Glycolysis lecture

Lecture Outline

Energy production - general overview

Glycolysis overview
Glycolysis - step by step process
Regulation of glycolysis in cancer cells

Central Importance of Glucose

Major pathways for glucose utilization

Glucose is an excellent fuel

Yields good amount of energy upon oxidation
Can be efficiently stored in the polymeric form
Many organisms and tissues can meet their energy needs
on glucose only
Glucose is a versatile biochemical precursor
Bacteria can use glucose to build the carbon skeletons of:
All the amino acids
Membrane lipids
Nucleotides in DNA and RNA
Cofactors needed for the metabolism

Why is glycolysis so important?

2
net

glycolysis

ATP
2 pyruvate

Mitochondrion

2 acetyl CoA

2 CO2

ATP

NADH

NADH

NADH

FADH2

Citric acid
cycle
4 CO2

6 O2
subtotal

Electron transport chain

Cytoplasm

glucose

4 or 6

ATP

ATP

Glycolysis
ATP
18
4

ATP

32
or 34

ATP

6 H2O
subtotal

ATP
36 or 38
total

ATP

Glycolysis is considered one of the core metabolic pathways

because
1. Glycolytic enzymes are highly conserved among all living
organisms.
2. Glycolysis is the primary pathway for ATP generation under
anaerobic conditions and in cells lacking mitochondria such
as erythrocytes.
3. Metabolites of glycolysis are precursors for a large number
of interdependent pathways, including mitochondrial ATP
synthesis.

Overview of the Glycolysis

1.What does glycolysis accomplish for the cell?
Generates a small amount of ATP which is critical under
anaerobic conditions.
Generates pyruvate, a precursor to acetyl CoA, lactate,
and ethanol (in yeast).
2.What is the overall net reaction of glycolysis?
Glucose + 2NAD+ + 2ADP + 2 Pi
= -35.8 kJ/mol

Overview of the Glycolysis

The complete oxidation of glucose to CO2 and H2O
summarized by the reaction:
Glucose (C6H12O6) + 6O2

is

6CO2 + 6H2O

= -2,840 kJ/mol
G = -2,937 kJ/mol

2 pyruvate + 2NADH + 2H+ + 2ATP + 2H2O

Overview of the Glycolysis

3.What are the key regulated enzymes in glycolysis?
Hexokinase, Phosphofructokinase, Pyruvate kinase
4.What are examples of glycolysis in real life?
A deficiency in the hexokinase-related enzyme, glucokinase,
leads to a rare form of diabetes, which is caused by the
inability of liver and pancreatic cells to phosphorylate
glucose inside cells when blood glucose levels are elevated.

Glycolysis has two phases

Glycolysis [Embden-Meyerhof-Parnas (EMF) pathway]

First metabolic pathway to be elucidated.
Otto Warburg & Euler-Chelpin (in yeast), Embden & Meyerhof (in
muscles).
Universal pathway - from bacteria and yeast to mammals.
In eukaryotes, glycolysis takes place in the cytosol.
Breakdown of glucose to pyruvate; anaerobic
+ O2, pyruvate is further oxidized to CO2.
- O2, pyruvate can be fermented to lactate or ethanol.
Net Reaction:
Glucose + 2NAD+ + 2 Pi + 2 ADP = 2 Pyruvate + 2 ATP + 2 NADH + 2
H2O

Preparatory phase

First Priming Reaction

Regulation of Hexokinase
Hexokinase catalyzed phosphorylation of glucose is the first
irreversible step of glycolysis.
G-6-P is required for other pathway
including the pentose phosphate shunt
and glycogen synthesis.
So, hexokinase step is not inhibited
unless G-6-P accumulates.
Liver, the site of glycogen synthesis, has a homologous enzyme
called glucokinase. This has a high Km for glucose.
This allows brain and muscle to utilize glucose prior to its storage
as glycogen.

Regulated mainly by substrate inhibition

Second Priming Reaction

second irreversible
reaction in glycolysis

Reaction 2: Isomerization of glucose-6-phosphate to fructose 6-phosphate.

The aldose sugar is converted into the ketose isoform.
phosphoglucoisomerase.
The isomerization is catalyzed by the active-site glutamate, via general
acid/base catalysis
This is a reversible reaction. The fructose-6-phosphate is quickly
consumed and the forward reaction is favored.

Reaction 3: Phosphorylation of the hydroxyl gp on C1 forming fructose-1,6bisphosphate which is committed to glycolysis.

Enzyme: phosphofructokinase. This allosteric enzyme regulates the pace
of glycolysis.
Reaction is coupled to the hydrolysis of an ATP to ADP and Pi.

Regulation of Phosphofructokinase

Go = 23.8 kJ/mol

The phosphofructokinase step is rate-limiting step of glycolysis.

High AMP/ADP levels are activators of this enzyme, while high ATP
levels are inhibitory (energy charge).
In addition, feedback inhibition by citrate, an intermediate of the
TCA cycle.

Reaction 4:
Fructose-1,6-bisphosphate is split into 2 3-carbon molecules, one aldehyde
and one ketone: dihyroxyacetone phosphate (DHAP) and glyceraldehyde
3-phosphate (GAP).
Enzyme: aldolase; reversible
Thermodynamically unfavorable/reversible; GAP concentration kept low to
pull reaction forward

Preparatory phase

Reaction 5: DHAP and GAP are isomers of each other and can readily
inter-convert by the action of the enzyme triose-phosphate isomerase.
GAP is a substrate for the next step in glycolysis so all of the DHAP is
eventually depleted.
Thus, 2 molecules of GAP are formed from each molecule of glucose

Payoff phase

Reaction 6: GAP is oxidized to 1,3-bisphosphoglycerate; NAD+ is reduced

to NADH.
Oxidation of CHO gp to COOH gp is coupled to the phosphorylation of
C1 carbon to form a mixed anhydride called acyl phosphate.
- The acyl phosphate formed here is 1,3-bisphosphoglycerate.
Enzyme: glyceraldehyde 3-phosphate dehydrogenase (GAPDH).

Substrate level phosphorylation

Reaction 7: Substrate-level phosphorylation

1

BPG converted to 3-phosphoglycerate; ATP

released.
BPG is a mixed anhydride with a high
energy bond at C1.
This high energy bond is hydrolyzed to a
carboxylic acid and the energy released is
used to generate ATP from ADP.
Enzyme: phosphoglycerate kinase.

The formation of ATP by phosphoryl gp transfer from a substrate is

referred to as substrate level phosphorylation to distinguish this
mechanism from oxidative phosphorylation.

G3P
3PG

1,3 bisphosphoglycerate
(endergonic)

(exergonic)

The sum of the reactions 6&7 is:

Glyceraldehyde 3-phosphate + ADP + Pi + NAD+

3-phosphoglycerate + ATP + NADH + H+

o = -12.5 kJ/mol

The outcome of the coupled reactions is that the energy released during
oxidation of an aldehyde to a carboxyl gp is conserved by the coupled
formation of ATP from ADP and Pi.

(Low energy compound)

(High energy compound)

Reaction 9: Dehydration of 2-Phosphogylcerate

Reaction 8: The phosphate shifts from C3 to C2 to form 2-phosphoglycerate.
Enzyme: phosphoglycerate mutase (migration of phosphate group)
Thermodynamically unfavorable/reversible; Reactant concentration kept
high by PGK to push forward

Reaction 10: 2nd Substrate-level phosphorylation

Enolphosphate has a high energy bond.
It is hydrolyzed to form the enolic form of
pyruvate with the synthesis of ATP.

A water molecule is removed to form phosphoenolpyruvate which has a

double bond between C2 and C3.
Enzyme: enolase.
Slightly thermodynamically unfavorable/reversible; Product concentration

kept low to pull forward

Regulation of pyruvate kinase

If glycolysis gets past the phosphofructokinase step, then
regulation is at the pyruvate kinase step.

The irreversible reaction is catalyzed by the

enzyme pyruvate kinase.

Pyruvate kinase
activity is inhibited

Enol pyruvate quickly changes to keto pyruvate

which is far more stable.

F-1,6-BiP acts a allosteric feed

forward activator and drives the
pyruvate
kinase
reaction
forward.

Balance Sheet
Reaction 1: - 1 ATP
Reaction 3: -1 ATP
Reaction 6: +2 NADH
Reaction 7 : +2 ATP
Reaction 10 : +2 ATP
Total/ molecule of glucose: +2 ATP, +2 NADH
Glucose + 2NAD+ + 2 Pi + 2 ADP = 2 Pyruvate + 2 ATP + 2 NADH + 2 H2O
Glucose + 2NAD+ + 2 Pi + 2 ADP = 2 Pyruvate + 2 ATP + 2 NADH + 2 H2O

What are three regulatory steps in glycolysis?

Regulation of Glycolysis
Enzyme
Hexokinase

Activator
AMP/ADP

Phosphofructokinase
(F6P to F1-6)

AMP/ADP,
Fructose-2,6-bisphosphate

Pyruvate kinase

AMP/ADP
Fructose-1,6-bisphosphate

Enzyme
Hexokinase
Phosphofructokinase
Pyruvate kinase

Inhibitor
Glucose-6-phosphate
ATP, Citrate
ATP, Acetyl CoA, Alanine

Feeder pathways of glycolysis

Starch

Carbohydrate catabolism-I
Glycogen

-amylase

Phosphorylase

Dextrin

D-Galactose

Glucose
UDP-glucose
or
Fructose
or their phosphates

Maltose
Lactose
Sucrose

D-Mannose

Trehalose

ADP
ATP

Glycolysis

Pyruvate

Glycogen

Glucose 1-phosphate

Fructose
is
phosphorylated
by
fructokinase
(liver)
or
hexokinase
(adipose) on the 1 or 6 positions
respectively.
Fructose-6-phosphate is an intermediate
of glycolysis.
Fructose-1-phosphate is acted upon by
an aldolase-like enzyme that gives DHAP
and glyceraldehyde.
DHAP is a glycolysis intermediate and
glyceraldehyde can be phosphorylated to
glyceraldehyde-3-P.

Feeder pathways of glycolysis

Galactose has a slightly complicated multistep pathway for conversion to glucose-1phosphate.
Gal
Gal-1-P
Glc-1-P.

UDP-Gal

UDP-Glc

If this pathway is disrupted because of

defect in one or more enzyme involved in the
conversion of gal to glc-1-P,
then galactose accumulates in the blood
and the subject suffers from galactosemia
which is a genetic disorder, an inborn error of
metabolism.

Fate of pyruvate is different under aerobic and anaerobic conditions

Fate of pyruvate is different under aerobic and anaerobic conditions

Anaerobic conditions

Aerobic conditions (Electrons are passed on from NADH to O2)

NADH is formed from NAD+ during

glycolysis.
NAD+ has to be regenerated for
further energy yielding cycles of
glycolysis to continue.

OR
2 Lactic acid

In very active muscles, submerged plants and lactic acid bacteria,

pyruvate is reduced to lactate with a concomitant oxidation of NADH to NAD+.
In yeast, pyruvate is converted to ethanol accompanied by the
oxidation of NADH to NAD+.

Pyruvate is converted to acetyl CoA which enters TCA cycle and gets
completely oxidized to CO2.
NAD+ is regenerated from NADH by ETC in mitochondria

Cancer and glucose catabolism

Glucose uptake and utilization proceed 10X faster in
solid tumor vs normal.
Tumor cells are hypoxic, low oxygen, depend on
anaerobic generation of ATP.
Otto Warburg - Warburg Effect
most cancer cells predominantly produce energy
by a high rate of glycolysis.

Glucose transporters and most of the

glycolytic enzymes are overproduced in
tumors

Compounds that inhibit hexokinase,

glucose 6-phosphate dehydrogenase, or
transketolase block ATP production by
glycolysis, thus depriving the cancer cell
of energy and killing it.

Ethanol fermentation
Fermentation: Processes that extract energy (in the form
of ATP) from glucose but do not consume oxygen or
NAD+.

Zn++

There is no net change in oxidation state of sugars.

Regeneration of NAD+ for further glycolysis under
anaerobic conditions.
Reduction of pyruvate to another product
Basis for production of food from beer to bread.

Fermentation of glucose to ethanol is catalyzed by 2 enzymes:

1. Pyruvate decarboxylase catalyzes the first reversible reaction
to form acetaldehyde:
CH3-CO-COOH CH3-CHO + CO2
2. Acetaldehyde is reduced by alcohol dehydrogenase is a
reversible reaction:
CH3-CHO + NADH + H+ CH3CH2OH + NAD+
Ethanol fermentation is used during bread and wine making.

TPP is a common acetaldehyde carrier

Lactate Fermentation

Formation of lactate catalyzed by lactate dehydrogenase:

CH3-CO-COOH + NADH + H+
CH3-CHOH-COOH + NAD+

Lactate Fermentation
In highly active muscle and RBCs, there is anaerobic glycolysis
because the supply of O2 cannot keep up with the demand for ATP.
Increased lactate lowers pH which inactivates glycolytic enzymes.
Energy deprivation and cell death
the symptoms being pain and fatigue of the muscle.
Lactate is transported to the liver where it can be reconverted to pyruvate
by the LDH reverse reaction.
Thus, there is no net change in NAD+ and NADH concentration during
lactate fermentation.

Cori Cycle
Lactate is formed in the active muscle to
regenerate NAD+ from NADH so that
glycolysis can continue.
The muscle cannot spare NAD+ for
re-conversion of lactate back to pyruvate.
Thus, lactate is transported to the liver,
where, in the presence of oxygen, it
undergoes
gluconeogenesis to form
glucose.
The glucose is supplied by the liver to
various tissues including muscle.
This inter-organ cooperation during high
muscular activity is called as the Cori cycle.

Gluconeogenesis

Precursors for Gluconeogenesis

Animals can produce glucose from sugars or
proteins

Sugars: pyruvate, lactate, or oxaloacetate

Protein: from amino acids that can be converted to
citric acid cycle intermediates (or glucogenic amino
acids)

Animals cannot produce glucose from fatty acids

Product of fatty acid degradation is acetyl-CoA

Cannot have a net conversion of acetyl-CoA to
oxaloacetate
Plants, yeast, and many bacteria can do this, thus
producing glucose from fatty acids

Precursors for Gluconeogenesis

Glycolysis vs. Gluconeogenesis

Glycolysis and gluconeogenesis are reciprocally regulated to prevent wasteful

operation of both pathways at the same time.

Gluconeogenesis
Gluconeogenesis: Synthesis of glucose from noncarbohydrate precursors including pyruvate, lactate, glycerol
and amino acids.
Location: liver and kidney; provides glucose for use by
brain, muscles and erythrocytes.
In animals, gluconeogenesis is for the most part the
reverse of glycolysis except for three steps.

Gluconeogenesis
Lacate is converted to pyruvate
by LDH.
Amino acids are converted to
either pyruvate or oxaloacetate
prior to gluconeogenesis.

- There are substitute or bypass reactions for the three

irreversible steps of glycolysis.

Gluconeogenesis

Steps different in glycolysis and gluconeogenesis

Three irreversible steps of glycolysis require bypass
reactions for gluconeogenesis:
1. Conversion of pyruvate to PEP
2. Dephosphorylation of fructose 1,6 bis-phosphate
3. Dephosphorylation of glucose 6-phosphate

Steps different in glycolysis and gluconeogenesis

Bypass for pyruvate kinase: Requires two energy-consuming
steps.
1. Pyruvate carboxylase converts pyruvate to oxaloacetate
Pyruvate synthesized by glycolysis or from amino acids moves
to mitochondria.
One carbon is supplied by CO2 to form the 4-C oxaloacetate.
Coupled to ATP hydrolysis; carboxylation used biotin cofactor

Bypass for pyruvate kinase

1. PEP can be converted to fructose1,6 bisphosphate by reverse
glycolysis.
2. F-1-6 BP
F-6-P cannot proceed
by reverse glycolysis since the
PFK reaction is irreversible.

Steps different in glycolysis and gluconeogenesis

Bypass for pyruvate kinase

2. Phosphoenolpyruvate carboxykinase converts oxaloacetate to

PEP.
Oxaloacetate is shuttled out to the cytoplasm where the glycolytic
enzymes are located.
CO2 is removed and energy in the form of GTP is utilized.
Two high energy molecules (ATP and GTP) with a total free
energy change of 62 kJ/mol are used up for the formation of PEP.

Additional bypass for PFK and Hexokinase

3. Reverse glycolysis continues to
form glucose-6-P
glucose-6-P
glucose
catalyzed by glucose-6phosphatase
since
the
hexokinase
reaction
is
irreversible.

Instead a different enzyme

called
as
fructose-1,6
bisphosphatase is used. This
removes the phosphate gp from
the 1 position.
However, no ATP is formed.

Balance Sheet
Net reaction of gluconeogenesis
2 Pyruvate + 4 ATP + 2 GTP + 2 NADH + 2 H+ + 4 H2O
Glucose + 2 NAD+ + 4 ADP + 2 GDP + 6 Pi.

Gluconeogenesis is expensive

Costs 4 ATP, 2 GTP, and 2 NADH

BUT hysiologically necessary
Brain, nervous system, and red blood cells generate
ATP ONLY from glucose.

Regulation of Gluconeogenesis
1. Fructose 1-6-bisphosphatase is coordinately regulated with
phosphofructokinase.
Thus, citrate is a positive effector and AMP and F-2,6-BP are
negative effectors.
When glucose levels are high, F-2,6-BP is high
gluconeogenesis is inhibited while glycolysis is favored.

and

When glucose levels are low, F-2,6-BP is low and glycolysis is

inhibited.

When glycogen stores are depleted we need to get

glucose from somewhere.
During starvation or vigorous exercise.

Regulation of Gluconeogenesis
2. Pyruvate carboxylase is an imp regulatory step in gluconeogenesis.
Acetyl CoA and ATP are positive effectors while AMP/ADP are
inhibitors.
3.

Glycolysis and gluconeogenesis are regulated by hormones.

Insulin stimulates synthesis and activity of glycolytic enzymes
while glucagon turns on gluconeogenic enzymes.

Pentose phosphate pathway

The major catabolic fate of glucose in mammalian cells is glycolytic
breakdown to pyruvate.
A small portion of glucose in rapidly growing animal cells is not
catabolized by glycolysis, but is instead oxidized to pentose phosphate
sugars and NADPH.
Role of the players
1. NADPH: reducing power for biosynthetic reactions
2. Pentose sugar, ribose-5 phosphate: provides C-skeleton for
biosynthesis of nucleotides, coenzymes and nucleic acids.

Pentose phosphate pathway

or

Phosphogluconate pathway
or

Hexose monophosphate shunt

Pentose phosphate pathway

PP can be divided into two phases:
(1) Oxidative phase: oxidation of glucose 6-phosphate to ribulose5 phosphate in a two step reaction with generates NADPH.
(2) Non-oxidative phase

Pentose phosphate pathway

Pentose phosphate pathway

1. Oxidative reactions of pentose phosphate pathway

2. Non-oxidative phase recycles pentose phosphates to

glucose 6-phosphates in the tissues where the primary
product of PPP is NADPH.
Transaldolases and transketolases catalyzes interconversion of 3, 4,
5, 6 or 7-carbon sugars.

NADPH regulates partitioning into glycolysis vs.

pentose phosphate pathway
Entry of glucose 6-phosphate either
into glycolysis or pentose phosphate
pathway is largely determined by
relative concentrations of NADP+ and
NADPH.

Glycogen Synthesis

G-6-P Dehydrogenase Deficiency

Can be fatal in cases of high oxidative stress
Certain drugs, herbicides, and some foods
Resistance to malaria due to high oxidative stress in red
blood cells.

Glycogen Synthesis - regulation

Glycogen Synthesis - regulation

Chapter 14 Summary
Glycolysis, a process by which cells can extract a limited amount
of energy from glucose under anaerobic conditions.
Gluconeogenesis, a process by which cells can use a variety of
metabolites for the synthesis of glucose
The differences between glycolysis and gluconeogenesis

How they are both made thermodynamically favorable

How they are differentially regulated to avoid a futile cycle

Pentose phosphate pathway, a process by which cells can
generate reducing power (NADPH) that is needed for the
biosynthesis of various compounds.
Glycogen synthesis

Glycogen
Synthesis
regulation

Low sugar