glycolysis - ksu · •glycolysis is degradation of glucose to generate energy (atp) and to provide...
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GlycolysisBCH 340 lecture 3
Chapter 8 in Lippincott 5th edition
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• 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.
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3
Glycolysis takes place in the cytoplasm of all cells in the body but it is of physiological importance in:
• Tissues with no mitochondria: mature RBCs, cornea and lens
• Tissues with few mitochondria: Testis, leucocytes, medulla of the kidney, retina, skin and gastrointestinal tract
• Tissues undergo frequent oxygen lack: skeletal muscles especially during exercise
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Biological importance of glycolysis:
1. Energy production:
• Under anaerobic conditions: glycolysis gives 2 ATP
• Under aerobic: glycolysis gives 8 ATP
2. Oxygenation of tissues:
Through formation of 2,3 bisphosphoglycerate, which decreases the affinity of Hemoglobin to O2:
Pure hemoglobin releases only 8% of oxygen to the tissues, however hemoglobin with 2,3-BPG allows it to release 66% of the oxygen to the tissues. It is for this reason that hemoglobin, and not myoglobin, is more used in transferring oxygen between tissues and the lungs.
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3. Provides important intermediates:
Dihydroxyacetone phosphate: can give glycerol-
3phosphate, 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.
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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-3-phosphate. In this phase there is an investment of two molecules of ATP
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.
Steps: There are 10 enzyme-catalyzed reactions in glycolysis
There are two stages
Thus there is a net gain of two ATP molecules per molecule of Glucose in glycolysis.
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H O
OH
H
OHH
OH
CH2OH
H
OH
H H O
OH
H
OHH
OH
CH2OPO32
H
OH
H
23
4
5
6
1 1
6
5
4
3 2
ATP ADP
Mg2+
glucose glucose-6-phosphate
Hexokinase
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
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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
H O
OH
H
OHH
OH
CH2OH
H
OH
H H O
OH
H
OHH
OH
CH2OPO32
H
OH
H
23
4
5
6
1 1
6
5
4
3 2
ATP ADP
Mg2+
glucose glucose-6-phosphate
Hexokinase
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Comparison between glucokinase and hexokinase enzymes:
HexokinaseGlucokinase
All tissue cellsLiver onlySite
High affinity (low km) i.e. it acts
even in the presence of low blood
glucose concentration.
Low affinity (high km) i.e. it
acts only in the presence of
high blood glucose
concentration.
Affinity to glucose
Glucose, galactose and fructoseGlucose onlySubstrate
No effect Induces synthesis of
glucokinase.
Effect of insulin
Allosterically inhibits hexokinaseNo effectEffect of glucose-6-p
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.
Acts in liver after meals. It
removes glucose coming in
portal circulation, converting
it into glucose -6-phosphate.
Function
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
H O
OH
H
OHH
OH
CH2OPO32
H
OH
H
1
6
5
4
3 2
CH2OPO32
OH
CH2OH
H
OH H
H HO
O
6
5
4 3
2
1
glucose-6-phosphate fructose-6-phosphate
Phosphoglucose Isomerase
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 allostericallyby elevated levels of ATP
CH2OPO32
OH
CH2OH
H
OH H
H HO
O
6
5
4 3
2
1 CH2OPO32
OH
CH2OPO32
H
OH H
H HO
O
6
5
4 3
2
1
ATP ADP
Mg2+
fructose-6-phosphate fructose-1,6-bisphosphate
Phosphofructokinase
4. Cleavage of fructose-1,6-bisP:
Aldolase catalyzes:fructose-1,6-bisphosphatedihydroxyacetone-P + glyceraldehyde-3-P
The reaction is reversible
Aldolase A occurs in most tissues
Aldolase B occurs in liver and kidney
6
5
4
3
2
1CH2OPO32
C
C
C
C
CH2OPO32
O
HO H
H OH
H OH
3
2
1
CH2OPO32
C
CH2OH
O
C
C
CH2OPO32
H O
H OH+
1
2
3
fructose-1,6- bisphosphate
Aldolase
dihydroxyacetone glyceraldehyde-3- phosphate phosphate
Triosephosphate Isomerase
5. Isomerization of dihydroxyacetone phosphate:
Triose Phosphate Isomerase (TIM) interconverts:
dihydroxyacetone-P glyceraldehyde-3-P
Two molecules of glyceraldehyde-3-Pproduced for each glucose
6
5
4
3
2
1CH2OPO32
C
C
C
C
CH2OPO32
O
HO H
H OH
H OH
3
2
1
CH2OPO32
C
CH2OH
O
C
C
CH2OPO32
H O
H OH+
1
2
3
fructose-1,6- bisphosphate
Aldolase
dihydroxyacetone glyceraldehyde-3- phosphate phosphate
Triosephosphate Isomerase
Glucose + 2 ATP ——> 2 GA3P + 2 ADP + 2 H+
Recall that there are 2 GAP per glucose
Summary of First Stage of Glycolysis (Energy Investment)
C
C
CH2OPO32
H O
H OH
C
C
CH2OPO32
O OPO32
H OH+ Pi
+ H+
NAD+ NADH
1
2
3
2
3
1
glyceraldehyde- 1,3-bisphospho- 3-phosphate glycerate
Glyceraldehyde-3-phosphate Dehydrogenase
6. Oxidation of glyceraldehyde-3-phosphate
Glyceraldehyde-3-phosphate Dehydrogenase catalyzes:
glyceraldehyde-3-P + NAD+ + Pi 1,3-bisphosphoglycerate + NADH +H+
High energy compound
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 (one SH in its active site)
Glyceraldehyde-3-P Dehydrogenase is inhibited by iodoacetate
C
C
CH2OPO32
H O
H OH
C
C
CH2OPO32
O OPO32
H OH+ Pi
+ H+
NAD+ NADH
1
2
3
2
3
1
glyceraldehyde- 1,3-bisphospho- 3-phosphate glycerate
Glyceraldehyde-3-phosphate Dehydrogenase
C
C
CH2OPO32
O OPO32
H OH
C
C
CH2OPO32
O O
H OH
ADP ATP
1
22
3 3
1
Mg2+
1,3-bisphospho- 3-phosphoglycerate glycerate
Phosphoglycerate Kinase
7. Formation of ATP from 1,3 BPG and ADP
Phosphoglycerate Kinase catalyzes the Transfer of
phosphoryl group from 1,3 bisphosphoglycerate to ADP generating ATP:
1,3-bisphosphoglycerate + ADP 3-phosphoglycerate + ATP
This phosphate transfer is reversible, since one ~Pbond is cleaved & another synthesized
C
C
CH2OPO32
O OPO32
H OH
C
C
CH2OPO32
O O
H OH
ADP ATP
1
22
3 3
1
Mg2+
1,3-bisphospho- 3-phosphoglycerate glycerate
Phosphoglycerate Kinase
2 molecules of ATP are produced (by Substrate-levelphosphorylation)
Recall every molecule of glucose gives rise to 2 trioses!!!
This means phosphorylation of ADP to ATP at the reaction itself
In glycolysis there are 2 examples:
o 1.3 BPG + ADP 3 Phosphoglycerate + ATP
o PEP + ADP pyruvate + ATP
Substrate level phosphorylation
C
C
CH2OH
O O
H OPO32
2
3
1C
C
CH2OPO32
O O
H OH2
3
1
3-phosphoglycerate 2-phosphoglycerate
Phosphoglycerate Mutase
8. Shift of the P group from C3 to C2
Phosphoglycerate Mutase catalyzes the Conversionof 3-phosphoglycerate to 2-phosphoglycerate (2-PG).
3-phosphoglycerate 2-phosphoglycerate
It is a freely reversible reaction
9. Dehydration of 2-P-glycerate to phosphoenolpyruvate
Enolase catalyzes:
2-phosphoglycerate phosphoenolpyruvate + H2O
This 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
C
C
CH2OH
O O
H OPO32
C
C
CH2OH
O O
OPO32
C
C
CH2
O O
OPO32
OH
2
3
1
2
3
1
H
2-phosphoglycerate enolate intermediate phosphoenolpyruvate
Enolase
High energy compound
10. Formation of pyruvate
Pyruvate Kinase catalyzes the transfer of phosphorylgroup from PEP to ADP generating ATP and Pyruvate
phosphoenolpyruvate + ADP pyruvate + ATP
This enzyme requires Mg++ and K+
Irreversible reaction
C
C
CH3
O O
O2
3
1
ADP ATPC
C
CH2
O O
OPO32
2
3
1
phosphoenolpyruvate pyruvate
Pyruvate Kinase
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 phosphorylationreaction of glycolysis
C
C
CH3
O O
O2
3
1
ADP ATPC
C
CH2
O O
OPO32
2
3
1
phosphoenolpyruvate pyruvate
Pyruvate Kinase
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!
Glycolysis
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.) ________
Net production of ~P bonds of ATP per glucose:
________
2
4
2
Pyruvate is catabolizedfurther in mitochondria through pyruvatedehydrogenase 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 the aerobic condition:
In absence of oxygen, NADH+ H+ is not oxidized by the respiratory chain.
Pyruvate is converted to Lactate in homolacticfermentation 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).
Under anaerobic condition:
C
C
CH3
O
O
O
C
HC
CH3
O
OH
O
NADH + H+ NAD
+
Lactate Dehydrogenase
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 (step 6)
Cell membranes contain carrier proteins that facilitate transport of lactate
HomolacticFermentation:
C
C
CH3
O
O
O
C
HC
CH3
O
OH
O
NADH + H+ NAD
+
Lactate Dehydrogenase
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 topyruvate, which may be oxidized in Krebs Cycle or (in liver) converted back to glucose via gluconeogenesis
C
C
CH3
O
O
O
C
HC
CH3
O
OH
O
NADH + H+ NAD
+
Lactate Dehydrogenase
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.
C
C
CH3
O
O
O
C
CH3
OHC
CH3
OH H
H
NADH + H+ NAD
+CO2
Pyruvate Alcohol Decarboxylase Dehydrogenase
pyruvate acetaldehyde 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.
Alcoholic fermentation
C
C
CH3
O
O
O
C
CH3
OHC
CH3
OH H
H
NADH + H+ NAD
+CO2
Pyruvate Alcohol Decarboxylase Dehydrogenase
pyruvate acetaldehyde ethanol
It is a two step process:
1. Pyruvate decarboxylase (PDC) 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+
Mature RBCs contain no mitochondria, thus:
o They depend only upon glycolysis for energy production
(=2 ATP).
o Lactate is always the end product.
Special features of glycolysis in RBCs
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.