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.