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KAPITOLA 9
Energetický metabolismus II
• cyklus trikarboxylových kyselin
• dýchací řetězec
• aerobní fosforylace
Catabolism of proteins, fats, and carbohydrates occurs
in the three stages of cellular respiration.
Stage 1: Oxidation of fatty acids, glucose, and some amino
acids yields acetyl-CoA.
Stage 2: Oxidation of acetyl groups via the citric acid
cycle includes four steps in which electrons are
abstracted.
Stage 3: Electrons carried by NADH and FADH2 are
funneled into a chain of mitochondrial (or plasma
membrane-bound, in bacteria) electron carriers the respiratory chain - ultimately reducing O2 to
H2O. This electron flow drives the synthesis of
ATP, in the process of oxidative phosphorylation.
Each turn of the cytric acid cycle
produces three NADH and one
FADH2 as well as one GTP (or
ATP). Two CO2 are released in
oxidative decarboxylation reactions.
The reactions of the citric acid cycle.
The carbon atoms are those originally derived
from the acetate of acetyl-CoA in the first turn
of the cycle. These carbons are not the ones
released as CO2 in the first turn. Note that in
fumarate, the two-carbon group derived from
acetate can no longer be specifically denoted;
because succinate and fumarate are symmetric
molecules, C-1 and C-2 are indistinguishable
from C-4 and C-3. The number beside each
reaction step corresponds to a numbered
heading in the text.
Steps 1, 3, and 4 are essentially
irreversible in the cell; all of
the others are reversible.
Steps in the oxidative decarboxylation of
pyruvate to acetyl-CoA by the pyruvate
dehydrogenase complex.
In step 1 pyruvate reacts with the bound
thiamine pyrophosphate (TPP) of pyruvate
dehydrogenase (E1), undergoing decarboxylation to form the hydroxyethyl derivate.
Pyruvate dehydrogenase also carries out step
2, the transfer of two electrons and the acetyl
group from TPP to the oxidized form of the
lipoyllysyl group of the core enzyme,
dihydrolipoyl transacetylase (E2), to form the
acetyl thioester of the reduced lipoyl group.
Step 3 is a transesterification in which the -SH
group of CoA replaces the -SH group of E2 to
yield acetyl-CoA and the fully reduced
(dithiol) form of the lipoyl group. In step 4
dihydrolipoyl dehydrogenase (E3) promotes
transfer of two hydrogen atoms from the
reduced lipoyl groups of E2 to the FAD
prosthetic group of E3, restoring the oxidized
form of the lipoyllysyl group of E2 (in bold
box). In step 5 the reduced FADH2 group on
E3 transfers a hydride ion to NAD+, forming
NADH. The enzyme complex is now ready
for another catalytic cycle.
E1 - pyruvate dehydrogenase
E2 - dihydrolipoyl transacetylase
E3 - dihydrolipoyl dehydrogenase
The noncyclic reactions that provide biosynthetic
precursors in anaerobically growing bacteria. These
cells lack -ketoglutarate dehydrogenase and therefore
cannot carry out the complete cytric acid cycle. Ketoglutarate and succinyl-CoA serve as precursors in a
variety of biosynthetic reactions.
Intermediates of the citric acid cycle are drawn off as
precursors in many biosynthetic pathways, yielding the
products in the shaded areas. Four anaplerotic reactions
that replenish depleted intermediates of the cytric acid
cycle (broken arrows).
Regulation of metabolite flow from
pyruvate through the citric acid
cycle. The pyruvate dehydrogenase
complex is allosterically inhibited at
high [ATP]/[ADP], [NADH]/[NAD+],
and [acetyl-CoA]/[CoA] rations, all of
which indicate the energy-sufficent
metabolic state. When these rations
decrease, allosteric activation of pyruvate oxidation results. The rate of flow
through the citric acid cycle can be
limited by the availability of the
substrates oxaloacetate and acetyl-CoA
or by the depletion of NAD+ by its
conversion to NADH, which slows the
three oxidation steps for which NAD+
is the cofactor. Feedback inhibition by
succinyl-CoA, citrate, and ATP also
slows the cycle by inhibiting early
steps. In muscle tissue, Ca2+ signals
contraction and stimulates energyyielding metabolism to replace the ATP
consumed by contraction.
A summary of the points of entry of the
standart amino acids into the citric acid cycle.
Some amino acids are listed more than once; these
are broken down to yield different fragments, each
of which enters the citric acid cycle at a different
point. This scheme represents the major catabolic
pathways in vertebrate animals, but there are
minor variations from organism to organism.
Biochemical anatomy of a mitochondrion.
The convolutions (cristae) of the inner membrane
give it a very large surface area. The inner
membrane of a single liver mitochondrion may
have over 10 000 sets of electron transfer system
(respiratory chains) and ATP synthase molecules,
distributed over the whole surface of the inner
membrane. Heart mitochondria, which have very
profuse cristae and thus a much larger area of inner
membrane, contain over three times as many sets of
electron transfer systems as liver mitochondria. The
mitochondrial pool of coenzymes and intermediates
is functionally separated from the cytosolic pool.
The mitochondria of invertebrates, plants, and
microbial eukaryotes are similar to those shown
here, although there is much variation in size,
shape, and degree of convolution of the inner
membrane.
Path of electrons from NADH,
succinate, fatty acyl-CoA, and
glycerol-3-phosphate to ubiquinone
(UQ). Electrons from NADH pass
through a flavoprotein to a series of
iron-sulfur proteins (in Complex I )
and then to UQ. Electrons from
succinate pass through a flavoprotein
and several Fe-S centers (in Complex
II) on the way to UQ. Glycerol-3phosphate donates electrons to a
flavoprotein
(glycerol-3-phosphate
dehydrogenase) on the outer face of
the inner mitochondrial membrane,
from which they pass through Fe-S
centers to UQ. Acyl-CoA dehydrogenase (the first enzyme in b
oxidation) transfers electrons to
electron-transferring
flavoprotein
(ETFP), from which they pass via
ETFP-ubiquinone oxidoreductase to
UQ.
The path of electrons through the
Complex III probably involves a "Q
cycle" such as that shown here (blue
arrows). The broken arrows represent
diffusion of UQH2 (ubiquinol) or its
oxidized form UQ across the
membrane. Notice that the electron
transfers between cytochromes and
ubiquinone are one-electron reactions,
producing the semiquinone radical as
an intermediate. The net effects of the
reactions here are movement of
electrons from UQH2 to cytochrome c,
and movement of protons from the
inside (matrix) to the outer (cytosolic)
side of the inner membrane (the
intermembrane space).
Path of electrons through Complex IV.
CuA (Cu2+) and cyt a (Fe2+) from one
bimetallic redox center capable of
accepting two electrons; CuB and cyt a3
constitute a second two-electron redox
center. The detailed path of electron flow
between cyt c and O2 is not known with
certainty; apparently electrons first move
from cyt c to CuA or cyt a, which are in
rapid redox equilibrium with each other.
This bimetallic center then donates
electrons to CuB and cyt a3, also in redox
equilibrium, which in turn donate the
electrons that reduce O2 to H2O. The four
protons used in the reduction of O2 to
H2O are taken up from the matrix side of
the inner mitochondrial membrane.
Consequently, cytochrom oxidase pumps
protons out of the matrix as electrons are
transferred to O2.
The ATP synthase complex from mitochondria.
(a) Electon micrographs the knoblike protrusions from the mitochondrial inner membrane.
(b) Schematic diagram showing the likely organization of the subunits to form the protonconducting F0 portion, and the ATP-synthesizing F1 unit. The F1 complex consists of three ,
three , and one each of ,, and  subunits.
Summary of the flow of electrons and protons through the four complexes of the
respiratory chain.
Electrons reach UQ via Complexes I and II. UQH2 (ubiquinol) serves as a mobile carrier of
electrons and protons. It passes electrons to Complex III, which passes them to another
mobile connecting link, cytochrome c. Complex IV transfers electrons from reduced
cytochrome c to O2. Electron flow through Complexes I, III, and IV is accompanied by
proton flow from the matrix to the intermembrane space. Recall that electrons from fatty
acid b oxidation can also enter the respiratory chain through UQ.
In this simple version of the chemiosmotic theory applied to mitochondria, electrons from NADH and other oxidizable
substrates pass through a chain of carriers (cytochromes, etc.) arranged asymmetrically in the membrane. Electron flow is
accompanied by proton transfer across the mitochondrial membrane, producing both a chemical (pH) and an electrical
() gradient. The inner mitochondrial membrane is impermeable to protons; protons can reenter the matrix only through
proton-specific channels (F0). The proton-motive force that drives protons back into the matrix provides the energy for ATP
synthesis, catalyzed by the F1 complex associated with F0.
The malate-asparate shuttle for
transporting reducing equivalents
from cytosolic NADH into the
mitochondrial matrix.
1 NADH in the cytosol (intermembrane space) passes two reducing equivalents to oxaloacetate, producing malate.
2 Malate is transported across the inner membrane by the malate--ketoglutarate transporter.
3 In the matrix, malate passes two reducing equivalents to NAD+; the resulting matrix NADH is oxidized by the
mitochondrial respiratory chain. The oxaloacetate formed from malate cannot pass directly into the cytosol. It is
first transaminated to form asparate 4, which can leave via the glutamate-aspartate transporter 5. Oxaloacetate is
regenerated in the cytosol 6, completing the cycle.
The glycerol-3-phosphate shuttle,
an alternative means of moving
reducing equivalents from the cytosol to the mitochondrial matrix.
Dihydroxyacetone phosphate in the
cytosol accepts two reducing equivalents from cytosolic NADH in a
reaction catalyzed by cytosolic
glycerol-3-phosphate
dehydrogenase. A membrane-bound
isozyme of glycerol-3-phosphate
dehydrogenase, located on the outer
face of the inner membrane,
transfers two reducing equivalents
from glycerol-3-phosphate in the
intermembrane space to ubiquinone.
Note that this shuttle does not
involve membrane transport systems.
The uncoupling protein (thermogenin)
of brown fat mitochondria, by providing
an alternative route for protons to reenter
the mitochondrial matrix, causes the
energy conserved by proton pumping to
be dissipated as heat.
Interlocking regulation of glycosis, pyruvate oxidation, the citric acid cycle, and oxidative phosphorylation by the relative
concentrations of ATP, ADP, and AMP, and by NADH. High [ATP] (or low [ADP] and [AMP]) produces low rates of glycolysis,
pyruvate oxidation, acetate oxidation via the citric acid cycle, and oxidative phosphorylation. All four pathways are accelerated when
there is an increase in the rate of ATP utilization and increased formation of ADP, AMP, and Pi. Interlocking of glycolysis and the
citric acid cycle by citrate, which inhibits glycolysis, supplements the action of the adenine nucleotide system. In addition, increased
levels of NADH and acetyl-CoA also inhibit the oxidation of pyruvate to acetyl-CoA, and high [NADH]/[NAD+] ratios inhibit the
dehydrogenase reactions of the citric acid cycle.
Protein components of the mitochondrial electron transfer
chain
Mass
(kDa)
Number of
subunits
Prosthetic
group(s)
850
>25
FMN, Fe-S
II Succinate
dehydrogenase
140
4
FAD, Fe-S
III Ubiquinonecytochrome c
oxidoreductase
250
10
Hemes, Fe-S
13
1
Heme
160
6-13
Hemes; CuA, CuB
Enzyme complex*
I NADH
dehydrogenase
Cytochrome c
IV Cytochrome
oxidase
Sources: DePierre, J.W. & Ernster, L. (1977) Enzyme topology of intracellular
membranes. Annu. Rev. Biochem. 46, 201-262; Hafeti, Y. (1985) The
mitochondrial electron transport and oxidative phosphorylation system. Annu. Rev.
Biochem. 54, 1015-1069.
* Note that cytochrome c is not part of an enzyme complex, but moves between
Complexes III and IV as a freely soluble protein.
The stoichiometry of coenzyme reduction and ATP
formation in the aerobic oxidation of a molecule of
glucose via glycolysis, the pyruvate dehydrogenase
reaction, and the citric acid cycle
Number of ATP
or reduced
coenzymes
directly
formed
Number of ATP
ultimately
formed*
Glucose  glucose-6-hosphate
-1 ATP
-1
Fructose-6-phosphate 
fructose-1,6-bisphosphate
-1 ATP
-1
2 NADH
6
2 ATP
2
2 ATP
2
2 NADH
6
2 NADH
6
2 NADH
6
2 ATP (or 2
GTP)
2
2 Succinate  2 fumarate
2 FADH2
4
2 Malate  2 oxaloacetate
2 NADH
6
Reaction
2 Glyceraldehyde-3-phosphate
 2 1,3-bisphosphoglycerate
2 1,3-Bisphosphoglycerate
 2 3-phosphoglycerate
2 Phosphoenolpyruvate
 2 pyruvate
2 Pyruvate  2 acetyl-CoA
2 Isocitrate
 2 -ketoglutarate
2 -Ketoglutarate
 2 succinyl-CoA
2 Succinyl-CoA  2 succinate
Total
38
* This is calculated as 3 ATP per NADH and 2 per ATP per FADH2. A negative
value indicates consumption.
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