GLYCOLYSIS

Learning objectives:

List the enzymes and intermediates involved in glycolysis

List the irreversible and regulated steps of glycolysis

Discuss regulation of glycolysis

 Blood glucose concentrations
Normal plasma glucose concentrations roughly
3.9 – 8.3 mM

Glucose transporters
Name Tissue location Km .

GLUT1 All mammalian tissues 1 mM Basal glucose uptake

GLUT2 Liver and pancreatic β cells 15-20 mM Uptake approximately

proportional to glucose conc.

GLUT3 All mammalian tissues 1 mM Basal glucose uptake

GLUT4 Skeletal muscle and fat 5 mM Recruited to cell membrane

in insulin dependent manner
 Glucose transporters
Normal physiological range

100
Km: 1 mM
90
Transport rate (% of Vmax)

80 Km: 5 mM GLUT1 and GLUT3

70 GLUT2
60 GLUT4
50 Km: 20 mM

40
30
20
10
0
0 5 10 15 20 25
Glucose concentration (mM)
GLUT4 is recruited to cell membrane by
insulin-dependent translocation

The increase in number of GLUT4 transporters in the cell membrane

increases the speed of glucose transport.
Insulin stimulates glucose uptake in fat tissue and skeletal muscle
 Glycolysis
(Emden-Meyerhof glycolytic pathway)

Stoichiometry
1 Glucose + 2 ADP + 2 Pi + 2 NAD+ → 2 Pyruvate + 2 ATP + 2 H2O + 2 NADH + 2 H+

Glycolysis is used to provide energy (in the form of ATP)

and intermediates for other metabolic pathways.
Glycolysis occurs in the cytoplasm.

Aerobic conditions: Oxygen is required to re-oxidize NADH to NAD+ via the

electron transport chain

Anaerobic conditions: NADH is re-oxidized to NAD+ in a reaction in which

pyruvate is reduced to lactate (humans)
or ethanol (yeast and other microorganisms)
 O O-
C
GLYCOLYSIS
C6H12O6 C O
TCA CYCLE &
Glucose OXIDATIVE PHOSPHORYLATION
CH3
Pyruvate
+ O2
FERMENTATION

CO2 + H2O
O O-
C H

HO C H HO C H

CH3 CH3

Lactate Ethanol
 O H O OH O O-
CH2OH C C C

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

CH2OH CH2OH CH2OH CH2OH

Glycerol Glyceraldehyde Glyceric acid Glycerate

O O-
CH2OH C

C O C O

CH2OH CH3

Dihydroxyacetone Pyruvate
(α-keto carboxylic acid)
 Glucokinase
Hexokinase

Glucose + ATP Glucose 6-phosphate + ADP

Irreversible
Highly exergonic (ΔG = -33.5 kJ/mol)
Regulated step in glycolysis

R kinase
R-OH + ATP R-OPO32- + ADP
 Glucokinase is a glucose sensor

120

100 Vmax
Km = 0.1 uM
Reaction velocity v

80

60
Km = 10 uM
40

20
Normal physiological
range
0
0 5 10 15 20

Glucose concentration (mM)

 Glucose-stimulated insulin secretion in
pancreatic beta cells
Glucose metabolism determines secretion of insulin
Glucose

GLUT2
GK Insulin
G-6-P Pyruvate

Ca 2+
K+ ATP
Depolarization ADP

Ca 2+
GLUT2 is a high Km, high efficiency glucose transporter
(not rate-limiting)

Glucokinase has a high Km (is rate-limiting)

- reaction velocity is roughly proportional to glucose
concentration
- therefore glucose metabolism and insulin secretion
in beta cells is roughly proportional to glucose
concentration
 Phosphoglucose
isomerase
Glucose 6-phosphate Fructose 6-phosphate
 Phosphofructokinase

Fructose 6-phosphate + ATP Fructose 1,6-bisphosphate + ADP

Irreversible
Highly exergonic (ΔG = -22.2 kJ/mol)
Regulated step in glycolysis
The main regulatory enzyme of the glycolytic pathway

ATP balance:
2 ATP spent in phosphorylating glucose and fructose 6-phosphate
 CH2OPO32-
Ι
C=O Aldolase CH2OPO32- CHO
Ι Ι Ι
HO – C – H C=O + H – C – OH
Ι Ι Ι
H – C – OH CH2OH CH2OPO32-
Ι
H – C – OH Dihydroxyacetone Glyceraldehyde
Ι phosphate 3-phosphate
CH2OPO32-

Fructose 1,6-
bisphosphate
 Triose phosphate
isomerase
CH2OPO32- CHO
Ι Ι
C=O H – C – OH
Ι Ι
CH2OH CH2OPO32-

Dihydroxyacetone Glyceraldehyde
phosphate 3-phosphate
 Mixed anhydride of
phosphoric acid and a
carboxylic acid
High energy bond
Glyceraldehyde
3-phosphate
2-
O3PO O
CHO dehydrogenase
Ι C
H – C – OH + NAD + Pi
+ Ι + NADH + H +
Ι H – C – OH
CH2OPO32- Ι
CH2OPO32-
Glyceraldehyde
3-phosphate 1,3-Bisphosphoglycerate

Energy from oxidation of glyceraldehyde 3-phosphate to the carboxylic acid is

“captured” in the formation of the acyl~phosphate linkage
 Phosphoglycerate
kinase
1,3-Bisphosphoglycerate + ADP 3-Phosphoglycerate + ATP

Substrate-level phosphorylation

ATP balance:
2 ATP spent in phosphorylating glucose and fructose 6-phosphate
2 x 1 ATP generated in the phosphoglycerate kinase step
 Phosphoglycerate
mutase

3-Phosphoglycerate 2-Phosphoglycerate

Enolase
2-Phosphoglycerate Phosphoenolpyruvate + H 2O
 Pyruvate kinase

Phosphoenolpyruvate + ADP Pyruvate + ATP

Substrate-level phosphorylation

Irreversible
Highly exergonic (ΔG = -16.7 kJ/mol)
Regulated step in glycolysis
 Pyruvate kinase

Phosphoenolpyruvate + ADP Pyruvate + ATP

ATP balance:
2 ATP spent in phosphorylating glucose and fructose 6-phosphate
2 x 1 ATP generated in the phosphoglycerate kinase step
2 x 1 ATP generated in the pyruvate kinase step

2 molecules of ATP are generated from 1 molecule of glucose in glycolysis

 Stoichiometry
1 Glucose + 2 ADP + 2 Pi + 2 NAD+ → 2 Pyruvate + 2 ATP + 2 H2O + 2 NADH + 2 H+

The 3 Irreversible Enzyme-catalyzed Reactions

1. Hexokinase (or glucokinase)
2. Phosphofructokinase
3. Pyruvate kinase
In yeast and many other microorganisms:

Pyruvate
decarboxylase
Pyruvate + H+ Acetaldehyde + CO 2

Alcohol
dehydrogenase

Acetaldehyde + NADH + H+ Ethanol + NAD +

 In mammals:

Lactate
dehydrogenase Under anaerobic conditions,
e.g. in rapidly contracting
Pyruvate + NADH + H+ Lactate + NAD + muscle

In erythrocytes

NADH + H+ + ½ O2 NAD+ + H2O Under aerobic conditions

Electron
transport
chain
Fates of pyruvate generated by glycolysis

PEP

Pyruvate Lactate

Alanine Oxaloacetate Acetyl CoA

Oxidation via Fatty acid

TCA cycle synthesis
 Regulation of metabolic pathways

The flow of intermediates through metabolic pathways is

controlled by four mechanisms

1. Substrate availability
2. Allosteric activation or inhibition
3. Covalent modification of enzymes
4. Induction/repression of enzyme synthesis
 Signals

Energy state
ATP high-energy signal
ADP, AMP low-energy signal

Availability of biosynthetic intermediates

Citrate
Alanine

Metabolic state of the whole organism

Insulin – signal of the fed state
High blood glucose – signal of the fed state
Glucagon – signal of the fasted state
Epinephrine – signal of stress

Metabolic regulation of biochemical pathways makes “physiological sense”

Control of flow though a pathway is often distributed among several enzymatic

steps in the pathway
 Liver cell - Hepatocyte

Fed state
Insulin Insulin receptor
Glycogen
Hepatic
Lipids glucose
GLUT 2 GK P
uptake and
Pyruvate utilization
glucose glucose glucose-6-phosphate
Glycolysis

Fasted state
Receptor
Glycogen
Glucagon
Hepatic
Gluconeogenesis
glucose
production
GLUT 2 G6Pase
P

glucose glucose glucose-6-phosphate

Glycolysis
 Skeletal muscle cell

Rapidly
Low ATP Glycogen
contracting state
High AMP

Hexokinase Glycolysis
GLUT 4 P

ATP
glucose glucose glucose-6-phosphate

Resting state High ATP Glycogen

Low AMP

GLUT 4 P Glycolysis
ATP
glucose glucose glucose-6-phosphate
 Glucokinase
Hexokinase

Glucose + ATP Glucose 6-phosphate + ADP

Hexokinase

- Glucose 6-phosphate (low phosphofructokinase activity)

Glucokinase

+ High blood glucose (release from GKRP, High Km)

+ Insulin stimulates gene transcription (only in liver)

Glucokinase (GK) can be bound to the Glucokinase
Regulatory Protein (GKRP).

When GK is bound to the GKRP, it is sequestered in the

nucleus and is inactive.

Glucose promotes dissociation of GK from the GKRP,

allowing GK to be transported to the cytoplasm where it is
active in glycolysis.
 Phosphofructokinase

Fructose 6-phosphate + ATP Fructose 1,6-bisphosphate + ADP

The main regulatory step of glycolysis

- ATP (high-energy state)

- Citrate (abundance of biosynthetic building blocks)

- H+ (build-up of lactate)

+ AMP (low-energy state)

+ Fructose 2,6-bisphosphate (fed state, high insulin/glucagon ratio)

 Phosphofructokinase

Fructose 6-phosphate + ATP Fructose 1,6-bisphosphate + ADP

ATP is a substrate for phosphofructokinase

ATP can also bind to an allosteric site
 Pyruvate kinase

Phosphoenolpyruvate + ADP Pyruvate + ATP

- ATP (high-energy state)

- Alanine (abundance of alanine which is synthesized from pyruvate

and is an important gluconeogenic substrate)

- Glucagon (fasted state)

+ Fructose 1,6-bisphosphate (feed-forward mechanism)

+ Glucose (stimulates gene transcription)

Glucagon binding to the glucagon receptor 
Activation of adenylyl cyclase 
Increase in [cAMP] 
Activation of protein kinase A 
Phosphorylation and inactivation of pyruvate kinase

Occurs in liver in the fasted state