GLYCOLYSIS
Definition: from Greek “glykys”
(sweet) & “lysis” (splitting)

“Living organisms, like machines,
conform to the law of the conservation
of energy, and must pay for all their
activities in the currency of catabolism”

Ernest Baldwin, Dynamic Aspects of Biochemistry
(1952)
I. BACKGROUND

Glycolysis
 Carried out by nearly every living cell


Catabolic process


In cytosol of eukaryotes
Releases energy stored in covalent bonds
Stepwise degradation


Glucose
Other simple sugars
I. Background, cont…

Anaerobic process

Evolved in an environment lacking O2


Early, important pathway




Primitive earth … millions of years ago
Provided means to extract energy from nutrient
molecules
Central role in anaerobic metabolism
For the first 2 billion years of biological evolution on earth
Modern organisms


Provides precursors for aerobic catabolic pathways
Short term anaerobic energy source
Background, cont…

Glucose is a precursor
 Supplies metabolic intermediates
 Three fates
 Storage
 Oxidation to pyruvate
 Oxidation to pentoses
Background, cont…

beta D-Glucose is the major fuel
 Rich in potential energy




Stored in bonds
Is literally solar energy
ΔG01= -2840 kJ/mole
Advantages to glucose


Catabolism  ATP
Can be stored


Eg: Polysaccarides, sucrose
Can be transported


Blood glucose
Organism to organism
Background, cont…

History


Began with Pasteur: Mid- nineteenth century
Eduard Buchner: 1897


Arthur Harden and William Young: 1905




Discover phosphate is required for glucose fermentation
Gustov Embden, Otto Meyerhof and Jocob Parnas


Fermentation in broken extracts of yeast cells
Seminal work
Often called the Embden-Meyerhof-Parnas pathway
Elucitated in 1940
Fritz Lipmann and Herman Kalckar: 1941

Metabolic role of high energy compounds like ATP
II. GLYCOLYSIS

“Most completely understood biochemical
pathway”




Sequence of 10 enzymatic pathways
1 molecule of glucose is converted to 2 3-carbon
pyruvate molecules
Concomitant generation of 2 ATP
Key role in energy metabolism


Provides free energy for organisms
Prepares glucose (and other molecules) for further
oxidative degradation
Function, Glycolysis, cont…

Most carbon in cells follows this
pathway


Only source of energy for many
tissues
Rates and Regulation vary among
species

Most significant difference is the way
that pyruvate is utilized
Glycolosis, cont…

The fates of pyruvate

Aerobic

Oxidative decarboxylation to acetyl





2-cabon molecule
Forms acetyl-coenzyme A
To Krebs cycle
Electrons to ETS
Anaerobic


To lactate
To ethanol
Glycolysis, cont…

Overview of glycolysis in animal metabolism
 Glucose in the blood
 From breakdown of polysaccharides



Gluconeogenesis


Liver glycogen
Dietary sources
Synthesis from noncarbohydrate precursors
Glucose enters cells

Specific transporters
Glycolosis, cont…

Enzymes of glycolysis in cytosol




Glucose converted into 2 3-carbon unites (pyruvate)
Free energy harvested to synthesis ATP from ADP
and Pi
Pathway of chemically coupled
phosphorylation reactions
10 reactions broken into 2 phases
 Preparatory phase (energy investment)


Reactions 1 – 5
Payoff phase (energy recovery)

Reactions 6 - 10
Glycolosis, cont…

Preparatory phase (energy investment)
 Hexose glucose is phosphorylated
by ATP
 C3-C4 bond broken
 yields 2 triose phosphates
(glyceraldehyde -3-phosphate)
 Requires 2 ATP to “prime” glucose
for cleavage
Glycolosis, cont…

Payoff Phase (energy recovery)
 Each triose phosphate is oxidized
 Energy is conserved





by reduction of NAD+
Phosphate is transferred to ADP  ATP
Net gain: 2 ATP
2 Glyceraldehyde-3-phosphate molecules
are converted to 2 molecules of pyruvate
NadH must be reoxidized
Glycolosis, cont…


ATP formation is coupled to glycolysis
 Glucose  pyruvate generates 2 ATP (net)
 Involves coupled reactions
 Makes glycolysis irreversible under
intracellular conditions
Most energy remains in pyruvate
 Glycolysis releases ~ 5%
 Oxidation via TCA cycle releases the rest
Glycolosis, cont…

Phosphorylated intermediates are
important
 Each intermediate is phosphorylated
 Phosphate has 3 functions:
 Prevent diffusion of the
intermediates out of the cell
 Can donate Pi to ADP  ATP
 Provide binding energy to increase
specificity of enzymes
The Reactions of Glycolysis




10 enzymes
9 Intermediates
Cost (2 ATP)
Payment




4 ATP
2 NADH +H+
End products
Metabolic crossroads
Reaction 1

Hexokinase: First ATP Utilization
 Transfer of a phosphoryl group

From ATP

To glucose (at C-6)

Intermediate formed: Glucose-6-

phosphate (G6P)
Enzyme: Hexokinase



Allosterically inhibited by product
REGULATION SITE (one of three)
Reaction is irreversible
Reaction 1, cont…

Kinase: enzymes that transfers phosphoryl
groups between ATP and a metabolite


Name of metabolite acceptor is in prefix of the
kinase name
E.g.:


glucokinase (in liver) is specific for glucose
Hexokinase: ubiquitous, relatively nonspecific for hexoses



D-glucose
D-mannose
D-fructose
Reaction 1, cont…

Second substrate for kinases (including
hexokinase)

Mg2+ -ATP complex




Mg2+ is essential
Uncomplexed ATP is a potent inhibitor of
hexokinase
Mg2+ masks negative charge on phosphate
oxygen atoms
Makes nucleophilic attack by C6-OH group on
gamma-phosphorus atom more possible
Reaction 1, cont…
Substrate induced conformational
changes in yeast hexokinase
Glucose (magenta) induces significant change … like jaws … this places
ATP in close proximity to the C6-H2OH group and excludes water (which
prevents ATP hydrolysis)
Reaction 1, cont…



Begins glycolysis
Is first of 2 priming reactions
Reaction is favorable under cellular
conditions



Hydrolysis of ATP: liberates 30.5 kJ/mol
Phosphylation of glucose: costs 13.8kJ/mol
Delta G= -16.7 kJ/mol
Reaction 1, cont…

Importance of phosphorylating glucose

Keeps substrate in the cell

Glucose enters cell via specific transporters



G6P is negatively charged, thus can not pass through
plasma membrane
Rapid phosphorylation of glucose keeps
intercellular concentrations of glucose low


The transporter does not bind to G6P
Favors diffusion into cell
Regulatory control can be imposed only on
reactions not at equilibrium

Large negative free energy change make this an
important site for regulation
Reaction 1, cont…

Glucokinase


In liver
Carries out same reaction, but is glucose
specific (high Km for glucose)


Not inhibited by the product
Important when blood glucose levels are high



Glucose to G6P to stored glycogen
Inducible by insulin
When blood glucose levels are low, liver
uses hexokinase
Reaction 2


Phosphoglucose Isomerase (PGI)

Conversion of G6P to Fructose-6-phosphate

Isomerization of an aldose to a ketose
Intermediate formed: Fructose-6phosphate (F6P)

Enzyme: Phosphoglucose Isomerase

Reversible reaction
Reaction 2, cont…

Common reaction: isomerization of a sugar




Requires ring of G6P to open
Isomerization
Ring of F6P closes
Prep for next reactions



R3: Phosphorylation at C-1
R4: cleavage between C-3 and C-4
PGI in humans



Requires Mg2+
Highly specific for G6P
Reaction is near equilibrium, easily reversible

Small delta G value
Reaction 3

Phosphofructokinase: second ATP utilization

Phosphorylation of F6P to Fructose-1,6bisphosphate
bis not di: phosphates not together)
 ATP donates a phosphate
Intermediate formed: Fructose-1,6bisphosphate (FBP or F1,6P)
Enzyme: Phosphofructokinase (PFK-1)

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REGULATION SITE (two of three)
Irreversible reaction
Reaction 3, cont…

Similar to Hexokinase reaction


PFK plays central role in control of
glycolysis


Nucleophilic attack by C1-OH of F6P on
Mg2+ -ATP complex
Catalyzes one of the pathway’s ratedetermining reactions
Allosteric regulation of PFK in many
organisms
Reaction 4

Aldolase

Cleavage of Fructose-1,6-bisphosphate

Forms two trioses


Glyceraldehyde-3-phosphate (GAP)
Dihydroxyacetone phosphate (DHAP)

Intermediates formed: GAP and DHAP

Enzyme: aldolase

Reversible reaction
Reaction 4, cont…

A cleavage between C-3 and C-4
 two molecules from one
 Requires:


A carbonyl at C-2
A hydroxyl at C-4
Hence the “logic” at reaction 2
2 classes of aldolases




Class I: in animal tissues
Class II: in bacteria and fungi

Require a active-site metal, normally zinc Zn 2+
Reaction 5

Triose phosphate isomerase

Interconversion of DHAP and GAP (triose
phosphates)

Isomerization of aldose-ketose isomers
Intermediate formed:
Glyceraldehyde-3-phosphate

Enzyme: Triose phosphate isomerase

Reversible reaction

Reaction 5, cont …

Only glyceraldehyde-3-P can
continue in glycolysis

Dihydroxyacetone-P is rapidly
converted
Taking Stock so far

Investment phase: Produces 2 triose
phoshates
 One glucose  2 glyceraldehyde-3-P
 Costs 2 ATP
 Now, need a little chemical “artistry”
to convert low energy GAP to high
energy compounds and synthesis
ATP
Next …

Payoff phase: Produces ATP
 One glucose  2 glyceraldehyde-3-P
 Conversion to pyruvate  4 ATP
 Also 2 reduced NADH
Reaction 6

Glyceraldehyde-3-phosphate Dehydrogenase:
First “High-energy” Intermediate Formation
+
 Oxidation of GAP by NAD and Pi

Intermediate formed: 1,3bisphosphoglycerate

Enzyme:

Reaction is reversible

Energy-conserving reaction
GLYCERALDEHYDE-3PHOSPHATE DEHYDROGENASE
Reaction 6, cont …
 Aldehyde is dehydrogenated to an
acyl phosphate with a high
standard free energy of hydrolysis
(ΔG01 = -49.3 kJ/mole)
+
 NAD serves as hydrogen
acceptor:
NAD+  NADH + H+
Reaction 7

Phosphoglycerate kinase: first ATP generation
 Transfer of a phosphate to ATP
 Yields ATP & 3-phosphoglycerate
Intermediate formed: 3-phosphoglycerate
 Enzyme: PHOSPHOGLYCERATE
KINASE

Energy-coupling reactions 6 & 7
 A substrate-level phosphorylation

Reaction 8

Conversion of 3 PG to 2phosphoglycerate (2PG)
Intermediate formed:
 Enzyme: PHOSPHOGLYCERATE

MUTASE (PGM)

Reversible phosphate shift
Reaction 9

Dehydration to Phosphoenol Pyruvate
(PEP)

Intermediate formed:
phosphoenol pyruvate

Enzyme: ENOLASE
Energy-conserving reaction
 Reversible reaction

Reaction 10

Pyruvate kinase: Second ATP generation

Transfer of a phosphate to  ATP
Product: pyruvate
 Enzyme: Pyruvate kinase

Irreversible reaction
 Substrate-level phosphorylation
 “enol” spontaneously tautomerizes to
“keto” form

Glycolosis, cont…

Overall balance sheet:

Anaerobic:




net gain of 2 ATP
Must “free” reduced NAD from reaction 6
In humans: lactic acid pathway
Aerobic:




NADH re-oxidized to NAD+ via respiratory chain in
mitochondria
e- transfer provides energy for ATP synthesis
2.5 ATP/ reduced NAD
Therefore: 5 more ATPs if go aerobic
Glycolosis, cont…

Anaerobic alternatives for pyruvate

Must oxidize NAD



Lactic acid pathway
Fermentation
Aerobic alternatives for pyruvate

Hydrogens from reduced NAD transported
to ETS in mitochondria

Transporters in mitochondrial membrane
Glycolosis, cont…

Dietary polysaccharides:


must by hydrolyzed to monosaccarides
Dietary Disaccharides:

must by hydrolyzed to monosaccarides

Disaccharides cannot enter glycolytic
pathway
Glycolosis, cont…
Hexoses can enter glycolysis
 Hydrolytic enzyes are attached to
epithelial cells in intestines
 Monosaccharides  intestinal cells
 blood  liver (phosphorylation) 
glycolysis

III. REGULATION of
CARBOHYDRATE CATABOLISM

Regulatory enzymes act as metabolic valves


Substrate-limited reactions are determined by [S]
Enzyme-limited reactions are RATE-LIMITING
STEPS
Irreversible reactions
 Exergonic
 regulatory

Regulation of Carbohydrate Catabolism, cont…

Regulation of glucose metabolism differs in
muscle & liver
 Muscle: Object is ATP production
Enzyme: GLYCOGEN PHOSPHORYLASE
Enzyme is allosterically regulated
Skeletal muscle signalled to  ATP by
EPINEPHRINE
Both enzyme & hormone influence ATP
production




Regulation of Carbohydrate Catabolism, cont…

Liver: object is maintenance of blood
glucose levels
 Regulated by GLUCAGON & [blood
glucose]
 Enzyme: GLUCOSE-6-PHOSPHATE
↓
GLUCOSE-6-P + H2O  GLUCOSE + Pi
Regulation of Carbohydrate Catabolism, cont…

Other regulatory enzymes
 Hexokinase: catalyzes entry of free
glucose into gycolysis
 Pyruvate kinase: catalyzes last step
in glycolysis
 Inhibited by ATP, excess fuel
Regulation of Carbohydrate Catabolism, cont…

Phosphofructokinase-1: commits cell to
passage of glucose through glycolysis
 Irreversible reaction
 Allosterically inhibited by ↑ [ATP]


When ATP levels are sufficiently high,
glycolysis is turned down
Inhibition relieved by allosteric action of
ADP & AMP

Rate of glycolysis increases when ATP levels
are low
Regulation of Carbohydrate Catabolism, cont…

Phosphofructokinase-1: links glycolysis and
citric acid cycle (CAC)
 Allosterically inhibited by citrate



An intermediate in CAC
When citrate accumulates, glycolysis slows
down
Phosphofructokinase-1also regulated by
beta-D-fructose-2,6-bisphosphate


Allosteric activator
Increases affinity of PFK for F6P
Regulation of Carbohydrate Catabolism, cont…

Futile Cycling: simultaneous production
& consumption of glucose by the cell


Gluconeogenesis: conversion of pyruvate
 glucose (opposite of glycolysis)
Uses some of the same enzymes as
glucolysis
Regulation of Carbohydrate Catabolism, cont…

Both sets of reactions are substrate
limited

Some glycolytic reactions are
irreversible (3, catalyzed by
regulatory enzymes)
Regulation of Carbohydrate Catabolism, cont…

These reactions are by-passed in
gluconeogenesis by different
enzymes

To prevent FUTILE CYCLING,
enzymes limited reactions are subject
to reciprocal allosteric control
IV. SECONDARY PATHWAYS of
GLUCOSE OXIDATION
 Pentose Phosphate pathway
 Produces NADPH & ribose-5phosphate
 NADPH used in biosynthesis of fatty
acids, steroids
 Pentoses used in nucleic acid
synthesis
Secondary Pathways of Glucose Oxidation, cont…
 Transformation into Glucuromic Acid &
Ascorbic Acid
 D-glucuronate: used to convert non-polar
toxins to polar derivatives
 L-ascorbic acid: cannot be accomplished
by humans