5. Glycolytic pathway, is the oxidation of
glucose to pyruvate to produce energy
(ATP and NADH + H) and other
metabolic intermediates.
6. The process of glucose oxidation to
pyruvate is called glycolysis.
Glycolysis is a process that takes place
both aerobic and anaerobic conditions.
The fate of pyruvate is determined by
the presence of oxygen in the tissue
and the presence or absence of
mitochondria.
7. Aerobic/Anaerobic Glycolysis
In fact, under aerobic and unaerobic
conditions the reactions are the same
until the production of pyruvate from
glucose.
The end product of glycolysis in cells
with mitochondria and enough oxygen
is is pyruvate (aerobic glycolysis).
Conversion of glucose to lactate is the
anaerobic glycolysis.
8. 6 CH OPO 2â
2
3
5
O
H
4
OH
H
OH
3
H
H
2
H
1
OH
OH
glucose-6-phosphate
Glycolysis takes place in the cytosol of cells.
Glucose enters the Glycolysis pathway by
conversion to glucose-6-phosphate.
9. âą Glucose gets phosphorylated rapidly after
entering the cell.
âą Phosphorylation of glucose prevents it from
getting out of the cell.
10. 6 CH2OH
5
H
4
OH
O
H
OH
H
2
3
H
OH
glucose
ATP
H
1
OH
6 CH OPO 2â
2
3
ADP
H
Mg2+
5
4
OH
Hexokinase
O
H
OH
3
H
H
1
H
2
OH
OH
glucose-6-phosphate
âą ATP is required for this rxn. Glucose gets a
phosphate group from ATP and becomes
glucose-6-phosphate.
11. 6 CH2OH
5
H
4
OH
O
H
OH
H
2
3
H
OH
glucose
6 CH OPO 2â
2
3
5
O
ATP ADP
H
H
1
OH
Mg2+
4
OH
H
OH
3
H
2
H
1
OH
Hexokinase H
OH
glucose-6-phosphate
1. Hexokinase catalyzes:
Glucose + ATP ï glucose-6-P + ADP
The reaction is between hydroxyl O of C6 of glucose
and the terminal phosphate of ATP.
ATP binds to the enzyme as a complex with Mg++.
13. 6 CH OPO 2â
2
3
5
O
H
4
OH
H
OH
3
H
H
2
OH
H
1
OH
6 CH OPO 2â
2
3
1CH2OH
O
5
H
H
4
OH
HO
2
3 OH
H
Phosphoglucose Isomerase
glucose-6-phosphate
fructose-6-phosphate
2. Phosphoglucose Isomerase catalyzes:
glucose-6-P (aldose) ïï fructose-6-P (ketose)
The mechanism involves acid/base catalysis, with ring
opening, isomerization, and then ring closure. A
similar reaction catalyzed by Triosephosphate
Isomerase will be presented in detail.
14. 6 CH OPO 2â
2
3
5
H
4
OH
O
H
OH
3
H
H
2
OH
H
1
OH
6 CH OPO 2â
2
3
1 CH2OH
O
5
H
H
4
OH
HO
3
H
2
OH
Phosphoglucose Isomerase
glucose-6-phosphate
fructose-6-phosphate
âą Isomerization of an aldose sugar (glucose 6
phosphate) to a ketose sugar (fructose 6phosphate)
15. Phosphofructokinase
6 CH OPO 2â
2
3
1CH2OH
O
5
H
H
4
OH
ATP ADP
HO
2
3 OH
H
fructose-6-phosphate
6 CH OPO 2â
2
3
O
5
Mg2+
1CH2OPO32â
H
H
4
OH
HO
2
3 OH
H
fructose-1,6-bisphosphate
3. Phosphofructokinase catalyzes:
fructose-6-P + ATP ï 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.
The enzyme is highly regulated, as will be discussed later.
16. A phosphate group is
transferred to the first C of
fructose 6 phosphate from
ATP.
Phosphofructokinase
6 CH OPO 2â
2
3
1CH2OH
O
5
H
H
4
OH
ATP ADP
HO
2
3 OH
H
fructose-6-phosphate
6 CH OPO 2â
2
3
O
5
Mg2+
1CH2OPO32â
H
H
4
OH
HO
2
3 OH
H
fructose-1,6-bisphosphate
20. âą dihydroxyacetone-P must be isomerirized to
glyceraldehyde-3-P for futher metabolism in
the glycolytic pathway.
âą This isomerization results in the production of
2 mol. of glyceraldehyde-3-P from the
cleavage products of fructose 1,6 biphosphate..
26. Phosphoglycerate Kinase
Oâ
OPO32âADP ATP O
O
1C
H 2C OH
Mg2+
2â
3 CH2OPO3
1,3-bisphosphoglycerate
C
1
H 2C OH
2â
3 CH2OPO3
3-phosphoglycerate
7. Phosphoglycerate Kinase catalyzes:
1,3-bisphosphoglycerate + ADP ïï
3-phosphoglycerate + ATP
The high energy phophate of 1,3-bisphosphoglycerate is used to
synthesize ATP from ADP. 2 mol. of 1,3-bisphosphoglycerate
are formed from each glucose therefore 2 ATP mol. Are
replaced that were used first during the formation of glucose 6
phosphate and fructose 1,6 biphophosphate.
27. Phosphoglycerate Mutase
Oâ
O
C
1
Oâ
O
C
1
H 2C OH
CH2OPO32â
3
H 2C OPO32â
3 CH2OH
3-phosphoglycerate
2-phosphoglycerate
8. Phosphoglycerate Mutase catalyzes:
3-phosphoglycerate ïï 2-phosphoglycerate
Phosphate is shifted from the OH on C3 to the
OH on C2.
28. Phosphoglycerate Mutase
Oâ
O
C
1
histidine
Oâ
O
C
1
H 2C OH
CH2OPO32â
3
H 2C OPO32â
3 CH2OH
3-phosphoglycerate
2-phosphoglycerate
An active site histidine side-chain
participates in Pi transfer, by
donating & accepting phosphate.
The process involves a
2,3-bisphosphate intermediate.
Oâ
O
C
1
H 2C OPO32â
2â
3 CH2OPO3
2,3-bisphosphoglycerate
29. Enolase
â
O
O
C
1
H 2 C OPO32â
3 CH2OH
H+ â
O
â
O
C
C
OHâ
Oâ
O
1
OPO32â
CH2OH
C
2C
OPO32â
3 CH2
2-phosphoglycerate enolate intermediate phosphoenolpyruvate
9. Enolase catalyzes:
2-phosphoglycerate ïï phosphoenolpyruvate + H2O
The dehydration of 2-phosphoglycerate by enolase reaction
is Mg++-dependent.
2 Mg++ ions interact with oxygen atoms of the substrate
carboxyl group at the active site.
33. glyceraldehyde-3-phosphate
NAD+ + Pi
Glyceraldehyde-3-phosphate
Dehydrogenase
NADH + H+
Glycolysis
continued.
Recall that
there are 2
GAP per
glucose.
1,3-bisphosphoglycerate
ADP
Phosphoglycerate Kinase
ATP
3-phosphoglycerate
Phosphoglycerate Mutase
2-phosphoglycerate
Enolase
H2O
phosphoenolpyruvate
ADP
Pyruvate Kinase
ATP
pyruvate
34. Glycolysis
Balance sheet for ~P bonds of ATP:
2
ï· How many ATP ~P bonds expended? ________
ï· How many ~P bonds of ATP produced? (Remember
4
there are two 3C fragments from glucose.) ________
ï· Net production of ~P bonds of ATP per glucose:
2
________
35. Balance sheet for ~P bonds of ATP:
ï· 2 ATP expended
ï· 4 ATP produced (2 from each of two 3C fragments
from glucose)
ï· Net production of 2 ~P bonds of ATP per glucose.
Glycolysis - total pathway, omitting H+:
glucose + 2 NAD+ + 2 ADP + 2 Pi ï
2 pyruvate + 2 NADH + 2 ATP
In aerobic organisms:
ï· pyruvate produced in Glycolysis is oxidized to CO2
via Krebs Cycle
ï· NADH produced in Glycolysis & Krebs Cycle is
reoxidized via the respiratory chain, with production
of much additional ATP.
36. Lactate Dehydrogenase
Oâ
O
C
C
NADH + H+ NAD+
O
Oâ
O
C
HC
OH
CH3
CH3
pyruvate
lactate
E.g., Lactate Dehydrogenase catalyzes reduction of
the keto in pyruvate to a hydroxyl, yielding lactate, as
NADH is oxidized to NAD+.
Lactate, in addition to being an end-product of
fermentation, serves as a mobile form of nutrient energy,
& possibly as a signal molecule in mammalian organisms.
Cell membranes contain carrier proteins that facilitate
transport of lactate.
37. Lactate Dehydrogenase
Oâ
O
C
C
NADH + H+ NAD+
O
Oâ
O
C
HC
OH
CH3
CH3
pyruvate
lactate
Skeletal muscles ferment glucose to lactate during
exercise, when the exertion is brief and intense.
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
38. Lactate Dehydrogenase
Oâ
O
C
C
NADH + H+ NAD+
O
Oâ
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.
Lactate taken up by adjacent neurons is converted to
pyruvate that is oxidized via Krebs Cycle.
39. Pyruvate
Decarboxylase
Alcohol
Dehydrogenase
CO2
NADH + H+ NAD+
Oâ
O
C
C
O
CH3
pyruvate
H
O
C
CH3
acetaldehyde
H
H
C
OH
CH3
ethanol
Some anaerobic organisms metabolize pyruvate to
ethanol, which is excreted as a waste product.
NADH is converted to NAD+ in the reaction
catalyzed by Alcohol Dehydrogenase.
40. Glycolysis, omitting H+:
glucose + 2 NAD+ + 2 ADP + 2 Pi ï
2 pyruvate + 2 NADH + 2 ATP
Fermentation, from glucose to lactate:
glucose + 2 ADP + 2 Pi ï 2 lactate + 2 ATP
Anaerobic catabolism of glucose yields only 2 âhigh
energyâ bonds of ATP.
41. Flux through the Glycolysis pathway is regulated by
control of 3 enzymes that catalyze spontaneous reactions:
Hexokinase, Phosphofructokinase & Pyruvate Kinase.
ï· Local control of metabolism involves regulatory effects
of varied concentrations of pathway substrates or
intermediates, to benefit the cell.
ï· Global control is for the benefit of the whole organism,
& often involves hormone-activated signal cascades.
Liver cells have major roles in metabolism, including
maintaining blood levels various of nutrients such as
glucose. Thus global control especially involves liver.
Some aspects of global control by hormone-activated
signal cascades will be discussed later.
42. 6 CH2OH
5
H
4
OH
O
H
OH
H
2
3
H
OH
glucose
6 CH OPO 2â
2
3
5
O
ATP ADP
H
H
1
OH
Mg2+
4
OH
H
OH
3
H
2
H
1
OH
Hexokinase H
OH
glucose-6-phosphate
Hexokinase is inhibited by product glucose-6-phosphate:
ï· by competition at the active site
ï· by allosteric interaction at a separate enzyme site.
Cells trap glucose by phosphorylating it, preventing exit
on glucose carriers.
Product inhibition of Hexokinase ensures that cells will
not continue to accumulate glucose from the blood, if
43. 6 CH2OH
5
H
Glucokinase
is a variant of
Hexokinase
found in liver.
4
OH
O
H
OH
H
2
3
H
OH
glucose
6 CH OPO 2â
2
3
5
O
ATP ADP
H
H
1
OH
Mg2+
4
OH
H
OH
3
H
1
H
2
OH
Hexokinase H
OH
glucose-6-phosphate
ï· Glucokinase has a high KM for glucose.
It is active only at high [glucose].
ï· One effect of insulin, a hormone produced when blood
glucose is high, is activation in liver of transcription of
the gene that encodes the Glucokinase enzyme.
ï· Glucokinase is not subject to product inhibition by
glucose-6-phosphate. Liver will take up & phosphorylate
glucose even when liver [glucose-6-phosphate] is high.
44. ï· Glucokinase is subject to inhibition by glucokinase
regulatory protein (GKRP).
The ratio of Glucokinase to GKRP in liver changes in
different metabolic states, providing a mechanism for
modulating glucose phosphorylation.
45. Glycogen
Glucose
Hexokinase or Glucokinase
Glucose-6-Pase
Glucose-6-P
Glucose + Pi
Glycolysis
Pathway
Glucokinase,
with high KM
Glucose-1-P
for glucose,
allows liver to
store glucose
Pyruvate
as glycogen in
Glucose metabolism in liver.
the fed state
when
blood [glucose] is high. catalyzes hydrolytic release of P
Glucose-6-phosphatase
i
from glucose-6-P. Thus glucose is released from the liver
to the blood as needed to maintain blood [glucose].
The enzymes Glucokinase & Glucose-6-phosphatase, both
found in liver but not in most other body cells, allow the
liver to control blood [glucose].
46. Pyruvate Kinase
â
O
O
Pyruvate Kinase, the
ADP
C
last step Glycolysis, is
1
C OPO32â
controlled in liver partly
2
by modulation of the
3 CH2
amount of enzyme.
phosphoenolpyruvate
ATP
Oâ
O
C
1
C
2
O
3 CH3
pyruvate
High [glucose] within liver cells causes a transcription
factor carbohydrate responsive element binding protein
(ChREBP) to be transferred into the nucleus, where it
activates transcription of the gene for Pyruvate Kinase.
This facilitates converting excess glucose to pyruvate,
which is metabolized to acetyl-CoA, the main precursor
for synthesis of fatty acids, for long term energy storage.
47. Phosphofructokinase
6 CH OPO 2â
2
3
1CH2OH
O
5
H
H
4
OH
ATP ADP
HO
2
3 OH
H
fructose-6-phosphate
6 CH OPO 2â
2
3
O
5
Mg2+
1CH2OPO32â
H
H
4
OH
HO
2
3 OH
H
fructose-1,6-bisphosphate
Phosphofructokinase is usually the rate-limiting step of
the Glycolysis pathway.
Phosphofructokinase is allosterically inhibited by ATP.
ï· At low concentration, the substrate ATP binds only at
the active site.
ï· At high concentration, ATP binds also at a low-affinity
regulatory site, promoting the tense conformation.
48. 60
low [ATP]
PFK Activity
50
40
30
high [ATP]
20
10
0
0
0.5
1
1.5
[Fructose-6-phosphate] mM
2
The tense conformation of PFK, at high [ATP], has lower
affinity for the other substrate, fructose-6-P. 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.
49. Glycogen
Glucose-1-P
Glucose
Hexokinase or Glucokinase
Glucose-6-Pase
Glucose-6-P
Glucose + Pi
Glycolysis
Pathway
Pyruvate
Glucose metabolism in liver.
Inhibition of the Glycolysis enzyme Phosphofructokinase
when [ATP] is high prevents breakdown of glucose in a
pathway whose main role is to make ATP.
It is more useful to the cell to store glucose as glycogen
when ATP is plentiful.
50. Glycolysis:
Specific tissue functions
RBCâs
ïŹ
Rely exclusively for energy
Skeletal muscle
ïŹ
Source of energy during exercise, particularly high
intensity exercise
Adipose tissue
Source of glycerol-P for TG synthesis
ïŹ Source of acetyl-CoA for FA synthesis
ïŹ
Liver
Source of acetyl-CoA for FA synthesis
ïŹ Source of glycerol-P for TG synthesis
ïŹ