Carbohydrates

Carbohydrates
• Named so because many have formula Cn(H2O)n
• Produced from CO2 and H2O via photosynthesis in plants
• Range from as small as glyceraldehyde (Mw = 90 g/mol)
to as large as amylopectin (Mw = 200,000,000 g/mol)
• Fulfill a variety of functions including:
– energy source and energy storage
– structural component of cell walls and exoskeleton
– informational molecules in cell-cell signaling
• Often covalently linked with proteins or lipids to form
glycoconjugates – glycoproteins, proteoglycans,
glycolipids
 Aldoses and Ketoses

Two principal groups

An aldose contains an aldehyde functionality

A ketose contains a ketone functionality

The simplest carbohydrates – aldehydes or
ketones with two hydroxyl groups
Aldose - from triose to hexose
Asymmetric centre in monosaccharides

• Enantiomers: stereoisomers that are non-

superimposable mirror images
 Enantiomers
• Sugars may contain many chiral centers, only the most
distant from the carbonyl carbon is designated as D or L
• D and L isomers of a sugar are enantiomers
 Enantiomers
• Enantiomers: stereoisomers that are non-superimposable
mirror images
• In sugars that contain many chiral centers, only the one
that is most distant from the carbonyl carbon is designated
as D or L
• D and L isomers of a sugar are enantiomers
– e.g. L and D glucose have the same water solubility
• Most hexoses in living organisms are D stereoisomers
• Some simple sugars occur in the L-form, such as L-
arabinose
 Cyclic structure of monosaccharides

Hemiacetals and Hemiketals

• Aldehyde and ketone carbons are electrophilic

• Alcohol oxygen atom is a nucleophilic

Aldehydes form hemiacetals with alcohols

Ketones form hemiketals with alcohols

 Cyclization of Monosaccharides
• Pentoses and hexoses undergo intramolecular cyclization
 Cyclization of Monosaccharides

• The former carbonyl carbon becomes a new chiral center,

called the anomeric carbon
• The former carbonyl oxygen becomes a hydroxyl group; the
position of this group determines if the anomer is  or 
• If the hydroxyl group is on the opposite side (trans) of the
ring as the CH2OH moiety the configuration is 
• In the hydroxyl group is on the same side (cis) of the ring as
the CH2OH moiety, the configuration is 
The ring forms exist in equilibrium with the open-chain forms
Important Hexose Derivatives
 The Glycosidic Bond
• Reaction of anomeric carbon with hydroxyl or amino group
forms O-glycosidic or N-glycosidic bond
• Two sugar molecules can be joined via a glycosidic bond
between an anomeric carbon and a hydroxyl carbon -
disacharides
• The glycosidic bond between monomers (an acetal bond) is
less reactive than the hemiacetal (at the second monomer)

Maltose
• The disaccharide formed upon condensation of two glucose
molecules via 1  4 bond is called maltose
Polysaccharides
 Polysaccharides
• Natural carbohydrates are usually found as
polymers
• These polysaccharides (glycans) can be
– homopolysaccharides
– heteropolysaccharides
• Polysaccharides do not have a defined molecular
weight.
– This is in contrast to proteins because unlike
proteins, no template is used to make
polysaccharides
Storage function of polysaccharides
Homopolysaccharides – how to effectively
store the cellular fuel

Glycogen – in animals
Starch – in plants

Heavily hydrated – hydrogen bonding with

water
 Glycogen
• Glycogen is a branched homopolysaccharide of
glucose
– Glucose monomers form (1  4) linked chains
extensively branched
– Branch-points with (1  6) linkers every 8-12
residues
– Molecular weight reaches several millions
– Functions as the main storage polysaccharide in
animals
Glycogen
 Starch
• Starch is a mixture of two homopolysaccharides of
glucose
• Amylose is unbranched polymer of (1  4)
linked residues
• Amylopectin is branched like glycogen but the
branch-points with (1  6) linkers occur every
24-30 residues
• Molecular weight of amylopectin is up to 200
million
• Starch is the main storage homopolysaccharide in
plants
Starch
 Metabolism of Glycogen and
Starch
• Glycogen and starch often form granules in cells
• Granules contain enzymes that synthesize and
degrade these polymers
• Glycogen and amylopectin have one reducing
end but many non-reducing ends
• Enzymatic processing occurs simultaneously in
many non-reducing ends
Structural function of polysaccharides
Both homo- and heteropolysaccharides

Cellulose – in plants
Agar – in marine algae
Chitin – in arthropods - insects, lobsters,
crabs and fungi (mushroom)
Glycosaminoglycans – extracellular matrix
 Cellulose
• Cellulose is a homopolysaccharide of glucose

– Most abundant polysaccharide in nature

– Glucose monomers form (1  4) linked linear
chains
– Hydrogen bonds form between adjacent
monomers
– Additional H-bonds between chains
– Structure is tough and water-insoluble
– Cotton is nearly pure fibrous cellulose
Additional H-bonds between chains
 Cellulose Metabolism
• The fibrous structure, and water-insolubility makes
cellulose a difficult substrate to act on
• Fungi, bacteria, and protozoa secrete cellulase, which
allows them to use wood as source of glucose
• Most animals cannot use cellulose as a fuel source
because they lack the enzyme to hydrolyze (1 4)
linkages
• Ruminants and termites live symbiotically with a
microorganisms that produces cellulase
• Cellulases hold promise in the fermentation of biomass
into biofuels
 Chitin
• Chitin is a homopolysaccharide of N-acetylglucosamine
– N-acetylglucosamine monomers form (1  4) linked
linear chains
– Forms extended fibers that are similar to those of cellulose
– Hard, insoluble, cannot be digested by vertebrates
– Structure is tough but flexible, and water-insoluble
– Found in cell walls in mushrooms, and in exoskeletons of
insects, spiders, crabs, and other arthropods
Glycoconjugates
 Glycoconjugates
1. Glycoproteins – proteins with small
oligosaccharides attached
2. Proteoglycans – large polychaccharides bound
to peptides or proteins
3. Glycolipids – conjugate of oligosacharides and
lipids

The sugar code

• Variety of monosacharides linked in linear or branched
chains
• Bilions of combinations
 Glycoproteins
• A protein with small oligosaccharides
attached
– Carbohydrate attached via its anomeric carbon to serin,
asparagin or threonin
– About half of mammalian proteins are glycoproteins
– Very variable
– Function as enzymes, transport, receptors, hormones,
structural proteins
– Carbohydrates play role in protein-protein recognition
Oligosaccharide linkages in glycoproteins
 Glycolipids

• A lipid with covalently bound

oligosaccharide
– Parts of plant and animal cell membranes
– Recognition function on membrane surfaces
Bacterial lipopolysaccharide
 Proteoglycans

• Different polysacharides
- glucosaminoglycans -
linked to the core protein
• Many are secreted into extracellular matrix
• Some are membrane bound
 Glycosaminoglycans
• Linear polymers of repeating disaccharide units

• Various amino saccharides (on C2)

– N-acetyl-glucosamine or
– N-acetyl-galactosamine
• Negatively charged groups (on C6)
– Uronic acids (C6 oxidation)
– Sulfate esters

• Extended hydrated molecule, minimal charge repulsion

• Forms meshwork with fibrous proteins to form extracellular
matrix
– Connective tissue, cartilage, tendon, cornea, bones
– Lubrication of joints
 Repeating units of some common
glycosaminoglycans of extracellular matrix
 Proteoglycan Aggregates of
Extracellular Matrix
• Core proteins e.g. hyaluronan or aggrecan with
covalently bound glycosaminoglycans form huge
(Mr > 2•108) non-covalent aggregates
• Hold lots of water (1000 X its weight); provides
lubrication
• Very low friction or elastic material
• Covers joint surfaces: articular cartilage
– Reduced friction
– Load balancing
Proteoglycan aggregate of the extracellular matrix
 Proteoglycans
as the structural polysaccharides
of extracellular matrix
 Extracellular Matrix (ECM)
• Material outside the cell
• Strength, elasticity, and physical barrier in tissues
• Main components:
– Proteoglycan aggregates (Heparan, Chondroitin, Keratan)
– Polysaccharides (Hyaluronic acid)
– Fibrous proteins (Collagen, Elastin)
Glucose Catabolism
Glycogen Metabolism
Gluconeogenesis
 Central Importance of Glucose
• 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
 Major Pathways of Glucose
Utilization
• When there’s plenty of excess energy, glucose can
be stored in the polymeric form (starch, glycogen)
• Short-term energy needs are met by oxidation of
glucose via glycolysis to ATP and NADH
• Pentose phosphate pathway generates NADPH that
is used for detoxification, and for the biosynthesis of
lipids and nucleotides
• Structural polysaccharides (e.g. in cell walls of
bacteria, fungi, and plants) and ribose are derived
from glucose
 Glycolysis

Almost universal
central pathway
 Glycolysis: Importance
• Glycolysis is a sequence of enzyme-
catalyzed reactions by which glucose is
converted/oxidized into pyruvate
• Pyruvate can be further aerobically oxidized
• Pyruvate can be used as a precursor in biosynthesis

• In this process, some of the oxidation free

energy is captured by the synthesis of
ATP and NADH
 Glycolysis: Overview
• In the evolution of life, glycolysis was probably one
of the earliest energy-yielding pathways
• It developed before photosynthesis, when the
atmosphere was still anaerobic
• Thus, the task upon early organisms was how to
extract free energy from glucose anaerobically?
• The solution
– Activate it first by transferring couple of
phosphates to it
– Collect energy later form the high-energy
metabolites of the activated glucose
The two phases of glycolysis

the preparatory
phase

the payoff phase

 Phosphorylated intermediates:
Each of the nine glycolytic intermediates is phosphorylated

1. release of energy upon hydrolysis of

phosphoester bonds, substrate phosphorylation
2. lack of membrane transporters of
phosphorylated sugars, no need to spend
energy to keep them inside the cell
Glycolysis: The Preparatory Phase
 1) The Hexokinase Reaction
• The first step, of glucose, is catalyzed by
hexokinase in eukaryotes, and by glucokinase in
prokaryotes
• This process uses the energy of ATP
2) Phosphohexose Isomerization
• An aldose isomerize into ketose
• Rearangement of hydroxyl and carbonyl groups
at C1 and C2 cardons
• Carbonyl at C2 serves for further phophorylation
3) The Second Priming Reaction;
The First Commitment
• ATP is the donor of the second phosphate group
• From this point the product, fructose 1,6-
bisphosphate is committed to become pyruvate
and yield energy
• This is an
irreversible step
 4) Cleavage of 6-Carbon Sugars
Reaction products:
glyceraldehyde 3-phosphate (GAP)
dihydroxyacetone phosphate (DAP)
• Strongly positive DG’o favors the reverse reaction
• Low concentration of products makes the reaction
reversible
 5) Triose Phosphate Interconversion
• Two triose phosphates: DAP and GAP
• Only GAP is the substrate for the next enzyme
• DAP is converted enzymatically to GAP by triose
phosphate isomerase
• C1, C2, C3 indistinguishable from C4, C5, C6
• Reversible reaction driven by consumption of GAP
During preparatory phase of glycolysis

glucose has been phosphorylated at C1

and C6
and then

cleaved into two identical molecules of

glyceraldehyde 3-phosphate
Glycolysis: The Payoff Phase
 6) Glyceraldehyde 3-Phosphate
Dehydrogenase Reaction

• First energy-yielding
step in glycolysis
• Oxidation of
aldehyde with NAD+
gives NADH
• Phosphorylation
yields an high-energy
reaction product
7) First Substrate-Level Phosphorylation

• 1,3-bisphosphoglycerate
is a high-energy
compound
• Substrate level
phosphorylation
• The reaction is reversible
• Kinases - enzymes that
transfer phosphate groups
from ATP to various
substrates
The overall oxidation of glyceraldehyde 3-
phosphate to 3-phosphoglycerate is energy
coupling process with 1,3-bisphosphoglycer-
aldehyde as an intermediate
The enegry of aldehyde oxidation is here
conserved in NADH and ATP
Reversible reaction with DG’o = - 12.2 kJ/mol
 8) Conversion of 3-Phosphoglycerate
to 2-Phosphoglycerate
• Reversible isomerization reaction
• Enzymes that shift functional groups around are
called mutases
 9) Dehydration of 2-Phosphoglycerate
• The goal of this step is to create a better
phosphoryl donor
• Loss of phosphate from 2-phosphoglycerate would
give a secondary alcohol with no further
stabilization …
10) Second Substrate-Level Phosphorylation

• … but loss of
phosphate from
phosphoenolpyruvate
yields to an enol that
spontaneously
tautomerizes into
ketone
• The tautomerization effectively lowers the
concentration of the reaction product and drives
the reaction toward ATP formation
Feeder Pathways for Glycolysis
 Feeder Pathways for Glycolysis
• Dietary dichaccharides and polysaccharides (sucrose,
lactose, starch, glycogen)
- hydrolytic digestion to monosaccharides
- transport to target cells
• Endogenous glycogen or starch
- phosphorolytic generation of glucose 1-phosphate by
a Pi attach on the 1-4 glycosidic bond of last residues
by phosphorylase
- phosphorylated monosaccharide can not be
transferred through membrane
- glucose 1-phosphate is converget to glucose 6-
phosphate by phosphoglucomutase
 Konec prvni casti
Ruzne tezky test pro chemiky a pro biofyziky a
bioinformatiky
The two phases of glycolysis

the preparatory
phase

the payoff phase

Fate of Pyruvate
Pyruvate metabolism under aerobic
and anaerobic conditions
• Aerobic conditions – pyruvate is oxidised to
acetylCoA
• NADH is further oxidised to NAD+ in electron
transport chain by oxygen
• Under anaerobic condition NADH cannot be
reoxidized
• Energy yielding glycolysis would stop due to the
lack of NAD+
 Under Anaerobic Conditions, Animals
Reduce Pyruvate to Lactate
• During long exercise (demand for ATP exceeds the oxidation
capacity – not enough of ATP produced in NADH oxidation)
• Organism regenerates more NAD+ to produce ATP in glycolysis
lactate builds up in the muscle
Under Anaerobic Conditions, Animals
Reduce Pyruvate to Lactate

• The acidification of muscle prevents its continuous

intensive work
• The lactate can be transported to liver and
converted to glucose there
 The Cory Cycle

• Lactate produced after

exercise in muscles returns to
the liver
• Lactate in liver is converted to
glucose which moves back to
muscles to be converted to
glycogen
Under Anaerobic Conditions, Yeast
Ferments Glucose to Ethanol
• Both steps require cofactors
– pyruvate decarboxylase Mg++ and thiamine
pyrophosphate
– alcohol dehydrogenase Zn++ and NAD+ in
 Pyruvate metabolism under
aerobic conditions
Conversion of Pyruvate to Acetyl-CoA

• the reaction: oxidative decarboxylation of pyruvate

• acetyl-CoA can then enter the citric acid cycle and
yield energy
• acetyl-CoA can be used to synthesize storage
lipids
• catalyzed by the pyruvate decarboxylase complex
• requires five coenzymes and three enzymes
Overall reaction catalyzed by the
pyruvate dehydrogenase complex

- oxidative decarboxylation -
 Five coenzymes

Thiamine pyrophosphate – TPP

Flavin adenine dinucleotide – FAD
Coenzyme A – CoA, CoA-SH
Nicotine adenine dinucleotide – NAD
Lipoic acid – Lipoate
 Pyruvate Dehydrogenase
Complex (PDC)

PDC is a large (Mr = 7.8 × 106 Da) multienzyme complex

- pyruvate dehydrogenase (E1)
- dihydrolipoyl transacetylase (E2)
- dihydrolipoyl dehydrogenase (E3)
short distance between catalytic sites allows channeling
of substrates from one catalytic site to another
channeling minimizes side reactions
Three-dimensional Reconstruction
from Cryo-EM data
Gluconeogenesis
Gluconeogenesis: Precursors for
Carbohydrates

• Glucose – central role in metabolism

• Universal in living organisms
• Some tissues depends only on glucose
• Brain requires about 120 g of glucose pear day
• Glucose supply varies over a day
• Need for glucose synthesis from noncarbohydrate
precursors
Carbohydrate synthesis from simple precursors
Mammals (vertebrates)
cannot convert
Fatty acids (Acetyl-CoA)
to
Sugars (Glucose)
Glycolysis vs. Gluconeogenesis

• Glycolysis occurs mainly in the muscle and brain

• Gluconeogenesis occurs mainly in the liver

• They are not identical pathways

• 7 of the 10 are reversible glycolytic reactions

1. Conversion of glucose to Glc
6-phosphate
2. Phosphorylation of fructose 6-
phosphate
3. Conversion of
phosphoenolpyruvate to
pyruvate

Large negative DG
The three reactions are bypassed
by a separate set of enzymatic
reactions
Both pathways occurs largely in
cytosol
 Synthesis of Oxaloacetate
• Conversion of pyruvate to energy-rich
phosphoenolpyruvate requires two energy-
consuming steps

• In the first step, pyruvate is

transported into
mitochondria and converted
into oxaloacetate by
pyruvate carboxylase
• Pyruvate carboxylase reaction is the first site
of regulation of gluconeogenetic pathway

• It is positively effected by Acetyl-CoA

(product of fatty acid catabolism)

• Pyruvate carboxylase reaction can replenish

intermediates in other metabolic pathways
 Oxalacetate is converted to
phosphoenolpyruvate
In the second step,
oxaloacetate is converted to
phosphoenolpyruvate

Oxaloacetate picks up
phosphate from GTP

The phosphoenolpyruvate
carboxykinase reaction
occurs either in the cytosol or
the mitochondria
 From Pyruvate to
Phosphoenolpyruvate
Mitochondrial membrane has no
transporters for oxaloacetate

In mitochondria:
Oxaloacetate has to be first
reduced to malate by malate
dehydrogenase
Malate is transported from
mitochondria and reoxidized to
oxaloacetate
 From Pyruvate to
Phosphoenolpyruvate
Transport of malate from
mitochondria is accompanied by
the transport of reducing “power” of
NADH from mitochondria to cytosol

[NADH]/[NAD+] ratio in cytosol is

about 105 times lower than in
mitochondria
NADH is consumed in
gluconeogenesis – need for NADH
in cytosol
 Second Bypasse

• Conversion of Fructose 1,6-bisphosphate to

Fructose 6-phosphate by Fructose 1,6-
bisphosphatase (FBPase-1)
• Release of an inorganic phosphate
• Resonance stabilization of phosphate

DG’o = - 16.3 kJ/mol

1. Conversion of glucose to Glc
6-phosphate
2. Phosphorylation of fructose 6-
phosphate
3. Conversion of
phosphoenolpyruvate to
pyruvate

Large negative DG
The three reactions are bypassed
by a separate set of enzymatic
reactions
Both pathways occurs largely in
cytosol
 Third Bypasse
• Conversion of Glucose 6-phosphate to Glucose
• Reaction does not synthesis ATP - simple
hydrolysis of phosphate ester to inorganic
phosphate
• Resonance stabilization of phosphate

DG’o = - 13.8 kJ/mol

Gluconeogenesis – energetically expensive
Requires 4ATP, 2GTP, 2NADH for one molecule of Glucose
Only 2ATP and 2NADH are produced in glycolysis
Glycolysis and Gluconeogenesis are reciprocally regulated
- 16,7 kJ/mol - 13.8 kJ/mol

- 14,2 kJ/mol - 16.3 kJ/mol

- 31,4 kJ/mol 0,9 kJ/mol

Pentose Phosphate Pathway
 Pentose Phosphate Pathway
• The main goals are to produce NADPH for
anabolic reactions and ribose 5-phosphate for
nucleotides

In rapidly dividing cells - high demand for

ribose 5-phosphate

Cells with high reductive biosynthesis -

demand for NADPH

Demand of NADPH in oxygen exposed cells

NAD and NADP in metabolism

NAD+/NADH - catabolism, further in ATP

production

NADP+/NADPH – anabolism, biosynthetic

reactions
Pentose Phosphate Pathway
 Oxidative phase

1. Oxidation of Glucose 6-phosphate by glucose 6-

phosphate dehydrogenase to 6-phophogluconate

2. Oxidation and decarboxylation of

6-phophogluconate by 6-phophogluconate
dehydrogenase to Ribulose 5-phosphate

3. Isomerisation of Ribulose 5-phosphate to Ribose

5-phosphate by phosphopentose isomeraze
Pentose Phosphate Pathway
 Nonoxidative phase

In tissues requiring only NADPH Ribulose 5-phosphate is

regenerated to Glucose 6-phosphate in nonoxidative paths

In a series of reactions catalysed by transketolases and

transaldolases 6 pentoses are transformed in to 5 hexoses

6 x C5 = 5 x C6
 NADPH Regulates Pentose
Phosphate Pathway

NADPH inhibits glucose-6-

phosphate dehydrogenase
 Learning objectives

– Carbohydrates, their chemistry and function

– Glycoconjugates, glycoproteins, peptidoglycans,
glycolipids
– Glycolysis preparatory and payoff phase principles of
energy extraction
– Pyruvate, anaerobic and aerobic metabolism
– Regeneration of NAD+
– Acetyl-CoA production
– Gluconeogenesis
– Importance of the first bypass reaction in metabolism
– Pentose phosphate cycle
– Production od NADPH and ribose