CATABOLISM OF GLUCOSE

UNIVERSITY OF ZAMBIA
SCHOOL OF NATURAL SCIENCES
CHEMISTRY DEPARTMENT
JUNE 2020

METABOLISM OF GLUCOSE

LECTURE NO 2
CATABOLISM OF GLUCOSE

GLYCOLYSIS
Phase II
What are the stages of Glycolysis?

 The second phase of glycolysis is made up of last 5 steps.

 It is also called the pay off phase , which includes the energy conserving
phosphorylation steps in which some of the free energy of the glucose is
conserved in the form of 𝐴𝑇𝑃.

 The two molecules of glyceraldehyde 3-phosphate produced in the

preparatory phase of glycolysis are oxidized and phosphorylated to
produces 4𝐴𝑇𝑃 𝑎𝑛𝑑 2𝑁𝐴𝐷𝐻.

 Therefore overall 2ATP molecules are produced (4ATP-2ATP).

What is step 1 of payoff Phase of glycolysis?-
the sixth step of glycolysis
 Step 1 of the payoff phase or the sixth step of of glycolysis
is the reversible oxidative phosphorylation of the two
molecules of Glyceraldehyde-3-Phosphate to form two molecules of
high energy intermediate compound called 1,3–bisphosphoglycerate.
 The conversation of glyceraldehyde 3-phosphate to 1,3 -
bisphosphoglycerate by glyceraldehyde 3- phosphate dehydrogenase is
the first oxidation reduction reaction of glycolysis .
 Glyceraldehyde-3 -phosphate dehydrogenase (GAPDH)
is a key enzyme in the glycolytic pathway, catalyzing the reversible
oxidative phosphorylation of glyceraldehyde-3-phosphate (GAP) to 1,3
-bisphosphoglycerate using NAD+ as a redox cofactor.
 GAPDH has a reactive cysteine residue at position 149 which is readily
acylated in the first step of the reaction sequence.
 Because of its reactive cysteine residue, GAPDH is particularly
vulnerable to the effects of oxidants.
What is step 1 of payoff Phase of glycolysis?-
the sixth step of glycolysis

 What happens in this reaction is that the aldehyde group of

glyceradehyde -3-phosphate is dehydrogenated to a carboxylic acid
anhydride with phosphoric acid .
 This type of anhydride is called an acyl phosphate .
 This reaction has very high free energy of hydrolysis of ∆𝐺 0 = −49.3𝐾𝑗/
𝑚𝑜𝑙.
 The hydrogen ion removed from Glyceradehyde -3-phosphate is accepted
by 𝑁𝐴𝐷 + to form 𝑁𝐴𝐷𝐻 and Glyceradehyde -3-phosphate becomes
1,3 – bisphosphoglycerate.
What is step 1 of payoff Phase of glycolysis?-
the sixth step of glycolysis
• Note that this reaction is catalyzed by multifunctional
enzyme, glyceraldehyde 3-phosphate dehydrogenase.
• This is the first of the two energy-conserving reactions of
glycolysis that eventually leads to the formation of ATP
• Oxidative phosphorylation is when ATP is generated from the
oxidation of NADH and FADH2 and the subsequent transfer of
electrons and pumping of protons
O
O H NADH + H+ –
O NAD+ O O P O
C
C –
HCOH O – O
+ HO P O HCOH O
–
CH2 O P O –
–
O
–
Glyceraldehyde CH2 O P O
O
3-phosphate –
Glyceraldehyde Inorganic
O

phosphate dehydrogenase1, 3-
3-phosphate
bisphosphoglycerate
ΔG’O = 6.3 kJ/mol
6
What is step 1 of payoff Phase of glycolysis?-
the sixth step of glycolysis

 The mechanism of this reaction is that the aldehyde group

of Glyceradehyde-3-phosphate reacts with the 𝑆𝐻 group of a cysteine
residue on the active site of enzyme to form a thiohemiacetal that is
converted to a thioester bond by oxidation.
 The hydrogen ions removed being transferred to 𝑁𝐴𝐷 + bound to
enzyme.
 The formed thioester is a high energy bond.
 After phosphorolysis this bond becomes a high energy phosphate bound
at position C1.
 The high energy phosphate group at carbon 1 of 1,3 -
bisphosphoglyccerate (1,3 − 𝐵𝑃𝐺) conserves much of the free energy
produced by the oxidation of Glyceradehyde – 3 – phosphate.
 The energy of this high energy phosphate drives the synthesis of 𝐴𝑇𝑃 in
the next reaction of glycolysis.
What are some of the other functions of
Glyceraldehyde-3-Phosphate dehydrogenase?
 Glyceraldehyde-3 -phosphate dehydrogenase (GAPDH)
is located mainly in the cytoplasm.

 It primarily exists as a tetrameric structure composed of four identical

37 kDa subunits.

 GAPDH has other functions such as

 Regulating cell proliferation
 DNA repair
 tRNA export
 Membrane fusion and transport,
 Endocytosis and nuclear membrane assembly
 Cell death
 Involved in immunity and senescence
What is the effect of oxidants on glyceraldehyde
3- phosphate dehydrogenase?
 Oxidants can oxidize he reactive thiol group of cysteine-149
of Glyceraldehyde-3 -phosphate dehydrogenase.

 The oxidants include compounds such as

 Hydrogen peroxide,

 Other reactive oxygen species,

 Nitric oxide or compounds releasing nitric oxide.

 Oxidation of the thiol group of cysteine-149 of Glyceraldehyde-3 -

phosphate dehydrogenase by oxidants leads to loss of enzymatic
activities of of Glyceraldehyde-3 -phosphate dehydrogenase.
 T

.
What is the effect of arsenate on
glyceraldehyde 3- phosphate dehydrogenase?
 Arsenate is a very common environmental pollutant.
 It has the structure similar to inorganic phosphate.
 Therefore in the presence of arsenate, there is competition between
arsenate and inorganic phosphate for Glyceraldehyde – 3 p
dehydrogenase.

 Arsenate may bind Glyceraldehyde–3-P dehydrogenase and the enzyme

arsenylates Glyceraldehyde – 3-P forming a complex 1-asenate-
Glyceraldehyde–3-P which spontaneously hydrolyses to form 3 –
phosphoglycerate.

 Therefore 1,3 - bisphosphoglyccerate (1,3 − 𝐵𝑃𝐺) is not formed

consequently ATP and NADH are not formed.

 In other words the pentavalent arsenic (arsenate) prevents net

𝐴𝑇𝑃 𝑎𝑛𝑑 𝑁𝐴𝐷𝐻 production by glycolysis.
What is the 2,3-bisphosphoglycerate shunt
 2,3 − BPG Shunt

 In the red blood cell is an enzyme called 2,3 −bisphosphoglycerate

mutase.

 This enzyme isomerises 1,3 - bisphosphosphoglycerate converting it to

2, 3 – bisphosphoglycerate (2,3 − 𝐵𝑃𝐺).

 Therefore 2,3 − 𝐵𝑃𝐺 is found in large amounts in the erythrocytes and is

 found in only traces amounts in most cells.

 2,3 𝐵𝑃𝐺 is hydrolyzed by a phosphatase to 3 – phosphoglycerate, which is

also an intermediate in glycolysis.

 In 𝑅𝐵𝐶, glycolysis is modified by inclusion of this shunt reactions.

What is the importance of 2,3-BPG shunt in red
blood cells

 2,3 − 𝐵𝑃𝐺 lowers the oxygen affinity of hemoglobin.

 𝐵𝑃𝐺 lowers the oxygen affinity of hemoglobin, which is essential in

enabling hemoglobin to unload oxygen in tissue capillaries.

 𝐵𝑃𝐺 diminishes the oxygen affinity of hemoglobin by binding to

deoxyhemoglobin, stabilizing the 𝑇 − 𝑠𝑡𝑟𝑢𝑐𝑡𝑢𝑟𝑒 but not the oxygenated
form.

 Hemoglobin tetramer binds one molecule of 𝐵𝑃𝐺 in the central cavity

formed by its four subunits.
What is step 2 of payoff Phase of glycolysis?-
the seventh step of glycolysis

 Step 7 is the reversible synthesis of 3 phosphoglycerate.

 In this reaction a phosphoryl transfer from 1,3 bisphosphoglycerate to
𝐴𝐷𝑃 𝑜𝑐𝑐𝑢𝑟𝑠 𝑟𝑒𝑠𝑢𝑙𝑡𝑖𝑛𝑔 𝑖𝑛 𝑝𝑟𝑜𝑑𝑢𝑐𝑡𝑖𝑜𝑛 𝑜𝑓 𝐴𝑇𝑃.
 The enzyme phosphoglycerate kinase transfers the high energy
phosphoryl group from carbonyl group of 1,3-phosphhoglycerate to 𝐴𝐷𝑃,
forming 𝐴𝑇𝑃 𝑎𝑛𝑑 3 − 𝑝ℎ𝑜𝑠𝑝ℎ𝑜𝑔𝑙𝑦𝑐𝑒𝑟𝑎𝑡𝑒.
 The formation of 𝐴𝑇𝑃 by phosphoryl group 1,3-bis phosphoglycerate is
referred to as substrate level phosphorylation.
 It actual involves soluble enzymes and chemical intermediates.
 Substrate-level phosphorylation is directly phosphorylating ADP with a
phosphate and energy provided from a coupled reaction.
 Note that because two molecules of 1,3 𝐵𝑃𝐺 are formed from each glucose
molecule, this kinase reaction produces 2ATP molecules thus replaces
the two 𝐴𝑇𝑃 molecules consumed by the earlier formation of glucose 6-
phosphate and fructose 1,6 bisphosphate.
What is step 2 of payoff Phase of glycolysis?-
the seventh step of glycolysis

 Note that respiratory linked phosphorylation involves

membrane bound enzymes and transmembrane gradients of protons.

 Oxidative phosphorylation is when ATP is generated from the oxidation

of NADH and FADH2 and the subsequent transfer of electrons and
pumping of protons.

 Step 7 is an example of substrate level phosphorylation in which the

production of a high energy phosphate is coupled directly to the oxidation
of a substrate instead of resulting from oxidative phosphorylation via the
electron transport chain
What is step 2 of payoff Phase of glycolysis?-the
seventh step
O
of glycolysis – O
–

–
P O O
–
Mg 2+ O O
O O P O C P
C – P +
O + Phosphoglycerate HCOH O
–
P
HCOH O O
– kinase CH2 O P O
CH2 O P O – P
Rib Adenine O
–
O O

1, 3-bisphosphoglycerate ADP 3-phosphoglycerate Rib Adenine

ATP
ΔG’O = – 18.5 kJ/mol
• The enzyme phosphoglycerate kinase transfers the high-energy
phosphoryl group from carboxyl group of 1, 3-
bisphosphoglycerate to ADP, forming ATP and 3-
phosphoglycerate
• The formation of ATP by phosphoryl group transfer from a
substrate such as 1, 3 bisphosphoglycerate is referred to as
substrate-level phosphorylation
– Involves soluble enzymes and chemical intermediates
• Respiratory-linked phosphorylation involves membrane bound
enzymes and transmembrane gradients of protons
15
What is step 3 of payoff Phase of glycolysis?-
the eighth step of glycolysis

 Step 8 is the reversible conversion of 3-phosphoglycerate

to 2-phosphoglycerate.

 This reaction occurs in two steps.

 The phosphoryl group initially attached to a histidine residue of the

mutase is transferred to the hydroxyl group at 𝐶 − 2 of 3 -
phosphoglycerate forming 2,3 bisphosphoglycerate (𝐵𝑃𝐺).

 The phosphoryl group at c3 of BPG is then transferred to the same

histidine residue producing 2 phosphorylated and regenerating the
phosphorylated enzyme
What is step 3 of payoff Phase of glycolysis?-
the eighth
–
step of glycolysis
O O
–

O O C O
C Mg 2+
–
HC O P O
HCOH O Phosphoglycerate –
– mutase CH2OH O
CH2 O P O
–
O
3-phosphoglycerate 2-phosphoglycerate

ΔG’O = 4.4 kJ/mol

• The reaction occurs in two steps

– A phosphoryl group initially attached to a histidine residue of
the mutase is transferred to the hydroxyl group at C-2 of 3-
phosphoglycerate, forming 2, 3 bisphosphoglycerate (BPG)
– The phosphoryl group at C-3 of BPG is then transferred to the
same histidine residue, producing 2-phosphoglycerate and
regenerating the phosphorylated enzyme

17
What is step 4 of payoff Phase of glycolysis?-
the ninth step of glycolysis

 Despite the relatively small free energy change of reaction

there is a very large difference in the standard free energy of hydrolysis
between the reactant and product −17.6𝐾𝑗/𝑚𝑜𝑙 for
2 - phosphoglycerate and −16.9𝐾𝑗/𝑚𝑜𝑙 for phosphoenol pyruvate.

 The loss of water from 2-phosphoglycelate causes a redistribution of

energy within the molecule resulting in increased standard free energy
of hydrolysis of the phosphate group enolase is inhibited by fluoride.

 The fluoride ions inhibits the enolase enzyme by combining with Mg2+.

 It is for this reason that fluoride ions are often included in blood
collection tubes to prevent metabolism of glucose during blood
transport and storage in clinical laboratory.
What is step 4 of payoff Phase of glycolysis?-
the ninth– step of glycolysis –
O O O O
C O C O
– –
HC O P O C O P O
H2O
– –
CH2OH O CH2 O

2-phosphoglycerate Enolase

Phosphoenolpyruvate
ΔG’O = 7.5 kJ/mol

• Despite the relatively small free-energy change of the reaction, there is a very
large difference in the standard free-energy of hydrolysis between the reactant
and product: – 17.6 kJ/mol for 2-phosphoglycerate and – 61.9 kJ/mol for
phosphoenolpyruvate

19
What is step 5 of payoff Phase of glycolysis?-
the tenth step of glycolysis

 The last step involves the transfer of a phosphoryl group from

phosphoenol pyruvate to 𝐴𝐷𝑃 catalyzed pyruvate kinase.
 The enzyme requires 𝐾 + , 𝑀𝑔2+ , 𝑀𝑛2+ second substrate level
phosphorylation stage in glycolysis.

 Overall reaction has a large negative standard free energy change due in
part to spontaneous conversion of the enol form of pyruvate to the keto
form.

 Of the overall free energy change for hydrolysis of phosphoenol pyruvate

(−61.9𝐾𝑗/𝑚𝑜𝑙) about half (−30.5𝐾𝑗/𝑚𝑜𝑙) is conserved in formation of
phosphohydride bonds of ATP and the rest constitutes a large driving
force pushing the reaction towards ATP formation.
 The reaction under cellular cautions is irreversible.
 The overall there is a net gain of ATP
What is step 5 of payoff Phase of glycolysis?-
the tenth step of glycolysis
–
– O O
– O
O O O C
C O ADP C
– Mg 2+, K+ C O + ATP
C O O C OH
– Pyruvate Tautomerization CH3
CH2 O CH2
kinase
Pyruvate Pyruvate
Phosphoenolpyruvate
(enol form) (keto form)
ΔG’O = –31.4 kJ/mol

• The last step involves the transfer of a phosphoryl group from

phosphoenolpyruvate to ADP, catalyzed by pyruvate kinase

• Pyruvate kinase requires cofactors such as K+, Mg 2+ or Mn 2+

• This is a Second substrate-level phosphorylation stage in glycolysis

21
What is step 5 of payoff Phase of glycolysis?-
the tenth step of glycolysis
–
– O O
– O
O O O C
C O ADP C
– Mg 2+, K+ C O + ATP
C O O C OH
– Pyruvate Tautomerization CH3
CH2 O CH2
kinase
Pyruvate Pyruvate
Phosphoenolpyruvate
(enol form) (keto form)
ΔG’O = –31.4 kJ/mol
• Overall reaction has a large, negative standard free-energy change due
in part to the spontaneous conversion of the enol form of pyruvate to
the keto form

• Of the overall free-energy change for hydrolysis of

phosphoenolpyruvate (– 61.9 kJ/mol) about half (– 30.5 kJ/mol) is
conserved in the formation of phosphoanhydride bonds of ATP and
the rest constitutes a large driving force pushing the reaction towards
ATP formation.
• The reaction under cellular conditions is irreversible
22
What is the equation for Glycolysis?

Overall there is a net gain of ATP

Glucose + 2ATP + 2NAD+ + 4ADP + 2Pi 2 pyruvate + 2ADP + 2NADH + 2H+
+4ATP + 2H2O

Cancelling out the common terms on both sides gives the overall
equation for glycolysis under aerobic conditions as:

Glucose + 2NAD+ + 2ADP + 2Pi 2 pyruvate + 2NADH + 2H+

+2ATP + 2H2O
Two molecules of NADH formed by glycolysis in the cytosol are, under
aerobic conditions, re-oxidized to NAD+ by transfer of electrons to the
respiratory chain, located in the mitochondria and these electrons are
passed on to O2

23
What is function of glycolysis?
 The main function of glycolysis is energy (ATP) production.
 Glycolysis also provides substrate for further oxidation, pyruvate which is
further oxidized to produce NADH, FADH2 and GTP.
 The intermediates and products of Glycolysis can provide substrates for
other pathways.
 For instance dihydroxyacetone phosphate(DHAP) an intermediate of
glycolysis can used in adipose tissues converting it into glycerol 3-
phosphate, which is a critical substrate in the pathways of triacylglycerol
and glycerophospholipid synthesis.
 Red blood cells use 1,3-bisphosphoglycerate, another intermediate in
glycolysis, to generate 2,3-bisphosphoglycerate, which is an allosteric
regulator of the interaction of oxygen with hemoglobin.
 Pyruvate can be transminated to produce the amino acid alanine.
 In addition, acetyl-CoA, produced by the mitochondria1 oxidation of
pyruvate, is a substrate for the synthesis of both fatty acids and
cholesterol.
At what level do other carbohydrate enter the
of glycolytic pathway?
 Glucose is the most abundant fuel used in glycolysis

 However other carbohydrates such as:

 Glycogen

 Sucrose

 Lactose

 D-Galactose

 D-Mannose

 D-Fructose
 Feed into the glycolytic pathway at different entry points
 Feeder pathways for glycolysis
D-Galactose
Lactose Gylcogen
Lactase Pi UDP-galactose
Phosphorylase
UDP-glucose
Glucose 1-phosphate
D-Glucose Phosphohexose
(gluco)mutase)
Glucose 6-phosphate D-Mannose
Sucrose
Sucrase ATP
Hexokinase
D-Fructose ATP Fructose 6-phosphate
Mannose 6-
Fructokinase ATP phosphate
Fructose 1-phosphate Fructose 1, 6-bisphosphate

Fructose 1-phosphate
aldolase

Glyceraldehyde Dihydroxyacetone
phosphate
ATP Glyceraldehyde 3-bisphosphate

Triose kinase
26
D-Fructose feeder pathway for glycolysis?

 D-Fructose is formed by hydrolytic cleavage of sucrose in

 the small intestine using the sucrase enzyme.

 D-Fructose is phosphorylated at C6 by a kinase thus

Mg 2+
 Fructose + ATP Fructose 6-phosphate + ADP.

 This is major pathway for fructose entry into glycolysis in the muscle
and kidney.

 D-Fructose is also phosphorylation of fructose at C-1 rather than C-6 by

fructokinase thus
 Fructose + ATP Mg 2+ Fructose 1-phosphate + ADP.

 This the major pathway of fructose entry into glycolysis in the liver
D-Galactose feeder pathway for glycolysis?

 D-Galactose is formed by hydrolytic cleavage of lactose in

the small intestine using the lactase enzyme.

 D-Galactose is phosphorylated at C1 by galactokinase thus

Mg 2+
 D-Galactose + ATP Galactose-1-phosphate + ADP.

 Galactose 1- phosphate is then converted to its epimer at C-4, glucose 1-

phosphate, by a set of reactions in which uridine diphosphate (UDP)
functions as a coenzyme-like carrier of hexose groups
D-Galactose feeder pathway for glycolysis?

Galactose
ATP Galactokinase
Mg 2+
ADP
Galactose 1-phosphate
Galactose 1-phosphate uridyl transferase

UDP-galactose Glucose 1-phosphate

NAD+ UDP galactose 4-

epimerase

UDP-glucose

29
D-Galactose feeder pathway for glycolysis?

 Several genetic diseases result in disordered galactose

metabolism.

 One of the diseases is called galactosemia, in which there is excess

accumulation of galactose in the blood.

 In the most common form of galactosemia, the enzyme galactose 1-

phosphate uridyl transferase is genetically defective.

 In this condition the enzyme Aldose reductase in the eye can reduce
galactose to galactitol, which accumulate and crystallises on the eye lens
causing cataract.

 Other forms of galactoseamia result from genetic defect of either

galactokinase or UDP galactose 4-epimerase
D-Mannose feeder pathway for glycolysis?

 Mannose can be phosphorylated at C-6 by hexokinase thus;

Mg 2+
 Mannose + ATP Mannose 6-phosphate +ADP.

 Mannose 6-phosphate is isomerized by phosphomannose isomerase to

yield fructose 6- phosphate, an intermediate of glycolysis