Download Anaerobic and aerobic oxidation of glucose

Anaerobic and aerobic oxidation of glucose
For centuries,
bakeries and
breweries have
exploited the
conversion of
glucose to ethanol
and CO2 by
glycolysis in yeast
DIGESTION OF CARBOHYDRATES
Glycogen, starch and disaccharides (sucrose,
lactose and maltose) are hydrolyzed to
monosaccharide units in the gastrointestinal tract.
The process of digestion starts in the mouth by the
salivary enzyme –amilase.
The time for digestion in mouth is limited.
Salivary -amilase is inhibited in stomach due to the
action of hydrochloric acid.
Another -amilase is produced in pancreas and is
available in the intestine.
-amilase hydrolyzes the -1-4-glycosidic
bonds randomly to produce smaller subunits like
maltose, dextrines and unbranched
oligosaccharides.
-amilase
The intestinal juice contains enzymes hydrolyzing
disaccharides into monosaccharides (they are produced
in the intestinal wall)
Sucrase hydrolyses sucrose into glucose and fructose
Glucose
sucrase
Fructose
Sucrose
Galactose
lactase
Glucose
Lactase hydrolyses
lactose into glucose
and galactose
Lactose
Glucose
maltase
Maltase hydrolyses
maltose into two
glucose molecules
Maltose
Glucose
ABSORPTION OF CARBOHYDRATES
Only monosaccharides are absorbed
The rate of absorption: galactose > glucose > fructose
Glucose and galactose from the intestine into endothelial
cells are absorbed by secondary active transport
Na+
Protein
Glucose
Protein
Carrier protein is specific for D-glucose or
D-galactose.
L-forms are not transported.
There are competition between glucose and
galactose for the same carrier molecule;
thus glucose can inhibit absorption of
galactose.
Fructose is absorbed from intestine into
intestinal cells by facilitated diffusion.
Absorption of glucose from intestinal cells
into bloodstream is by facilitated diffusion.
Transport of glucose from blood into cells of
different organs is mainly by facilitated diffusion.
The protein facilitating the glucose transport is
called glucose transporter (GluT).
GluT are of 5 types.
GluT2 is located mainly in hepatocytes membranes (it
transport glucose into cells when blood sugar is high);
GluT1 is seen in erythrocytes and endothelial cells;
GluT3 is located in neuronal cells (has higher affinity
to glucose);
GluT5 – in intestine and kidneys;
GluT4 - in muscles and fat cells.
The fate of glucose molecule in the cell
Glucose
Glycogenogenesis
(synthesis of
glycogen) is
activated in well
fed, resting state
Glucose-6phosphate
Pentose phosphate
pathway supplies
the NADPH for lipid
synthesis and
pentoses for nucleic
acid synthesis
Ribose,
NADPH
Glycogen
Pyruvate
Glycolysis
is activated if
energy is required
Glycolysis is the earliest discovered and most important
process of carbohydrates metabolism.
Glycolysis – metabolic pathway in which glucose is
transformed to pyruvate with production of a small
amount of energy in the form of ATP or NADH.
Glycolysis is an anaerobic process (it does not require
oxygen).
Glycolysis pathway is used by anaerobic as well as aerobic
organisms.
In glycolysis one molecule of glucose is converted into two
molecules of pyruvate.
In eukaryotic cells, glycolysis takes place in the cytosol.
Pyruvate can be further metabolized to:
(1) Lactate or ethanol (anaerobic conditions)
(2) Acetyl CoA (aerobic conditions)
• Acetyl CoA is further oxidized to CO2 and H2O via
the citric acid cycle
• Much more ATP is generated from the citric acid
cycle than from glycolysis
Acetyl CoA
• Catabolism of glucose in
aerobic conditions via
glycolysis and the citric
acid cycle
The glycolytic pathway consist of ten enzymecatalyzed reactions that begin with a glucose and
split it into two molecules of pyruvate
Glycolysis
(10 reactions)
can be
divided into
three stages
• In the 1st stage
(hexose stage)
2 ATP are
consumed per
glucose
• In the 3rd stage
(triose stage)
4 ATP are
produced per
glucose
• Net: 2 ATP
produced per
glucose
Stage 1, which is the conversion of glucose into fructose
1,6-bisphosphate, consists of three steps: a phosphorylation,
an isomerization, and a second phosphorylation reaction.
The strategy
of these
initial steps
in glycolysis
is to trap
the glucose
in the cell
and form a
compound
that can be
readily
cleaved into
phosphorylated
threecarbon units.
Stage 2 is the cleavage of the fructose
1,6-bisphosphate into two three-carbon fragments
dihydroxyacetone phosphate and glyceraldehyde 3phosphate.
Dihydroxyacetone phosphate and glyceraldehyde 3phosphate are readily interconvertible.
In stage 3,
ATP is
harvested
when the
threecarbon
fragments
are
oxidized to
pyruvate.
Glycolysis Has 10 Enzyme-Catalyzed Steps
• Each chemical reaction prepares a substrate for the next
step in the process
1. Hexokinase
• Transfers the g-phosphoryl of ATP to glucose C-6 oxygen to
generate glucose 6-phosphate (G6P)
• Four kinases in glycolysis: steps 1,3,7, and 10
• All four kinases require Mg2+ and have a similar mechanism
Properties of hexokinases
• Broad substrate specificity - hexokinases can
phosphorylate glucose, mannose and fructose
• Isozymes - multiple forms of hexokinase occur in
mammalian tissues and yeast
• Hexokinases I, II, III are active at normal glucose
concentrations
• Hexokinase IV (Glucokinase) is active at higher glucose
levels, allows the liver to respond to large increases in
blood glucose
• Hexokinases I, II and III are allosterically inhibited by
physiological concentrations of their immediate product,
glucose-6-phosphate, but glucokinase is not.
2. Glucose 6-Phosphate Isomerase
• Converts glucose 6-phosphate (G6P) (an aldose) to
fructose 6-phosphate (F6P) (a ketose)
• Enzyme preferentially binds the a-anomer of G6P
(converts to open chain form in the active site)
• Enzyme is highly stereospecific for G6P and F6P
• Isomerase reaction is near-equilibrium in cells
3. Phosphofructokinase-1 (PFK-1)
• Catalyzes transfer of a phosphoryl group from ATP to the
C-1 hydroxyl group of F6P to form fructose 1,6bisphosphate (F1,6BP)
• PFK-1 is metabolically irreversible and a critical regulatory
point for glycolysis in most cells
• A second phosphofructokinase (PFK-2) synthesizes
fructose 2,6-bisphosphate (F2,6BP)
4. Aldolase
• Aldolase cleaves the hexose F1,6BP into two triose
phosphates: glyceraldehyde 3-phosphate (GAP) and
dihydroxyacetone phosphate (DHAP)
• Reaction is near-equilibrium, not a control point
5. Triose Phosphate Isomerase (TPI)
• Conversion of DHAP into GAP
• Reaction is very fast, only the D-isomer of GAP is formed
• Reaction is reversible. At equilibrium, 96% of the triose
phosphate is DHAP. However, the reaction proceeds readily
from DHAP to GAP because the subsequent reactions of
glycolysis remove this product.
Fate of carbon atoms from hexose stage
to triose stage
6. Glyceraldehyde 3-Phosphate
Dehydrogenase (GAPDH)
• Conversion of GAP to 1,3-bisphosphoglycerate (1,3BPG)
• Molecule of NAD+ is reduced to NADH
• Energy from oxidation of GAP is conserved in acidanhydride linkage of 1,3BPG
• Next step of glycolysis uses the high-energy phosphate
of 1,3BPG to form ATP from ADP
7. Phosphoglycerate Kinase (PGK)
• Transfer of phosphoryl group from the energy-rich mixed
anhydride 1,3BPG to ADP yields ATP and
3-phosphoglycerate (3PG)
• Substrate-level phosphorylation - Steps 6 and 7 couple
oxidation of an aldehyde to a carboxylic acid with the
phosphorylation of ADP to ATP
8. Phosphoglycerate Mutase
• Catalyzes transfer of a phosphoryl group from one part
of a substrate molecule to another
• Reaction occurs without input of ATP energy
9. Enolase: 2PG to PEP
• 2-Phosphoglycerate (2PG) is dehydrated to
phosphoenolpyruvate (PEP)
• Elimination of water from C-2 and C-3 yields the enolphosphate PEP
• PEP has a very high phosphoryl group transfer potential
because it exists in its unstable enol form
10. Pyruvate Kinase (PK)
PEP + ADP  Pyruvate + ATP
• Catalyzes a substrate-level
phosphorylation
• Metabolically irreversible
reaction
• Regulation both by
allosteric modulators and
by covalent modification
• Pyruvate kinase gene can be
regulated by various
hormones and nutrients
Net reaction of glycolysis
During the convertion of glucose to pyruvate:
• Two molecules of ATP are produced
• Two molecules of NAD+ are reduced to NADH
Glucose + 2 ADP + 2 NAD+ + 2 Pi
2 Pyruvate + 2 ATP + 2 NADH + 2 H+ + 2 H2O
The Fate of Pyruvate
The sequence of reactions from glucose to pyruvate is
similar in most organisms and most types of cells.
The fate of pyruvate is variable.
Three reactions of pyruvate are of prime importance:
1. Aerobic conditions:
oxidation to acetyl CoA
which enters the citric acid
cycle for further oxidation
2. Anaerobic conditions
(muscles, red blood cells):
conversion to lactate
3. Anaerobic conditions
(microorganisms, yeast):
conversion to ethanol
Diverse
Fates of
Pyruvate
Metabolism of Pyruvate to Ethanol
Ethanol is formed from pyruvate in yeast and several other
microorganisms in anaerobic conditions.
Two reactions required:
The first step is the decarboxylation of pyruvate to
acetaldehyde.
Enzyme - pyruvate decarboxylase.
Coenzyme - thiamine pyrophosphate (derivative of the vitamin
thiamine B1)
The second step is the reduction of acetaldehyde to ethanol.
Enzyme - alcohol dehydrogenase (active site contains a zinc).
Coenzyme – NADH.
The conversion of glucose into ethanol is an example of alcoholic
fermentation.
The net result of alcoholic fermentation is:
Glucose+2Pi + 2ADP + 2H+  2 ethanol + 2CO2 + 2ATP + 2H2O
The ethanol formed in alcoholic fermentation provides a key
ingredient for brewing and winemaking.
There is no net NADH formation in the conversion of glucose
into ethanol.
NADH generated by the oxidation of glyceraldehyde 3-phosphate
is consumed in the reduction of acetaldehyde to ethanol.
Metabolism of Pyruvate to Lactate
Lactate is formed from pyruvate in an animal organism
and in a variety of microorganisms in anaerobic
conditions.
The conversion of glucose into lactate is called lactic
acid fermentation.
Enzyme - lactate dehydrogenase.
Coenzyme – NADH.
• Muscles of higher organisms and humans lack pyruvate
decarboxylase and cannot produce ethanol from pyruvate
• Muscle contain lactate dehydrogenase. During intense
activity when the amount of oxygen is limiting the lactic acid
can be accumulated in muscles (lactic acidosis).
• Lactate formed in skeletal muscles during exercise is
transported to the liver.
• Liver lactate dehydrogenase can reconvert lactate to
pyruvate.
Overall reaction in the conversion of glucose into lactate:
Glucose + 2 Pi + 2 ADP  2 lactate + 2 ATP + 2 H2O
As in alcoholic fermentation, there is no net NADH
formation.
NADH formed in the oxidation of glyceraldehyde 3phosphate is consumed in the reduction of pyruvate.
Metabolism of Pyruvate to Acetyl CoA
In aerobic conditions pyruvate is converted to acetyl coenzyme A (acetyl CoA).
Acetyl CoA enters citric acid cycle where degrades to CO2 and H2O and the
energy released during such oxidation is utilized in NADH and FADH2.
Pyruvate is converted to acetyl CoA in the matrix of mitochondria.
The overall reaction: Pyruvate + NAD+ + CoA  acetyl CoA + CO2 + NADH
Reaction is catalyzed by the pyruvate dehydrogenase complex (three
enzymes and five coenzymes).
If pyruvate is converted to acetyl CoA, NADH formed in the oxidation of
glyceraldehyde 3-phosphate ultimately transfers its electrons to O2 through
the electron-transport chain in mitochondria.
Other Sugars Can Enter Glycolysis
• Glucose is the main metabolic
fuel in most organisms
• Other sugars convert to
glycolytic intermediates
• Fructose and sucrose (contains
fructose) are major sweeteners
in many foods and beverages
• Galactose from milk lactose (a
disaccharide)
• Mannose from dietary
polysaccharides, glycoproteins
The Entry of Fructose into Glycolysis
Much of the ingested fructose is metabolized by the liver, using
the fructose 1-phosphate pathway.
The first step is the phosphorylation of fructose to fructose 1phosphate by fructokinase.
Fructose 1-phosphate is then
split into glyceraldehyde and
dihydroxyacetone phosphate, an
intermediate in glycolysis, by a
specific fructose 1 -phosphate
aldolase.
Glyceraldehyde is then
phosphorylated to glyceraldehyde
3-phosphate, a glycolytic
intermediate, by triose kinase.
Fructose Is Converted to Glyceraldehyde 3-Phosphate
Fructose can be phosphorylated to fructose 6-phosphate by
hexokinase.
However, the affinity of hexokinase for glucose is 20 times
as great as it is for fructose.
Little fructose 6-phosphate is formed in the liver because
glucose is so much more abundant in this organ.
Glucose, as the preferred fuel, is also trapped in the muscle
by the hexokinase reaction.
Because liver and muscle phosphorylate glucose rather than
fructose, adipose tissue is exposed to more fructose than
glucose.
Hence, the formation of fructose 6-phosphate in the adipose
tissue is not competitively inhibited to a biologically significant
extent, and most of the fructose in adipose tissue is
metabolized through fructose 6-phosphate.
The Entry of Galactose into Glycolysis
Galactose is converted into glucose 6-phosphate in four
steps.
The first reaction is the phosphorylation of galactose to
galactose 1-phosphate by galactokinase.
Galactose 1-phosphate react with uridine
diphosphate glucose (UDP-glucose).
UDP-galactose and glucose 1-phosphate are
formed.
Enzyme - galactose 1-phosphate uridyl
transferase.
The galactose moiety of UDP-galactose is then
epimerized to glucose.
The configuration of the hydroxyl group at carbon 4
is inverted by UDP-galactose 4-epimerase.
Glucose 1-phosphate, formed from galactose, is
isomerized to glucose 6-phosphate by
phosphoglucomutase.
The Entry of Mannose into Glycolysis
Mannose is converted to Fructose 6-Phosphate in two
steps.
Hexokinase catalyzes the convertion of mannose into
mannose 6-phosphate.
Isomerase converts mannose 6-phosphate into fructose
6-phosphate (metabolite of glycolysis).
Intolerance to Milk
Many people are unable to metabolize the
milk sugar lactose and experience gastrointestinal disturbances if they drink milk.
Lactose intolerance, or hypolactasia, is caused by a deficiency of the
enzyme lactase, which cleaves lactose into glucose and galactose.
Microorganisms in the colon ferment undigested lactose to lactic acid
generating methane (CH4) and hydrogen gas (H2). The gas produced
creates the uncomfortable feeling of gut distention and the annoying
problem of flatulence.
The lactic acid is osmotically active and draws water into the intestine,
as does any undigested lactose, resulting in diarrhea.
The gas and diarrhea hinder the absorption of other nutrients (fats
and proteins).
Treatment:
- to avoid the products containing lactose;
- the enzyme lactase can be ingested.
Galactosemia
The disruption of galactose metabolism is referred to as
galactosemia.
Classic galactosemia is an inherited deficiency in galactose
1-phosphate uridyl transferase activity.
Symptoms:
- vomiting, diarrhea after consuming milk,
- enlargement of the liver, jaundice,
sometimes cirrhosis,
- cataracts,
- lethargy and retarded mental
development,
- markedly elevated blood-galactose level
- galactose is found in the urine.
The absence of the transferase in red blood cells is a
definitive diagnostic criterion.
The most common treatment is to remove galactose (and
lactose) from the diet.
Regulation of Glycolysis
The rate glycolysis is regulated to meet two major cellular needs:
(1) the production of ATP, and
(2) the provision of building blocks for synthetic reactions.
There are three control sites in glycolysis - the reactions catalyzed by
hexokinase,
phosphofructokinase 1, and
pyruvate kinase
These reactions are irreversible.
Their activities are regulated
by the reversible binding of allosteric effectors
by covalent modification
by the regulation of transcription (change of the enzymes amounts).
The time required for allosteric control, regulation by phosphorylation,
and transcriptional control is typically in milliseconds, seconds, and
hours, respectively.
Phosphofructokinase 1 Is the Key Enzyme in
the Control of Glycolysis
Phosphofructokinase 1 is the most important control element
in the mammalian glycolytic pathway.
Phosphofructokinase 1
in the liver is a tetramer
of four identical
subunits.
The positions of the
catalytic and allosteric
sites are identical.
High levels of ATP allosterically inhibit the
phosphofructokinase 1 in the liver lowering its affinity for
fructose 6-phosphate.
AMP reverses the inhibitory action of ATP, and so the
activity of the enzyme increases when the ATP/AMP ratio
is lowered (glycolysis is stimulated as the energy charge
falls).
A fall in pH also inhibits phosphofructokinase 1 activity.
The inhibition of phosphofructokinase by H+ prevents
excessive formation of lactic acid and a precipitous drop in
blood pH (acidosis).
Phosphofructokinase 1 is inhibited by citrate, an early
intermediate in the citric acid cycle.
A high level of citrate means that biosynthetic precursors are
abundant and additional glucose should not be degraded for this
purpose.
Fructose 2,6-bisphosphate (F-2,6-BP) is a
potent activator of phosphofructokinase 1.
F-2,6-BP activates phosphofructokinase I by
increasing its affinity for fructose 6-phosphate
and diminishing the inhibitory effect of ATP.
Fructose 2,6-bisphosphate is formed in a reaction catalyzed by
phosphofructokinase 2 (PFK2), a different enzyme from
phosphofructokinase 1.
Fructose 2,6-bisphosphate
is hydrolyzed to fructose 6phosphate by a specific
phosphatase, fructose
bisphosphatase 2 (FBPase2).
Both PFK2 and FBPase2 are
present in a single
polypeptide chain
(bifunctional enzyme).
Regulation of Glycolysis by Fructose 2,6-bisphosphate
 When blood glucose
level is low the glucagon
is synthesized by
pancreas
 Glucagon binds to cell
receptors, stimulates
the protein kinase A
activity
 Protein kinase A
phosphorylates the
PFK-2 inhibiting its
kinase activity and
stimulating its
phosphatase activity
As result the amount
of F-2,6-BP is decreased and glycolysis is
slowed.
Regulation of Hexokinase
Hexokinase is inhibited by its
product, glucose 6-phosphate
(G-6-P).
High concentrations of G-6-P signal that the cell no longer
requires glucose for energy, for glycogen, or as a source of
biosynthetic precursors.
Glucose 6-phosphate levels increase when glycolysis is inhibited at
sites further along in the pathway.
Glucose 6-phosphate inhibits hexokinase isozymes I, II and III.
Glucokinase (isozyme IV) is not inhibited by glucose 6-phosphate.
The role of glucokinase is to provide glucose 6-phosphate for the
synthesis of glycogen.
Regulation of Pyruvate Kinase (PK)
Several isozymic
forms of pyruvate
kinase are present in
mammals (the L type
predominates in liver,
and the M type in
muscle and brain).
Fructose 1,6-bisphosphate allosterically activates pyruvate kinase.
ATP allosterically inhibits pyruvate kinase to slow glycolysis
when the energy charge is high.
Finally, alanine (synthesized in one step from pyruvate) also
allosterically inhibits the pyruvate kinases (signal that
building blocks are abundant).
The isozymic forms of pyruvate kinase differ in their
susceptibility to covalent modification.
The catalytic properties of the L (liver) form—but not of the
M (brain) form controlled by reversible phosphorylation.
When the
blood-glucose
level is low, the
glucagon leads
to the
phosphorylation of
pyruvate
kinase, which
diminishes its
activity.
Inhibition
1) PFK-1 is
inhibited by ATP
and citrate
2) Pyruvate
kinase is
inhibited by ATP
and alanine
3) Hexokinase is
inhibited by
excess glucose
6-phosphate
Regulation of
Glycolysis
Stimulation
1) AMP and fructose 2,6bisphosphate (F2,6BP) relieve
the inhibition of PFK-1 by ATP
2) F1,6BP stimulate the activity
of pyruvate kinase
Alanine
Regulation of Hexose Transporters
Several glucose transporters (GluT) mediate the thermodynamically downhill
movement of glucose across the plasma membranes of animal cells.
GluT is a family of 5 hexose transporters.
Each member of this protein family consists of a single polypeptide chain
forming 12 transmembrane segments.
GLUT1 and GLUT3,
present in erythrocytes,
endothelial, neuronal
and some others
mammalian cells, are
responsible for basal
glucose uptake. Their Km
value for glucose is about
1 mM.
GLUT1 and GLUT3
continually transport
glucose into cells at an
essentially constant rate.
GLUT2, present in liver and pancreatic -cells has a very
high Km value for glucose (15-20 mM).
Glucose enters these tissues at a biologically significant
rate only when there is much glucose in the blood.
GLUT4, which has a Km value of 5 mM, transports glucose
into muscle and fat cells.
The presence of insulin leads to a rapid increase in the
number of GLUT4 transporters in the plasma membrane.
Insulin promotes the uptake of glucose by muscle and fat.
The amount of this transporter present in muscle
membranes increases in response to endurance exercise
training.
GLUT5, present in the small intestine, functions primarily
as a fructose transporter.
The Pasteur Effect
Under anaerobic conditions
the conversion of glucose to
pyruvate is much higher than
under aerobic conditions
(yeast cells produce more
ethanol and muscle cells
accumulate lactate)
The Pasteur Effect is the
slowing of glycolysis in the
presence of oxygen.
• More ATP is produced under aerobic conditions than
under anaerobic conditions, therefore less glucose is
consumed aerobically.