2. Flow of Energy in Eukaryotes
Cells require a constant supply
of energy to generate and
maintain the biological order that
keeps them alive.
Plants make their organic
molecules by photosynthesis,
whereas animal cells obtain
them by eating other organisms.
Useful energy is derived from
the chemical bond energy as the
organic molecule is broken down
and oxidized to CO2 and H2O.
3. ATP: Energy for Cellular Work
1. Transport
2. Mechanical
3. Chemical
4. Food molecules are
broken down to
produce ATP in three
stages.
A chain of reactions
called glycolysis
converts one molecule
of glucose into 2 pyruvate,
meanwhile 2 ATP and
2 NADH are formed.
5. Burning versus stepwise oxidation
Burning (oxidizing) a molecule
releases energy in the form of
heat.
In the cell, enzymes catalyze
oxidation via a series of small
steps in which energy is
transferred to carrier molecules–
ATP and NADH.
The total free energy released is
exactly the same in (a) and (b).
6.
7.
8.
9. Major Pathways of Glucose Utilization
Glycogen,
starch, sucrose
Glucose
Ribose 5-phosphate Pyruvate
Oxidation
(glycolysis)
Oxidation
(pentose phosphate
pathway)
Storage
10. Glycolysis (glykys-sweet, lysis-
splitting) is a catabolic pathway.
In glycolysis, one molecule of
glucose or a glucosyl unit of
glycogen (6-C) is degraded to
yield two molecules of pyruvate
(3-C), two molecules of NADH
and two molecules of ATP.
Each reaction in the pathway is
catalyzed by an enzyme.
11. Glycolysis (Embden-Meyerhof pathway) breaks down glucose through
10 sequential reactions to form pyruvate.
None of the reactions requires oxygen, therefore glycolysis can occur both
aerobically and anaerobically.
Under anaerobic conditions, pyruvate cannot enter mitochondria, instead it
is converted to lactate in the cytosol.
Breakdown of glucose into two molecules of pyruvate requires cytosolic
reactions.
All intermediates between glucose and pyruvate are phosphorylated
molecules.
The first five reactions constitute the preparatory (energy investment)
phase.The rest of the reactions provide energy or the payoff (energy
generation) phase.
Glycolysis is exquisitely controlled at key points.
16. GA3P
BPG
3PG
2PG
Pyruvate
PEP
P
P
P
P
ATP
ATP
ADP
ADP
6. Oxidation and phosphorylation-GA3P dehydrogenase
7. Phosphorylation-Phosphoglycerate kinase
8. Isomerization-Phosphoglycerate mutase
9. Dehydration-Enolase
10. Phosphorylation-Pyruvate kinase
P
P
H2
O
NAD+ + Pi
NADH + H+
i
Phase 2
17.
18. Step 1: The first ATP investment: phosphorylation of glucose
Phosphorylation of glucose yields glucose 6-phosphate.
Glucose 6-phosphate is a charged molecule therefore it is entrapped
in the cell.
ATP is the source of the phosphate group.
The reaction is catalyzed by hexokinase (or glucokinase in the liver).
19. Importance of phosphorylated intermediates
Each of the nine glycolytic intermediates between glucose and
pyruvate is phosphorylated.
The phosphoryl groups appear to have three functions.
1. Because the plasma membrane generally lacks transporters for
phosphorylated sugars, the phosphorylated glycolytic
intermediates cannot leave the cell.
2. Phosphoryl groups are essential components in the enzymatic
conservation of metabolic energy. Energy released in the
breakage of phosphoanhydride bonds (i.e. those in ATP) is
partially conserved in the formation of phosphate esters.
3. Binding energy resulting from the binding of phosphate groups to
the active sites of enzymes lowers the activation energy and
increases the specificity of the enzymatic reactions. The
phosphate groups of ADP, ATP, and the glycolytic intermediates
form complexes with Mg+2. Most glycolytic enzymes require Mg+2
for activity.
20. Step 2: Isomerization of glucose 6-P
Glucose 6-phosphate (aldose) isomerizes to give fructose 6-
phosphate (ketose).
Phosphohexose (phoshoglucose) isomerase catalyzes the
reaction.
There is no net oxidation or reduction.
21.
22. Step 3: The second ATP investment:
phosphorylation of fructose 6-P
Phosphorylation of fructose 6-phosphate gives fructose 1,6-
bisphosphate.
ATP is the source of the phosphate group.
The reaction is irreversible, it is catalyzed by phosphofructokinase-1.
Phosphofructokinase-1 (PFK-1) is an allosteric enzyme.
23. Step 4: Cleavage of fructose 1,6-bis-phosphate
to two triose phosphates
The reaction is
reversal of aldol
condensation.
Aldolase is the
enzyme catalyzing
this reversible
reaction.
A Schiff base is
formed as the key
intermediate.
24. Step 5: Isomerization of dihydroxyacetone phosphate
Dihydroxyacetone phosphate is converted to glyceraldehyde
3-phosphate, another triose phosphate. Triose phosphate
isomerase catalyzes the reaction.
Only glyceraldehyde-3-phosphate can be carried through
the rest of glycolysis: two for every one glucose molecule.
25.
26. Step 6: Oxidation of glyceraldehyde 3-phosphate to 1,3-
bisphosphoglycerate
A phosphate group is added to glyceraldehyde 3-phosphate
along with oxidation of the aldehyde to carboxylic acid.
Glyceraldehyde 3-phosphate dehydrogenase catalyzes the
reaction.
27.
28. Step 7: Transfer of a phosphate group from 1,3-
bisphosphoglycerate to ADP
A phosphate group is transferred to ADP and the first
ATP generation occurs.
Phosphoglycerate kinase catalyzes the reaction.
29.
30. Step 8: Isomerization of 3-phosphoglycerate to 2-
phoshoglycerate
The phosphate group is transferred from carbon 3 to
carbon 2 to form 2-phosphoglycerate.
Phosphoglyceromutase catalyzes the reaction.
31.
32.
33. Step 9: Dehydration of 2-phosphoglycerate to
phosphoenolpyruvate
The removal of water from 2-phosphoglycerate
creates a high-energy enol phosphate linkage.
Enolase catalyzes the reaction.
34. Step 10: Transfer of a phosphate group from
phosphoenolpyruvate to ADP
This is the second ATP formation in
glycolysis; a phosphate group is
transferred to ADP.
Pyruvate kinase catalyzes the
reaction.
35. Catabolic fates of pyruvate
Glucose
2 Lactate
2 Ethanol + 2 CO2
2 Acetyl-CoA
4 CO2 + 4 H2O
2 Pyruvate
Glycolysis
anaerobic
aerobic
TCA
cycle
anaerobic
36. Alcohol fermentation
In alcohol fermentation
pyruvate produced by
glycolytic reactions is
converted to first
acetaldehyde and then
ethanol.
37. Lactic acid fermentation
Under anaerobic conditions
glycolysis can be
maintained by lactic acid
formation a reaction
catalyzed by lactate
dehydrogenase.
38. When oxygen is not available,
mitochondria cannot
regenerate NAD+ and use
carbons from glycolysis
and the system “backs up”.
Pyruvate lactic acid
(animals)
Pyruvate ethanol and CO2
(plants, fungi, microbes)
40. Cori cycle
2 NAD+
HC O
C
CH
OH
C
HO
C
OH
CH2OH
OH
H
H
H
glycolysis in muscle
2 pyruvate
per glucose
CH3
C
COOH
O
2 NADH
4 ATP
4 ADP
2 ADP
2 ATP
glucose
NAD+
lactate dehydrogenase
lactate
NADH
CH3
CHOH
COOH
glucose
H
H
H
HC O
C
CH
OH
C
HO
C
OH
CH2OH
OH
4 ATP
4 ADP
2 NADH
CH3
C
COOH
O
CH3
CHOH
COOH
NADH
lactate
pyruvate
lactate
dehydrogenase
gluconeogenesis in liver
2 GDP
2 GTP
glucose
lactate
NAD+
2
NAD+
43. Regulation of glycolysis
Points of regulation
◦ Glucose uptake / entry
◦ Three irreversible steps
Hexokinase reaction
Phosphofructokinase-1
reaction
Pyruvate kinase reaction
When ATP is needed, rate of
glycolysis is activated.
When ATP levels are sufficient, rate
of glycolysis is slowed down.
44. PFK-1
Committed step
Regulated by energy charge
(ATP, AMP ratio)
◦ [ATP] does not change much
Citrate
Fructose 2,6-bisphosphate
46. Allosteric Regulation of Phosphofructokinase
A high level of ATP inhibits the enzyme by decreasing its affinity for
fructose 6-phosphate. AMP diminishes and citrate enhances the inhibitory
effect of ATP.
47. Activation of Phosphofructokinase by Fructose 2,6-Bisphosphate
(A)The sigmoidal dependence of velocity on substrate concentration becomes
hyperbolic in the presence of 1 μM fructose 2,6-bisphosphate.
(B) (B) ATP, acting as a substrate, initially stimulates the reaction. As the
concentration of ATP increases, it acts as an allosteric inhibitor. The
inhibitory effect of ATP is reversed by fructose 2,6-bisphosphate.
48. Domain Structure of the Bifunctional Enzyme Phosphofructokinase 2
The kinase domain (purple) is fused to the phosphatase domain (red).
The kinase domain is a P-loop NTP hydrolase domain, as indicated by the
purple shading.
49. Control of the Synthesis and Degradation of Fructose 2,6-Bisphosphate
A low blood-glucose level as signaled by glucagon leads to the phosphorylation of the
bifunctional enzyme and hence to a lower level of fructose 2,6-bisphosphate, slowing
glycolysis. High levels of fructose 6-phosphate accelerate the formation of fructose 2,6-
bisphosphate by facilitating the dephosphorylation of the bifunctional enzyme.
50.
51.
52. Hexokinase
First irreversible reaction
Linked to glucose uptake
◦ Locks glucose in cell
Four isozymes
◦ Most are inhibited by glucose-
6-phosphate
◦ Product inhibition
Not a committed step
53. Glucokinase
Isozyme present in the liver
Has high Km for glucose
Is not inhibited by glucose-6-P
Liver serves as a reservoir for blood sugar
54. Hexokinase / glucokinase
Hexokinase
- inhibited by glucose 6-P
- low Km for glucose
- present in all tissues
Glucokinase
- not inhibited by glucose 6-P
- high Km value for glucose
- present only in the liver
- induced by insulin
0
20
40
60
80
100
120
0 100 200 300 400
[substrate], mmol /L
rate
of
reaction,
µmol
/min
GK
HK
55. Isozymes I,II and II have similar KM (important in muscle)
Normally at saturation
Hexokinase IV has much higher KM (important in liver)
Important when blood glucose is high
56.
57. Pyruvate Kinase
Third irreversible step
Activated by F1,6bP
◦ Feed-forward
activation
◦ Sigmoid curve
becomes hyperbolic
58. Pyruvate kinase
Controlled by both covalent modification
(phosphorylation/dephosphorylation) and allosteric
regulation
Allosteric regulators
◦ ATP (-), acetyl CoA (-), alanine (-)
◦ fructose 1,6-bisphosphate (+)
59. Control of the Catalytic Activity of Pyruvate Kinase
Pyruvate kinase is regulated by allosteric effectors and covalent modification.
60. Regulation of pyruvate kinase
Phosphopyruvate
Kinase
Pyruvate
Kinase
Protein
phosphatase
Protein
kinase
Alanine
ATP
Fructose
1,6-bisphosphate
- +
Glucagon
+
Glucagon
-
63. Glucose Entry into Cells
Tissues have unique function
Isozymes of glucose transporter, GLUT
◦ Insulin dependent in muscle
◦ Higher [glucose] in liver
64. Name Tissue location Km Comments
GLUT1 All mammalian tissues 1 mM Basal glucose uptake
GLUT2 Liver and pancreatic β
cells
15 – 20 mM In the pancreas, plays a role in
regulation of insulin
In the liver, removes excess
glucose from the blood
GLUT3 All mammalian tissues 1 mM Basal glucose uptake
GLUT4 Muscle and fat cells 5 mM Amount in muscle plasma
membrane increases with
endurance training
GLUT5 Small intestine — Primarily a fructose transporter
Family of glucose transporters
65. GLUT1
GLUT3
Hexokinase
Phosphofructokinase
Aldolase
Glyceraldehyde 3-phosphate dehydrogenase
Phosphoglycerate kinase
Enolase
Pyruvate kinase
Lactate dehydrogenase
Proteins in glucose metabolism that
are encoded by genes regulated by HIF
Alteration of Gene Expression in Tumors
Due to Hypoxia
The hypoxic conditions inside a tumor mass
lead to the activation of the hypoxia-
inducible transcription factor (HIF-1), which
induces metabolic adaptation (increase in
glycolytic enzymes) and activates angiogenic
factors that stimulate the growth of new
blood vessels.
