Glycolysis

Glycolysis
 Biological process in which glucose is converted into pyruvate to provide cells
with energy.

 Occurs in the cytosol of all cells

 All the intermediates in glycolysis have either 3 or 6 carbon atoms

Glycolysis
FIVE main categories of glycolytic reactions
• phosphoryl transfer

• phosphoryl shift

• Isomerization

• Dehydration

• aldol cleavage
Glycolysis
Three main stages in glycolysis
Stage 1
Glucose is trapped and destabilized.
Stage 2
Six carbon molecule is split into 2 three carbon molecules
Stage 3
ATP is generated
Glycolysis – Stage 1
STEP 1

6 CH2OH 6 CH OPO 2
2 3
ATP ADP
5 O 5 O
H H H H
H H
4 1 4 H 1
OH H OH
Mg2+
OH OH OH OH
3 2 3 2
H OH Hexokinase H OH
glucose glucose-6-phosphate

6 CH2OH 6 CH OPO 2
2 3
ATP ADP
5 O 5 O
H H H H
H H
4 1 4 H 1
OH H 2+ OH
Mg
OH OH OH OH
3 2 3 2
H OH Hexokinase H OH
glucose glucose-6-phosphate

6 CH2OH 6 CH OPO 2
2 3
ATP ADP
5 O 5 O
H H H H
H H
4 1 4 H 1
OH H OH
Mg2+

Glucose-6-phosphate
OH OH OH OH
3 2 3 2
H OH Hexokinase H OH
glucose glucose-6-phosphate

 Glucose enters the Glycolysis pathway by conversion to glucose-

6-phosphate.
 ATP utilization step.

 This step is irreversible

Glycolysis – Stage 1
Step 2

 Phosphoglucose Isomerase catalyzes:

glucose-6-P (aldose)  fructose-6-P (ketose)

 aldose is converted to ketose

Glycolysis – Stage 1
Step 3
Phosphofructokinase
6 CH OPO 2 1CH2OH 6 CH OPO 2 1CH2OPO32
2 3 2 3
O ATP ADP O
5 H HO 2 5 H HO 2
CH2OH
H 4 3 OH Mg2+ H 4 3 OH
OH H OH H C O
fructose-6-phosphate fructose-1,6-bisphosphate
HO C H

H C OH
Phosphofructokinase
6 CH OPO 2 6 CH OPO 2
H C OH
2 3 1CH2OH 2 3 1CH2OPO32
O ATP ADP O
5 H HO 2 5 H HO 2 CH2OH
H 4 3 OH Mg2+ H 4 3 OH D-fructose
OH H OH H
fructose-6-phosphate fructose-1,6-bisphosphate

fructose-1,6-bisPhosphate
 Phosphofructokinase (PFK) catalyzes:
fructose-6-P + ATP  fructose-1,6-bisP + ADP
 ATP utilization step.
 The PFK reaction is the rate-limiting step of Glycolysis.
 This is an irreversible step.
SUMMARY OF GLYCOLYSIS STAGE 1
(STEP1-3)
Glycolysis -stage 2

Step 4
Six carbon molecule split into TWO 3 carbon molecules-aldose and ketose
Aldolase catalyzes: fructose-1,6-bisphosphate 
dihydroxyacetone-P + glyceraldehyde-3-P

2
1 CH 2 O PO 3

2C O
H O
2
HO 3
C H A ld o la s e 3
CH 2 O PO 3 1C

H 4
C OH 2C O + H 2
C OH
2
H C OH 1 CH 2 O H 3 CH 2 O PO 3
5
2 d ih y d ro x y a c e to n e g ly c e r a ld e h y d e -3 -
6 CH 2 O PO 3
p h o s p h a te p h o s p h a te
f r u c to s e -1 ,6 -
b is p h o s p h a te
T r i o s e p h o s p h a t e Is o m e r a s e
Glycolysis -stage 2

Step 5
 Triose Phosphate Isomerase (TIM) catalyzes:
dihydroxyacetone-P  glyceraldehyde-3-P
 Glycolysis continues from glyceraldehyde-3-P.
2
1 CH 2 O PO 3

2C O
H O
2
HO 3
C H A ld o la s e 3
CH 2O PO 3 1C

H 4
C OH 2C O + H 2
C OH
2
H C OH 1 CH 2 O H 3 CH 2 O PO 3
5
2
6 CH 2 O PO 3 d ih y d r o x y a c e to n e g ly c e r a ld e h y d e -3 -
p h o s p h a te p h o s p h a te
f r u c to s e -1 ,6 -
b is p h o s p h a te
T r io s e p h o s p h a te Is o m e r a s e
Glycolysis -stage 3

Step 6
• Glyceraldehyde-3-phosphate Dehydrogenase catalyzes:
glyceraldehyde-3-P + NAD+ + Pi  1,3-bisphosphoglycerate + NADH + H+

 This is the only step in Glycolysis in which NAD+ is reduced to NADH.

G ly c e ra ld e h y d e -3 -p h o s p h a te
D e h y d ro g e n a se
2 
H O + H + O O PO 3
1 C N A D + N A D H 1 C
+ P i
H C O H H C O H
2 2
2  2 
3 CH 2 O PO 3 3 CH 2 O PO 3

g ly c e ra ld e h y d e - 1 ,3 -b is p h o s p h o -
3 -p h o s p h a te g ly c e ra te
Glycolysis -stage 3

Step 7
 Phosphoglycerate Kinase catalyzes:

1,3-bisphosphoglycerate + ADP  3-phosphoglycerate + ATP

P h o s p h o g ly c e ra te K in a s e
2 
O O PO 3 A D P A TP O O
1C 1
C
H 2
C O H H 2
C O H
2+
2 M g 2
3 CH 2 O PO 3 3 CH 2 O PO 3

1 ,3 -b is p h o s p h o - 3 -p h o s p h o g ly c e ra te
g ly c e ra te
Glycolysis -stage 3

Step 8
 Phosphoglycerate Mutase catalyzes:

3-phosphoglycerate2-phosphoglycerate

Phosphate is shifted from the OH on C3 to the OH on C2

P h o s p h o g ly c e ra te M u ta s e
O O  O O 
1
C 1
C
H 2
C O H H 2
C O PO 3 2 

3 CH 2 O PO 3 2  3 CH 2 O H
3 -p h o s p h o g ly c e ra te 2 -p h o s p h o g ly c e ra te
Glycolysis -stage 3
Step 9
Enolase catalyzes:
2-phosphoglycerate  phosphoenolpyruvate + H2O

E n o la s e
O  H  O  OH O
O O O
C C 1
C
1
H 2 C O P O 32 C O P O 32 2C O P O 32

3 C H 2O H C H 2O H 3CH2
2 -p h o s p h o g ly c e r a te e n o la te in te r m e d ia te p h o s p h o e n o lp y r u v a te
Glycolysis -stage 3

Step 10
 Pyruvate Kinase catalyzes:
phosphoenolpyruvate + ADP  pyruvate + ATP

Irreversible step
P y ru v a te K in a s e
 
O O O O
A D P A T P
1
C 1
C
2
2
C O PO 3 2
C O

3 CH 2 3 CH 3
p h o s p h o e n o lp y ru v a te p y ru v a te
 glucose Glycolysis
ATP UTILIZATION STEP
ATP
Hexokinase
ADP
glucose-6-phosphate
Phosphoglucose Isomerase
fructose-6-phosphate
ATP UTILIZATION STEP
ATP
Phosphofructokinase
ADP
fructose-1,6-bisphosphate
Aldolase

glyceraldehyde-3-phosphate + dihydroxyacetone-phosphate
Triosephosphate
Isomerase
Glycolysis continued
 glyceraldehyde-3-phosphate
NAD+ + Pi Glyceraldehyde-3-phosphate
NADH + H+ Dehydrogenase
1,3-bisphosphoglycerate
ADP ATP GENERATION STEP
Phosphoglycerate Kinase
ATP
3-phosphoglycerate
Phosphoglycerate Mutase
2-phosphoglycerate

H2O Enolase
phosphoenolpyruvate
ADP ATP GENERATION STEP
Pyruvate Kinase
ATP
pyruvate
Glycolysis pathway is regulated by control of 3 enzymes that catalyze
spontaneous reactions:

Hexokinase, Phosphofructokinase & Pyruvate Kinase.

THREE IRREVERSIBLE STEPS IN GLYCOLYSIS:

Hexokinase
1. Glucose Glucose-6-phosphate

Hexokinase has a high affinity for its substrate glucose

2. Phosphofructokinase
Fructose-6-P Fructose-1,6-bisPhosphate

PFK is the rate limiting enzyme in glycolysis

3. Pyruvate kinase
Phosphoenolpyruvate Pyruvate
Fate of Pyruvate
FATE OF PYRUVATE (ANAEROBIC)
1.ETHANOL
 Alcohol fermentation carried out by Yeast and other microorganisms

 NADH is converted to NAD+ in the reaction catalyzed by Alcohol Dehydrogenase ( NAD+ is

regenerated)

 Some anaerobic organisms metabolize pyruvate to ethanol, which is excreted as a waste product.

P y ru v a te A lc o h o l
D e c a rb o x y la s e D e h y d ro g e n a se
O O 
CO 2 H NADH + H+ NAD+ H
C O
C O C H C O H

CH 3 CH 3 CH 3
p y ru v a te a c e ta ld e h y d e e th a n o l
FATE OF PYRUVATE(ANAEROBIC)
2. LACTIC ACID FERMENTATION
 Lactate Dehydrogenase catalyzes reduction of the keto in pyruvate to
a hydroxyl, yielding lactate.
 NADH is oxidized to NAD+.
 Lactate serves as a fuel source for cardiac muscle as well as brain
neurons.

Lactate Dehydrogenase
O O O O
C NADH + H+ NAD+ C
C O HC OH
CH3 CH3
pyruvate lactate
FATE OF PYRUVATE(AEROBIC)
3. ACETYL COA

CH3COCOOH + HSCoA + NAD+

(Pyruvate)
Pyruvate dehydrogenase (CoA as
coenzyme)

CH3CO-SCoA + NADH
(Acetyl-CoA)
Medical and Biological Importance of Glycolysis

• Provides energy to cells. Anaerobic glycolysis meets energy requirement of

rapidly contracting skeletal muscle.
Energy production:degrades glucose to generate ATP
a) anaerobic glycolysis gives 2 ATP.
b) aerobic glycolysis gives 8 ATP.

• Since heart is mainly aerobic organ, myocardial ischemia decreases glycolytic

ability of cardiac muscle. As a result energy or ATP production in heart is
affected.
Medical and Biological importance of glycolysis:

• Deficiency of enzymes of erythrocyte glycolysis (pyruvate

kinase) causes haemolytic anemia. This is because
erythrocytes gets their energy from glycolysis.
Oxygenation of tissues:
Through formation of 2,3 bisphosphoglycerate, which
decreases the affinity of Hemoglobin to O2.
Medical and Biological Importance of
Glycolysis
• Deficiency of muscle phosphofructo kinase causes decreased
muscular performance and fatigue.

• Dietary fructose and galactose are also metabolized by this pathway.

• Glycolysis has amphibolic role : It provides precursors for the

formation of lipids and aminoacids. For example, pyruvate is
converted to alanine by transamination and dihydroxy acetone
phosphate serves as precursor for triglyceride formation.
Biological importance (functions) of
glycolysis:
Glycolysis has amphibolic role : Provides important intermediates/ provides
building blocks for synthesis of cellular components.
a) Dihydroxyacetone phosphate: can give glycerol-3phosphate, which is
used for synthesis of triacylglycerols and phospholipids (lipogenesis).
b) 3 Phosphoglycerate: which can be used for synthesis of amino acid serine.
c) Pyruvate: which can be used in synthesis of amino acid alanine.

4. Aerobic glycolysis provides the mitochondria with pyruvate, which gives

acetyl CoA Krebs' cycle.
Medical and Biological Importance of
Glycolysis
• Two glycolytic intermediates pyruvate and glyceraldehydes-3-
phosphate are used for the synthesis of cholesterol, thiamine and
pyridoxine in tuberculosis, malaria and gastritis causing organisms.

• In brain tumors lactate production is 10 times more.

Medical and Biological Importance of
Glycolysis
• Glycolysis is the major energy source for rapidly growing malarial
parasite in R.B.C.
Lactate is the end product of glycolysis in malarial parasite.
LDH of parasite is different from human enzyme. Unlike human LDH
parasite enzyme is not subjected to inhibition by substrate pyruvate.
This allows rapid formation of lactate from pyruvate and fast energy
production.
How can fructose be used for energy?-entry of fructose in to
glycolysis

To glycolysis
ENTRY POINT OF GALACTOSE TO
GLYCOLYTIC PATHWAY
Phosphorylation of galactose
CH2 OH ATP ADP CH2 OH
O O
OH H OH H
H H
OH H OH H
H OH Galactokinase H OPO3 =

H OH H OH

Galactose Galactose-1-P
ACTIVATION OF GALACTOSE

CH2 OH Glycolysis
O
H H
H
O O
OH H Glucose-6-P
OH O P O P O Uridine
O– O–
H OH
Phosphoglucomutase
UDP-Glucose
CH2 OH CH2 OH
O
Glucose-1-P
O UMP
OH H OH H
H H
O O
OH H Galactose-1-P OH H
H OPO3 = H O P O P O Uridine
Uridylyl Transferase
O– O–
H OH H OH

Galactose-1-P UDP-Galactose
EPIMERIZATION OF UDP-
GALACTOSE

CH2 OH CH2 OH
O O
OH H + H H
[NAD ]
H H
O O
OH H OH H
H O P UMP UDP-Galactose- OH O P UMP
4-Epimerase
O– O
–
H OH H OH

UDP-Galactose UDP-Glucose
 FORMATION OF GLUCOSE-1-P

CH2 OH PPi UTP CH2 OH

O O
H H H H
H H
O O
OH H OH H
OH O P UMP UDP-Glucose OH O P O–
Pyrophosphorylase
O– O–
H OH H OH

UDP-Glucose Glucose-1-P
FORMATION OF GLUCOSE-6-P

CH2 OH CH2 OPO3 2–

O O
H H H H
H H
O
OH H OH H
OH O P O– Phosphoglucomutase OH OH
O–
H OH H OH

Glucose-1-P Glucose-6-P

Glucose-6-P ——> Glycolysis

Diseases associated with impared glycolysis
Lactic acidosis
• Blood levels of lactic acid are normally less than 1.2 mM.
• In lactic acidosis, the values for blood lactate may be 5 mM or more.
• The high concentration of lactate results in lowered blood pH and bicarbonate
levels.
• High blood lactate levels can result from increased formation or decreased
utilization of lactate.
• Common cause of hyperlacticidemia is anoxia. Tissue anoxia may occur in shock
and other conditions that impair blood flow, in respiratory disorders, and in
severe anemia
Diseases associated with impared glycolysis
Hexokinase deficiency
• The red blood cells in these patients contain low concentrations of the glycolytic
intermediates including the precursor of 2,3-DPG.
• In consequence, the hemoglobin of these patients has an abnormally high oxygen
affinity.
• The oxygen saturation curves of red blood cells from a patient with hexokinase
deficiency are shifted to the left, which indicates that oxygen is less available for the
tissues.
Pyruvate kinase deficiency (hemolytic anemia):
• All red blood cells are completely dependent upon glycolytic activity for ATP production.
• Pyruvate kinase deficiency leads to decrease in the production of ATP resulting to
hemolysis of red cells.
• Inadequate production of ATP reduces the activity of the Na+ and K+ stimulated ATPase
ion pump.
Galactosemia
• In order for lactose to be used as an energy source, galactose must be converted to a
phosphorylated glucose molecule

• When enzymes necessary for this conversion are absent, the genetic disease
galactosemia results.

• People who lack the enzyme lactase (~20%) are unable to digest lactose and have the
condition lactose intolerance.
 Galactosemia is an inherited recessive deficiency in enzymes that metabolize
galactose

• Galactosemia = high level of plasma galactose

MILK
the primary source of galactose

http://learn.genetics.utah.edu/units/disorders/whataregd/galactosemia/images/galacpthwy.jpg
GALACTOSE METABOLISM
 NORMAL GALACTOSE METABOLISM
• Dietary lactose is digested into glucose and galactose, and absorbed through the
intestine

• Galactose is taken up by a RBC (carrier-mediated), it is phosphorylated to

Galactose-1-Phosphate (Gal-1-P) by Galactokinase (GALK)

• Gal-1-P is converted to Glucose-1-Phosphate (Glu-1P) using the epimerization of

UDP-Glucose to UDP-Galactose by the enzyme Galactose-1-Phosphate Uridyl
Transferase (GALT). That is:

Gal-1-P + UDP-Glucose GALT UDP-Galactose + Glu-1-P

• Glu-1-P proceeds on to glycolysis

• UDP-Galactose is recycled back to UDP-Glucose by Uridyl Diphosphate Galactose

4-Epimerase (GALE)
Mechanism
1. GALT Deficiency

Deficiency in GALT leads to a

build up of Gal-1-P and UDP-
Glucose.
1. GALT Deficiency

Effects
• Jaundice
• Vomiting & Diarrhea
• Lethargy
• Renal dysfunction
• Hepatomegaly
• Cataract
• Sepsis
• Ovarian failure
• Mental retardation
• Death
GALK DEFICIENCY
Mechanism

Deficiency of GALK leads to

a build up of galactose which
2. GALK Deficiency
can be converted to toxic
galactitol.
 3. GALE Deficiency
Mechanism

Deficiency in GALE leads to a

build up of Gal-1-P and UDP-
Galactose cannot be
converterted back to UDP-
Glucose

Symptoms similar to classic

galactosemia, sometimes with
hearing loss.
DIAGNOSIS
Tests
• Blood tests
• Enzyme activity in RBCs
Normal range for Galactose-1-phosphate uridyl
transferase activity is18.5 to 28.5 U/g Hb.
• Low blood sugar (hypoglycemia)

• Urine analysis
• Reducing substances accumulation (i.e. galactose &
galactose-1-P)
Signs & Symptoms
TREATMENT

• No pharmacological treatment is currently available

• Sources of galactose (especially lactose) must be eliminated from the

diet

• All dairy products (chesses, yoghurt, ice cream), breast milk, infant
formulas, sweeteners
• Foods with > 10mg galactose/100g fresh weight must be avoided;
dates, papaya, tomatoes, watermelon

• Calcium and vitamin supplementation (vitamin D)

 Glucose
synthesis(gluconeogenesis),
glycogenesis and glycogenolysis
Lecture 4
All cells are dependent on glucose.
Glucose level in blood plasma must be stable.
Brain is especially sensitive to the decrease of
glucose deficiency can impair the brain function.

Red blood cells use only glucose as a fuel.

Glucose needed daily by the whole body.

The amount of glucose present in body fluids is about 20 g,

and that readily available from glycogen is approximately 190
g.

During period of fasting glycogen in liver is mobilized but it

only lasts 12 to 24 hours and this source of glucose may not
fulfill metabolic need.

During a longer period of starvation organism must synthesize

glucose from smaller noncarbohydrate precursor molecules.
 Gluconeogenesis – synthesis of glucose from
noncarbohydrate precursors

 Liver and kidney are major sites of glucose synthesis

 Main precursors: lactate, pyruvate, glycerol and some amino acids

 Under fasting conditions, gluconeogenesis supplies almost all of the body’s

glucose
 Gluconeogenesis is not a Reversal Glycolysis
In glycolysis, glucose is converted into pyruvate.

In gluconeogenesis, pyruvate is converted into glucose.

However, gluconeogenesis is not a reversal of glycolysis.

The three irreversible reactions in glycolysis catalyzed by

hexokinase, phosphofructokinase, and pyruvate kinase.
1. Glucose + ATP  glucose-6-phosphate + ADP (hexokinase)

3. Fructose-6-phosphate + ATP  fructose-1,6-biphosphate + ADP

(phosphofructokinase)

10. Phosphoenolpyruvate + ADP  pyruvate + ATP (pyruvate kinase)

These three reactions must be bypassed in gluconeogenesis

 Bypassed Reactions in Gluconeogenesis
1. Phosphoenolpyruvate is formed from pyruvate by way of
oxaloacetate through the action of pyruvate carboxylase and
phosphoenolpyruvate carboxykinase.

Pyruvate + CO2 + ATP + H2O  oxaloacetate + ADP + Pi + 2H+

Oxaloacetate + GTP  phosphoenolpyruvate + GDP + CO2

2. Fructose 6-phosphate is formed from fructose 1,6-

bisphosphate.
Enzyme - fructose 1,6-bisphosphatase.

Fructose 1,6-bisphosphate + H2O  fructose 6-phosphate + Pi

3. Glucose is formed by hydrolysis of glucose 6-phosphate in a

reaction catalyzed by glucose 6-phosphatase.

Glucose 6-phosphate + H2O  glucose + Pi

 Gluconeogenesis
The distinctive reactions are shown in red.
 Bypass I: Step 1(gluconeogenesis)
Pyruvate  Phosphoenolpyruvate
Enzyme pyruvate carboxylase is present only in mitochondria.

The first step in gluconeogenesis is the carboxylation of

pyruvate to form oxaloacetate at the expense of a molecule
of ATP.

Pyruvate is transported into mitochondria from cytoplasm; the

part of pyruvate is formed in mitochondria from amino acids.

Essential cofactor of pyruvate carboxylase is biotin, which

serves as a carrier of CO2.
Pyruvate carboxylase is allosterically activated by acetyl CoA.

Accumulation of acetyl CoA from fatty acid oxidation signals abundant energy,
and directs pyruvate to oxaloacetate for gluconeogenesis.

Pyruvate carboxylase reaction

biotin

This reaction takes place in mitochondria matrix.

 Phosphoenolpyruvate carboxykinase reaction

Reaction takes place in the cytosol.

In decarboxylation reaction GTP donates a phosphoryl group.
Oxaloacetate is simultaneously decarboxylated and
phosphorylated by phosphoenolpyruvate carboxykinase.
One molecule of ATP and one molecule of GTP were spent to
lift pyruvate to the energy level of phosphoenlpyruvate.
Bypass II: Fructose 1,6-bisphosphate 
Fructose 6-phosphate
 A metabolically irreversible reaction
 The enzyme responsible for this step is fructose
1,6-bisphosphatase
 F1,6BPase is allosterically inhibited by AMP and
fructose 2,6-bisphosphate (F2,6BP)
Bypass III: Glucose 6-phosphate  glucose
In most tissues, gluconeogenesis ends with the formation of glucose 6-phosphate
(G-6P).

Glucose 6-phosphate, unlike free glucose, cannot diffuse out of the cell.

The generation of free glucose is controlled in two ways:

 enzyme responsible for the conversion of glucose 6-phos-phate into glucose,

glucose 6-phosphatase, is regulated;

 enzyme is present only in tissues whose metabolic duty is to maintain blood-

glucose homeostasis — liver and to a lesser extent kidney, pancreas, small
intestine.
The final step in the generation of glucose does not take place in the cytosol.

G-6-P is transported into the lumen of the endoplasmic reticulum, where it is

hydrolyzed by glucose 6-phosphatase, which is bound to the membrane.

Glucose 6-phosphatase reaction

 Subcellular Locations of
Gluconeogenic Enzymes
 Gluconeogenesis enzymes are cytosolic
except:
(1) Glucose 6-phosphatase (endoplasmic
reticulum)
(2) Pyruvate carboxylase (mitochondria)
(3) Phosphoenolpyruvate carboxykinase
(cytosol and/or mitochondria)
 Regulation of Gluconeogenesis
Gluconeogenesis and glycolysis are reciprocally regulated - within a cell one
pathway is relatively inactive while the other is highly active.

The amounts and activities of the distinctive enzymes of each pathway are
controlled.

The rate of glycolysis is determined by the concentration of glucose.

The rate of gluconeogenesis is determined by the concentrations of

precursors of glucose.
Regulation of the Enzymes Amount by Hormones

Hormones affect gene expression primarily by changing

the rate of transcription.

Insulin, which rises subsequent to eating, stimulates the

expression of phosphofructokinase and pyruvate kinase.

Glucagon, which rises during starvation, inhibits the

expression of these enzymes and stimulates the production
of phosphoenolpyruvate carboxykinase and fructose 1,6-
bisphosphatase.
 Precursors for Gluconeogenesis

 Any metabolite that can be converted to

pyruvate or oxaloacetate can be a glucose
precursor
 Major gluconeogenic precursors in mammals:
(1) Lactate
(2) Most amino acids (especially alanine),
(3) Glycerol (from triacylglycerol hydrolysis)
Regulation of Glycolysis and Gluconeogenesis
• High glucose levels and insulin promote glycolysis
• Low glucose levels and glucagon promote gluconeogenesis
Glycogenesis
Glycogen is a highly branched glucose polymer used for carbohydrate
storage in animals

Glycogen stores are used to keep the blood sugar level steady
between meals

Glycogenesis is the synthesis of glycogen from glucose-6-phosphate.

Glycogenesis
Glycogenesis pathways are also responsible for converting
noncarbohydrate precursors / glucose to glycogen.
 It occurs when high levels of glucose-6-phosphate are formed
in the first reaction of glycolysis.
 It does not operate when glycogen stores are full, which means
that additional glucose is converted to body fat.
 Diagram of Glycogenesis
• Glucose is converted to
glucose-6-phosphate,
using one ATP

• Glucose-6-phosphate is
converted to glucose-1-
phosphate, which is
activated by UTP, forming
UDP-glucose

• As UDP-glucose attaches
to the end of the glycogen
chain, UDP is released
(and converted to UTP by
ATP)
Glycogenolysis
• Breakdown of glycogen to glucose

• The glucose is phosphorylated as it is cleaved from the glycogen to form glucose-1-

phosphate

• Glucose-1-phosphate can be converted to glucose-6-phosphate, which can enter

glycolysis

• Phosphorylated glucose can’t be absorbed into cells

• In the liver and kidneys, glucose-6-phosphate can be hydrolized to glucose

Glycogenolysis
Glycogenolysis is activated by;
glucagon in the liver
epinephrine in muscles
 These hormones are produced when blood glucose levels are
low

Glycogenolysis is inhibited by;

insulin produced when blood glucose levels are high
Enzymes involved in glycogenolysis
Glycogen phosphorylase
-catalyzes cleavage of the 1→4 linkages of glycogen to yield glucose-1-
phosphate
α(1→4) glucan transferase
transfer a trisaccharides unit from one branch to the other.
Debranching enzyme
hydrolysis of the 1→6 linkages
Overview of Glycogen Synthesis and Breakdown
Glycogen Storage Diseases

• These are group of inherited (genetic) diseases of glycogen metabolism.

• In these diseases,there is an abnormal accumulation of large amount of glycogen or its
metabolites in the tissues due to deficiency or absence of enzymes of glycogen
metabolism.
• Some of them are not serious mild disorders but few of them are fatal.
• (a) Von Geirke’s disease (Type-1 glycogen storage disease)
• It is due to the deficiency or absence of glucose-6-phosphatase in liver, kidney and
intestine
• Lack of glucose-6-phosphatase cause accumulation of glycogen in liver and kidney and
enlargement of liver occurs
• Hypoglycemia is common symptom other symptoms are hyperuricemia, hyperlipemia
• and ketosis.
Glycogen Storage Diseases
b) Pompe’s disease
It is due to the deficiency of lysosomal α-glucosidase.
• Lysosomes can not utilize glycogen and accumulation of glycogen occurs in all tissues. Accumulation
of glycogen in heart leads to cardiomegaly.
• It is a fatal disorder and death occurs before second year of life due to cardio respiratory failure.
(c) Cori’s disease
It is due to deficiency or absence of debranching enzyme.
• Limit dextrin a metabolite of glycogenolysis accumulates in liver. Hence, this condition is also called
as limit dextrnosis.
(d) Anderson’s disease
It is a fatal disease. It is due to absence of branching enzyme.
• Amylopectin an intermediate of glycogenesis accumulates in liver, spleen and heart.
• Hence, this condition is called as amylopectinosis.
Glycogen Storage Diseases
(e) Mc Ardle’s syndrome
• It is due to the absence of muscle phosphorylase.
• Glycogen accumulates in muscle and lactic acid production in muscle
is not increased after exercise.
• Affected person suffer from painful muscle cramps and diminished
tolerance to exercise.
(f) Her’s disease
It is due to the absence of liver phosphorylase.
• Glycogenolysis is defective and glycogen accumulates in liver.
Cori Cycle
 When anaerobic conditions occur in active muscle, glycolysis
produces lactate

 The lactate moves through the blood stream to the liver, where it is
oxidized back to pyruvate.

 Gluconeogenesis converts pyruvate to glucose, which is carried back

to the muscles

 The Cori cycle is the flow of lactate and glucose between the muscles
and the liver
Pathways for Glucose
 Kreb’s Cycle
(aka, tricarboxylic acid (TCA)cycle, citric
acid cycle)
Overall goal
Makes ATP

Makes NADH

Makes FADH2

Requires some carbohydrate to run

Geography
• Glycolysis in the cytosol
• Krebs in mitochondrial matrix
• Mitochondrion
• Outer membrane very permeable
• Space between membranes called intermembrane space
• Inner membrane (cristae)
• Permeable to pyruvate,
• Impermeable to fatty acids, NAD, etc
• Matrix is inside inner membrane
Conversion of pyruvate to Acetyl CoA
NAD+ NADH HSCoA CO2 O
O
O SCoA
H3C H3C
O
pyruvate dehydrogenase complex acetyl CoA

pyruvate

• 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

Fates of Acetyl CoA
O
Kreb's
TAG's H3C SCoA
acetyl CoA CO2, ATP, NADH...energy

no CHO present

ketone bodies

 In the presence of CHO an using energy

• Metabolized to CO2, NADH, FADH2,GTP and, ultimately, ATP
 If energy not being used (Lots of ATP present)
• Made into fat
 If energy being used, but no CHO present
• Starvation
• Forms ketone bodies (see fat metabolism slides)
• Danger!
8 enzymes involved in TCA cycle reactions

• Citrate synthase
• Aconitase
• Iso-citrate dehydrogenase
• a ketoglutarate dehydrogenase
• Succinyl-Coenzyme A synthetase
• Succinate dehydrogenase
• Fumerase
• Malate dehydrogenase
Kreb’s Cycle
acetyl CoA
H3C SCoA
C
O O
O
CoASH C
O O O H2O
O + CH2 O
C
C NAD
NADH
HO C C O
C O citrate synthase
HC OH CH2
CH2
H2O CH2 malate C citrate
dehydrogenase
C O O
O O C O
O
fumarase O
C O oxaloacetate
C H malate
aconitase
H C fumarate

O
C
O Kreb's Cycle O O
FADH2 C
succinate
dehydrogenase HO CH O
HC C O
FAD alpha ketoglutarate CH2
O SCoA O C isocitrate
O NADH O O
C GTP NAD C
CoASH C NADH O O
CH2 CH2 C O CO2 NAD
CH2 GDP CH2
CH2 CoASH
C C CO2 CH2
succinyl CoA isocitrate dehydrogenase
O O synthetase O O alpha ketoglutarate C
succinate succinyl CoA
dehydrogenase
O
O
Step 1: Citrate Synthase Reaction
O O O
C O
C
C O H2O CoASH
CH2 CH2 O
H3C SCoA
C C HO C C O
O + O
O
citrate synthase CH2
C
O O
acetyl CoA oxaloacetate
citrate

Binding of Oxaloacetate to the enzyme results in conformational change.

 This facilitates the binding of the next substrate, the acetyl Coenzyme A thus
forming Citrate.
Step 2:Aconitase Reaction
O O O O
C C
CH2 O
HO CH O
HO C C O HC C O
CH2 aconitase CH2
C C
O O
O O

citrate isocitrate

 This enzyme catalyses the isomerization reaction by removing and then adding
back the water ( H and OH ) to cis-aconitate in at different positions.

 Goes through alkene intermediate (cis-aconitate)

elimination then addition

 Forms isocitrate
Step 3: Isocitrate Dehydrogenase
O O
O O C
C C O
HO CH O
NAD NADH CO2 CH2
HC C O CH2
CH2
C C
O O isocitrate dehydrogenase O O

isocitrate alpha ketoglutarate

• All dehydrogenase reactions make NADH or FADH2

• Oxidative decarboxylation
Step 4: α-ketoglutarate dehydrogenase
O O
C SCoA O
C O
C
CH2 CO2 NADH
CoASH NAD CH2
CH2
CH2
C C
O O O O
alpha ketoglutarate
dehydrogenase
alpha ketoglutarate succinyl CoA

 Same as pyruvate dehydrogenase reaction

 Formation of thioester
• endergonic
• driven by loss of CO2
 Converts alpha-Keto gluterate to Succinyl-COA

 NAD+ is an electron acceptor.

Step 5: Succinyl CoA synthetase
SCoA O O
O
C C
CH2 GTP CoASH CH2
GDP
CH2 CH2
C C
O O succinyl CoA O O
succinyl CoA synthetase
succinate

 Hydrolysis of thioester
• Releases CoASH
 Converts succinyl COA to succinate

 Coupled to synthesis of GTP

• GTP very similar to ATP and interconverted later
 Hydrolysis of the thioester bond leads to the formation of phosphoester bond with inorganic phosphate.
Step 6: Succinate dehydrogenase
O O O
O
C FADH2 C
CH2 FAD C H
CH2 H C
C succinyl CoA
C
O O
O O dehydrogenase

succinate fumarate
 Dehydrogenation
 Uses FAD (FAD dependent enzyme).

 NAD used to oxidize oxygen-containing groups

• Aldehydes
• alcohols
 FAD used to oxidize C-C bonds
 Oxidation of succinate to fumarate.
 The only citric acid cycle enzyme that is tightly bound to the inner mitochondrial membrane.
Step 7:Fumarase
O O O O
C H2 O C
C H HC OH
H C CH2
C fumarase
C
O O O
O
fumarate
malate

Addition of water to a double bond

Hydration of Fumarate to malate

Step 8: Malate Dehydrogenase
O O
C O O
HC OH C
NAD NADH C O
CH2
CH2
C C
O O malate O
dehydrogenase O
malate
oxaloacetate

Oxidation of secondary alcohol to ketone (Oxidation of malate to oxaloacetate)

Regenerates oxaloacetate for another round
Makes NADH
 It is an NAD+dependent enzyme.
Pentose phosphate
pathway(PPP)
BBY 1208-Lecture 4

92
Pentose Phosphate Pathway
Also known as:
Pentose shunt

Hexose monophosphate shunt

Phosphogluconate pathway

It occurs in the cytosol.

Pentose phosphate pathway as shunt.
The pathway begins with the glycolytic intermediate
glucose 6-P.
One fate of G6P is the
pentose pathway.
Pentose phosphate pathway as shunt.
 PPP reconnects with glycolysis because two of the end products of the
pentose pathway are glyceraldehyde 3-P and fructose 6-P two intermediates
further down in the glycolytic pathway.

 It is for this reason that the pentose pathway is often referred to as a shunt.
 Moderate glucose flux

Glycolysis
only
 Large glucose flux

Pentose
Phosphate
Pathway
Glycolysis
 ROLES OF PPP
 For formation of NADPH for synthesis of fatty acid and steroids.

 The pathway yields reducing potential in the form of NADPH to be used in

anabolic reactions requiring electrons.

 The pathway yields ribose 5-phosphate and ribose essential for nucleic acids
formation e.g.DNA , RNA and various cofactors (CoA, FAD, NAD+/NADP+).

99
PATHWAYS REQUIRING NADPH

100
TISSUES WITH ACTIVE PPP

101
TWO PHASES OF PPP
1. Oxidative Phase

102
 OXIDATIVE PHASE

NADPH + H+ is formed from two separate reactions.

The glucose 6-phosphate DH (G6PD) reaction is the

rate limiting step and is essentially irreversible.

There is a medical story for G6PD enzyme.

Cells have a greater need for NADPH than ribose 5-

phosphate.
 OXIDATIVE PHASE

G lucose-6-phosphate 6-Phospho- O O
D ehydrogenase glucono- 1C
6 CH 2 O PO 3 2 6 CH O PO 2 lactonase
+ 2 3
NADPH + H HC OH
H
5 O O H NADP
+
H
5 O H 2O 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 CH 2 O PO 3 2
6
glucose-6-phosphate 6-phoshogluconolactone 6-phosphogluconate

 Glucose-6-phosphate Dehydrogenase catalyzes oxidation of the aldehyde at

C1 of glucose-6-phosphate, to a carboxylic acid(lactone).

 NADP+ serves as electron acceptor.

 6-Phosphogluconolactonase catalyzes hydrolysis of the ester linkage,

resulting in ring opening. The product is 6-phosphogluconate.
 O O Phosphogluconate
1C Dehydrogenase
HC OH 1CH 2 O H
2 NADP +
NADP H + H +
HO CH
3
C O
2
HC OH HC OH
4 3
HC OH CO 2 HC OH
5 4
CH 2 O PO 3 2 CH 2 O PO 3 2
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.

2. Non oxidative phase
 Involves interconversion of sugars.
 Ribulose-5-P converted 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.
Transketolase : catalyze transfer of 2-C molecular
fragments from a ketose donor to an aldose acceptor.
Transaldolase: catalyze transfer of 3-C molecular
fragments from a ketose donor to an aldose
acceptor.
THE NON-OXIDATIVE PHASE
CH 2O H

C O
E pim erase
HO C H

CH H C OH
2O H
2
C O CH 2O P O 3
xylulose -5 -
H C OH ph osp hate
H C OH HC O
2 H C OH
CH 2O P O 3
ribulose -5 - H C OH
ph osp hate Iso m erase
H C OH
2
CH 2O P O 3
ribose -5-
ph osp hate

 Epimerase :inter-converts stereoisomers ribulose-5-P and xylulose-5-P.

 Isomerase :converts the ketose ribulose-5-P to the aldose ribose-5-P.
 Both reactions are reversible.
107
 CH 2 O H
T ran sk eto lase
C O

CH 2 O H 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

CH 2 O P O 3 2  CH 2 O P O 3 2  CH 2 O P O 3 2  CH 2 O P O 3 2 

x ylu lo se - rib o se - glyc e ra ld e h yd e - se d o h e p tu lo se -

5 -p h o sp h a te 5 -p h o sp h a te 3 -p h o sp h a te 7 -p h o sp h a te

 Transketolase
Transfers a 2-C fragment from xylulose-5-P to ribose-5-P to form
sedoheptulose-7-phosphate.
 C H 2O H
T ran sald o lase
C O H 2C 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
2 2 2 2
H 2C O PO 3 H 2C O PO 3 H 2C O PO 3 H 2C O PO 3

se d o h e p tu lo se - g ly c e ra ld e h y d e - e ry th ro se - fru c to se -
7 -p h o sp h a te 3 -p h o sp h a te 4 -p h o sp h a te 6 -p h o sp h a te

 Transaldolase
Catalyzes transfer of a 3-C dihydroxyacetone moiety, from
sedoheptulose-7-phosphate to glyceraldehyde-3-phosphate to yield
fructose-6-phosphate.
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:
 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.

2 NADP+ 2 NADPH + CO2

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

Pentose Phosphate Pathway producing

NADPH and ribose-5-phosphate
 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.

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.

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
OXIDATIVE AND NON-OXIDATIVE PHASES

113
Oxidative and non-oxidative Ppp
What happens if glucose 6-phosphate Dehyrogenase
is defective?

Insufficient production of NADPH.

Which translates into insufficient glutathione.

Is this a medical problem?

 YES
GLUTATHIONE AND
ERYTHROCYTES
GSH is extremely important particularly in the
highly oxidizing environment of the red blood cell.

Mature RBCs have no mitochondria and are totally

dependent on NADPH from the pentose
phosphate pathway to regenerate GSH from GSSG
via glutathione reductase.

10% of glucose consumption by erythrocytes is

mediated by the pentose pathway.
Glutathione and Erythrocytes
• The reduced form of glutathione serves as a
sulfhydryl buffer.
• It maintains cysteine residues in hemoglobin and
other proteins in a reduced state.
• GSH is essential for normal RBC structure and
keeping hemoglobin in Fe++ state.
GLUTATHIONE AND ERYTHROCYTES

Cells with low levels of GSH are susceptible to hemolysis.

Individuals with reduced GSH are subject to hemolysis.

This is often clinically seen as black urine under certain conditions.

CONDITIONS FOR HEMOLYTIC
ANEMIA RELATED G6PD DEFICIENCY.
The ingestion of oxidative agents that generate
peroxides or reactive oxygen species (ROS).
o Antimalarials - pamaquine
o purine glycoside from fava beans.

Individuals with G6PD deficiency can not produce

sufficient GSH to cope with the ROS.

Proteins become cross linked leading to cell lysis.

REGULATION OF THE PENTOSE
PATHWAY
Glucose 6-phosphate DH is the regulatory enzyme.

NADPH is a potent competitive inhibitor of the

enzyme.

Usually the ratio NADPH/NADP+ is high so the

enzyme is inhibited.

With increased demand for NADPH, the ratio

decreases and enzyme activity is stimulated.