Biochemistry I
Lecture 3 2008 (J.S.)
Basic concept and design of
metabolism
The glycolytic pathway
Oxidative decarboxylation of pyruvate
and other 2-oxocarboxylic acids
2
Living organisms require a continual input of free energy for three
major purposes:
– the performance of mechanical work in cellular movements,
– the active transport of molecules and ions across membranes,
– the synthesis of macromolecules and other biomolecules from
simple precursors.
The free energy used in these processes, which maintain an
organism in a state that is far from equilibrium,
is derived from the environment.
3
Catabolism (catabolic reactions) converts chemical energy by
decomposing foodstuffs into biologically useful forms.
Anabolism (anabolic reactions) requires energy – useful forms of
energy are employed to generate complex structures from simple
ones, or energy-rich states from energy-poor ones.
Metabolism is essentially a series of chemical
reactions that provides energy transformations:
Energy is being extracted from fuels (nutriments)
and used to power biosynthetic processes.
4
5
The Gibbs free-energy change ∆G
The maximal amount of useful energy that can be gained in the reaction (at
constant temperature and pressure).
a A + b B → c C + d D ∆G = GA+B – GC+D
∆Gº = – RT ln K
Reactions can occur spontaneously only if they are
exergonic (if ∆G, the change in free energy, is negative).
ΔG = ΔGº + RT ln
[A]a
[B]b
[C]c
[D]d
The ΔG of a reaction depends on the nature of the reactants (expressed by the ΔGº
term) and on their concentrations (expressed by the second term).
6
An endergonic reaction cannot proceed spontaneously,
but such a thermodynamically unfavourable reaction can be
driven by an exergonic reaction to which it is coupled.
Energetic coupling occurs because the two reactions share
a common reactant or intermediate.
Example:
The overall net free energy change is negative (ΔGº = – 13.4 kJ/mol),
the conversion of malate to aspartate is exergonic.
Malate
Fumarate
H2O
NH3
Aspartate
ΔGº1 = + 2 kJ/mol
ΔGº2 = – 15.4 kJ/mol
7
Glucose Glucose 6-phosphate
ATP ADP
The reaction which is used to drive endergonic ones is very
oft the hydrolysis of ATP.
Example:
∆Go´
= + 13.8 kJ mol–1
∆Go´
= – 30.5 kJ mol–1
= – 16.7 kJ mol–1
8
ATP + H2
O → ADP + Pi ∆G°´ (at pH 7) = – 30,5 kJ  mol–1
O
OH OH
2
O
O
CHOPOP
O
O
O ~ P
O
O
O ~
+ H+H
N
N
N
N
NH2
ATP
+ P O
O
O
HO2O
O
OH OH
N
N
N
N
NH2
2
ADP
P
O
O
O CHOP
O
O
O~
Adenosine triphosphate (ATP)
is a high-energy compound that serves as the "universal currency" of free
energy in biological systems. ATP hydrolysis drives metabolism by shifting
the equilibrium of coupled reactions.
9
The metabolic interplay of living organisms in our biosphere
Living organisms can be divided into two large groups according to the chemical form of
carbon they require from the environment.
Autotrophic cells ("self-feeding" cells)
– green leaf cells of plants and photosynthetic bacteria – utilize CO2 from the
atmosphere as the sole source of carbon for construction of all their carbon-containing
biomolecules.
They absorb radiant energy of the sun. The synthesis of organic compounds is
essentially the reduction (hydrogenation) of CO2 by means of hydrogen atoms,
produced by the photolysis of water (generated dioxygen O2 is released).
Heterotrophic cells
– cells of higher animals and most microorganisms – must obtain carbon in the form of
relatively complex organic molecules (nutrients such as glucose) formed by other cells.
They obtain their energy from the oxidative (mostly aerobic) degradation of organic
nutrients made by autotrophs and return CO2 to the atmosphere.
Carbon and oxygen are constantly cycled between the animal and plant
worlds, solar energy ultimately providing the driving force for this massive
process.
10
Photosynthetic
autotrophs
Heterotrophs
CO2
H2O
O2
Nutrients rich in H
hν
4 H
4 H+
O2 2 O2–
CO2Biological oxidations
(dehydrogenations)
Decarboxylations
Reducing equivalents
(reducing power)
4 e–
2 H2O
Nutrients rich in H
Heterotrophs:
11
Fatty acids of fats are a more efficient fuel source than saccharides
such as glucose because the carbon in fatty acids is more reduced
Most of the Gibbs´ free energy in the body originates in the exergonic
synthesis of water (2H2 + O2 → 2H2O, 25 °C): ΔG° = – 474.3 kJ mol–1
12
Stages in the extraction of energy from foodstuffs
The first stage of catabolism
Large molecules in food are broken
down into smaller units
Stage II
Degradation to a few amphibolic
intermediates
Stage III
The final common pathways –
most of the ATP is produced from
the complete oxidation of the acetyl
unit of acetyl CoA
13
High-energy compounds
is not a high-energy compound
(no anhydride bond)
GTP, CTP, UTP, TTP are quite analogous to ATP. as well as
GDP, CDP, UDP, TDP are analogous to ADP.
14
Anhydrides
di– and triphosphates ATP, ADP, UTP, CTP etc.
phosphosulfate phosphoadenosyl-phosphosulfate (PAPS)
acylphosphates 1,3-bisphosphoglycerate
Ester phosphoenolpyruvate
Thioesters acyl coenzymes A
Amides phosphocreatine
Different types of high-energy compounds
HN=C
NH–PO3
2–
N–CH2COOH
CH3
CH2
C–O–PO3
2–
COO
–
C
O
O– PO 3
2–
CH–OH
2CH –O– PO 3
2–
15
Factors contributing to the large change in ΔGº of hydrolysis:
1 Electrostatic repulsion of negatively charged groups
2 Products of hydrolysis are more stable than the reactant because
of greater resonance possibilities
3 and the groups in the products are more prone to isomerization or
they exhibit more states of ionization
Phosphoenolpyruvate–
Hydrogen phosphate 2–
+ pyruvate –
More negative el. charges and
tautomerization of enolpyruvate to the ketoform
R
CO–R H C–R
O
O
–+
16
Synthesis of ATP by phosphorylation of ADP in the cell
1 Oxidative phosphorylation in mitochondria
accounts for more than 90 % of ATP generated in animals.
The synthesis of ATP from ADP and Pi is driven by the
electrochemical potential of proton gradient across the inner
mitochondrial membrane.
This gradient is generated by the
terminal respiratory chain,
in which hydrogen atoms,
as NADH + H+ and FADH2
produced by the oxidation
of carbon fuels,
are oxidized to water.
The oxidation of hydrogen by O2
is coupled to ATP synthesis.
17
2 Phosphorylations of ADP on the substrate level
are provided by few reactions, in which a nucleoside triphosphate is
synthesized by utilization of the free energy of hydrolysis of
a soluble energy-rich compound.
- Energy released by certain carbon oxidations can be converted
into high phosphoryl-transfer potential and so the
favourable oxidation is coupled with the unfavourable synthesis
(phosphorylation) of ATP.
- The high phosphoryl-transfer potential of phosphoenolpyruvate
arises primarily from the large driving force of the
subsequent enol-ketone conversion. Dehydration of 2phosphoglycerate
"traps" the molecule of the product in its unstable
enol form.
18
Examples of substrate-level phosphorylations
phosphoenolpyruvate pyruvate
ADP ATP
pyruvate kinase
1,3-bisphosphoglycerate 3-phosphoglycerate
In glycolysis
ADP ATP
phosphoglycerate kinase
In the citrate cycle
succinyl coenzyme A succinate + CoA
GDP + Pi GTP
thiokinase
ADP + H+
ATP
creatine kinase
phosphocreatine creatine
In skeletal muscle phosphocreatine serves as a reservoir of high-potential
phosphoryl groups that can be readily transferred to ATP:
19
Control of metabolism
Metabolism is regulated by controlling
 catalytic activity of enzymes
allosteric and cooperative effects, reversible covalent
modification, substrate concentration
 the amount of enzymes
synthesis of adaptable enzymes
 the accessibility of substrates
compartmentalization segregates biosynthetic and degradative
pathways, the flux of substrates depends on controlled transfer from one
compartment of a cell to another
 the energy status of the cell
of which the energy charge or the phosphorylation potential
are used as indexes
 communication between cells
hormones, neurotransmitters, and other extracellular
molecular signals often regulate the reversible modification of key
enzymes
20
Energy charge =
[ATP] + ½[ADP]
[ATP] + [ADP] + [AMP]
can have a value ranging from 0 (all AMP) to 1 (all ATP).
Catabolic (ATP-generating) pathways are inhibited by an energy charge,
whereas anabolic (ATP-utilizing) pathways are stimulated by a high-energy
charge.
The energy charge of most cells ranges from 0.80 to 0.95.
Phosphorylation potential =
[ATP]
[ADP] × [Pi]
is an alternative index of the energy status of a cell.
In contrast with the energy charge, it depends on the concentration
of Pi and is directly related to the free energy storage available from ATP.
21
The glycolytic pathway
22
Glucose is an important and common nutrient for most organisms.
In mammals
glucose is the only fuel that the brain uses under non-starvation conditions
and the only fuel that red blood cells can use at all.
Some fates of glucose: ANAEROBIC
ANAEROBIC
GLYCOLYSIS
23
Glucose transporters
mediate the thermodynamically downhill movement of glucose across the plasma
membranes of animal cells.
The members of the family of transporters have distinctive roles.
24
Glucose transporter GLUT4
transports glucose into muscle and fat cells. The presence of insulin, which signals
the fed state leads to a rapid increase in the number of GLUT4 transporters in the
plasma membrane. Hence, insulin promotes the uptake of glucose by muscle and
adipose tissue.
insulin
Insulin
Insulin
receptor
"Sleeping“
GLUT4 in the membranes
of endosomes
GLUT4 are exposed
in the plasma membrane
25
The glycolytic pathway
(also known as the Embden-Meyerhof pathway)
The conversion of glucose into two molecules of pyruvate is anaerobic with the
concomitant net production of two molecules of ATP.
Under anaerobic conditions, pyruvate can be processed to lactate.
Under aerobic conditions, pyruvate can be decarboxylated to acetyl CoA
and completely oxidized to CO2, generating much more ATP.
Glycolysis is common to all types of cells.
In eukaryotic cells, glycolysis takes place in the cytosol.
Reactions of glycolysis are catalyzed by enzymes.
Three of them are irreversible. (In gluconeogenesis, pyruvate is converted to glucose:
those three reactions differ and are catalyzed by different enzymes.)
Fructose and galactose also enter into glycolysis.
26
The glycolysis can be thought of as comprising three stages:
Trapping the glucose in the cell and
destabilization by phosphorylation.
Cleavage into two
three-carbon units.
Oxidative stage in which new molecules of
ATP are formed by substrate-level
phosphorylation of ADP.
27
The phosphorylation of glucose by ATP:
Hexokinase reaction traps glucose in the cell, Glc-6-P cannot diffuse through the membrane,
because of its negative charges.
Conversion of Glc-6-P to glucose catalysed by glucose 6-phosphatase takes place
only in the liver (and to a lesser extent in the kidney).
The addition of the phosphoryl group begins to destabilize glucose,
thus facilitating its further metabolism:
- through further reactions of glycolysis,
but also through reactions starting
- synthesis of glycogen (glycogenesis)
- the pentose phosphate pathway (supplying NADPH),
- synthesis of other saccharides (e.g. mannose, galactose, amino sugars, glucuronic acid).
Glc-6-P
28
Glucokinase Hexokinases
In the liver, specific for glucose In extrahepatic tissues, broad specifity for hexoses
Not inhibited by Glc-6-P Inhibited by Glc-6-P
Low affinity for glucose High affinity for glucose
Inducible (in the liver) by insulin Not inducible by insulin
The phosphorylation of glucose in the cytosol accelerates the entry of glucose into the cell.
On the contrary to other tissues, the liver cells (and the pancreatic β-cells) comprise a specialized
isoenzyme of hexokinase called glucokinase. The enzyme is very efficient, but its affinity for glucose
is low (value of Michaelis constant is high, Km = 10 mmol/l). It means that the uptake of glucose by the
liver cells (as well as β-cells of pancreatic islets secreting insulin) shall predominate, if there is a steep
rise in blood glucose. The role of glucokinase is to provide glucose for the synthesis of glycogen and
for the formation of fatty acids. Glucose will not be wasted in other tissues when it is abundant.
Hexokinases present in the other tissues are inhibited by glucose 6-phosphate, the reaction product.
High concentration of this molecule signal that the cell no longer requires glucose for energy, for
storage in the form of glycogen, or as a source of biosynthetic precursors, and the glucose will be left in
the blood.
High affinities of hexokinases for glucose (Michaelis constant Km ≤ 0,1 mmol/l) will ensure the constant
and preferential flow of glucose into the extrahepatic tissues, if the blood glucose level is low..
29
The isomerization of Glc-6-P to fructose 6-phosphate
catalysed by phosphoglucose isomerase:
30
The second phosphorylation catalysed by phosphofructokinase
is the rate-limiting step and a major control point of glycolysis :
Common features of the rate-limiting step of a metabolic pathway:
- The molar activity (turnover number, kcat) of the particular enzyme is smaller than those of other
enzymes taking part in the metabolic pathway.
- The reaction rate does not usually depend on substrate concentration [S] because it reaches
the maximal value Vmax.
- The reaction is practically irreversible. The process can be reversed only by the catalytic action
of a separate enzyme.
Allosteric control of phosphofructokinase:
• allosteric inhibition by ATP and citrate,
• allosteric activation by AMP, ADP, and in the liver by fructose 2,6-bisphosphate
(Fru-6-P) (Fru-1,6-P2)
31
- summary
32
Stage 2
The splitting of fructose 1,6-bisphosphate into two triose phosphates
catalysed by aldolase:
CH2–OH
CH2 –O–PO 3
2–
C O
CH–OH
CH O
CH2 –O–PO 3
2–
33
In the following stage 3, only glyceraldehyde 3-phosphate is oxidized.
Dihydroxyacetone phosphate does not accumulate because it is continuously
converted to glyceraldehyde phosphate by triose phosphate isomerase:
Stage 2 – summary
Fructose 1,6-bisphosphate 2 molecules of glyceraldehyde 3-phosphate
CH2–OH
CH2–O–PO3
2–
C O CH–OH
CH O
CH2–O–PO3
2–
34
Stage 3
Oxidative stage – new molecules of ATP are formed
by substrate-level phosphorylation of ADP
The reaction is the only oxidative step in the glycolytic pathway, it produces NADH and is
highly exergonic. The product 1,3-BPG is a high-energy intermediate (a mixed
anhydride of 3-phosphoglycerate and phosphate).
This reaction is coupled energetically with the following step in which the large negative
free energy of hydrolysis of 1,3-BPG is utilized in an endergonic phosphorylation of ADP
to ATP.
Oxidation of GAP by NAD+
to 1,3-bisphosphoglycerate:
Anhydride bond
C
O
O–PO3
2–
CH–OH
CH2–O–PO3
2–
+ NADH + H+
Anhydride bond
+CH–OH
CH=O
CH2–O– PO 3
2–
35
The oxidation of GAP to 1,3-BPG thus drives the synthesis of ATP from ADP.
This is an example of substrate-level phosphorylation of ADP.
In red blood cells (the demand of ATP is lower when compared to other cells) the reaction can be
passed by without the gain of ATP:
In the reaction catalysed by phosphoglycerate kinase the energy-rich anhydride 1,3bisphosphoglycerate
is hydrolysed, and at the same time
the energy-rich ATP is formed by the phosphorylation of ADP:
C
O
O–PO3
2–
CH–OH
CH2–O–PO3
2–
COO
–
CH–OH
CH2–O–PO3
2–
36
The by-pass of phosphoglycerate kinase reaction in red blood cells:
mutase 2,3-bisphosphoglycerate
phosphatase
3-Phosphoglycerate + Pi
COO
–
CH2–O–PO3
2–
CH–O–PO3
2–
C
O
O–PO3
2–
CH–OH
CH2–O–PO3
2–
COO
–
CH–OH
CH2–O–PO3
2–
COO
–
CH–OH
CH2–O–PO3
2–
(phosphoglycerate kinase)
+ ATP
2,3-Bisphosphoglycerate
(3-Phosphoglycerate + ATP)
+ H2O
+ ADP
+ Pi
2,3-Bisphosphoglycerate is an important effector of oxygen binding by haemoglobin.
37
Formation of phosphoenolpyruvate
Both reactions are readily reversible.
The product phosphoenolpyruvate is a high-energy intermediate (an ester of the enol form of
pyruvate and phosphate).
COO
–
CH–OH
CH2–O–PO3
2–
CH2–OH
COO
–
CH–O–PO3
2–
Phosphoenolpyruvate
C–O–PO3
2–
COO
–
CH2
Ester bond
(PEP)
is catalysed by phosphoglycerate mutase and by enolase:
38
Pyruvate kinase reaction is the 3rd
control point of the glycolytic pathway. Pyruvate kinase is
- allosterically activated by fructose-1,6-bisphosphate (the product of an earlier step),
- and in liver cells inhibited by hormone glucagon through phosphorylation.
In the reaction catalysed by pyruvate kinase the energy-rich ester
phosphoenolpyruvate is hydrolysed, and at the same time
the energy-rich ATP is formed by the phosphorylation of ADP:
This reaction (essentially irreversible) is a substrate-level phosphorylation, the second
one of the 3rd
stage of glycolysis.
The synthesis of ATP from ADP is driven by the dehydration of 2-phosphoglycerate to
phosphoenolpyruvate (PEP) in the previous reaction.
COO
–
C O
CH3
C–O–PO3
2–
COO
–
CH2
COO
–
C–OH
CH2
ATP
ADP
Pyruvate
(enol form) (keto form)
39
2 ×
Stage 3
- summary
40
The diverse fates of pyruvate
Pyruvate is a pivotal intermediate in saccharide metabolism
CH–OH
COO
–
CH3
Lactate
Anaerobic glycolysis
NADH NAD+
Ethanol
(in microorganisms)
COO
–
C O
CH3
Pyruvate
Alanine
COO–
CH–NH2
CH3
Glu
2-OG
Oxaloacetate
CH2
COO–
COO–
C O
CO2 + ATP
ADP + Pi Acetyl CoA
CO–S–Co A
CH3
Oxidative decarboxylation
(mitochondria)
CoA + NAD+
CO2 + NADH
41
Pyruvate catabolism
in animals is an aerobic pathway located in the mitochondrial matrix.
Glucose
Oxidation in the citrate cycle to CO2
or conversion to fatty acids or cholesterol
+ CoA
The initial steps are
- transport into mitochondria
- oxidative decarboxylation
acetyl CoA
Anaerobic glycolysis:
42
COO
–
C O
CH3
CH–OH
COO
–
CH3
NADH + H+
Anaerobic glycolysis
When the oxidative decarboxylation of pyruvate is stopped under anaerobic
conditions, pyruvate is reduced to lactate. The reaction is catalysed by lactate
dehydrogenase, and it is readily reversible.:
The purpose of this final reduction is to regenerate NAD+
consumed in
dehydrogenation of 3-phosphoglyceraldehyde to 1,3-bisphosphoglycerate.
At insufficient concentration of NAD+
, molecules of glucose cannot enter the
glycolytic pathway.
43
Lactate
Reoxidation of NADH
in anaerobic glycolysis:
In fact, the anaerobic glycolysis produces
lactic acid (lactate anion as well as H+
).
The intense lactate production may be a cause
of its accumulation associated with a decrease in
pH that could stop the glycolytic pathway.
44
The total lactate formation in man (70 kg) ≈ 1.3 mol / d
Of this, 25 % comes from erythrocytes,
25 % from skin,
about 14 % each from muscle,
brain and
renal medulla,
8 % from intestinal mucosa.
The lactate concentration in blood is normally around 1 mmol / l; it can rise to about 30
mmol / l during vigorous exercise, but quickly falls when exercise ceases.
LIVER BLOOD MUSCLE
Glucose Glucose Glucose
Gluconeogenesis Glycolysis
Pyruvate Pyruvate
LD LD
Lactate Lactate Lactate
The reconversion of lactate (gluconeogenesis) in the liver - the Cori cycle
45
Alcoholic fermentation of glucose
in yeasts (obligatory anaerobic organisms) also produces pyruvate. The difference between
anaerobic glycolysis and alcoholic fermentation is in the process of reoxidation of NADH:
Pyruvate is a subject of simple decarboxylation
to acetaldehyde, NADH is reoxidized through
reduction of acetaldehyde to ethanol.
Pyruvate decarboxylase
Alcohol dehydrogenase
NAD+
46
Energetic yield of glycolysis and aerobic breakdown of glucose
GLYCOLYSIS
Stage 1: two molecules ATP are consumed
Stage 3: four molecules ATP are formed by substrate-level phosphorylations
Net yield: 2 molecules ATP / 1 molecule glucose (i.e. 2 pyruvates)
AEROBIC BREAKDOWN of glucose to CO2
Glycolysis: (by substrate-level phosphorylations) 2 molecules ATP
and 2 molecules NADH ∗)
⇒ 6 molecules ATP
The possible loss due to redox shuttle transport – 2 molecules ATP
Oxidative decarboxylation of two pyruvates:
2 molecules NADH ⇒ 6 molecules ATP
Decomposition of 2 acetyl CoA in the citrate cycle:
⇒ the overall yield 24 molecules ATP
Net yield: 36 – 38 molecules ATP / 1 molecule glucose
∗)
Supposing that reoxidation of NADH will give 3 ATP and FADH2 2 ATP
(in spite of the lower values are referred to in recent literature).
47
GLUCONEOGENESIS
GLYCOLYSIS
Hexokinase
The control of glycolysis
Three control points
are the three irreversible
reactions of glycolysis
catalysed by
1 hexokinase,
2 phosphofructokinase 1,
3 pyruvate kinase.
48
1 Hexokinase(s)
present in the extrahepatic tissues are inhibited by glucose 6phosphate,
the reaction product.
High concentration of this molecule signal that the cell no longer requires
glucose for energy, for storage in the form of glycogen, or as a source of
biosynthetic precursors, and the glucose will be left in the blood.
49
2 Phosphofructokinase
is the key enzyme in the control of glycolysis
Phosphofructokinase (PFK) in
the liver is a tetramer of four
identical subunits.
The positions of catalytic and
allosteric sites are indicated.
50
Allosteric inhibition of PFK by ATP
ATP as a substrate of the PFK catalyzed reaction binds to the catalytic site.
At high concentration of ATP it also binds to a specific regulatory site that is distinct
from the catalytic site and allosterically inhibits the PFK activity.
AMP reverses the inhibitory action of ATP –
glycolysis is stimulated as the energy charge falls.
A fall in pH value also inhibits PFK activity – inhibition by H+
prevents
excessive formation of lactic acid and a drop in blood pH.
51
Allosteric activation of phosphofructokinase
by fructose 2,6-bisphosphate
52
(A) Allosteric activation of PFK by Fru-2,6-P2
(B) The inhibitory effect of ATP is reversed by Fru-2,6-P2
53
The concentration of Fru-2,6-P2 is controlled
by a regulated bifunctional enzyme.
Fru-2,6-P2 is formed in a reaction catalyzed by
phosphofructokinase 2,
and hydrolyzed to Fru-6-P by a specific phosphatase
fructose bisphosphatase 2.
Both activities are present in a single polypeptide chain:
54
Insulin
Control of the bifunctional enzyme by phosphorylation and dephosphorylation
55
3 Control of pyruvate kinase activity
- by phosphorylation and dephosphorylation
- by allosteric effectors
Insulin
Glucagon
56
Oxidative decarboxylation of pyruvate
and of other 2-oxocarboxylic acids
57
The synthesis of acetyl-CoA
by the pyruvate dehydrogenase complex
Is a key irreversible step in the metabolism of
glucose.
The oxidative decarboxylation of pyruvate
takes place within the matrix of
mitochondrion.
Under aerobic conditions, the pyruvate
is transported into mitochondria in exchange
for OH−
by the pyruvate carrier, an antiporter.
Pyruvate + CoA + NAD+
→
→ acetyl CoA + CO2 + NADH
58
59
Oxidative decarboxylation of pyruvate represents the link
between glycolysis and the citric acid cycle.
Pyruvate produced by glycolysis is
converted into acetyl CoA, the substrate
(fuel) for the citric acid cycle.
60
Electron micrograph of the pyruvate dehydrogenase complex from E. coli
61
Pyruvate dehydrogenase complex – schematic representation
The three enzymes of the complex:
E1 – the decarboxylating component of the dehydrogenase
E2 – the transacetylase core
E3 – dihydrolipoyl dehydrogenase
62
The enzyme complex requires the participation of five coenzymes:
Thiamine diphosphate
Lipoamide (lipoate attached to the E2 by an amide linkage to lysyl)
Coenzyme A
FAD (flavin adenine dinucleotide)
NAD+
63
Acetaldehyde
(as hydroxyethyl
bound to TDP)
Oxidation
by lipoamide
Acetyl
(bound to
DHlipoamide)
Decarboxylating component Transacetylase
E1 E2
Reoxidation of dihydrolipoamide to lipoamide
(2 hydrogen atoms accepted by FAD and then
by NAD+
resulting in NADH + H+
)
E3
Dihydrolipoyl dehydrogenase
Steps in the oxidative decarboxylation of pyruvate
64
(+)
(-)
Decarboxylating component of pyruvate dehydrogenase E1 contains bound
thiamine diphosphate (TDP):
The thiazole ring of the coenzyme TDP binds pyruvate. The product of
decarboxylation is acetaldehyde bound onto TDP in the form of α-hydroxyethyl:
E1 catalyses the transfer of α-hydroxyethyl to the lipoyl arm of transacetylase E2.
65
Transacetylase E2 contains bound lipoic acid that is attached to the amino group of
the side chain of certain lysyl residue. That is why it is named lipoamide.
Lipoamide (oxidized form, a disulfide) acts as an arm that
accepts the hydroxyethyl group from TDP. Hydroxyethyl
group ("activated acetaldehyde") reduces lipoamide to
dihydrolipoamide and thus is oxidized to acetyl bound as a
thioester – 6-acetyllipoamide.
The acetyl is then transferred to coenzyme A :
66
~
O
OH
CH2OP
O
O
O
N
N
N
N
NH2
O
P O
O
O
HO
P
O
O
OCH2C
HS CH2 CH2 HN
O
C CH2 CH2 HN
O
C CH
CH3
CH3
Cysteamine β-Alanine Pantoic acid
Pantothenic acid
3´–phospho ADP
Coenzyme A
Acyls are attached to the sulfanyl group
by means of a thioester bond.
67
TDP TDP
68
Dihydrolipoyl dehydrogenase
E3
The dihydrolipoyl arm then swings to E3, where it is reoxidized.
Dihydrolipoyl dehydrogenase E3 contains bound coenzyme FAD that
accepts two hydrogen atoms which are passed on to NAD+
.
69
In the citrate cycle, the oxidative decarboxylation of 2-oxoglutarate (to succinyl
CoA) closely resembles that of pyruvate:
The 2-oxoglutarate dehydrogenase complex consists of E1 (decarboxylating
2-oxoglutarate) and E2 (transsuccinylase) components different from
but homologous to the corresponding enzymes in the pyruvate dehydrogenase
complex, whereas E3 (dihydrolipoyl dehydrogenase) components of the two
complexes are identical.
70
Regulation of the pyruvate dehydrogenation complex
Inhibition - by the immediate products NADH and acetyl CoA,
- by ATP, and
- by phosphorylation (depending e.g. on glucagon)
Activation by dephosphorylation (depending on insulin)