Download 3-Glycolysis BCH340

Dr. Abir Alghanouchi
Biochemistry department
Sciences college
All carbohydrates to be catabolized must enter
the glycolytic pathway
Glycolysis is degradation of glucose to generate
energy (ATP) and to provide pyruvate (in the
presence of oxygen) or lactate (in the absence
of oxygen)
Glycolysis is central in generating both
energy and metabolic intermediates.
Site:
Glycolysis takes place in the cytoplasm of all cells in the
body but it is of physiological importance in:
1.
Tissues with no mitochondria: mature RBCs, cornea
and lens
2.
Tissues with few mitochondria: Testis, leucocytes,
medulla of the kidney, retina, skin and
gastrointestinal tract
3.
Tissues undergo frequent oxygen lack: skeletal
muscles especially during exercise
Biological importance of glycolysis:
glycolysis:
1. Energy production:
Under anaerobic conditions: glycolysis gives 2 ATP
Under aerobic glycolysis: glycolysis gives 8 ATP
2. Oxygenation of tissues:
Through formation of 2,3 bisphosphoglycerate,
which decreases the affinity of Hemoglobin to O2.
3. Provides important intermediates:
Dihydroxyacetone phosphate: can give glycerol3phosphate, which is used for synthesis of TGs and PLs
(lipogenesis).
3 Phosphoglycerate: which can be used for synthesis of
amino acid serine.
Pyruvate: which can be used in synthesis of amino acid
alanine.
4. Aerobic glycolysis provides the mitochondria with
pyruvate, which gives acetyl CoA Krebs' cycle.
Steps:
There are 10 enzyme-catalyzed reactions
There are two stages :
Stage 1: (Reactions 1-5) A preparatory stage in
which glucose is phosphorylated, converted to
fructose which is again phosphorylated and
cleaved into two molecules of glyceraldehyde-3phosphate. In this phase there is an investment
of two molecules of ATP
Steps:
Stage 2: (reactions 6-10) The two molecules of
glyceraldehyde-3-phosphate are converted to
pyruvate with concomitant generation of four
ATP molecules and two molecules of NADH.
Thus there is a net gain of two ATP molecules
per molecule of Glucose in glycolysis.
6 CH2OH
5
H
4
OH
O
H
OH
H
2
3
H
OH
glucose
6 CH OPO 2−
2
3
ATP ADP
H
H
1
OH
5
4
Mg2+
OH
Hexokinase
O
H
OH
3
H
H
2
H
1
OH
OH
glucose-6-phosphate
1.
Phosphorylation of glucose:
Hexokinase catalyzes:
Glucose + ATP glucose-6-P + ADP
ATP binds to the enzyme as a complex with Mg++
A phosphoanhydride bond of ATP (~P) is cleaved ADP
6 CH2OH
5
H
4
OH
O
H
OH
H
2
3
H
OH
glucose
6 CH OPO 2−
2
3
5
O
ATP ADP
H
H
1
OH
4
Mg2+
OH
H
OH
3
H
2
H
1
OH
Hexokinase H
OH
glucose-6-phosphate
The reaction catalyzed by Hexokinase is irreversible
(glucose-6-P can not diffuse out of the cell because there
are no specific carriers for phosphorylated sugars)
This reaction is catalyzed by several isoenzymes of
hexokinase and glucokinase: both requires Mg2+ as a
cofactor
Comparison between glucokinase and
hexokinase enzymes:
Glucokinase
Hexokinase
Site
Liver only
All tissue cells
Affinity to glucose
Low affinity (high km) i.e. it
acts only in the presence of
high blood glucose
concentration.
High affinity (low km) i.e. it acts
even in the presence of low blood
glucose concentration.
Substrate
Glucose only
Glucose, galactose and fructose
Effect of insulin
Induces synthesis of
glucokinase.
No effect
Effect of glucose-6-p
No effect
Allosterically inhibits hexokinase
Function
Acts in liver after meals. It
removes glucose coming in
portal circulation, converting
it into glucose -6-phosphate.
It phosphorylates glucose inside
the body cells. This makes glucose
concentration more in blood than
inside the cells. This leads to
continuous supply of glucose for
the tissues even in the presence of
low blood glucose concentration.
6 CH OPO 2−
2
3
5
O
H
4
OH
H
OH
3
H
H
2
OH
H
1
OH
6 CH OPO 2−
2
3
1CH2OH
O
5
H
H
4
OH
HO
2
3 OH
H
Phosphoglucose Isomerase
glucose-6-phosphate
fructose-6-phosphate
2.
Isomerization of glucose-6-P:
Phosphoglucose Isomerase catalyzes:
glucose-6-P (aldose) fructose-6-P (ketose)
It is not rate-limiting or regulated step
Phosphofructokinase
6 CH OPO 2−
2
3
O
5
H
H
4
OH
6 CH OPO 2−
2
3
1CH2OH
O
ATP ADP
HO
2
3 OH
H
fructose-6-phosphate
5
Mg2+
1CH2OPO32−
H
H
4
OH
HO
2
3 OH
H
fructose-1,6-bisphosphate
3. Phosphorylation of fructose-6-P
Phosphofructokinase 1 (PFK-1) catalyzes:
fructose-6-P + ATP fructose-1,6-bisP + ADP
Rate-limiting step
PFK-1 is an allosteric enzyme, it is inhibited allosterically
by elevated levels of ATP
2−
1CH2OPO3
2C
O
HO 3C
H 4C
H
H
H
2−
CH
OPO
2
3
3
Aldolase
OH
2C
OH
1CH2OH
2−
OPO
CH
2
3
6
dihydroxyacetone
phosphate
5
C
fructose-1,6bisphosphate
O
+
O
1C
H 2C OH
2−
3 CH2OPO3
glyceraldehyde-3phosphate
Triosephosphate Isomerase
4. Cleavage of fructose-1,6-bisP:
Aldolase catalyzes:
fructose-1,6-bisphosphate
dihydroxyacetone-P
+ glyceraldehyde-3-P
The reaction is reversible
Aldase A occurs in most tissues
Aldase
B occurs in liver and kidney
1CH2OPO3
2C
O
HO 3C
H 4C
H
H
2−
H
Aldolase
2−
CH
OPO
2
3
3
OH
2C
OH
1CH2OH
2−
CH
OPO
2
3
6
dihydroxyacetone
phosphate
5
C
fructose-1,6bisphosphate
O
+
O
1C
H 2C OH
2−
CH
OPO
3
2
3
glyceraldehyde-3phosphate
Triosephosphate Isomerase
5. Isomerization of dihydroxyacetone phosphate:
Triose Phosphate Isomerase (TIM) interconverts:
dihydroxyacetone-P glyceraldehyde-3-P
Two molecules of glyceraldehyde-3-P bare
produced for each glucose
Summary of Second Stage of
Glycolysis (Energy Investment)
Glucose + 2 ATP ——> 2 GA3P + 2 ADP + 2 H+
Recall that there are 2 GAP per glucose
Glyceraldehyde-3-phosphate
Dehydrogenase
H
O
1C
H
2
C
OH
OPO32−
+ H+ O
NAD+ NADH
1C
+ Pi
H C OH
2−
CH
OPO
2
3
3
glyceraldehyde3-phosphate
2
2−
OPO
CH
2
3
3
1,3-bisphosphoglycerate
6. Oxidation of glyceraldehyde-3-phosphate
Glyceraldehyde-3-phosphate Dehydrogenase catalyzes:
glyceraldehyde-3-P + NAD+ + Pi 1,3-bisphosphoglycerate + NADH +H+
High energy compound
Glyceraldehyde-3-phosphate
Dehydrogenase
H
O
1C
H
2
C
OH
OPO32−
+ H+ O
NAD+ NADH
1C
+ Pi
H C OH
2−
OPO
CH
2
3
3
glyceraldehyde3-phosphate
2
2−
CH
OPO
2
3
3
1,3-bisphosphoglycerate
This is the only step in Glycolysis in which NAD+ is reduced to NADH
NAD+ is the cofactor in this reaction which acts as an oxidizing agent
Glyceraldehyde-3-P Dehydrogenase is a tetrameric enzyme with 4SH
group on each polypeptide chain (one SH in its active site)
Glyceraldehyde-3-P Dehydrogenase is inhibited by iodoacetate
Phosphoglycerate Kinase
O−
OPO32− ADP ATP O
O
1C
H 2C OH
2−
3 CH2OPO3
1,3-bisphosphoglycerate
C
1
Mg
2+
H 2C OH
2−
3 CH2OPO3
3-phosphoglycerate
7. Formation of ATP from 1,3 BPG and ADP
Phosphoglycerate Kinase catalyzes the Transfer of
phosphoryl group fron 1,3 bisphosphoglycerate to ADP generating ATP:
1,3-bisphosphoglycerate + ADP 3-phosphoglycerate + ATP
phosphate transfer is reversible, since
one ~P bond is cleaved & another synthesized
This
Phosphoglycerate Kinase
1C
H 2C OH
2−
3 CH2OPO3
1,3-bisphosphoglycerate
O−
OPO32− ADP ATP O
O
C
1
Mg
2+
H 2C OH
2−
3 CH2OPO3
3-phosphoglycerate
2 molecules of ATP are produced (by Substrate-level
phosphorylation)
Recall every molecule of glucose gives rise to 2 trioses!!!
Substrate level phosphorylation
This
means phosphorylation of ADP to ATP at the reaction
itself
In glycolysis
there are 2 examples:
o 1.3 Bisphosphoglycerate +
o
ADP 3 Phosphoglycerate + ATP
Phospho-enol pyruvate + ADP
Enolpyruvate + ATP
Phosphoglycerate Mutase
O−
O
O−
O
C
C
1
1
H 2C OH
2−
3 CH2OPO3
H 2C OPO32−
3 CH2OH
3-phosphoglycerate
2-phosphoglycerate
8. Shift of the P group from C3 to C2
Phosphoglycerate Mutase catalyzes the Conversion
of 3-phosphoglycerate to 2-phosphoglycerate (2-PG).
3-phosphoglycerate 2-phosphoglycerate
It is a freely reversible reaction
Enolase
−
O
O
C
2
C
−
−
O
O
C
1
H
H+
OPO32−
3 CH 2OH
C
OH −
O
O
−
1C
OPO32−
CH 2OH
2C
O PO32−
3 CH 2
2-phosphoglycerate enolate intermediate phosphoenolpyruvate
9. Dehydration of 2-P-glycerate to phosphoenolpyruvate
Enolase catalyzes:
2-phosphoglycerate phosphoenolpyruvate + H2O
High energy compound
dehydration reaction is Mg++-dependent and reversible
Enolase is inhibited by fluoride
To measure glucose level in blood, fluoride is
added to inhibit Enolase and stop glycolysis
This
Pyruvate Kinase
O−
O
C
1
C
2
ADP ATP
O−
O
C
1
OPO32−
3 CH2
phosphoenolpyruvate
C
2
O
3 CH3
pyruvate
10. Formation of pyruvate
Pyruvate Kinase catalyzes the transfer of phosphoryl
group from PEP to ADP generating ATP and Pyruvate
phosphoenolpyruvate + ADP pyruvate + ATP
This enzyme requires Mg++ and K+
Irreversible reaction
Pyruvate Kinase
O−
O
C
1
C
2
ADP ATP
O−
O
C
1
OPO32−
3 CH2
phosphoenolpyruvate
C
2
O
3 CH3
pyruvate
This phosphate transfer from PEP to ADP is spontaneous
(the free energy of PEP hydrolysis is coupled to the
synthesis of ATP)
This is the second substrate level phosphorylation
reaction of glycolysis
Summary of Second Stage of
Glycolysis
2 GA3P + 2 NAD+ + 4 ADP + 2 Pi
2 Pyruvate + 2 NADH + 2 H+ + 4 ATP
Summary of Glycolysis
Glucose + 2 NAD+ + 2 ADP + 2 Pi
2 Pyruvate + 2 NADH + 2 H+ + 2 ATP
can directly be used for doing work
or synthesis
NOTE: NAD+ must be regenerated for glycolysis to proceed!
Balance sheet for ~P bonds of ATP:
How
many ATP ~P bonds expended? ________
How
many ~P bonds of ATP produced?
(Remember there are two 3C fragments from
glucose.) ________
production of ~P bonds of ATP per glucose:
________
Net
What happens next?
Under the aerobic condition:
pyruvate is catabolized
further in mitochondria
through pyruvate
dehydrogenase and citric
acid cycle where all the
carbon atoms are oxidized
to CO2.
The free energy
released is used in the
synthesis of ATP, NADH
and FADH2.
Under anaerobic condition:
In absence of oxygen, NADH+
H+ is not oxidized by the
respiratory chain.
Pyruvate is converted to
Lactate in homolactic
fermentation or in ethanol in
alcoholic fermentation to
regenerate NAD+.
This helps continuity of
glycolysis, as the generated
NAD+ will be used once more
for oxidation of another
glucose molecule (step6).
Homolactic
Fermentation:
Lactate Dehydrogenase
O−
O
C
C
NADH + H+ NAD+
O
O−
O
C
HC
OH
CH3
CH3
pyruvate
lactate
Skeletal muscles ferment glucose to lactate during exercise,
when the exertion is brief and intense.
Lactate dehydrogenase (LDH) reduces pyruvate to lactate
using NADH and thereby oxidizing it to NAD+
NAD+ is regenerated by lactic fermentation to carry out
GAPDH reaction of glycolysis
Cell membranes contain carrier proteins that facilitate
transport of lactate
Lactate Dehydrogenase
O−
O
C
C
NADH + H+ NAD+
O
O−
O
C
HC
OH
CH3
CH3
pyruvate
lactate
Lactate released to the blood may be taken up by
other tissues, or by skeletal muscle after exercise,
and converted via Lactate Dehydrogenase back to
pyruvate, which may be oxidized in Krebs Cycle or
(in liver) converted to back to glucose via
gluconeogenesis
Lactate Dehydrogenase
O−
O
C
C
NADH + H+ NAD+
O
O−
O
C
HC
OH
CH3
CH3
pyruvate
lactate
Lactate serves as a fuel source for cardiac muscle as
well as brain neurons.
Astrocytes,
which surround and protect neurons in the
brain, ferment glucose to lactate and release it.
Alcoholic fermentation
Pyruvate
Decarboxylase
Alcohol
Dehydrogenase
CO2
NADH + H+ NAD+
O−
O
C
C
O
CH3
pyruvate
H
O
C
CH3
acetaldehyde
H
H
C
OH
CH3
ethanol
Microorganisms and yeast convert pyruvate to ethanol,
which is excreted as a waste product, and carbon dioxide
to regenerate NAD+ for glycolysis
NADH is converted to NAD+ in the reaction catalyzed by
Alcohol Dehydrogenase.
Pyruvate
Decarboxylase
Alcohol
Dehydrogenase
CO2
NADH + H+ NAD+
O−
O
C
C
H
C
O
CH3
CH3
pyruvate
O
acetaldehyde
H
H
C
OH
CH3
ethanol
It is a two step process:
1. Pyruvate decarboxylase (PDC) reaction:
reaction: This enzyme is Mg++dependent and requires an enzyme-bound cofactor, thiamine
pyrophosphate (TPP). In this reaction a molecule of CO2 is
released producing acetaldehyde.
2. Alcohol dehydrogenase reaction: Acetaldehyde is reduced to
ethanol using NADH as reducing power, thus regenerating NAD+
Cytosol
Mitochondrion
Glycolysis
Glucose
2
Pyruvic
acid
2
AcetylCoA
Krebs
Cycle
Electron
Transport
Maximum
per
glucose:
by direct
synthesis
by
direct
synthesis
by
ATP
synthase
Energy production of glycolysis:
glycolysis:
ATP produced
ATP consumed
Net energy
In absence of oxygen
(anaerobic glycolysis)
4 ATP:
(Substrate level
phosphorylation)
2ATP from 1,3 DPG.
2ATP from
phosphoenol pyruvate
2ATP
2 ATP/
Glucose Molecule
From glucose to
glucose -6-p.
From fructose -6-p to
fructose 1,6 p.
In presence of oxygen
(aerobic glycolysis)
4 ATP
(substrate level
phosphorylati on)
2ATP from 1,3 BPG.
2ATP from
phosphoenol pyruvate.
+ 6ATP
(from oxidation of 2
NADH + H+ in
mitochondria).
2ATP
-From glucose to
glucose -6-p.
-From fructose -6-p
to fructose 1,6 p.
8 ATP/
Glucose Molecule
Differences between aerobic and
anaerobic glycolysis
Aerobic
Anaerobic
End product
Pyruvate
Lactate/ethanol
Energy
8 ATP
2 ATP
Regeneration of NAD+
Through respiration
chain in mitochondria
Availability to TCA in
mitochondria
Through Lactate
/ethanol formation
Available and 2 Pyruvate Not available as lactate
is cytoplasmic substrate
can oxidize to give 30
ATP
Special features of glycolysis in RBCs
Mature RBCs contain no mitochondria, thus:
o
They depend only upon glycolysis for energy production
(=2 ATP).
o
Lactate is always the end product.
Glucose uptake by RBCs is independent on insulin hormone.
Reduction of met-hemoglobin: Glycolysis produces NADH+H+,
which used for reduction of met-hemoglobin in red cells.
In most cells 2,3 bisphosphoglycerate is present in trace
amount, but in erythrocytes it is present in significant amount:
In red
cells 1,3 BPG is converted to 2,3BPG which unites
with oxy Hb and helps release of oxygen at tissues.
Regulation of Glycolysis
There
are three steps in glycolysis that have enzymes which
regulate the flux of glycolysis
These
enzymes catalyzes irreversible reactions of glycolysis
The hexokinase (HK)
II. The phoshofructokinase (PFK)
III. The pyruvate kinase
I.
They are regulatory enzymes which are regulated by
the level of ATP in the cell
I- PhosphofructokinasePhosphofructokinase-1 (PFK(PFK-1):
It is the most important regulatory enzyme which catalyzes the
first irreversible reaction unique to the glycolytic pathway (the
committed step)
It is an allosteric enzyme inhibited by elevated level of ATP, which:
is the end product of glycolysis as well as it is substrate for PFK-1
o At high [ATP], PFK
has lower affinity
for the other substrate, fructose-6-P.
ATP binds to inhibition site of PFK, and
thereby decreases the activity of
enzyme.
o Sigmoidal dependence
of reaction
rate on[fructose-6-P] is seen.
AMP, present at significant levels only when there is extensive
ATP hydrolysis, antagonizes effects of high ATP.
AMP, ADP and Fructose 2, 6 biphosphate act as allosteric
activators of PFK-1.
II
II-- Hexokinase
It
is allosterically inhibited by its product
Glucose 6 phosphate.
In
liver, glucokinase is inhibited by Fructose
6P and ATP (acts as a competitive inhibitor
of this enzyme)
III-- Pyruvate Kinase
III
It
is allosterically inhibited by ATP. ATP binding
to the inhibitor site of PK decreases its ability
to bind to PEP the substrate.
It
is also inhibited by Acetyl Coenzyme A and
long chain fatty acid because they are source rich
ATP which inhibits PK.
Hormonal regulation of glycolysis
Insulin and Glucagon (secreted by the pancreas) are
the main endocrine that modulate blood glucose levels
and they act antagonistically
Insulin is secreted in hyperglycemia and after
carbohydrates feeding, it causes:
1.
Induction for synthesis of glycolytic key enzyme
2.
Activation of protein phosphatase 1 producing
dephosphorylation and activation of glycolytic
key enzymes
Glucagon is secreted in hypoglycemia or in CHO
deficiency and it affects liver cells mainly as
follows:
1.
It acts as repressor of glycolytic key enzymes
(PFK1, Pyruvate kinase, glucokinase)
2.
It produces phosphorylation of specific enzymes
leading to inactivation of glycolytic key enzymes
2-deoxyglucose: inhibits hexokinase
Mercury and iodoacetate: inhibit glyceraldehyde-3-P
dehydrogenase
Fluoride: inhibits enolase by removal of Mg2+ as Mg
fluoride
Arsenate: is uncoupler of oxidation and phosphorylation,
it forms 1-arseno-3-phosphoglycerate which interferes
with ATP formation at substrate level
It is the inhibition of glycolysis by the presence of
oxygen
Explanation: Aerobic oxidation of glucose produces
increased amount of ATP and citrate. Those inhibit
PFK1