Biochemistry I
Lecture 10 2008 (J.S.)
Integration of metabolism pathways
Mitochondria
The citric acid cycle
Biosynthesis of haem
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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
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Relationships among the major energy metabolism pathways
GLYCOGEN STORES
MONOSACCHARIDES
FAT STORES
TRIACYLGLYCEROLS
FATTY ACIDS
PROTEINS
Glucogenic AA (non-essent.)
Glucogenic AA (essential)
Ketogenic AA (essential) ACETYL-CoA
Citrate cycle
OXIDATIVE PHOSPHORYLATION
ATP
KETONE BODIES
Glycerol
×
Pyruvate
×
×
× ×
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Summary of the previous picture
Saccharides are the most universal nutrients –
the overdose is transformed in the fat stores,
carbon skelet of non-.essential amino acids may originate from saccharides.
Triacylglycerols exhibit the highest energetic yield –
but fatty acids cannot convert into saccharides or the skelet of amino acids.
Amino acids represent the unique source of nitrogen for proteosynthesis
that serves as fuel rather when the organism is lacking in other nutrients glucogenic
amino acids can convert into glucose,
a overdose of diet protein may be transformes in fat stores.
The metabolism of nutrients is sophistically controlled with different mechanisms
in the well-fed state (absorptive phase),
short fasting (post-absorptive phase), and in
prolonged starvation.
It also depends on energy expenditure (predominantly muscular work) –
either of maximal intensity (anaerobic, of short duration only)
or aerobic work of much lower intensity (long duration).
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The tissues differ in their enzyme equipment:
Pathway Liver Kidney Muscle CNS RBC
Adipose
tissue
Glycolysis + + + + + +
FA β-oxidation + + + 0 0 0
Utilization of
ketone bodies 0 + + (+) 0 +
Ketogenesis + 0 0 0 0 0
Gluconeogenesis + + 0 0 0 0
FA synthesis + ± ± ± 0 +
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Cellular compartmentation of the major metabolic pathways
Plasma membrane Transport in and out of cells, signal transduction
Nucleus DNA replication, RNA synthesis (DNA transcription)
Cytosol
Glycolysis, pentose phosphate pathway, FA
synthesis, proteosynthesis on ribosomes, etc.
Mitochondrion
Citrate cycle, FA β-oxidation, aerobic oxidation of
α-ketoacids, oxidative phosphorylation
Endoplasmic reticulum
Lipid and glycoprotein synthesis, FA desaturation,
hydroxylation of xenobiotics, etc..
Golgi complex
Protein glycosylation, intracellular sorting of proteins,
secretion vesicles
Lysosome Degradation of biopolymers by hydrolysis
Peroxisome Oxidations, production and degradation of H2
O2
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Compartmentation of the major pathways of metabolism
8
Mitochondria
Mitochondria are semiautonomous organelles that live in an endosymbiotic relation
with the host cell.
Oxidative phosphorylation (terminal respiratory chain producing a proton gradient
that drives the phosphorylation of ADP) in eukaryotes takes place in mitochondria.
These organells also contain the enzymes of the citric acid cycle, β-oxidation of
fatty acids, and other important metabolic pathways.
9
The outer membrane is quite permeable for small molecules and ions – it
contains many copies of mitochondrial porin (voltage-dependent anion channel,
VDAC).
The inner membrane is intrinsically impermeable to nearly all ions and polar
molecules, but there are many specific transporters which shuttles metabolites
(e.g. pyruvate, malate, citrate, ATP) and protons across the membrane.
The external side of this membrane is called cytosolic (C side, also P side because
of the positive membrane potential), the inner side of the inner mitochondrial
membrane is the matrix side (M side, also N from the negative membrane potential).
(the C side and the opposite M side)
10
Mitochondria are the result of endosymbiosis
– a free-living organism capable of oxidative phosphorylation was engulfed
by another cell.
These organelles – have the double membrane,
– cardiolipin, the typical phospholipid of bacteria, is constituent of the inner membrane,
– mitochondria contain their own circular DNA and the mitochondrial-specific
transcription and translation machinery.
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The citric acid cycle
(also known as the tricarboxylic acid (TCA) cycle or Krebs cycle)
is the final common pathway for the oxidation of nutrients –
saccharides, fatty acids, and amino acids.
Most of the intermediates enter the cycle as acetyl-CoA.
The overall result of this cycle can be summarized in the following simplified form:
The acetyl group of acetyl-CoA is oxidized – two molecules of
carbon dioxide leave the cycle and eight electrons gained (in four
dehydrogenations, represented as 8 H* in the equation) serve to form
3 molecules of NADH + H+
and a molecule of FADH2.
CH3-CO-S-CoA + 3 H2O 2 CO2 + 8 H* + CoA-SH
Carbon dioxide is expired. The four molecules of reduced coenzymes serve as
substrates for terminal respiratory chain.
The direct energy yield is not large – oxidation of one acetyl-CoA in the cycle
yields only one molecule of GTP formed by substrate-level phosphorylation of
GDP.
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1 GTP
13
~
OH
CH2OP
O
O
O
O
N
N
N
N
NH2
O
P O
O
O
HO
P
O
O
OCH2C
S CH2 CH2 HN
O
C CH2 CH2 HN
O
C CH
CH3
CH3
Cysteamine
Pantothenic acid
3´–Phospho ADP
CH3-CO–
Acetyl-CoA, the substrate for the citric acid cycle, is formed
from
the breakdown of
– saccharides (oxidative decarboxylation of pyruvate),
– fatty acids (β-oxidation) and ketone bodies, and
– many amino acids.
14
Two carbon atoms enter the cycle in the condensation of an acetyl
unit (from acetyl CoA) with oxaloacetate.
Two carbon atoms leave the cycle in the form of CO2, oxaloacetate
is the end-product.
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16
1 Condensation of acetyl CoA and oxaloacetate
The reaction is an aldol condensation
and is irreversible in mitochondrial matrix.
Oxaloacetate Acetyl coenzyme A Citrate
CH2-COOH
O=C–COOH
+
S–CoA
CH3–C
O
CH2–COOH
HO–C–COOH
CH2–COOHCoA-SHH2O
is catalysed by citrate synthase:
17
2 Isomerization of citrate into isocitrate
is catalysed by aconitase (cofactor FeS-protein). The isomerization of
citrate is accomplished by a dehydratation step followed by a hydratation:
Secondary
alcoholic groupTertiary
alcoholic group
CH2–COOH
HO–C–COOH
CH2–COOH
Citrate Isocitrate
CH2–COOH
CH–COOH
HO–CH–COOH
C
HC
CH2-COOH
COOH
COOH
cis-Aconitate
H2O H2O
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3 Isocitrate is oxidized and decarboxylated to 2-oxoglutarate
CH2–COOH
CH–COOH
HO–CH–COOH
Isocitrate 2-Oxoglutarate
CH2
CH2–COOH
O=C–COOH
(Oxalosuccinate)
CH2–COOH
CH–COOH
O=C–COOH
NAD+
NADH + H+
CO2
The first of four oxidation reactions in the citrate cycle is catalysed by
isocitrate dehydrogenase, the cofactor is NAD+
.
The intermediate in this reaction is unstable oxalosuccinate which loses
CO2, while bound to the enzyme, to form 2-oxoglutarate.
The reaction is irreversible.
The rate of this reaction
is important in determining
the overall rate of the cycle.
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4 Oxidative decarboxylation of 2-oxoglutarate
to succinyl-CoA
The second oxidative step and decarboxylation in the cycle is closely
analogous to the oxidative decarboxylation of pyruvate.
The 2-oxoglutarate dehydrogenase complex requires also the same five
cofactors – TDP, lipoate, coenzyme A, FAD and NAD+
.
Succinyl-CoA
CH2
CH2–COOH
O=C–S–CoA
2-Oxoglutarate
CH2
CH2–COOH
O=C–COOH
HS–CoA
CO2
NADH + H+
NAD+
FAD FADH2
Succinyl-CoA is the high-energy
thioester compound.
20
5 The cleavage of succinyl-CoA is coupled to
the phosphorylation of GDP
CH2–COOH
CH2–COOH
Succinyl-CoA Succinate
CH2
CH2–COOH
O=C–S–CoA
Pi
CoA-SH
GDP GTP
ATP ADP
In the reaction catalysed by succinyl-CoA synthetase (succinate
thiokinase), the energy inherent in the thioester molecule is transformed
into phosphoryl-group transfer.
This substrate-level phosphorylation is the only step in the citrate
cycle that directly yields a high-energy compound.
GDP is phosphorylated to GTP n three steps,
succinyl phosphate and phosphohistidyl
residue of the enzyme are the intermediates. The
mechanism has got the nickname "passing a hot
potato".
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6 Oxidation of succinate to fumarate
CH2–COOH
CH2–COOH
Succinate Fumarate
FAD FADH2
HC
CH
COOH
HOOC
is the third oxidative step, catalysed by succinate dehydrogenase.
The prosthetic group FAD accepts two atoms of hydrogen from
succinate.
Succinate dehydrogenase differs from other enzymes in the citrate cycle
in being embedded in the inner mitochondrial membrane.
The enzyme is directly associated with the terminal respiratory chain as
the component of the complex II, which transfers a reducing
equivalent (in the form of two electrons) to ubiquinone.
Succinate dehydrogenase, like aconitase, is a non-haem iron protein. In addition to the
flavin prosthetic group, it contains three different types of Fe-S clusters that také part in the
electron transport.
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7 – 8 Oxaloacetate is regenerated by hydratation of
fumarate and oxidation of malate
L-Malate
CH2–COOH
HO–CH–COOH
CH2-COOH
O=C–COOH
Oxaloacetate
NAD+
NADH + H+
HC
CH
COOH
HOOC
H2O
Fumarate
Fumarase catalyses a stereospecific trans addition
of water, only the L-enantiomer of malate is formed.
The dehydrogenation of malate catalysed by malate
dehydrogenase is the fourth oxidative step in the
cycle. It is driven by the utilization of the products –
oxaloacetate by citrate synthase and NADH by the
terminal respiratory chain .
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Recapitulation:
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25
GTP
OXIDATIVE PHOSPHORYLATION
+ SUBSTRATE-LEVEL PHOSPHORYLATION
~ 3 ATP
~ 3 ATP
~ 3 ATP
~ 2 ATP
The energetic yield:
Total approx.12 molecules ATP from the oxidation of 1 acetyl-CoA
- about 11 ATP due to reoxidation of reduced coenzymes in the terminal respiratory chain,
- 1 GTP direct yield through a substrate-level phosphorylation in the citrate cycle.
26
Inhibitors of isocitrate DH:
ATP
NADH
Activator: ADP
Inhibitors of 2-oxoglutarate DH:
ATP
NADH
Succinyl-CoA
Inhibitors of pyruvate DH:
ATP
NADH
Acetyl CoA
Regulation of the citrate cycle
Molecular oxygen does not
participate directly in the citrate
cycle.
However, the cycle can operate
only under aerobic conditions
because it requires a supply of
(re)oxidized NAD+
and FAD.
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Not only acetyl-CoA is oxidized in the citrate cycle, but also
other compounds, which are metabolized to the cycle intermediates,
can also serve as substrates of the cycle.
The catabolic role of the citrate cycle
Glu, Gln, Arg, His, ProPropionyl-CoA
Ile, Val, Met, Thr
Phe, Tyr
Asp, Asn
The entries into the cycle:
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The anabolic role of the citrate cycle
The citric acid cycle also provides intermediates for biosyntheses –
thus it exhibits an amphibolic character.
Porphyrins, haem
Pyruvate
Asn, Aspartate
Pyrimidines
Phosphoenolpyruvate
Glutamate
Gln, Arg, Pro, His
Glucose
Malate
Fatty acids, steroids
Intermediates drawn off for biosyntheses are replenished by the
anaplerotic reactions.
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Anaplerotic reactions
lead to the net synthesis, or replenishment, of pathway components.
The most important of them is the
formation of new oxaloacetate by carboxylation of pyruvate,
a crucial step in gluconeogenesis.
+ Biotin–COOH + Biotin-H
H3C–C–COOH
O
Pyruvate
O
HOOC–CH2–C–COOH
Oxaloacetate
Pyruvate carboxylase
If the energy charge of the cell is low, oxaloacetate replenishes the citric acid cycle.
If the energy charge is high, oxaloacetate is converted into glucose.
There are also other anaplerotic reactions of less importance, e.g.
reductive carboxylation of pyruvate to malate,
transamination of aspartate that gives oxaloacetate,
transamination of glutamate to 2-oxoglutarate,
as well as the other reaction drawn in the picture 27
(the catabolic role of the cycle).
Biosynthesis of haem
31
The first reaction in the biosynthesis of porphyrins
is the condensation of succinyl-coenzyme A and glycine in
mitochondria:
The enzyme has pyridoxal phosphate as a prosthetic group.
5-aminolaevulinate (5-ALA, δ-aminolaevulinate) is transported into the cytosol.
Succinyl-CoA
5-Aminolaevulinate
(5-amino-4-oxobutanoic acid)
Glycine CH2–COOH
NH2
CO–S–CoA
COOH
CH2
CH2
5-ALA synthase
CO2
HS-CoA
COOH
CH2
CH2
C=O
CH2
NH2
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In the cytosol, two molecules of 5-aminolaevulinate undergo the
condensation to form a pyrrole derivative – porphobilinogen:
Two molecules of
5-aminolaevulinate
2 H2O
Porphobilinogen
COOH
CH2
CH2
C=O
CH2
NH2
COOH
CH2
CH2
C=O
CH2
NH2 N
H
NH2
COOHCOOH
CH2–CH2
CH2
CH2
Acetic acid
Propionic acid
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Methylene bridge
Four molecules of
porphobilinogen
N
H
NH2
COOHCOOH
4
4 NH3
Uroporphyrinogen III
Four molecules of porphobilinogen then condense "head to tail" to form
a linear tetrapyrrole in a reaction catalysed by porphobilinogen deaminase.
The product cyclizes to form the tetrapyrrole ring of uroporphyrinogen.
Under physiological circumstances due to the presence of a protein modifier
called co-synthase, uroporphyrinogen III with an asymmetrical arrangement
of side chains of the ring D is formed. Only traces of symmetrical
uroporphyrinogen I are produced.
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In subsequent reactions, the side chains and the degree of saturation of the
porphyrin ring are modified:
Uroporphyrinogen III
(eight carboxylic groups of
four acetates and four propionates)
Coproporphyrinogen III
(four carboxylic groups of propionates)
4 CO2
This reaction takes place in the cytosol. Coproporphyrinogen is then
transported into the mitochondria, where the biosynthesis of haem is
completed by two oxidative steps and a ferrous cation is built in.
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Protoporphyrinogen IX
(colourless)
Coproporphyrinogen III
O2
2 H2O
2 CO2
3 O
3 H2O
The isomer numbers: Uro- and coproporphyrinogen has theoretically only four isomers
that differ in the position of acetates and propionates, type III and I are natural products.
Protoporphyrin has three different types of substituents, we may imagine 15 isomers.
Product of the biosynthesis that originates from coproporphyrin III, is called
protoporphyrin IX.
The conversion of two of the propionate side chains
into vinyl groups yields protoporphyrinogen IX. then
follows the desaturation of the porphyrin ring.
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The desaturation of the ring (methylene bridges are converted into methene bridges)
forms a fully conjugated system of double bonds – the product is of intensive colour.
(coloured)
Protoporphyrin IX
methene bridge,
3 O
3 H2O
Haem
Fe2+
2 H+
Ascorbate
Ferrochelatase
Fe3+
-ferritin
The chelation of iron finally gives haem, the
prosthetic group of haemoglobin, myoglobin,
cytochromes, catalase, and peroxidase.
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Haem
Approx. 0.4 mmol synthesized
per day
Proteosynthesis of
globin α- and β-chains
HAEMOGLOBIN
monomers (1 Fe)
HAEMOGLOBIN
α2β2 (4 Fe)
Myoglobin
Cytochromes
Catalase, peroxidase
The rate-limiting step of the overall biosynthesis is synthesis of 5-ALA.
In the liver, where only 15 % of haem are synthesized; the feedback
control (inhibition) depends of the availability of haem or its FeIII
oxidation product haemin.
85 % of the body's haem groups are synthesized in the erythroid cells.
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MITOCHONDRION
CYTOSOL Uroporphyrinogen III
Uroporphyrinogen I
Glycine Succinyl-CoA
5-Aminolaevulinate
Porphobilinogen
5-Aminolaevulinate
HAEM
Protoporphyrin IX
Coproporphyrinogen III
Coproporphyrinogen III
Protoporphyrinogen IX
- CO2
- 2 H2O
- 4 NH3- 4 CO2
-2 CO2
+ O2
- 3 H2O
+ 3 O
MITOCHONDRION
Recapitulation:
Coproporphyrinogen I
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Porphyrias
The porphyrias are mostly genetic diseases characterized by defects in
haem synthesis.
In spite of haem synthesis may provide enough haem for the body due
to reduced feedback inhibition, there is often the overproduction of
porphyrins or their precursors – increased excretion of porphyrins in
the urine and faeces.
The various symptoms depend on the type of enzyme defect.
Some of them are
– skin lesions on exposure to sunlight
(porphyrins are photosensitizing agents) – erythemas, scarring,
– disturbances of erythropoiesis,
– disturbances of the liver functions,
– neuropsychiatric disturbances.
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MITOCHONDRION
Uroporphyrinogen III
Uroporphyrinogen I
Glycine Succinyl-CoA
5-Aminolaevulinate
Porphobilinogen
5-Aminolaevulinate
HAEM
Protoporphyrin IX
Coproporphyrinogen III
Coproporphyrinogen III
Protoporphyrinogen IX
– CO2
– 2 H2O
– 4 NH3
– 4 CO2
– 2 CO2
+ O2
– 3 H2O
+ 3 O
SIDEROBLASTIC
ANAEMIA
PBG DEFICIENCY
PORPHYRIA
ACUTE INTERMITTENT
PORPHYRIA
CONGEN. ERYTHROPOIETIC
PORPHYRIAPORPHYRIA
CUTANEA TARDA
TOXIC PORPHYRIAS
ERYTHROPOIETIC
PROTOPORPHYRIA
VARIEGATE
PORPHYRIA
HEREDITARY
COPROPORPHYRIA
Porphyrias
Coproporphyrinogen I
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Haem
Degradation of haemoglobin in the reticuloendothelial system:
Haemoxygenase
(cyt P450)
Biliverdin reductase