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三羧酸循环
The Citric Acid Cycle
The tricarboxylic acid (TCA) cycle
The TCA Cycle—A Brief
Summary
• The entry of new carbon units into the
cycle is through acetyl-CoA.
• This entry metabolite can be formed either
from pyruvate (from glycolysis) or from
oxidation of fatty acids.
• In the process, metabolic energy is
captured in the form of ATP, NADH, and
enzyme-bound FADH2 (symbolized as
[FADH2]).
• The citric acid cycle is the central
metabolic hub of the cell.
• It is the gateway to the aerobic metabolism
of any molecule that can be transformed
into an acetyl group or dicarboxylic acid.
• The cycle is also an important source of
precursors, not only for the storage forms
of fuels, but also for the building blocks of
many other molecules such as amino
acids, nucleotide bases, cholesterol, and
porphyrin (the organic component of
heme).
• The citric acid cycle is the final
common pathway for the
oxidation of fuel molecules—
amino acids, fatty acids, and
carbohydrates.
• Most fuel molecules enter the
cycle as acetyl coenzyme A.
• citric acid cycle, also called the
tricarboxylic acid (TCA) cycle or
the Krebs cycle (after its
discoverer, Hans Krebs).
Under aerobic conditions, the pyruvate generated from glucose is
oxidatively decarboxylated to form acetyl CoA.
In eukaryotes, the reactions of the citric acid cycle take place inside
mitochondria, in contrast with those of glycolysis, which take place
in the cytosol.
The Bridging Step: Oxidative
Decarboxylation of Pyruvate
oxidative decarboxylation reaction
• pyruvate dehydrogenase complex(PDC)
a cluster of enzymes—multiple
copies of each of three enzymes—
located in the mitochondria of
eukaryotic cells and in the cytosol of
prokaryotes.
• It contains three enzymes: pyruvate
dehydrogenase (E1), dihydrolipoyl
transacetylase 二氢硫辛酸转乙酰基酶
(E2), and dihydrolipoyl dehydrogenase
二氢硫辛酸脱氢酶 (E3)—each present
in multiple copies.
• The overall reaction involves a total of five
coenzymes: thiamine pyrophosphate焦磷酸
硫胺素, coenzyme A, lipoic acid硫辛酸, NAD,
and FAD.
硫辛酸同酶分
子中Lys残基
的ε-NH2以酰
胺键共价结合
Flexible Linkages Allow Lipoamide to Move
Between Different Active Sites
Thiamine-deficient
• As one might predict, mutations in the genes for the
subunits of the PDH complex, or a dietary thiamine
deficiency, can have severe consequences.
• Thiamine-deficient animals are unable to oxidize pyruvate
normally. This is of particular importance to the brain, which
usually obtains all its energy from the aerobic oxidation of
glucose in a pathway that necessarily includes the oxidation
of pyruvate.
• Beriberi脚气病, a disease that results from thiamine
deficiency, is characterized by loss of neural function. This
disease occurs primarily in populations that rely on a diet
consisting mainly of white (polished) rice, which lacks the
hulls in which most of the thiamine of rice is found.
• People who habitually consume large amounts of alcohol can
also develop thiamine deficiency, because much of their
dietary intake consists of the vitamin-free “empty calories”
of distilled spirits.
• An elevated level of pyruvate in the blood is often an
indicator of defects in pyruvate oxidation due to one of these
causes.
硫辛酸
(Thioctic Acid, Lipoic Acid)
• 酵母和微生物等必需的生长因子，含硫的
八碳酸，对于动物不能算是一个维生素，
但是一个辅酶，通过氧化还原作用参与生
物体的递氢和传递乙酰基的作用。
• 以硫辛酸（酰胺）为辅酶的酶有：硫辛酸
转乙酰酶、二氢硫辛酸脱氢酶、丙酮酸脱
氢酶、 α-酮戊二酸脱氢酶，与糖代谢关
系密切。
Entry into the Cycle: The Citrate
Synthase Reaction
Synthases and Synthetases
• Synthases合酶 catalyze condensation
reactions in which no nucleoside triphosphate
(ATP, GTP, and so forth) is required as an
energy source.
• Synthetases合成酶 catalyze condensations
that do use ATP or another nucleoside
triphosphate as a source of energy for the
synthetic reaction.
The Isomerization of Citrate by
Aconitase乌头酸酶
aconitate hydratase乌头酸水合酶
The active site of aconitase. The
iron-sulfur cluster (red) is
coordinated by cysteines (yellow)
and isocitrate (white).
Fluoroacetate氟乙酸 Blocks the
TCA Cycle
• trojan horse inhibitor.
• Citrate synthase converts
fluoroacetate to inhibitory
fluorocitrate for its TCA cycle partner,
aconitase, blocking the cycle.
Isocitrate Dehydrogenase—The First
Oxidation in the Cycle
There are two different forms of
isocitrate dehydrogenase in all cells,
one requiring NAD as electron
acceptor and the other requiring
NADP.
In eukaryotic cells, the NADdependent enzyme occurs in the
mitochondrial matrix and serves
in the citric acid cycle.
The main function of the
NADP-dependent enzyme,
found in both the mitochondrial
matrix and the cytosol, may be
the generation of NADPH,
which is essential for reductive
anabolic reactions.
Citrate: A Symmetrical Molecule
That Reacts Asymmetrically
2
α-Ketoglutarate Dehydrogenase—
A Second Decarboxylation
• Like the pyruvate dehydrogenase
complex, α-ketoglutarate
dehydrogenase is a multienzyme
complex—consisting of αketoglutarate dehydrogenase,
dihydrolipoyl transsuccinylase, and
dihydrolipoyl dehydrogenase—that
employs five different coenzymes.
Succinyl-CoA Synthetase—A SubstrateLevel Phosphorylation
• Succinyl-CoA is itself a high-energy
intermediate and is utilized in this step
of the TCA cycle to drive the
phosphorylation of GDP to GTP (in
mammals) or ADP to ATP (in plants
and bacteria).
• The reaction is catalyzed by succinylCoA synthetase, sometimes called
succinate Thiokinase琥珀酸硫激酶.
Succinate Dehydrogenase—An
Oxidation Involving FAD
• The oxidation of succinate to
fumarate is carried out by succinate
dehydrogenase, a membrane-bound
enzyme that is located in the innermembrane of mitochondrial .
• It is actually part of the electron
transport chain.
• Succinate dehydrogenase, like
aconitase, is an iron–sulfur protein铁硫
蛋白.
• Indeed, succinate dehydrogenase
contains three different kinds of
iron–sulfur clusters, 2Fe-2S (two iron
atoms bonded to two inorganic
sulfides), 3Fe-4S, and 4Fe-4S.
Fumarase Catalyzes TransHydration of Fumarate
This enzyme is highly stereospecific; it catalyzes hydration of
the trans double bond of fumarate but not the cis double bond
of maleate (the cis isomer of fumarate).
In the reverse direction (from L-malate to fumarate),
fumarase is equally stereospecific: D-malate is not a substrate.
马来酸
Malate Dehydrogenase—
Completing the Cycle
Binding of NAD causes a conformational change in the 20-residue
segment that connects the D and E βstrands of the β-sheet.
The change is triggered by an interaction between the adenosine
phosphate moiety of NAD and an arginine residue in this loop region.
A Summary of the Cycle
The Fate of the Carbon Atoms of Acetyl-CoA in
the TCA Cycle
The TCA Cycle Provides Intermediates
for Biosynthetic Pathways
• In aerobic organisms, the citric acid cycle is
an amphibolic pathway, one that serves in
both catabolic and anabolic processes.
• Besides its role in the oxidative catabolism of
carbohydrates, fatty acids, and amino acids,
• the cycle provides precursors for many
biosynthetic pathways.
malic enzyme
Why Is the Oxidation of Acetate So Complicated?
Anaplerotic Reactions Pathway
回补途径
• As intermediates of the citric acid
cycle are removed to serve as
biosynthetic precursors, they are
replenished by anaplerotic reactions.
Pyruvate carboxylase exists
in the mitochondria of animal
cells but not in plants.
PEP carboxylase occurs in
yeast, bacteria, and higher
plants, but not in animals.
Malic enzyme苹果酸酶 is
found in the cytosol or
mitochondria of many
animal and plant cells.
• The pyruvate carboxylase reaction
requires the vitamin biotin生物素 ,
which is the prosthetic group of the
enzyme.
• Biotin plays a key role in many
carboxylation reactions. It is a
specialized carrier of one-carbon
groups in their most oxidized form:
CO2.
Regulation of the Citric Acid Cycle
Regulation of the Citric Acid Cycle
• The flow of carbon atoms from
pyruvate into and through the citric
acid cycle is under tight regulation at
four sites:
– The conversion of pyruvate to acetyl-CoA,
the starting material for the cycle (the
pyruvate dehydrogenase complex
reaction).
– The entry of acetyl-CoA into the cycle
(the citrate synthase reaction).
– The isocitrate dehydrogenase reaction.
–α-ketoglutarate dehydrogenase reactions.
Regulation of Pyruvate
Dehydrogenase Complex
• Pyruvate Dehydrogenase
Complex is allosterically
inhibited by ATP and by
acetyl-CoA and NADH.
• The allosteric inhibition of
pyruvate oxidation is
greatly enhanced when
long-chain fatty acids are
available.
• AMP, CoA, and NAD+
allosterically activate the
Pyruvate Dehydrogenase
Complex(PDC).
• Acetyl-CoA specifically blocks
dihydrolipoyl transacetylase, and
NADH acts on dihydrolipoyl
dehydrogenase.
Mg2+ is
needed
The mammalian pyruvate
dehydrogenase is also
regulated by covalent
modifications.
Hans Krebs and the Discovery of
the TCA Cycle
• 1932 Krebs was studying the rates of
oxidation of succinate, fumarate,
acetate, malate, and citrate by kidney
and liver tissue.
• In 1935 in Hungary, Albert SzentGyörgyi was studying the oxidation of
similar organic substrates by pigeon
breast muscle.
• he observed that addition of any of
three four-carbon dicarboxylic acids—
fumarate, succinate, or malate—caused
the consumption of much more oxygen
than was required for the oxidation of
the added substance itself.
• Carl Martius and Franz
Knoop found that citric acid
could be converted to
isocitrate and then to αketoglutarate.
• This finding was significant
because it was already
known that α-ketoglutarate
could be enzymatically
oxidized to succinate.
• In 1937 Krebs found
that citrate could be
formed in muscle
suspensions if
oxaloacetate and
either pyruvate or
acetate were added.
• It is a cycle.
Steric Preferences in NAD-Dependent
Dehydrogenases
Fool’s Gold and the Reductive Citric Acid
Cycle—The First Metabolic Pathway?
• How did life arise on the planet Earth? It was
once supposed that a reducing atmosphere,
together with random synthesis of organic
compounds, gave rise to a prebiotic “soup,”
in which the first living things appeared.
However, certain key compounds, such as
arginine, lysine, and histidine, the straightchain fatty acids, porphyrins, and essential
coenzymes, have not been convincingly
synthesized under simulated prebiotic
conditions.
• This and other problems have led
researchers to consider other models for the
evolution of life.
• One of these alternate models, postulated by
Günter Wächtershäuser, involves an archaic
version of the TCA cycle running in the reverse
(reductive) direction.
• Reversal of the TCA cycle results in
assimilation of CO2 and fixation of carbon as
shown.
• For each turn of the reversed cycle, two
carbons are fixed in the formation of isocitrate
and two more are fixed in the reductive
transformation of acetyl-CoA to oxaloacetate.
Thus, for every succinate that enters the
reversed cycle, two succinates are returned,
making the cycle highly autocatalytic.
• Because TCA cycle intermediates are involved
in many biosynthetic pathways, a reversed TCA
cycle would be a bountifuland broad source of
metabolic substrates.
A reversed, reductive TCA cycle would
require energy input to drive it. What
might have been the thermodynamic
driving force for such a cycle?
Wächtershäuser hypothesizes that the
anaerobic reaction of FeS and H2S to
form insoluble FeS2 (pyrite黄铁矿,also
known as fool’s gold) in the prebiotic
milieu could have been the driving
reaction:
Substrate Channeling through Multienzyme
Complexes May Occur in the Citric Acid Cycle
• Although the enzymes of the citric acid
cycle are usually described as soluble
components of the mitochondrial matrix
(except for succinate dehydrogenase,
which is membrane-bound), growing
evidence suggests that within the
mitochondrion these enzymes exist as
multienzyme complexes.