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Biochemistry of Metabolism
Glycolysis
Glycolysis is also called as the EmbdenMeyerhof pathway.
Glycolysis Is the process of the catabolism of
glucose.
Glycolysis takes place in the cytosol of cells.
Glucose enters glycolysis by phosphorylation to
glucose 6-phosphate, catalyzed by hexokinase,
using ATP as the phosphate donor.
1. Hexokinase catalyzes:
Glucose + ATP  glucose-6-P + ADP
The reaction involves nucleophilic attack of the
C6 hydroxyl O of glucose on P of the terminal
phosphate of ATP.
Glucose is activated, irreversibly to glucose 6phosphate by glucokinase or hexokinase in the
presence of ATP.
ATP binds to the enzyme as a complex with
Mg++.
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
O
H
OH
3
H
1
H
2
OH
Hexokinase H
OH
glucose-6-phosphate
The reaction catalyzed by Hexokinase is highly
spontaneous. A phosphoanhydride bond of ATP (~P) is
cleaved.
Hexokinase is inhibited allosterically by its product,
glucose 6-phosphate.
Hexokinase has a high affinity (low K m ) for
glucose, and in the liver it is saturated under
normal conditions, and so acts at a constant
rate to provide glucose 6-phosphate to meet
the cell's need. Liver cells also contain an
isoenzyme of hexokinase, glucokinase, which
has a Km very much higher than the normal
intracellular concentration of glucose. The
function of glucokinase in the liver is to
remove glucose from the blood following a
meal, providing glucose 6-phosphate in excess
of requirements for glycolysis, which is used
for glycogen synthesis and lipogenesis.
Glucose 6-phosphate is an important
compound at the junction of several
metabolic pathways: glycolysis,
gluconeogenesis, the pentose phosphate
pathway, glycogenesis, and
glycogenolysis. In glycolysis it is
converted to fructose 6-phosphate by
phosphohexose isomerase, which
involves an aldose-ketose isomerization.
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
1 CH2OH
O
5
H
H
4
OH
HO
2
3 OH
H
Phosphohexose Isomerase
glucose-6-phosphate
fructose-6-phosphate
2. Phosphohexose Isomerase catalyzes:
glucose-6-P (aldose)  fructose-6-P (ketose)
This reaction is followed by another
phosphorylation catalyzed by the enzyme
phosphofructokinase (phosphofructokinase-1)
forming fructose 1,6-bisphosphate. The
phosphofructokinase reaction may be
considered to be functionally irreversible
under physiologic conditions; it is both
inducible and subject to allosteric regulation,
and has a major role in regulating the rate of
glycolysis.
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. Phosphofructokinase catalyzes:
fructose-6-P + ATP  fructose-1,6-bisP + ADP
This highly spontaneous reaction has a mechanism
similar to that of Hexokinase.
The Phosphofructokinase reaction is the rate-limiting
step of Glycolysis.
Fructose 1,6-bisphosphate is cleaved by
aldolase (fructose 1,6-bisphosphate aldolase)
into two triose phosphates, glyceraldehyde 3phosphate and dihydroxyacetone phosphate.
Glyceraldehyde 3-phosphate and
dihydroxyacetone phosphate are interconvert
by the enzyme phosphotriose isomerase.
2
1CH2OPO3
2C
O
HO 3C
H 4C
H
H
H
Aldolase
2
CH
OPO
2
3
3
OH
2C
OH
1CH2OH
2
CH
OPO
2
3
6
dihydroxyacetone
phosphate
C
5
fructose-1,6bisphosphate
O
+
O
1C
H 2C OH
2
CH
OPO
3
2
3
glyceraldehyde-3phosphate
Phosphotriose Isomerase
4. Aldolase catalyzes: fructose-1,6-bisphosphate 
dihydroxyacetone-P + glyceraldehyde-3-P
.
2
1CH2OPO3
2C
O
HO 3C
H 4C
H
OH
2C
C
OH
1CH2OH
2
OPO
CH
3
2
6
dihydroxyacetone
phosphate
H
5
O
H
Aldolase
fructose-1,6bisphosphate
2
OPO
CH
3
2
3
O
+
1C
H 2C OH
2
3 CH2OPO3
glyceraldehyde-3phosphate
Phosphotriose Isomerase
5. Phosphotriose Isomerase catalyzes:
dihydroxyacetone-P  glyceraldehyde-3-P
• Glycolysis continues with the oxidation of
glyceraldehyde 3-phosphate to 1,3bisphosphoglycerate. The enzyme
catalyzing this oxidation, glyceraldehyde
3-phosphate dehydrogenase, is NADdependent. This is the only step in
Glycolysis in which NAD+ is reduced to
NADH.
Glyceraldehyde-3-phosphate
Dehydrogenase
H
O
NAD+
1C
H
2
C
OH
+ Pi
2
CH
OPO
2
3
3
glyceraldehyde3-phosphate
OPO32
+ H+ O
NADH
1C
H
C
2
OH
2
CH
OPO
2
3
3
1,3-bisphosphoglycerate
6. Glyceraldehyde-3-phosphate Dehydrogenase
catalyzes:
glyceraldehyde-3-P + NAD+ + Pi 
1,3-bisphosphoglycerate + NADH + H+
H
O
H
H
C
C
NH2
+
N
R
NAD+
O

NH2
+
2e + H
N
R
NADH
Recall that NAD+ accepts 2 e plus one H+ (a hydride)
in going to its reduced form.
In the next reaction, catalyzed by phosphoglycerate kinase,
phosphate is transferred from 1,3-bisphosphoglycerate onto
ADP, forming ATP (substrate-level phosphorylation) and 3phosphoglycerate.
Phosphoglycerate Kinase
O
OPO32 ADP ATP O
O
1C
H 2C OH
2
CH
OPO
2
3
3
1,3-bisphosphoglycerate
C
1
Mg
2+
H 2C OH
2
CH
OPO
2
3
3
3-phosphoglycerate
7. Phosphoglycerate Kinase catalyzes:
1,3-bisphosphoglycerate + ADP 
3-phosphoglycerate + ATP
3-Phosphoglycerate is isomerized to 2-phosphoglycerate by
phosphoglycerate mutase
.
Phosphoglycerate Mutase
O
O
C
C
1
H
C
2
O
O
1
OH
3 CH2OPO3
H
2
3-phosphoglycerate
C
2
OPO32
3 CH2OH
2-phosphoglycerate
8. Phosphoglycerate Mutase catalyzes:
3-phosphoglycerate  2-phosphoglycerate
Phosphate is shifted from the OH on C3 to
the OH on C2.
The subsequent step is catalyzed by
enolase and involves a dehydration,
forming phosphoenolpyruvate. Enolase is
inhibited by fluoride, and when blood
samples are taken for measurement of
glucose, it is collected in tubes containing
fluoride to inhibit glycolysis. The enzyme
is also dependent on the presence of
either Mg2+ or Mn2+.
Enolase
O
O

C
2
C
O

C
1
H
H 
O
OPO32
3 CH2OH
C
OH
O
O
1
OPO32
CH2OH
C
2C
OPO32
3 CH2
2-phosphoglycerate enolate intermediate phosphoenolpyruvate
9. Enolase catalyzes:
2-phosphoglycerate  phosphoenolpyruvate + H2O
This dehydration reaction is Mg++-dependent.
The phosphate of phosphoenolpyruvate
is transferred to ADP by pyruvate
kinase to form two molecules of ATP
per molecule of glucose oxidized.
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. Pyruvate Kinase catalyzes:
phosphoenolpyruvate + ADP  pyruvate + ATP
Pyruvate Kinase
O
O
ADP ATP
C
1
C
2
O
O
C
C
1
OPO32
3 CH2
phosphoenolpyruvate
C
2
O
O
1
OH
3 CH2
enolpyruvate
C
2
O
3 CH3
pyruvate
This phosphate transfer from PEP to ADP is spontaneous.
 PEP has a larger ∆G of phosphate hydrolysis than ATP.
 Removal of Pi from PEP yields an unstable enol, which
spontaneously converts to the keto form of pyruvate.
Required inorganic cations K+ and Mg++ bind to anionic
residues at the active site of Pyruvate Kinase.
glucose
Glycolysis
ATP
Hexokinase
ADP
glucose-6-phosphate
Phosphoglucose Isomerase
fructose-6-phosphate
ATP
Phosphofructokinase
ADP
fructose-1,6-bisphosphate
Aldolase
glyceraldehyde-3-phosphate + dihydroxyacetone-phosphate
Triosephosphate
Isomerase
Glycolysis continued
glyceraldehyde-3-phosphate
NAD+ + Pi
Glyceraldehyde-3-phosphate
Dehydrogenase
NADH + H+
Glycolysis
continued.
Recall that
there are 2
GA3P per
glucose.
1,3-bisphosphoglycerate
ADP
Phosphoglycerate Kinase
ATP
3-phosphoglycerate
Phosphoglycerate Mutase
2-phosphoglycerate
Enolase
H2O
phosphoenolpyruvate
ADP
Pyruvate Kinase
ATP
pyruvate
Glycolysis
Balance sheet for ~P bonds of ATP:
2
 How many ATP ~P bonds expended? ________
 How many ~P bonds of ATP produced? (Remember
4
there are two 3C fragments from glucose.) ________
 Net production of ~P bonds of ATP per glucose:
2
________
Balance sheet for ~P bonds of ATP:
 2 ATP expended
 4 ATP produced (2 from each of two 3C fragments from
glucose)
 Net production of 2 ~P bonds of ATP per glucose.
Glycolysis - total pathway, omitting H+:
glucose + 2 NAD+ + 2 ADP + 2 Pi 
2 pyruvate + 2 NADH + 2 ATP
In aerobic organisms:
 pyruvate produced in Glycolysis is oxidized to CO2 via
Krebs Cycle
 NADH produced in Glycolysis & Krebs Cycle is
reoxidized via the respiratory chain, with production
of much additional ATP.