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GLYCOLYSIS (Embden-Meyerhof Pathway)

principal route for glucose metabolism


main pathway for the metabolism of fructose,
galactose, and other carbohydrates derived from
the diet
main Pathway for Glucose Oxidation
used by all cells to extract energy from glucose
all enzymes are in the cytosol
generates ATP (substrate level phosphrylation:
only 2 ATPs)
oxidation of glucose yields up to 38 ATPs in
Aerobic Conditions but only 2 ATP when O2 is
absent
end product pyruvate
for complete oxidation of pyruvate, the following
are needed:

Product: 4 ATPs in GLYCOLYSIS minus () 2 ATPs


used = 2 ATP TOTAL
Aerobic Glycolysis
-converts Glucose to Pyruvate in the presence
of Oxygen (Product: Pyruvate)
-sets the stage for complete oxidation of
Glucose to CO2 and H2O
-if Lactate is formed (from Pyruvate) which is
temporary, it is converted back to
Pyruvate
-Pyruvate is then converted to Acetyl CoA to
enter citric acid cycle

1 Glucose 2 Pyruvate

Acetyl CoA

Enzyme used: NAD =


3 ATPs produced from
conversion of Pyruvate to Acetyl CoA

1. molecular oxygen
2. mitochonfrial enzymes
- pyruvate dehydrogenase complex
- citric acid
- respiratory chain
2

Phases of Glycolsis:

**Importance of Formation of Pyruvate / Oxidation


of Glucose
-can produce more ATP if Glucose is oxidized
by O2 because it will pass the ETC
-oxidation of glucose can produce as much as
38 ATPs
-if it is unoxidized, ATP production will be very
small (only 2-ATPs)
Table 1: 3 Main Stages of Glycolysis

1. Preparatory or investment phase- from phosphorylation


of glucose to splitting into 2 triose phosphates
2. Payoff phase- conversion of glyceraldehyde 3phosphate to pyruvate with ATP formation

3 MAIN STA

Conversion of gluc

Overall Equation:
Glucose + 2 ADP + 2Pi 2 Lactate + 2 ATP + 2 H20
Has two types: Aerobic (in presence of oxidation) and
Anaerobic (e.g. erythrocytes)

Anaerobic glycolysis
- erythocytes do not have mitochondria;
they metabolize glucose thru the
anaerobic pathway
allows skeletal muscles to perform at
very high levels when oxygen supply
is inefficient, and it allows tissues to
survive anoxic episodes
glycogen disappears and lactate
appears
limits the amount of ATP formed per
mole of glucose oxidized. Therefore,
more glucose must be metabolized
when oxygen is admitted, aerobic
recovery takes place and lactate
disappears
erythrocytes
metabolize
glucose
anaerobically as they do not have
mitochondria
Anaerobic Degradation of Glucose to
Lactate
-forms lactic acid or lactate
-6-Carbon Glucose will yield 2

Pyruvate, which will then yield 2 Lactate

D-Glu

2 Pyruvate

2 Lactate

3.

- To trap glucose in the cell and form a compoun

Cleavage of Fructose 1,6-

Harvesting of ATP when 3

PYRUVATE
AEROBIC- CO2 + H2O through Citric Cycle
and Electron Transport Chain
ANAEROBIC- lactic acid or ethanol
- less energy is generated
Lactic Acid - in skeletal muscle, when
energy needs outspace the ability to
transport oxygen
Why use Fermentation/Anaerobic?

oxygen is not needed to create ATP


STEP 3 (IRREVERSIBLE)

RELATIONSHIP BETWEEN LACTATE AND PYRUVATE


Pyruvate is the end product of aerobic glycolysis
as noted earlier. In the end of anaerobic glycolysis,
lactate is formed from pyruvate. This reaction
takes place in the cells of higher organisms when
the amount of oxygen is limiting, as in muscle
during intense activity.
The reduction of pyruvate by NADH to form lactate
is catalyzed by LACTATE DEHYDROHENASE.
The regeneration of NAD + in the reduction of
pyruvate to lactate sustains the continued
operation of glycolysis under anaerobic conditions.

Fructose 6-phosphate + ATP Fructose 1,6bisphosphate +ADP + H

REACTIONS OF GLYCOLYSIS

STEP 1 (IRREVERSIBLE)
Glucose + ATP glucose 6-phosphate + ADP + H

HEXOKINASE OR GLUCOKINASE converts


glucose into glucose 6-phosphate through
PHOSPHORYLATION
Hexokinase is found in all cells
Glucokinase is found in the liver
Glucokinase is activated when cells within the
body are saturated with glucose

Fructose 1,6-bisphosphate dihydroxyacetone


phophate (DHAP) +
glyceraldehyde 3phosphate (GAP)

ALDOLASE is the enzyme used to form the 3


carbon fragments.
o Aldolase splites F-1,6-bis-P into two
seperate molecules, glyceraldehyde3-P and dihydroxyacetone phosphate
(DHAP). Only glyceraldehyde-3phosphate can continue in glycolysis.

reversible but has a strongly positive standard


free-energy change in the direction of
cleavage

CLEAVAGE of the 6-carbon molecule


triose phosphates are removed quickly by the
next 2 steps pulling the reaction in the
direction of cleavage

- 1 ATP is consumed during this reaction ; Requires mg2+


- Reaction is irreversible
Comparison between Hexokinase and Glucokinase
Hexokinse
in all tissues
10-2 mmol/L

Glucokinase
only in liver
20 mmol/L

high

low

Specificity

acts on:
glucose;fructose

Induction

not induced

Function

glucose is utilized
by cells even if
blood sugar level is
low

Acts on:
mannose;
glucose
induced by
insulin and
glucose
acts only
when blood
glucose level
is more
than
100mg/dl;glu
cose then is
taken up by
liver cells for
glycogen
synthesis

STEP 2 (REVERSIBLE)
specific for glucose 6-phosphate and fructose 6PO4

STEP 5
Dihydroxyacetone phosphate (DHAP)
glyceraldehyde 3-phosphate (G3P)

MUTASE/ISOMERASE is the enzyme responsible


for changing the configuration
G6P
is
converted
to
F6P
through
ISOMERIZATION
Convert aldolase/pyranose to ketose/furanose

Enzyme
TPI
(TRIOSE
PHOSPHATE
ISOMERASE) is the enzyme that allows the
conversion of DHAP to G3P.
G3P proceeds on the direct pathway of
glycolysis, DHAP does NOT. But TPI is there
to
convert
DHAP
to
G3P
through
ISOMERIZATION
Although the reaction is reversible, the
reaction proceeds readily in forming G3P from
DHAP.
TPI catalyzes the transfer of a hydrogen atom
in converting DHAP into G3P which is an
OXIDATION-REDUCTION reaction.

Everthing occurs in duplicate after this


step occurs

C1,C2 and C3 of glucose becomes


Dihydroxyacetone phosphate

C4,C5 and C6 becomes glyceraldehyde


phosphate

Glucose 6-Phosphate + ATP Fructose 6-Phosphate

utilizes 1 ATP as the source of the


phosphate transferred to fructose

STEP 4

Hexokinase and glucokinase are irreversible steps, and


produce G-6-P, which is trapped in the cell.

Occurrence
Km value
Affinity for
substrate

PHOSPHOFRUCTOKINASE
(PFK),
an
allosteric enzyme that sets the pace of
glycolysis
PHOSPHORYLATION of ATP to form another
monophosphate which is attached to C1 of the
fructose
the prefix bis in bisphosphate means two
seperate monophosphate groups are present
- inhibited by ATP; stimulated by
increase in ADP and AMP

STEP 6

NOTE:
o At this point, glycolysis has transformed one
molecule of glucose into 2 molecules of
glyceraldehyde 3-phosphate
o HARVEST TIME!!!

- arsenic- competes with inorganic PO4 in the reaction

Glyceraldehyde 3-phosphate + Pi + NAD+ 1,3bisphosphoglycerate + NADH

GLYCERALDEHYDE
3-PHOSPHATE
DEHYDROGENASE converts glyceraldehyde 3phosphate into 1,3-bisphosphoglycerate, by
PHOSPHORYLATION COUPLED TO OXIDATION
glyceraldehyde-3-phosphate
dehydrogenase- using NAD+ as a
cofactor and generating NADH worth 3
ATP or x 2= 6

- Oxidation of glyceraldehyde phospate to


1,3 bisphosphoglycerate

- 3-Bisphosphoglycerate is an acyl
phosphate and is a high energy
compound.

STEP 9
2-phosphoglycerate phosphoenolpyruvate + H2O

Phosphoenolpyruvate + ADP + H+ pyruvate + ATP

- NADH formed is reoxidized to NAD

PHOSPHOGLYCERATE KINASE catalyzes the


transfer of the phosphoryl group from the acyl
phosphate of 1,3-bisphosphoglycerate to ADP
through
the
process
of
PHOSPHORYL
TRANSFER
Formation of ATP is referred to as substrate-level
phosphorylation because the phosphate donor,
1,3-BPG is a substrate with high phosphoryltransfer potential.

ENOLASE catalyzes the formation of


phosphoenolpyruvate (PEP)
elevates the transfer potential of the
phosphoryl group

STEP 10 (IRREVERSIBLE)

- reaction is similar to hemiacetal formation but product


is thiohemiacetal

Phosphoglucomutase moves the


phosphate from the 3 position to the 2
position.

aldehyde group.of glyceraldehyde 3phosphate is dehydrogenated to a


carboxylic anhydride with phosphoric acid

STEP 7
1,3-bisphosphoglycerate + ADP 3- phosphoglycerate +
ATP

Transfers phosphate from 3 BPG to


ADP(substrate level phosphorylation)

Reactions 9 and 10

Enolase splits off a water and forms


a second high energy phosphate
called phosphoenolpyruvate (PEP).

Inhibited by fluoride

The energy from PEP is used to


phosphorylate ADP to form ATP. The
enzyme involved is pyruvate kinase,
and the hydrolysis product is pyruvate

- phosphate from phosphoenolpyruvate is


transferred to ADP (substrate level
phosphorylation)

STEP 8
3-phosphoglycerate 2-phosphoglycerate

the enzyme used in the reaction is a


MUTASE
alters the confuguration by changing the position
of the phosphoryl group
phosphoenolpyruvate is formed by dehydration of
2-phosphoglycerate
Reactions 7 and 8

Phosphoglycerate kinase assists in the


hydrolysis of 1,3-bisphosphoglycerate,
and uses the energy to form one ATP (2 to
double).

PYRUVATE KINASE is the enzyme used in this reaction.


Table 2: ATP Production

Table 3: Net Production of ATP

STEP
1
3
6NADH
7
10
Total

ATP (used -; produced +)

+1 * 2 = 2

-1
-1
+3 * 2 = 6

+1 * 2 = 2
8 ATP

1. Glucose produces two pyruvate molecules


because of Step 4, glucose (6 carbon molecule) was cleaved into TWO 3-carbon molecules
2. ATP is initially required
ATP was used in Steps 1 and 3
3. ATP is produced
NADH was produced in Step 6
ATP was produced in Steps 7 & 10
4. Fate of NADH+
Enters the Electron Transport Chain to produce 3 ATP

Note:steps 6, 7, and 10 were multiplied by two to account for the two 3 carbon sugars formed in Step 4
Pathway of Glycolysis:

Glucose
ATP
ADP
Hexokinase / Glucokinase
Glucose 6-Phosphate
Phosphohexose Isomerase

Fructose 6-Phosphate
ATP
ADP
Phosphofructokinase (PFK1)
Fructose-1,6-Biphosphate

Aldolase

DHAP
GLYC-3-P
Phosphotriose Isomerase

Phosphoric Acid
NAD
NADH (lost and used later)
Glyceraldehyde-3-Phosphate Dehydrogenase

OXIDATION STEP

1,3-BPG

SUBSTRATE LEVEL
PHOSPHORYLATION

3 Phosphoglycerate
ISOMERIZATION

Phosphoglycerate Mutase
(transfer of Phosphate)

2 Phosphoglycerate
DEHYDRATION

ADP
ATP (ATP is formed)
Phosphoglycerate Kinase

H2O
Enolase

Phosphoenolpyruvate
SUBSTRATE LEVEL

ADP + Pi

PHOSPHRYLATION

ATP
Pyruvate kinase

PYRUVATE
REDUCTION

Lactate Dehydrogenase
NADH (regenerated)
NAD
LACTATE

Summary of glycolytic pathway:


1. Glucose is Phosphorylated to Glucose 6-P
- uses ATP and liberates ADP
2. Glucose-6-Phosphate is converted to Fructose6-phosphate
-Enzyme: Phosphohexose Isomerase
-involves an aldose-ketose isomerization

3. Fructose-6-Phosphate is converted to Fructose1,6-Biphosphate


-phosphorylation with ATP catalyzed by
Phosphofructokinase (PFK-1)
-functionally irreversible and inducible (subject
to allosteric regulation)
-major role in regulating the rate of glycolysis
4. Fructose 1,6-Biphosphate cleaved into:
Glyceraldehyde-3-phosphate and
dihydroxyacetone Phosphate
-cleaved by Aldolase (fructose 1,6-biphosphate
aldolase)

5. DHAP and GLYC-3P are inter-converted by the


enzyme Phosphotriose Isomerase

6. Glyceraldehyde-3-Phosphate Oxidized to 1,3Biphosphoglycerate


Glyceraldehyde is first
Oxidized into an Acid (intermediate)
b.
Acid is then converted into 1,3
Bisphosphoglycerate (High Energy Compound)

-Enzyme: Glyceraldehyde-3-Phosphate
Dehydrogenase

NAD-dependent

Consists of 4 identical
polypeptides (monomers) forming a tetramer
with -SH groups present on each polypeptide
one of the SH groups is the active site of the
enzyme
combines with the substrate forming a
thiolhemiacetal that is oxidized to a thiol ester
-Hydrogens removed in this oxidation are
transferred to NAD+
-Pi is added forming 1,3-biphosphoglycerate, and
the SH group is reconstituted

7. 1,3-Biphosphoglycerate forms 3Phosphoglycerate

- phosphate transferred from 1,3biphosphoglycerate onto ADP, forming ATP


(substrate-level phosphorylation) and 3phoshoglycerate

- Enzyme: Phosphoglycerate Kinase

- 1,3-BPG is a High Energy Compound (with


ADP + Kinase, ATP can be formed

- 2 molecules of triose phosphate are formed


per molecule of glucose, 2 molecules
of ATP are
generated at this stage per molecule of glucose
undergoing glycolysis

- Toxicity of Arsenic is due to competition of


arsenate with inorganic phosphate (Pi) in the
above reactions to give 1-arseno-3phosphoglycerate, which hydrolyzes
spontaneously to give 3- phosphoglycerate
plus heat, without generating ATP

**Biphosphoglycerate Mutase-catalyzes the


conversion of 1,3-biphosphoglycerate to 2,3Biphosphoglycerate which is converted to 3-phosphoglycerate
by Phosphoglycerate Mutase (next step) - this alternative
pathway serve to provide 2,3-biphosphoglycerate

**2,3-Biphosphoglycerate (BPG) -binds to


hemoglobin, decreasing its affinity
for
oxygen

8. 3-Phosphoglycerate is Isomerized to 2Phosphoglycerate

-Enzyme: Phosphoglycerate Mutase

-2,3-biphosphoglycerate is an intermediate of
this reaction (2,3 BPG)

9. 2-Phosphoglycerate forms
Phosphoenolpyruvate

-dehydration of 2-phosphoglycerate forming


phosphoenolpyruvate (loss of H2O)

-Enzyme: Enolase

-inhibited by fluoride

10. Phosphoenolpyruvate Forms Pyruvate


Acid

-phosphate of phosphoenolpyruvate
transferred to ADP

-Enzyme: Pyruvate kinase

-irreversible in physiological conditions

-product of enzyme-catalyzed reaction


enolpyruvate, undergoes spontaneous
isomerization to pyruvate not available to
undergo the reverse reaction

11. Pyruvate forms Lactic Acid

-Enzyme: Lactate Dehydrogenase

-Pyruvate uses NADH lost earlier to form


Lactic Acid

REGULATION OF GLYCOLYSIS

-Glycolysis is regulated at Three Steps


Involving Nonequilibrium Reactions

-most of the reactions of glycolysis are


reversible

-3 are markedly Exergonic; Considered


Irreversible:
Hexokinase and Glucokinaseinhibited by its own product, G-6-P, in high levels

o
o

Phosphofructokinase (PFK1)- stimulated by high


levels of AMP or by insulin, inhibited by high levels
of ATP or citrate; inhibition relieved by 5AMP
Pyruvate kinase- inhibited by high levels of ATP

- In erythrocytes, reaction catalyzed by


phosphoglycerate kinase may be bypassed
by reaction of bisphosphoglycerate mutase
(conversion of 1,3-bisphosphoglycerate to 2,3bisphosphoglycerate)
and
2,3bisphosphoglycerate
phosphatase
(hydrolysis to 3-phosphoglycerate and P i).
There is therefore no net yield of ATP.
Oxidation of Pyruvate to Acetyl-CoA is the
irreversible route from Aerobic Glycolysis to the
citric acid cycle
Pyruvate formed in cytosol,
transported into mitochondrion by a
proton symporter
Inside mitochondrion, pyruvate is
oxidatively decarboxylated to acetylCoA by multienzyme complex,
associated with the inner
mitochondrial membrane
Pyruvate Dehydrogenase Complex is
analogous to Alpha-Ketoglutarate
Dehydrogenase complex of citric acid
cycle
Pyruvate is decarboxylated by
Pyruvate Dehydrogenase to a
Hydroxyethyl Derivative to thiazole
ring of enzyme-bound Thiamin
Diphosphate
Hydroxyethyl reacts with Oxidized
Lipoamide (prosthetic group of
dihydrolipoyl transacetylase) to form
Acetyl Lipoamide
Acetyl lipoamide reacts with
Coenzyme A to form Acetyl-CoA and
Reduced Lipoamide
Cycle of reaction is completed when
the reduced lipoamide is reoxidized by
a flavoprotein: dihydrolipoyl
dehydrogenase, containing FAD
Reduced flavoprotein is oxidized by
NAD+, transfers reducing equivalents
to the respiratory chain:

Pyruvate + NAD+ + CoA Acetyl-CoA + NADH +


H+ +CO2

**Pyruvate Dehydrogenase Complex


-consists of a number of polypeptide chains of
each of the three component enzymes, all
organized in a regular spatial configuration
-inhibited by its products acetyl-CoA and NADH

Figure 1: 2,3-Bisphosphoglycerate pathway in


erythrocytes
(Image
taken
from:
http://web.indstate.edu/thcme/mwking/hemogl
obin-myoglobin.html).

GLYCOLYSIS TO TCAC
irreversible route oxidation of pyruvate to
acetyl-CoA
pyruvate from the cytosol is transported into
the mitochondrion and is oxidatively
decarboxylated
by
a
pyruvate
dehydrogenase complex
pyruvate dehydrogenase is regulated by:
o inhibition by its products, acetyl-CoA and
NADH
o phosphorylation by a kinase of three serine
residues, resulting in decreased activity
o dephosphorylation by a phosphatase, causing
increased activity

CLINICAL ASPECTS

inhibition of pyruvate dehydrogenase may


result to lactic acidosis. This may be caused
by any of the following:

Arsenite and mercuric ions react with


the SH group of lipoic acid and inhibit pyruvate
dehydrogenase, as does a dietary deficiency of
thiamin, allowing pyruvate to accumulate
nutritionally deprived alcoholics are
thiamin-deficient, may develop potentially fatal
pyruvic and lactic acidosis
inherited pyruvate dehydrogenase
deficiency due to defects in one or more of the
components of the enzyme complex, present with
lactic acidosis after a glucose load
because of its dependence on glucose
as a fuel, the brain is a prominent tissue where
these metabolic defects manifest themselves in
neurologic disturbances
inherited aldose A deficiency and
pyruvate kinase deficiency in erythrocyte cause
hemolytic anemia
exercise capacity of patients with
muscle phosphofructokinase deficiency is low
especially in low carbohydrate diets
by providing an alternative lipid fuel
when blood free fatty acids and ketone bodies are
increase, work capacity is improved

Hemolytic Anemias diseases in which


enzymes of glycolysis are deficient
Fatigue if defect affects skeletal muscle
Lactate is used for gluconeogenesis in the
liver, an energy-expensive process

responsible for much of the hypermetabolism seen


in cancer cachexia

Lactic Acidosis results from impaired activity


of pyruvate dehydrogenase

Table 4: Summary

Reaction

Enzyme

Reaction
Type

Glucose + ATP glucose 6-phophate + ADP +


H+
Glucose 6-phosphate fructose 6-phophate +
ADP +H+
Fructose 6-phosphate + ATP fructose 1,6
bisphophate + ADP + H+
Fructose 1,6-bisphosphate dihydroxyacetone
phosphate + glyceraldehyde 3-phosphate
Dihydroxyacetone phosphate glyceraldehydes
3-phosphate

Hexokinase/Gluc
okinase
Isomerase

Phosphoryl
transfer
Isomerization

Phosphofructoki
nase
Aldolase

Glyceraldehyde 3-phosphate + Pi + NAD 1,3bisphosphoglycerate + NADH+ + H+

1,3-bisphosphoglycerate + ADP 3phosphoglycerate + ATP


3-phosphoglycerate 2-phosphoglycerate

Triose
phosphate
isomerase
Glyceraldehyde
3-phopsphate
dehydrogenase
Phosphoglycerat
e kinase
Mutase

2-phosphoglycerate phosphoenolpyruvate +
H2O
Phosphoenolpyruvate + ADP + H+ pyruvate +
ATP

Enolase

Phosphoryl
transfer
Aldol
cleavage
Isomerization

Phosphorylati
on coupled to
oxidation
Phosphoryl
transfer
Phosphoryl
shift
Dehydration