UNIT 4: CARBOHYDRATES

Objectives
 Define carbohydrates in chemical terms
• Classify carbohydrates in to three major groups with
examples of each group
• List the monosaccharides of biological importance and
learn their properties
• List the disaccharides of biological importance and
learn their properties
• List the polysaccharides of biological importance and
learn their properties
• Study the chemistry and functions of glycoproteins
 Introduction
• Carbohydrates are the most abundant macromolecules in
nature.
• They are the main source and storage of energy in the
body.
• They serve also as structural component of cell
membrane.
• The molecular formula of carbohydrate is CnH2nOn or
(CH2O)n, where n > 3.
• Chemically, they contain the elements Carbon, hydrogen
and oxygen. Thus they are Carbon compounds that
contain large quantities of Hydroxyl groups.
• Carbohydrates in general are polyhydroxy aldehydes or
ketones or compounds which give these substances on
hydrolysis.
 Chemistry of Carbohydrates
• Classification and Structure
Classification
There are three major classes of carbohydrates:
• Monosaccharides (Greek, mono = one)
• Oligosaccharides (Greek, oligo= few) 2-10
monosaccharide units.
• Polysaccharides (Greek, Poly = many) >10
monosaccharide units.
Monosaccharides
Monosaccharides also called simple sugars.
• They consist of a single polyhydroxy aldehyde or
ketone units.
• The most abundant monosaccharides in nature are the 6-
carbon sugars like D-glucose and fructose.
 Cont.….

• There are two families of monosaccharides.

• Monosaccharides having aldehyde groups are
called Aldoses and monosaccharides with Ketone group
are Ketoses.
• Depending on the number of carbon atoms, the
monosaccharides are named trioses (C3),
tetroses (C4), pentoses (C5), hexoses (C6), heptoses (C7)
Cont.…..
 Physical properties
• Physical properties of Monosaccharides
• They are colorless, crystalline compounds, readily soluble
in water. Their solutions are optically active and exhibit
the phenomenon of autorotation.
• Carbohydrates spontaneously change between the α and β
configuration.
Asymmetric Center and Stereoisomerism
Asymmetric carbon is a carbon that has four different
groups or atoms attached to it and having optically
activity in solution.
 Cont.….
• All the monosaccharides except dihydroxyacetone
contain one or more asymmetric or chiral carbon atoms
and thus occur in optically active isomeric forms.
• Monosaccharides with n number of asymmetric centers
will have (2n) isomeric forms. (n= number of
asymmetric carbon atoms).
 Cont.….

• The designation of a sugar isomer as the D- form or of its

mirror images the L- form is determined by the spatial
relationship to the parent compound of the carbohydrate
family.
• The orientation of- OH and- H groups around the carbon
atom adjacent to the terminal primary alcohol carbon
determines its D or L form .
• When the - OH group on this carbon is on the right, the
sugar is a member of the Dseries, when it is on the left, it
is a member of the L-series. These D and L configuration
are also called Enantiomers.
 Cont.…..

• The presence of asymmetric carbon atom causes optical

activity.
• When a beam of plane polarized light is passed through a
solution of carbohydrate it will rotate the light either to right
or to left.
• Depending on the rotation, molecules are called
dextrorotatory (+) (d) or levorotatory (-)(l).
• Thus, D- glucose is dextrorotatory but D- fructose is
levorotatory.
• When equal amounts of D and L isomers are present, the
resulting mixture has no optical activity, since the activities of
each isomer cancel one another.
• Such a mixture is called racemic or DL mixture.
 Epimers
• When sugars are different from one another, only
in configuration with regard to a single carbon
atom (around one carbon atom) they are called
epimers of each other.
• For example glucose and mannose are epimers.
• They differ only in configuration around C2.
• Mannose and Galactose are epimers of Glucose
 Anomers
• The two stereoisomers at the hemiacetal (anomeric)
carbon are:
o The alpha anomer: Where- OH group is down
(Haworth)
o The beta anomer:Where- OH group is up (Haworth)
• Anomers are diastereomers (having different physical
properties)
Cyclization of monosaccharides
• Monosaccharides with five or more carbon atoms in the
backbone usually occur in solution as cyclic or ring
structure, in which the carbonyl group is not free as
written on the open chain structure but has formed a
covalent bond with one of the hydroxyl group along the
chain to form a hemiacetal or hemiketal ring.
• In general, an aldehyde can react with an alcohol to form
a hemiacetal or acetal.
 Cont.….
• The C-1 aldehyde in the open-chain form of glucose
reacts with the -5th carbon atom containing hydroxyl
group to form an intramolecular hemiacetal.
• The resulting six membered ring is called pyranose
because of its similarity to organic molecule Pyran.
• Similarly, a ketone can react with an alcohol to form a
hemiketal or ketal.
• The C-2 keto group in the open chain form of fructose
can react with the 5th carbon atom containing hydroxyl
group to form an intramolecular hemiketal.
• This five membered ring is called furanose because of its
similarity to organic molecule furan
 Oligosaccharides
• Oligosaccharides contain 2 to 10 monosaccharide units.
• Disaccharides
• When two monosaccharides are covalently bonded
together by glycosidic linkages a disaccharide is formed.
• Glycosidic bond is formed when the hydroxyl group on
one of the sugars reacts with the anomeric carbon on the
second sugar.
• Biologically important disaccharides are sucrose, maltose,
and Lactose.
Maltose
Maltose contains two D glucose residues joined by a
glycosidic linkage between OH at the first carbon atom of
the first glucose residues and OH at the fourth carbon
atom of the second glucose forming a α-(1,4) glycosidic.
•
 Cont.…..

• Maltose is the major degradative product of Starch.

• Maltose is hydrolyzed to two molecules of D- glucose by
the intestinal enzyme maltase, which is specific for the α-
(1, 4) glycosidic bond.
 Sucrose (Cane sugar)
• Sucrose is a disaccharide of α- D- glucose and β-D-
fructose. It is obtained from cane sugar.
• It is also present in various fruits. In contrast to other
disaccharides sucrose contains no free anomeric carbon
atom. Since the anomeric carbons of both its component
monosaccharide units are linked to each other. For this
reason sucrose is non reducing sugar.
 Polysaccharides
• Most of the carbohydrates found in nature occur in the
form of high molecular polymers called polysaccharides.
There are two types of polysaccharides .These are:
• Homopolysaccharides that contain only one type of
monosaccharide building blocks.
• Heteropolysaccharides, which contain two or more
different kinds monosaccharide building blocks.
Homopolysaccharides
Example of Homopolysaccharides: Starch, glycogen,
Cellulose and dextrins.
 Starch
• It is one of the most important storage polysaccharide in
plant cells.It is especially abundant in tubers, such as
potatoes and in seeds such as cereals.
• Starch consists of two polymeric units made of glucose
called Amylose and Amylopectin but they differ in
molecular architecture.
• Amylose is unbranched with 250 to 300 D-Glucose units
linked by α-(1, 4) linkages Amylopectin consists of long
branched glucose residue (units) with higher molecular
weight.
 Cont.…..
• The inner part of glucose units in amylopectin are
joined by α-(1,4) glycosidic linkage as in amylose ,
but the branch points of amylopectin are α- (1,6)
linkages.
• The branch points repeat about every 20 to 30 (1-4)
linkages
Glycogen
- Glycogen is the main storage polysaccharide of
animal cells (Animal starch).
- It is present in liver and in skeletal muscle.
- Like amylopectin glycogen is a branched
polysaccharide of D-glucose units in α - (1, 4)
linkages, but it is highly branched.
 cont……
• The branches are formed by α -(1,6) glycosidic linkage
that occurs after every 8 -12 residues.
• Therefore liver cell can store glycogen within a small
space.
• Multiple terminals of branch points release many glucose
units in short time.
 Cellulose
• Cellulose is the most abundant structural polysaccharide
in plants. It is fibrous, tough, water insoluble.
• Cellulose is a linear unbranched homopolysaccharide of
10,000 or more D- glucose units connected by β-(1, 4)
glycosidic bonds.
• Humans cannot use cellulose because they lack of
enzyme (cellulase) to hydrolyze the β-( 1-4) linkages.
 Dextrins
• These are highly branched homopolymers of glucose
units with α-(1, 6), α-(1, 4) and α-(1, 3) linkages.
• Since they do not easily go out of vascular compartment
they are used for Intravenous infusion as plasma volume
expander in the treatment of hypovolumic shock.
Hetero polysaccharides
• These are polysaccharides containing more than one type
of sugar residues
1. Glycosaminoglycans, (GAGs or mucopolysaccharides)
They are long, usually unbranched, composed of a
repeating disaccharide units
* They are negatively charged heteropolysaccharid chains
(polyanions)
 Cont.….

• The amino sugar is either D-glucosamine or D-

galactosamine in which the amino group is
usually acetylated, thus eliminating its positive charges.
• The amino sugar may also be sulfated on carbon 4, 6, or on
a monoacetylated nitrogen.
• The acidic sugar is either D-glucuronic acid or its carbon
6 epimer, L-uronic acid. For
example Hayluronic acid, Heparin and chondatin sulphate.
 Function of Glycosammoglycans. (GAGS)
• 1. They have the special ability to bind large amounts of water,
there by producing the gel-like matrix that forms the basis of the
body’s ground substance.
2. Since they are negatively charged, for example, in bone,
glycosaminoglycans attract and tightly bind cattions like ca++,
they also take-up Na+and K+
3. GAGs stabilize and support cellular and fibrous components of
tissue while helping maintain the water and salt balance of the
body.
4. Its essential components of the extra cellular matrix, GAGs’
play an important role in mediating cell-cell interactions
• Ground substance is a part of connective tissue, which is a gel
like substance containing water, salt, proteins and
polysaccharides.
• An example of specialized ground substance is the synovial fluid,
which serves as a lubricant in joints, and tendon sheaths.
 3. Heparin
• contains a repeating unit of D-glucuronic and D-
gluconsamine, with sulfate groups on some of the
hydroxyl and amine-groups
• It is an important anticoagulation, prevents the clotting
of blood by inhibiting the conversion of prothrombin to
thrombin.
• Thrombin is an enzyme that acts on the conversion of
plasma fibrinogen into the fibrin.
• It is found in mast cells in lung, liver skin and intestinal
mucosa.
 Glycoproteins (Mucoproteins)
• Glycoprotiens are proteins to which oligosaccharides are
covalently attached. They differ from the
glycosaminoglycans in that the length of the
glycoproteins carbohydrate chain is relatively
short (usually two to ten sugar residues in length,
although they can be longer), whereas it can be very long
in the glycosaminoglycans.
The glycoprotein carbohydrate chains are often branched
instead of linear and may or may not be negatively
charged. For example:
- Glycophorin, a glycoprotein found in human red cell
membranes.
- Human gastric glycoprotein (mucin).
- Many protein hormones,receptors are glycoproteins
 Cont.……

• Proteoglycans
When glycosamnoglycans are attached to a protein
molecule the compound is called proteoglycan
[proteoglycans = Glycosaminoglycans + proteins]
 METABOLISM OF CARBOHYDRATES
 Objectives
• study utilization of glucose and other carbohydrates in the body
• study the various mechanisms and fate of glucose in the body
• study the energetics of the various mechanisms
Digestion of Carbohydrates
 Dietary carbohydrates principally consist of the
polysaccharides: starch and glycogen.
 It also contains disaccharides: sucrose, lactose, maltose
and in small amounts monosaccharides like fructose and
pentoses.
 Liquid food materials like milk, soup, fruit juice escape
digestion in mouth as they are swallowed, but solid
foodstuffs are masticated thoroughly before they are
swallowed.
 1. Digestion in Mouth
• Digestion of carbohydrates starts at the mouth, where they
come in contact with saliva during mastication.
• Saliva contains a carbohydrate splitting enzyme called salivary
amylase (ptyalin).
Action of ptyalin (salivary amylase)
• It is α - amylase, requires Cl- ion for activation and optimum
pH 6-7.
• The enzyme hydrolyzes α- (1,4) glycosidic linkage at random,
from molecules like starch, glycogen and dextrins, producing
smaller molecules maltose, glucose and disaccharides
maltotriose.
• Ptyalin action stops in stomach when pH falls to 3.0
 Cont.….
• 2. Digestion in Stomach
• No carbohydrate splitting enzymes are available in gastric juice.
• HCl may hydrolyze some dietary sucrose to equal amounts of
glucose and fructose.
3. Digestion in Duodenum
• Food reaches the duodenum from stomach where it meets the
pancreatic juice.
• Pancreatic juice contains a carbohydrate-splitting enzyme
pancreatic amylase.
• Action of pancreatic Amylase
• It is also an α - amylase, optimum pH 7.1. Like ptyalin it also
requires Cl- for activity.
• The enzyme hydrolyzes α-(1,4) glycosidic linkage situated well
inside polysaccharide molecule.
• Other criteria and end products of action are similar of ptyalin.
 Cont.…..

• 1. Digestion in Small Intestine

Action of Intestinal Juice
a. pancreatic amylase:
• It hydrolyzes terminal α-(1,4), glycosidic linkage in
polysaccharides and Oligosaccharide molecules liberating
free glucose molecules.
b. Lactase
It is a β- glycosidase, its pH range is 5.4 to 6.0. Lactose is
hydrolyzed to glucose and galactose.
 Cont.….

• Lactose Intolerance
• Lactose is hydrolyzed to galactose and glucose by lactase
in humans (by β- Galactosidase in Bacteria).
• Some adults do not have lactase. Such adults cannot digest
the sugar.
• It remains in the intestines and gets fermented by the
bacteria. The condition is called as Lactose intolerance.
• Such patients suffer from watery diarrhea, abnormal
intestinal flow.
• They are advised to avoid the consumption of Lactose
containing foods like Milk.
 Absorption of Carbohydrates
• Products of digestion of dietary carbohydrates are
practically completely absorbed almost entirely from
the small intestine.
• Absorption from proximal jejunum is three times
grater than that of distal ileum.
• It is also proved that some disaccharides, which
escape digestion, may enter the cells of the intestinal
lumen by “pinocytosis” and are hydrolyzed within
these cells.
• No carbohydrates higher than the monosaccharides
can be absorbed directly in to the blood stream.
 Mechanism of Absorption
• Two mechanisms are involved:
1. Simple Diffusion
• This is dependent on sugar concentration gradients
between the intestinal lumen.
• Mucosal cells and blood plasma.
• All the monosaccharides are probably absorbed to some
extent by simple ‘passive’ diffusion.
 Cont.…..

• 2. “Active “Transport Mechanisms

• Glucose and galactose are absorbed very rapidly and
hence it has been suggested that they are absorbed
actively and it requires energy.
• Fructose absorption is also rapid but not so much as
compared to glucose and galactose but it is definitely
faster than pentoses.
• Hence fructose is not absorbed by simple diffusion alone
and it is suggested that some mechanism facilitates its
transport, called as” facilitated transport”.
 GLYCOLYSIS
• Glycolysis is a set of reactions that converts glucose to
pyruvate or lactate. This is the first metabolic pathway to
be elucidated and hence is considered as a paradigm of
metabolic pathways.
• Glycolysis is also called Embden-Meyerhoff pathway.
• The complete set of reactions occurs in the cytoplasm of
virtually every animal cell. The entire process occurs
without molecular oxygen.
 Cont.……

• Glycolysis consumes 2 ATP and generates 4ATP. Thus, the

process results in the generation of 2 net ATP.
• The process also generates 2 NADH.
• What happens to pyruvate depends upon the presence or
absence of mitochondria in the cell or upon the
availability of oxygen in mitochondria containing cells.
 Cont.…..
• For the glycolytic pathway to continue, NAD+ has to
be regenerated.
• In erythrocytes (no mitochondria) and in
mitochondria possessing cells under anaerobic
conditions, NAD+ is regenerated from NADH during
the conversion of pyruvate to lactate.

In mitochondria – possessing cells under aerobic conditions,

NAD+ is regenerated by either malate – aspartate shuttle or
α-glycerophosphate shuttle, which transfer the reducing
equivalents from NADH into mitochondria for electron
transport chain, thus regenerating NAD+ in the cytoplasm.
 Reactions
• Glycolysis consists of two phases.
• In the first phase, glucose is broken down to two
molecules of glyceraldehyde-3-phosphate in a series of
five reactions.
• In the second phase, another series of five reactions
convert these two molecules of glyceraldehyde-3-
phosphate into two molecules of pyruvate.
• Phase I consumes 2 ATP and
• Phase II generates 4 ATP.
• The net ATP production in the entire process is 2.
 Individual Reactions
• Reaction # 1: Hexokinase/Glucokinase
• This is the first intracellular reaction of glycolysis
(remember all reactions are in the cytoplasm).
b. Requires an ATP (Mg). This is one of the
investment reactions.
c. The phosphorylation of glucose traps the glucose
inside the cell.
d. The reaction is considered irreversible.
e. Hexokinase has a low Km for glucose of less than
0.1 mM (high affinity). It is also inhibited by the
product glucose-6-phosphate
f. Liver hepatocytes and pancreatic β cells contain
another enzyme
 Glucokinase.
• Glucokinase has a high Km for glucose of about 10 mM
(low affinity).
• Glucokinase is not inhibited by its product glucose-6-
phosphate.
• The levels of glucokinase in the liver of untreated Type 1
diabetics are lower than normal.
• Hexokinase and glucokinase are isoenzymes.
 Reaction #2: Phosphoglucose isomerase.
• a. This reaction is readily reversible (not a
controlling step) and functions in both glycolysis
and gluconeogenesis.
b. Conversion of an aldose to a ketose.
 Reaction #3: 6-Phosphofructo-1-kinase (PFK-1) or
Phosphofructokinase-1.
• a. Reaction is the rate-limiting step of glycolysis.
b. It is irreversible, and the committed step.
• It is an allosteric enzyme and also a major
regulatory enzyme.
c. We have invested our second ATP molecule.
•
 Reaction #4: Aldolase.
• a. We now have two phosphorylated trioses.
b. Only glyceraldehyde-3-phosphate is used in glycolysis.
Therefore, dihydroxyacetone phosphate has to be
converted into glyceraldehyde-3-phosphate.
 Reaction #5: Triose phosphate isomerase
• a. Catalyzes the interconversion of dihydroxyacetone
phosphate and glyceraldehyde-3-phosphate.
b. Because of the interconversion, one glucose molecule
can be converted to two glyceraldehyde-3-phosphate
molecules.
•
 Reaction # 6: Glyceraldehyde-3-phosphate dehydrogenase
• a. The enzyme oxidizes the number one carbon aldehyde
and then adds a phosphate group.
• We have an acid anhydride in the product 1,3-
bisphophoglycerate.
• b. The phosphate on the number 3 carbon is not an high-
energy bond
•
 Cont.…..
• c. We have used an NAD+ for the oxidation reaction.
• The cell has limited amounts of NAD+, so somewhere
along the line we have to regenerate it or glycolysis will
stop.
d. This reaction is a target for Arsenate (AsO43-).
• The arsenate resembles inorganic phosphate (Pi).
• In the presence of arsenate, the product of the reaction is
1-arseno- 3-phosphoglycerate.
• This product is unstable and decomposes into arsenate and
3- phosphoglycerate with no ATP formation.
• After this step, glycolysis continues.
e. The enzyme contains an essential thiol (cysteine-SH)
group at the active site.
• Iodoacetic acid (ICH2COOH) is also an inhibitor of this
reaction. It reacts with the active site SH group and
inhibits the enzyme.
 Reaction #7: Phosphoglycerate kinase
• a. This is the first step of energy production.
b. This is referred to as substrate-level phosphorylation as
opposed to oxidative phosphorylation that occurs in
mitochondrial ATP production.
d. We have recovered both ATP that were invested.
Remember that each glucose gives 2 phosphoglycerate
molecules.
•
Cont.…..
 Reaction#9: Enolase
• a. Catalyzes the dehydration of 2-phosphoglycerate to
form phosphoenolpyruvate (PEP).
•
 Reaction # 10: Pyruvate Kinase

a. Catalyzes the transfer of the phosphate from PEP to

ADP to generate ATP and 9 pyruvate.
b. Again, this is substrate-level phosphorylation.
c. This reaction completes that part of glycolysis that is
common to both anaerobic and aerobic metabolism.
 Cont.….

• d. Under aerobic conditions and the presence of

mitochondria, pyruvate can enter the citric acid
cycle. NAD+ is regenerated by malate/aspartate
shuttle or by α- glycerophosphate shuttle
e. Under anaerobic conditions or in the absence of
mitochondria, pyruvate is reduced to lactate and
regenerate NAD+.
 Anerobic Glycolysis
• When pyruvate cannot be oxidized within mitochondria
for some reason (e.g., hypoxia, genetic defects in pyruvate
dehydrogenase or citric acid cycle enzymes, genetic
defects in electron transport chain), pyruvate is reduced
to lactate by lactate dehydrogenase.

•
 Cont.….
• a. All cells contain a catalytic amount of 2,3-BPG.
b. Red blood cells contain a high concentration of 2,3-
BPG (~ 4mM).
• It facilitates the release of oxygen from hemoglobin.
c. ~20% of glucose goes through this shunt in RBC.
d. No net ATP is generated if 1,3-BPG is converted to 3-
PG via 2,3-BPG.
• If the entire glycolytic pathway in RBC occurs via the
formation of 2,3-BPG, glycolysis will yield no net ATP
because the 2 ATP produced by pyruvate kinase will be
equal to 2 ATP consumed in Phase I of glycolysis.
 Cont.…..
• e. 2, 3-BPG levels in erythrocytes increase in high
altitude where partial pressure of oxygen is low.
f. Loss-of-function mutations in pyruvate kinase
increase the levels of 2,3-BPG in RBC, thus shifting the
sigmoidal curve of oxygen binding to hemoglobin to the
right.
• If loss-of-function mutations occurs in any of the
enzymes upstream of the step involved in the formation
of 1,3-BPG, the levels of 2,3-BPG in RBC will be lower
than normal, thus shifting the curve to the left.
 Control of Glycolysis
• It is obvious that glycolysis must be controlled.
 Cont.…..

• There are only three major points of control of glycolysis.

Hexokinase and glucokinase: Reaction # 1
• Hexokinase is found in most tissues.
1. It has a low Km for glucose (<0.1mM).
a. Allows for the phosphorylation of glucose when blood
levels of glucose are low.
b. This permits tissues that rely on glycolysis extensively
to carry out the phosphorylation.
c. Hexokinase is strongly inhibited by its product,
glucose-6-Phosphate (G6P).
2. Liver parenchymal cells (hepatocytes) contain an
isozyme of hexokinase, glucokinase.
a. High Km for glucose (10 mM).
 Cont.….
• b. Concentration in cell reflects blood concentration.
c. Liver uses glucose at a significant rate only when blood
glucose is elevated.
3. Glucokinase is not inhibited by G6P.
4. It is inhibited by fructose-6-phosphate (F6P) [which is
generated by reaction # 2] and stimulated by fructose-1-
phosphate (F1P).
a. Regulation occurs via an inhibitory protein.
1). F6P stimulates the binding of the inhibitory protein.
2). F1P stimulates the dissociation of the inhibitory
protein.
 Cont.……
• 5. Glucokinase is inducible in liver by insulin. cAMP
decreases glucokinase levels in liver.
6. Glucokinase is also present in pancreatic β cells where
it plays an essential role in glucose-induced insulin
secretion.
• Because of the low affinity, this enzyme serves as a sensor
of blood glucose level to co-ordinate insulin secretion
appropriately.
 Cont.…..

• 6-Phosphofructo-1-kinase (PFK-1) reaction # 3

1. This is the rate-limiting step of glycolysis.
2. First committed step of glycolysis.
3. Subject to the greatest degree of regulation by allosteric
effectors.
4. Positive effectors:
a. Pi and AMP are positive effectors of PFK-1.
b. AMP is a negative effector of F1,6BPase, which
catalyzes the reverse reaction, converting F1, 6BP into F6P
and Pi.
c. Fructose 2,6-bisphosphate.
• This is a positive effector of PFK-1 and a negative effector
of F1,6BPase.
 Cont.…..

• 5. Negative effectors.
a. ATP: ATP is an allosteric inhibitor of PFK-1.
b. Citrate: Glycolysis and citric acid cycle are coupled
via PFK-1 because citrate, an intermediate in citric
acid cycle, is an allosteric inhibitor of PFK-1.
• When the citric acid cycle is saturated with high
levels of citrate, citrate leaves mitochondria via a
transporter (tricarboxylate transporter) and inhibits
PFK-1.
• This prevents generation of pyruvate, which feeds
into citric acid cycle.
d. Pasteur effect. Oxygen inhibits glycolysis.
The consumption of glucose in the presence of oxygen
generates much more ATP. The inhibition is probably
due to the inhibition of PFK-1 by ATP.
 Cont.….
• C. Pyruvate kinase: reaction # 10
1. Inhibited by high concentrations of ATP.
2. Isoenzyme found in liver is activated by fructose 1,6-
bisphosphate.
3. Liver enzyme is subject to phosphorylation.
a. Active in the dephosphorylated state.
b. Inactive in the phosphorylated state.
Inactivation by phosphorylation is a function of cAMP-
dependent protein kinase in liver.
• 4. Enzyme is inducible by high carbohydrate concentration and
also high insulin levels.
 Cont.…..

• Control of 6-Phosphofructo-1-kinase (PFK-1) by Fructose

2,6-bisphosphate [Regulation by cAMP]
A. Let's talk about its effect in liver.
1. Fructose 2,6-bisphosphate behaves like AMP.
a. Positive effector of 6-phosphofructo-1-kinase (PFK-1).
• The positive effect involves an increase in the affinity of
PFK-1 for F6P and also a decrease in the inhibitory
effect of ATP.
b. Negative allosteric effector of fructose 1,6-
bisphosphatase.
2. This compound is not a product of glycolysis.
a. In fact, its only known function is as a regulator.
b. It is found in all cells.
B. Hormonal control of fructose 2,6-bisphosphate in liver.
Cont.…..
 Cont.….
• 1. Glucagon (a 29-aa hormonal peptide) is released by
the α-cells of the pancreas and interacts with a glucagon
receptor on the surface of the liver plasma membrane.
2. The binding of glucagon to the receptor is "sensed' by
adenylate cyclase (enzyme located on the inner surface of
the plasma membrane).
a. Adenylate cyclase catalyzes the conversion of cytosolic
ATP to cAMP and PPi.
3. cAMP ultimately causes a decrease in the levels of
fructose 2,6-bisphosphate.
 Cont.…..
• a. cAMP acts as a second messenger.
b. Note, cAMP acts to inhibit glycolysis in liver, but in
most other cells cAMP stimulates glycolysis.
• The difference is that liver is the primary organ, which
controls blood glucose levels.
• Liver can make glucose (gluconeogenesis) as well as
degrade glucose (glycolysis) as needed.
• cAMP stimulates gluconeogenesis in liver and inhibits
glycolysis.
 Cont.….
• cAMP-induced decrease in fructose 2, 6-
bisphosphate levels is at least partly responsible for these
effects.
• In some cells, cAMP actually stimulates the synthesis of
fructose 2,6-bisphosphate (e.g., heart muscle).
• In skeletal muscle, cAMP does not affect the synthesis of
fructose-2, 6- bisphosphate.
C. Synthesis of Fructose 2,6-bisphosphate
 Cont.……

• 1. Fructose 2,6-bisphosphate is synthesized from fructose

6-phosphate (an intermediate in glycolysis) via the
enzyme 6-phosphofructo 2-kinase (PFK-2). This is not
the same as 6-phosphofructo 1-kinase (PFK-1).
2. Fructose 2,6-bisphosphate is destroyed in cells by the
enzyme fructose 2,6-bisphosphatase.
3. Both of the above enzyme activities are located on the
same protein.
• It is a bifunctional enzyme and is referred to as 6-
phosphofructo-2-kinase/fructose 2,6- bisphosphatase.
4. cAMP is responsible for the regulation of fructose 2,6-
bisphosphate levels in liver.
 Cont.…..
• D. The PKF-2/F2, 6BPase is regulated by protein kinase A
and phosphoprotein phosphatase as follows:
•
 Cont.….

• 1. Phosphorylation causes inactivation of the active site

responsible for the synthesis of fructose 2,6-bisphosphate
and activation of the site responsible for the hydrolysis.
2. Dephosphorylation has the opposite effect.
3. Increased levels of glucagon cause an increase in cAMP
levels.
a. This second messenger activates cAMP dependent
protein kinase.
b. This phosphorylates 6-phosphofructo-2 kinase/fructose
2,6-bisphosphatase.
c. The synthesis of fructose 2,6-bisphosphate is inhibited
and its degradation is promoted.
 Cont.…..

• d. A decrease in fructose 2,6-bisphosphate makes 6-

phosphofructo-1-kinase less effective and fructose 1,6-
bisphosphatase more effective.
e. Glycolysis is inhibited.
4. Insulin opposes the action of glucagon.
• Glucagon and insulin clearly act in opposition to one
another, and the insulin/glucagon ratio of blood
determines the intracellular levels of fructose 2,6-
bisphosphate and therefore the rate of glycolysis.
 Cont.……
• 5. Different tissues express different isoforms of the
bifunctional enzyme PFK-2K/F-2, 6BPase, encoded by
different genes.
• The liver isoform possesses sites for protein Kinase A-
dependent phosphorylation.
• cAMP inactivates PFK-2 and activates F-2, 6-BPase and
thus reduces F-2, 6-BP levels.
• The heart isoform has phosphorylation sites for protein
kinase A, but at different part of the molecule.
• cAMP activates PFK-2 and has no effect on F-2, 6-BPase
and hence increases F-2, 6-BP levels.
• The skeletal muscle isoform does not possess
phosphorylation sites for protein kinase A and hence has
no effect on F-2, 6- BP levels.
 Clinical considerations
• A. Lactic acidosis
1. Characterized by high blood levels of lactate (generally
greater than 5 mM, While normal levels are usually less
than 1.2 mM).
• Blood pH can be less than 7.1 in severe cases.
2. Most tissues can convert lactate to CO2 and H2O
through the TCA cycle.
a. If the oxygen supply is inadequate, cells must rely on
glycolysis for ATP production.
b. A decrease in ATP enhances glycolysis at the level of
PFK-1 and produces more pyruvate and hence lactate.
 Cont.….

• 3. Accumulation of plasma lactate may be secondary to

tissue hypoxia (circulatory insufficiency due to shock,
heart failure), severe anemia, mitochondrial enzyme
defects and inhibitors of oxygen transport (carbon
monoxide, cyanide), liver disease, and ethanol.
4. Bicarbonate is usually administered to control acidosis
associated with lactate overproduction.
• Dichloroacetate is used as a drug to treat lactic acidosis
because it activates pyruvate dehydrogenase in
mitochondria, which facilitates the conversion of
pyruvate to acetyl CoA so that lactate production is
decreased.
5. Strenuous exercise leads to anerobic glycolysis.
This causes overproduction of lactate.
 B. Diabetes
• In diabetes, insulin activity is low (type 1 – no insulin;
type 2 – insulin resistance).
• Consequently, glucagon levels increase.
• Thus, glucagon/insulin ratio is higher in diabetes.
• There is no change in glucose uptake in liver because this
tissue does not express GLUT4, the insulin-responsive
facilitative glucose transporter.
• In skeletal muscle and adipocytes, glucose uptake
decreases because of the absence of insulin-dependent
recruitment of GLUT4 into the plasma membrane.
• In liver, glucose is not metabolized effectively because:
 Cont.…..

• This also results in an increase in gluconeogenesis,

producing more glucose from gluconeogenic precursors
alanine and glycerol.
• These precursers come from muscle and adipocytes as a
result of increased protein and aminoacid breakdown and
lipolysis to provide energy because of the decreased utility
of glucose as the energy source.
 Cont.…..

• C. Glucokinase deficiency: Mutations in glucokinase gene

leading to inactivation of the enzyme are associated with
a form of non-insulin-dependent diabetes mellitus (type
2) called Maturity Onset diabetes of the Young (MODY).
Complete absence of the enzyme activity will lead to type
1 diabetes because of the lack of insulin secretion in
response to blood glucose.
• This condition is associated with severe hyperglycemia.
• Mutations in the gene leading to increased activity of the
enzyme will cause hyperinsulinemic hypoglycemia.
D. Pyruvate kinase deficiency and hemolytic anemia
• 1. Genetic defect causing a 5-20% reduction in red cell pyruvate
kinase levels.
a. It's rare but the most common genetic disease associated with the
glycolytic pathway.
2. Results in markedly lower ATP concentrations in erythrocytes.
3. Cells swell and lyse.
4. Reticulocytes are unaffected because these "immature" red cells
contain mitochondria and are able to generate ATP through
oxidative phosphorylation.
5. The levels of 2, 3-bisphosphoglycerate are high in erythrocytes.
6. No Heinz bodies
7. The 2nd most common genetic cause of hemolytic anemia, only
next to glucose
6-phosphate dehydrogenase deficiency.
 E. Other glycolytic enzyme defects
• Deficiencies in the activities of phosphofructokinase,
phopshoglycerate kinase, phosphoglycerate mutase, and
lactate dehydrogenase represent important genetic defects
in glycolysis.
• All of these have certain common clinical features:
exercise intolerance, myoglobinuria, hemolytic anemia,
absence of lactate increase in forearm exercise test, and
increased glycogen deposition in the liver and skeletal
muscle.