO4 Glucose 1-PO4

Fructose 6-PO4

Glycolysis
 Some important facts about
glycolysis
Glycolysis occurs in the cytosol of the cell.
ATP Is Initially Required.
ATP is subsequently Produced.
Fate of NADH + H+ to ETC.
Glucose (C6) Produces Two Pyruvate (C3)
Molecules.
Anaerobic Pyruvate lactate (2C)
Aerobic Pyruvate Acetyl CoA (2C) TCA .
 The Glycolysis Pathway
Glycolysis means oxidation of glucose
Major anaerobic pathway in all cells
NAD+ is the major oxidant
Initially Requires ATP
Generates 2 ATPs per glucose oxidized
End product is lactate (anaerobic) or pyruvate
(aerobic)
Very imp in tissues with no mitochondria: mature RBCs,
cornea and lens and skeletal muscles especially during
exercise
Connects with Krebs cycle via pyruvate
 Glycolysis CH2OH
O
Two Stages -D-Glucose
OH
1. Hexose stage
1. Phase 1 ATP
2. Phase 2 Hexokinase
2. Triose CH2OPO3
stage O
Glucose-6-Phosphate
Hexose OH

Phosphogluco-
isomerase CH OPO
2 3

O CH2OH

Fructose-6-Phosphate
OH
 CH2OPO3

O CH2OH

Fructose-6-Phosphate OH

ATP
Phosphofructo-
kinase-I CH2OPO3

O CH2OPO3
Fructose 1,6-Bisphosphate
CH2OPO3 OH
CHO
C=O
H-C-OH
CH2OH
Aldolase
CH2OPO3
Dihydroxyacetone-Phosphate
Glyceraldehyde-3-
Phosphate
Phase 2 of first stage of
Glycolysis

fructose-1,6-bisphosphate is
split

2 molecules of glyceraldehyde-
3-phosphate provided for second
half of glycolysis
 Glucos
e
Hexokinas AT
e P
or AD
Glucokina P Fructose-6-
Glucose-
se Phosphogluc P
6-P AT
ose P
Phosphofructokin
isomerase
Phase ase-1
ADP
one Fructose-
Phase
1,6-BPAldolase A
two

Dihydroxyacetone P +
Glyceraldehyde-3-P
Triose
phosphate
isomerase
Glyceraldehyd
e-3-P
2 molecules of
Glyceraldehyde-3-P
Second half of glycolysis
(Phase three)
The phases of the glycolytic pathway
 Triose Stage
DihydroxyCH OPO 2 3 CHO
Glyceraldehyd
acetone C=O H-C-OH 3-phosphate
phosphateCH OH CH2OPO3
2
(DHAP)
Triose phosphate isomerase

O Phosphoglycerate
Kinase COO
CHO C ~OPO
PO4 3

H-C-OH H-C-OH H-C-OH

ADP ATP
CH2OPO3 NAD+ CH2OPO3 CH2OPO3
NADH
Glyceraldehyde-3-P + H+ 3-phospho-
1,3 bisphospho-
Dehydrogenase glycerate
glycerate
Glyceraldehyde-3-P
Dehydrogenase Features
Glyceraldehyde-3-P
(from first half of
glycolysis)
NAD+ + Glyceraldehyde
Pi -3-P
dehydrogenase
NADH +
H+
1,3-
BisPglycerate

reduction-oxidation reaction
NAD+ cofactor derived from niacin
product has high-energy phosphate
bond
 COO COO COO
-H2O
H-C-OH H-C-OPO3 C~OPO3
PEP
CH2OPO3 CH2OH CH2

3-PGA 2-PGA ADP

Enolase
Phosphoglycero-
mutase Pyruvate kinase
ATP

COO COO
NADH + H+
Back to Glycolysis
HO-C-H C=O

CH3 CH3
NAD+

L-lactateLDH Pyruvate
Integration of Glycolysis
NAD+/NADH cycles
ATP/ADP cycles
Other hexoses

Other anaerobic pathways

The ratio of NAD to NADH is balanced in favour of
O
NAD inCHO
normal cells.
C ~OPO
3
H-C-OH PO4 COO
H-C-OH
CH2OPO3 C=O
CH2OPO3
Pyruvate
CH3
NADH + H+
NAD+

COO
NAD+
HO-C-H

CH3

NAD L-lactate
NAD // NADH
++
NADH
Shift
Shift
Accumulation of lactate in tumours implies
an increase of NADH relative to NAD+.
ATP - ADP
Glucose 6-P
CYCLE CHO CHO
C hexose C
C kinase C
C C PEP
C C COO-
CH2OH CH2OPO3
| | ~OPO3
C
Glucose CH2
ATP ADP ADP pyruvate
kinase
ATP
COO-
C=O
ATP CH3 Pyruvate
 Glycolysis in RBC
There is anaerobic glycolysis
in erythrocytes.
Also , Bis Phospho Glycerate
mutase catalyzes the conversion
of 1,3-bis-phosphoglycerate to
2,3-bisphosphoglycerate.
2,3-bisphosphoglycerate, binds
to hemoglobin, decreasing its
affinity for oxygen and so making
oxygen more readily available to
tissues
2,3-bisphosphoglycerate is
converted to 3-phosphoglycerate
by 2,3-bisphospho-glycerate
phosphatase.
This alternative pathway involves
no net yield of ATP from glycolysis
but has role in oxygenation of
tissues by Hb.
Storing blood for loner time results in decrease of 2,3-BPG
leading to high oxygen affinity of Hb leading to oxygen trap.
(2) Carboxylated
Fate of Pyruvate Aerob
in (1) Decarboxylated to Acetyl C
gluconeogenesis
Pyruvate dehydrogenase
(3) H3C-C-COO-
Acetyl CoA
Reduced O
to
Lactate dehydrogenase (anaerobic-cytosol)
Lactate

Each is catalyzed by a specific enzyme:

Remove
(1) Pyruvate dehydrogenase (aerobic-mitochondria)
as CO2
(2) Pyruvate carboxylase (mitochondria)

(3) Lactate dehydrogenase (anaerobic-cytosol)

 The Energy Story of Glycolysis

Overall ANAEROBIC (no O2)

Glucose + 2ADP + 2Pi 2 Lactate +

2ATP + 2H2O
In Yeast:
Glucose + 2ADP + 2Pi 2 Ethanol + 2CO2 +2ATP + 2H2O

Overall AEROBIC

Glucose + 2ADP + 2Pi + 2NAD+

2 Pyruvate + 2ATP + 2NADH +

2H+ + 2H2O
6 ATPs
Glycolysis supply Intermediates for
other pathways:

Many of the metabolites in the glycolytic

pathway are also used by anabolic pathways.

Flux through the pathway is critical to

maintain a supply of carbon skeletons for
biosynthesis.

Not all carbon entering the pathway leaves

as pyruvate and may be extracted at earlier
stages to provide carbon compounds for
other pathways.
Monosaccharides, such as fructose and
galactose, can be converted to one of
the intermediates of glycolysis.
The intermediates may also be directly
useful, eg. Dihydroxyacetone
phosphate (DHAP) is a source of the
glycerol that combines with fatty acids
to form fat.
http://www.wiley.com/college/boyer/0470003790/animations/glycolysis
/glycolysis.html
Regulation of Glycolysis
 Glycolys Glucose
Anaerobic
is Galactose
Lactate Fructose
Mannose
Lactate
Dehydrogenase pyruvate
Aerobic
Fatty
Acetyl-Coenzyme A Acids
Aerobic Amino Acids
Krebs Cycle
Pyruvate dehydrogenase Complex
1Pyruvate
FADHdehydrogenase
2
Thiamin pyrophosphate

Dihydrolipoyl transacetylase Lipoic acid

3 NADH

Dihydrolipoyl dehydrogenase Coenzyme A O2

Oxidative phosphorylation
FAD

NAD
H2
REGULATION

1. Pyruvate dehydrogenase Complex

Inhibited
Inhibitedby
byNADH
NADHand
andAcetyl-CoA
Acetyl-CoA

NADH Acetyl-CoA
[NAD+] HS-CoA
High NADH means that the cell is experiencing a
surplus of oxidative substrates and should not produce
more. Carbon flow should be redirected towards synthesis.
High Acetyl-CoA means that carbon flow into the Krebs
cycle is abundant and should be shut down and rechanneled
towards biosynthesis
Mechanism:
1. By Competitive Inhibition
NADH and acetyl-CoA reverse the pyruvate dehydrogenase
reaction by competing with NAD+ and HS-CoA

2. By Covalent Modification (second level regulation)

E-1 subunits of PDH complex is subject to phosphorylation
Epinephrine
TPP FAD Active
Active
Glucagon
HPO4= E1-OH ATP
1 3
2
PDH PDH Cyclic-AMP
Insulin phosphatase kinase protein kinase

H2O E1-OPO3 ADP ATP

Inactive
Inactive
 TCA
Occurs in the matrix of the mitochondrion.
A key metabolic pathway that unifies
carbohydrate, fat and protein metabolism.
The oxidation of acetyl CoA derived from
carbohydrates, fats and proteins
metabolisms.
Carbon dioxide is liberated.
Energy in the form of Guanosine
triphosphate (GTP) are produced.
Provides precursors.
Generates the reducing potentials NADH and
FADH2 .
 The reducing potentials
The NADH and FADH2 generated by the
citric acid cycle are in turn used by the
oxidative phosphorylation pathway to
generate energy-rich adenosine
triphosphate (ATP).
NADH = 3 OR 2.5 ATPs
FADH2 = 2 OR 1.5 ATPs
 CH3 ~ S-CoA
8 steps C O Acetyl CoA ENERGY
ENERGYYIELD
YIELD
1
4 Oxaloacetate Citrate 6C
NADH + H+
8 3 ATP 2
4 Malate 3 ATP Isocitrate 6
1 ATP
7 2 ATP 3 NADH CO+2 H+
4 Fumarate 3 ATP -ketoglutarate 5
FADH2 6 Total = 12 ATP 4 CO2+ H+
NADH
4 Succinate 5 Succinyl-CoA 4
GTP
 CH3C ~ S-CoA CARBON BALANCE
O Acetyl CoA 2

4 Oxaloacetate Citrate 6

22 carbons
carbons in
in
4 Malate 22 carbons
carbons out
out
Isocitrate 6

CO2
4 Fumarate -ketoglutarate 5

CO2
4 Succinate Succinyl-CoA 4
 CH3-C~SCoA
1
Acetyl-CoA
O
COO-
COO -
-
OOC-CH2- C-OH
C=O HS-CoA CH2 COO-
CH2
COO- Citrate Synthase or Lyase
Oxaloacetate CH2COO-
(OAA) HO-C-COO- Acetyl-CoA

CH2COO-

Citric Acid or Citrate

 Isocitrate Formation
2

CH2COO- CH2COO- CH2COO-

-H2O +H2O
HO-C-COO- C-COO- H-C-COO-
H-C-COO- H C-COO- HO-C-COO-
H H

Citrate cis-Aconitate Isocitrate

Aconitase
 CH2COO- CO2 COO- 3
H-C-COO- CH2
HO-C-COO- CH2
H NAD+ NADH + H+ C=O
COO-
Isocitrate -Ketoglutarate

Isocitrate Dehydrogenase +decarboxylase)

 NAD+
FAD 4
COO-
Lipoic acid COO-
CH2 HS-CoA CH2
CH2 TPP CH2
C=O C~SCoA
COO- O
CO2
-Ketoglutarate Succinyl-CoA
-Ketoglutarate
dehydrogenase
Complex
Thioester
Thioesterbond
bondenergy
energyconserved
conserved as
as GTP
GTP
Pi
COO- + 5
GDP GTP COO -

CH2 CH2
CH2 CH2
C~SCoA HS-CoA
COO-
O
Succinyl-CoA Succinate

Succinyl-CoA Synthetase
 Succinate DH Malate DH
FAD FADH2 NAD+ NADH + H+
H2O
COOH COOH COOH COOH
C C H C OH C=O
C H C H C C
COOH COOH
COOH COOH
Succinate Fumarate Malate Oxaloacetate
6 7 8
Pyruvate Pyruvate dehydrogenase
carboxylase
Amino Acids that Become Kreb Cycle Intermediates
Anaplerotic Reactions (To fill)

COOH COOH
+
COOH COOH H3N C H C=O
+
H3N C H C=O CH2 CH2
CH2 CH2 CH2 CH2
COOH COOH COOH COOH
aspa rtate OAA glutamate keto gluta rate

-ketoacids are analogues of -amino acids

 CH3 ~ S-CoA
C O ENERGY
ENERGYYIELD
YIELD
4 Oxaloacetate Citrate 6
NADH + H+
3 ATP
4 Malate 3 ATP Isocitrate 6
1 ATP
2 ATP CO+2 H+
NADH
4 Fumarate 3 ATP -ketoglutarate 5
FADH2 Total = 12 ATP
CO2+ H+
NADH
4 Succinate Succinyl-CoA 4
GTP
 Summary of Reactions
Substrate or Cofactor Energy Feature

Citrate synthase Acetyl CoA, OAA

Aconitase ( H2O)

Isocitrate dehydrogenase NAD+ 3 ATP

releases CO2

-Ketoglutarate dehydrogenase NAD+ 3 ATP

releases CO2

Succinyl-CoA synthetase GDP, Pi 1 ATP GTP

Succinate dehydrogenase FAD 2 ATP

Fumarase (H2O)

Malate dehydrogenase NAD+ 3 ATP

TOTAL 12 ATP

Table 1. Summary of Enzymes and Specific cofactor or products in the Krebs Cycle
Citrate + 3NAD+ + FAD + GDP + Pi + H2O [1]
Oxaloacetate + 3NADH + 3H+ + FADH2 + 2CO2 + GTP
Regulation of the Citric Acid Cycle

Primary modes:
1. Substrate availability
2. Product inhibition
3. Feedback inhibition (competitive)
Key regulators:
1. Acetyl-CoA (controls citrate synthase)

2. OAA (controls citrate synthase, regulated by NADH)

3. NADH (controls citrate synthase, isocitrate dehydrogenase

4. Calcium (stimulates NADH production)
 Equilibria to Consider

O2 consumption
NADH oxidation
ATP production Controls NADH and is
controlled by
Tightly coupled: affect one is to affect all NADH

Malate + NAD+ OAA + NADH

A
Aworking
working muscle
muscle will
will increase
increase respiration
respiration and
and
oxidize
oxidize NADH.
NADH. This
This stimulates
stimulates OAAOAAsynthesis
synthesis
which
which stimulates
stimulates citrate
citrate synthase
synthase and
and isocitrate
isocitrate
dehydrogenase
dehydrogenase reactions.
reactions.
 Pyruvate Dehydrogenase

Citrate Synthase

No
Regulation

Isocitrate
Dehydrogenase

-Ketoglutarate
Dehydrogenase
 Inhibitors of TCA cycle
Arsenite poison at Pyruvate dehydrogenase and
alpha-ketoglutarate dehydrogenase.
Fluoroacetate and fluorocitrate at aconitase.
Malonate as competitive inhibitor of succinate
dehydrogenase.
Oxaloacetate at malate dehydrogenese.
Hydrogen Peroxide at a-Ketoglutarate Dehydrogenase
(Reduced NADH Production under Oxidative Stress).
Arsenic poisoning: Arsenite forms a stable complex
with enzyme-bound lipoic acid inhibiting Pyruvate
dehydrogenase and alpha-ketoglutarate
dehydrogenase.
 References
Lippincotts Illustrated Biochemistry, 5th edn.
Text Book of Biochemistry for Medical
Students by DM Vasudevan, 6th edn.
Harpers Biochemistry 26th edition.