Download Citric acid cycle

General Biochemistry
Part II-d
Prof. Dr. S. Beeckmans
Prof. Dr. E. Van Driessche
[email protected]
Protein Chemistry Lab
Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, room E.5.23
Pictures are taken from:
Lehninger PRINCIPLES OF BIOCHEMISTRY
(4th Edition) − 2005
by: David L. Nelson & Michael M. Cox
W.H. Freeman and Comp., New York
BIOCHEMISTRY
(5th Edition) − 2002
by: Jeremy M Berg, John L Tymoczko & Lubert Stryer
W.H. Freeman and Comp., New York
BIOCHEMISTRY
Prof. Dr. Sonia Beeckmans
(3rd Edition) − 2003
by: Donald Voet & Judith G Voet
John Wiley and Sons
I.
The foundations of biochemistry
1.
2.
The cell and subcellular organelles
Short overview of biomolecules:
proteins, nucleic acids, lipids, polysaccharides, and their building blocks
Fundamentals of energy and energy transfer
3.
II. Metabolism and its regulation
Prof. Dr. Sonia Beeckmans
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
Strategies to regulate metabolic pathways
Glycolysis and gluconeogenesis
Pentose-phosphate pathway and Calvin cycle
Glycogen metabolism
Fatty acid degradation (β-oxidation and ω-oxidation)
Fatty acid biosynthesis and ketone bodies
Pyruvate dehydrogenase, citric acid cycle and anaplerotic reactions
Amino acid degradation and urea cycle
Nitrogen metabolism: biosynthesis of amino acids, nucleotides and related molecules
Oxidative phosphorylation and ATP synthesis
Photosynthesis (light reactions)
III. Intra- and intercellular organization
15.
16.
17.
18.
19.
20.
Biomembranes (including transport systems)
The cytoskeleton
Metabolons: physical organization of metabolic pathways
Protein trafficking
Protein degradation
Integration of metabolism: from molecules to cells to organs
Overview of metabolism
Proteins
Fats
Saccharides
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FOOD
Prof. Dr. Sonia Beeckmans
Acetyl-CoA is an
energy-rich molecule
1 kcal = 4.18 kJ
Prof. Dr. Sonia Beeckmans
ATP
Prof. Dr. Sonia Beeckmans
Mitochondria
The conversion of pyruvate to acetyl-CoA is
catalyzed by a complex
Cryoelectron micrograph of bovine PDHC.
Prof. Dr. Sonia Beeckmans
Samples are viewed at extremely low
temperatures, thus avoiding potential
artefacts introduced by the usual process
of dehydrating + fixing + staining.
Prof. Dr. Sonia Beeckmans
The pyruvate dehydrogenase complex
consists of 3 enzymes that cooperate
and form a multienzyme complex
The pyruvate dehydrogenase complex
requires five coenzymes
Prof. Dr. Sonia Beeckmans
1.5 nm
swinging arm
Prof. Dr. Sonia Beeckmans
The pyruvate dehydrogenase complex
requires five coenzymes
Prof. Dr. Sonia Beeckmans
The pyruvate dehydrogenase complex
requires five coenzymes
3D model of bovine PDHC
E1 (pyruvate dehydrogenase) is at the outside.
The core consists of 60 molecules E2
(dihydrolipoyl transacetylase).
The lipoyl domain of E2 reaches outward
touching the active sites of molecules E1.
Prof. Dr. Sonia Beeckmans
E3 (dihydrolipoyl dehydrogenase) is also
supposed to be bound to the core.
E2 consists of three types of
domains connected by short
linkers:
Consecutive reactions of PDHC
+ TPP
HETPP + CO2
E1
HETPP + lipoamide
Prof. Dr. Sonia Beeckmans
E2
TPP + Acetyl-lipoamide
Acetyl-lipoamide + CoA-SH
Acetyl-CoA + Dihydrolipoamide
Dihydrolipoamide + FAD
Lipoamide + FADH2
NAD+
E3
NADH + H+
PROBLEM !!!
For energetic reasons this reaction is not possible:
FAD
FADH2
NAD+
NADH + H+
Solution of this problem:
FAD
E3
S
Prof. Dr. Sonia Beeckmans
S
FAD°H
S°
NADH + H+
NAD+
SH
Prof. Dr. Sonia Beeckmans
—S-S—
•
—S
—SH
x
!!!
Prof. Dr. Sonia Beeckmans
Generation of Acetyl-CoA
??
Prof. Dr. Sonia Beeckmans
Chapter: The citric acid cycle
Prof. Dr. Sonia Beeckmans
When you study this
reaction pathway,
try to remember the
formulas of the
intermediates,
not their names !!!!!
Preview
The citric acid cycle
‰ Stoichiometry:
Acetyl-CoA + 3NAD+ + FAD + ADP + Pi + 2H2O
2CO2 + 3(NADH + H+) + FADH2 + ATP + CoASH
‰ 2CO2 leaving are different from those entering
‰ 4 pairs of H atoms leave the cycle
Prof. Dr. Sonia Beeckmans
‰ 1 high-energy phosphate bond is formed
‰ NADH and FADH2 will be reoxidized in the next
pathway (OP), and ATP will be generated as
electrons are transferred from these carriers to O2,
the ultimate electron acceptor
‰ the cycle operates as a real cycle only under
aerobic conditions, i.e. when OP pathway occurs
Action of citrate synthase
Prof. Dr. Sonia Beeckmans
Citrate synthase is the driving force for
the citric acid cycle
(this is why an energy-rich acetyl-CoA
molecule is consumed,
instead of performing the reaction:
CH3-COOH + OAA
citrate).
Prof. Dr. Sonia Beeckmans
Action of citrate synthase
Action of aconitase
Prof. Dr. Sonia Beeckmans
Substrate binding involves a
“non-haem iron sulfur cluster”.
Action of isocitrate dehydrogenase
Mn2+ in the enzyme’s
active site interacts with
the carbonyl group of
the intermediate oxalosuccinate.
Prof. Dr. Sonia Beeckmans
It also stabilizes the
enol formed transiently
by decarboxylation.
There are two different
ICDHases, one acting
with NAD (found in
mitochondria) and
another one acting with
NADP (both in mito and
in cytoplasm).
The function of the latter may be generation of NADPH for anabolism.
Prof. Dr. Sonia Beeckmans
Action of α-ketoglutarate dehydrogenase
A complex built up of E1, E2 and E3, just like the pyruvate
dehydrogenase complex.
Enzyme E3 is even shared by both multi-enzyme complexes.
Action of succinyl-CoA synthetase
ATP
ADP
Nucleoside diphosphate kinase
Coenzyme A
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phosphate
The enzyme is built up of 2 different
subunits. The active site includes part
of both the α- (blue) and the β-subunit
(brown).
Prof. Dr. Sonia Beeckmans
Action of succinyl-CoA synthetase
Such process is called
“substrate-level phosphorylation”.
Prof. Dr. Sonia Beeckmans
Action of succinate dehydrogenase
SDH is an inner
membrane
enzyme.
All other CAC
enzymes are in
the mitochondrial
matrix.
Prof. Dr. Sonia Beeckmans
Action of fumarase
Fumarase is highly stereospecific:
maleate is not a substrate and
neither is D-malate.
Action of malate dehydrogenase
ΔG = –2.303*RT*logKeq
Keq = 0.00000618
Prof. Dr. Sonia Beeckmans
=
[OAA] [NADH]
[malate] [NAD+]
The equilibrium of this reaction lies far to the left under standard
conditions. In cells, the concentration of OAA is extremely low
(<10–6 M), pulling the malate dehydrogenase reaction towards
the formation of OAA.
Prof. Dr. Sonia Beeckmans
Citrate: a symmetrical molecule
that reacts asymmetrically
Prof. Dr. Sonia Beeckmans
Enzymes of the citric acid cycle
Anaerobic bacteria have an incomplete cycle
They miss the α-ketoglutarate
dehydrogenase complex.
Prof. Dr. Sonia Beeckmans
The lower branch now runs in
opposite direction, and will be
pushed forward by the
ΔG = –7.1 kcal/mol
of malate dehydrogenase.
The citric acid cycle is called
an amphibolic pathway
(serves 2 different purposes):
– source of energy
– source of intermediates for
anabolism.
Regulation of PDHC and citric acid cycle
Only in vitro, using free ATP !!!
Prof. Dr. Sonia Beeckmans
But: inside cells, ATP and ADP
are chelated
Regulation of PDHC and citric acid cycle
acetyl-CoA
NADH
CO2
Prof. Dr. Sonia Beeckmans
NAD+
pyruvate
coenzyme A
Role of citric acid cycle in anabolism
Prof. Dr. Sonia Beeckmans
Gluconeogenesis
Fatty acid
biosynthesis
Biosynthesis of purines/pyrimidines
Biosynthesis of amino acids
In order to remove intermediates,
intermediates need to be
replenished
Prof. Dr. Sonia Beeckmans
Role of citric acid cycle in anabolism
ANAPLEROTIC REACTIONS
Pyruvate carboxylase
ATP
HCO3–
pyruvate
Prof. Dr. Sonia Beeckmans
Acetyl-CoA
An allosteric activator
prevents waste of ATP:
the enzyme is inactive in
the absence of acetyl-CoA.
A long flexible arm
allows the prosthetic
group to rotate from
site 1 to site 2.
A high-energy substrate
Other anaplerotic pathways:
Prof. Dr. Sonia Beeckmans
‰ Glyoxylic acid cycle
‰ Amino acid degradation
The glyoxylic acid cycle
Allows the synthesis of sugars
exclusively from fatty acids.
Occurs in bacteria/yeasts, when
they grow on e.g. ethanol, ...
Prof. Dr. Sonia Beeckmans
Occurs in some plant seedlings.
Citric acid cycle
Prof. Dr. Sonia Beeckmans
In plant seedlings
In bacteria
Prof. Dr. Sonia Beeckmans
Coordinated regulation of glyoxylic
acid cycle and citric acid cycle
occurs through phosphorylation of
ICDH.
A high-energy substrate
Other anaplerotic pathways:
Prof. Dr. Sonia Beeckmans
‰ Glyoxylic acid cycle
‰ Amino acid degradation