Title: Tricarboxylic Acid Cycle
1Tricarboxylic Acid Cycle
- UNIT II
- Intermediary Metabolism
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3Figure 9.1. The tricarboxylic acid cycle shown as
a part of the central pathways of energy
metabolism.
4Overview
- TCA cycle (a.k.a Krebs cycle or citric acid
cycle) plays several roles in metabolism - It is the final pathway where oxidative
metabolism of CHOs, aas fatty acids converge,
their C skeletons being converted to CO2 H2O.
This oxidation provides energy for production of
majority of ATP. - The cycle occurs in mitoch is in close
proximity to the reactions of e-transport, which
oxidize the reduced coenzymes produced by the
cycle - The TCA cycle is thus an aerobic pathway, because
O2 is required as final e-acceptor - The cycle also participates in a number of
synthetic reactions. E.g., it functions in
formation of gluc from C skeletons of some aas,
it provides building blocks for synthesis of
some aas heme. - Intermediates of TCA cycle can also be
synthesized by catabolism of some aas. - This cycle should not be viewed as a closed
circle, but instead as a traffic circle with cpds
entering leaving as required.
5- II. Reactions of the TCA cycle
- In TCA cycle, OAA is first condensed with an
acetyl group from acetyl CoA, then is
regenerated as the cycle is completed. Thus,
entry of one acetyl CoA into one round of the
cycle does not lead to the net production or
consumption of intermediates - A. Oxidative decarboxylation of pyruvate
- Pyruvate, must be transported to mitoch before it
can enter TCA cycle. This is accomplished by a
specific pyruvate transporter - Once in matrix, pyruvate is converted to acetyl
CoA by pyruvate dehydrogenase complex - Note irreversibility of reaction precludes
formation of pyruvate from acetyl CoA, and
explains why gluc cant be formed from acetyl CoA
via gluconeogenesis
6Figure 9.2 Oxidative decarboxylation of pyruvate.
7- Strictly speaking, pyruvate dehydrogenase complex
is not part of TCA cycle proper, but is a major
source of acetyl CoA, the 2C substrate of the
cycle - 1. Component enzymes
- Pyruvate dehydrogenase complex is a
multimolecular aggregate of 3 enzs pyruvate
dehydrogenase (E1, a.k.a a decarboxylase),
dihydrolipoyl transacetylase (E2),
dihydrolipoyl dehydrogenase (E3). - Each is present in multiple copies, and each
catalyzes a part of the overall reaction. - Their physical association links the reactions in
proper sequence without release of intermediates - In addition to the enzymes participating in
conversion of pyruvate to acetyl CoA, the complex
also contains 2 tightly bound regulatory enzymes,
protein kinase phosphoprotein phosphatase
8Figure 9.3 Mechanism of action of the pyruvate
dehydrogenase complex. TPP thiamine
pyrophosphate L lipoic acid.
9- 2. Coenzymes
- - The pyruvate dehydrogenase complex contains 5
coenzymes that act as carriers or oxidants for
the intermediates of the reactions shown in Fig
9-3. - E1 requires thiamine pyrophosphate, E2 requires
lipoic acid coenzyme A, and E3 requires FAD
NAD - Note deficiencies of thiamine or niacin can
cause serious CNS problems. This is because brain
cells are unable to produce sufficient ATP (via
TCA cycle) for proper function if pyruvate
dehydrogenase is inactive - 3. Regulation of pyruvate dehydrogenase complex
- - The 2 regulatory enzymes that are part of the
complex alternately activate inactivate E1 the
cAMP-independent protein kinase phosphorylates
and, thereby, inhibits E1, whereas phosphoprotein
phosphatase activates E1
10- The kinase is allosterically activated by ATP,
acetyl CoA, and NADH. Therefore, in presence of
these high-energy signals, the pyruvate
dehydrogenase complex is turned off - Acetyl CoA and NADH also allosterically inhibit
the dephosphorylated (active) form of E1. - Protein kinase is allosterically inactivated by
NAD and coenzyme A, low energy signals that thus
turn pyruvate dehydrogenase on - Pyruvate is also a potent inhibitor of protein
kinase. Therefore, if pyruvate concs are
elevated, E1 will be maximally active - Calcium is a strong activator of protein
phosphatase, stimulating E1 activity. - Note this is particularly important in skeletal
muscle, where release of Ca2 during contraction
stimulates the pyruvate dehydrogenase complex,
and thereby energy production
11Figure 9.4 Regulation of pyruvate dehydrogenase
complex.
12- 4. Pyruvate dehydrogenase deficiency
- A deficiency in the pyruvate dehydrogenase
complex is the most common biochemical cause of
congenital lactic acidosis - This enz deficiency results in an inability to
convert pyruvate to acetyl CoA ? pyruvate shunted
to lactic acid via lactate dehydrogenase - This causes particular problems for the brain,
which relies on TCA cycle for most its energy,
is particularly sensitive to acidosis - The most severe form of this deficiency causes
overwhelming lactic acidosis with neonatal death - A 2nd form produces moderate lactic acidosis, but
causes profound psychomotor retardation, with
damage to cerebral cortex, basal ganglia and
brain stem ? death in infancy
13- A 3rd form causes episodic ataxia (an inability
to coordinate voluntary muscles) that is induced
by a CHO-rich meal - The E1 defect is X-linked, but because the
importance of the enz in the brain, it affects
both males females. Therefore, the defect is
classified as X-linked dominant - There is no proven treatment for pyruvate
dehydrogenase complex deficiency, although a
ketogenic diet (one low in CHO enriched in
fats) has been shown in some cases to be of
benefit. Such a diet provides an alternate fuel
supply in form of ketone bodies that can be used
by most tissues including the brain, but not the
liver
14- 5. Mechanism of arsenic poisoning
- As previously described, arsenic can interfere
with glycolysis at glyceraldehyde-3P step,
thereby decreasing ATP production - Arsenic poisoning is, however, due primarily to
inhibition of enzs that require lipoic acid as a
cofactor, including pyruvate dehydrogenase,
a-ketoglutarate dehydrogenase, and branched-chain
a-keto acid dehydrogenase - Arsenite (the trivalent form of arsenic) forms a
stable complex with thiol (-SH) groups of lipoic
acid, making that cpd unavailable to serve as a
coenzyme - When it binds to lipoic acid in pyruvate
dehydrogenase complex, pyruvate (and consequently
lactate) accumulate. Like pyruvate dehydrogenase
complex deficiency, this particularly affects
brain causing neurologic disturbances and death
15- B. Synthesis of citrate from acetyl CoA and OAA
- Condensation of acetyl CoA OAA to form citrate
is catalyzed by citrate synthase. This aldol
condensation has an equil far in direction of
citrate synthesis - Citrate synthase is allosterically activated by
Ca2 ADP, inhibited by ATP, NADH, succinyl
CoA, fatty acyl CoA derivatives - However, primary mode of regulation is also
determined by availability of its substrates,
acetyl CoA OAA - Note
- - Citrate, in addition to being an intermediate
in TCA cycle, provides a source of acetyl CoA for
cytosolic synthesis of fatty acids - - Citrate also inhibits PFK, the rate-setting enz
of glycolysis, activates acetyl CoA carboxylase
(the rate-limiting enz of fatty acid synthesis)
16Figure 9.5 Formation of a-ketoglutarate from
acetyl CoA and oxaloacetate.
17- C. Isomerization of citrate
- Citrate is isomerized to isocitrate by aconitase
- Note
- Aconitase is inhibited by fluoroacetate, a cpd
that is used as a rat poison. Fluoroacetate is
converted to fluoroacetyl CoA, which condenses
with OAA to form fluorocitrate, a potent
inhibitor of aconitase, resulting in citrate
accumulation - D. Oxidation and decarboxylation of isocitrate
- - Isocitrate dehydrogenase catalyzes the
irreversible oxidative decarboxylation of
isocitrate, yielding the 1st of three NADH
molecules produced by the cycle, 1st release of
CO2 - - This is one of rate-limiting steps of TCA
cycle. The enz is allosterically activated by ADP
(low energy signal) and Ca2, and is inhibited by
ATP and NADH, whose levels are elevated when cell
has abundant energy stores
18- E. Oxidative decarboxylation of a-KG
- Conversion of a-KG to succinyl CoA is catalyzed
by the a-KG dehydrogenase complex, which consists
of 3 enzymatic activities - The mechanism of this oxidative decarboxylation
is very similar to that used for conversion of
pyruvate to acetyl CoA - The reaction releases the 2nd CO2 and produces
the 2nd NADH of the cycle. - The coenzymes required are thiamine
pyrophosphate, lipoic acid, FAD, NAD, and
coenzyme A. each functions as part of the
catalytic mechanism in a way analogous to that
described for pyruvate dehydrogenase complex
19- The equil of reaction is far in direction of
succinyl CoA, a high-energy thioester similar to
acetyl CoA. - a-KG dehydrogenase complex is inhibited by ATP,
GTP, NADH, and succinyl CoA, and activated by
Ca2 - However, it is not regulated by phospho/dephospho
reactions as described for pyruvate dehydrogenase
complex - Note a-KG is also produced by oxidative
deamination or transamination the aa glu
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21- F. Cleavage of succinyl CoA
- Succinate thiokinase (a.k.a succinyl CoA
synthetase) cleaves the high-energy thioester
bond of succinyl CoA - This reaction is coupled to phospho of GDP to
GTP. GTP and ATP are energetically
interconvertible by the nucleoside diphosphate
kinase reaction - GTP ADP ? GDP ATP
- - Generation of GTP by succinate thiokinase is
another e.g. of substrate-level phospho. - Note succinyl CoA is also produced from
propionyl CoA derived from metabolism of fatty
acids with an odd of C atoms, from metabolism
of several aas (e.g., ile, val)
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23- G. Oxidation of succinate
- Succinate is oxidized to fumarate by succinate
dehydrogenase, producing reduced coenzyme FADH2
(FAD rather than NAD, is the e-acceptor because
the reducing power of succinate is not sufficient
to reduce NAD) - Succinate dehydrogenase is inhibited by OAA
- H. Hydration of fumarate
- Fumarate is hydrated to malate in a freely
reversible reaction catalyzed by fumarase (
fumarate hydratase) - Note fumarate is also produced by urea cycle, in
purine synthesis, and during catabolism of the
aas, phe tyr. -
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25- I. Oxidation of malate
- Malate is oxidized to OAA by malate
dehydrogenase. This reaction produces 3rd and
final NADH of the cycle. - Note OAA is also produced by transamination of
the aa, Asp.
26Figure 9.7. Formation of oxaloacetate from malate.
27- III. Energy produced by the TCA cycle
- Two C atoms enter the cycle as acetyl CoA leave
as CO2. - The cycle does not involve net consumption or
production of OAA or any other intermediate - Four pairs of es are transferred during one turn
of the cycle 3 pairs of es reducing NAD to
NADH one reducing FAD to FADH2. - Oxidation of one NADH by ETC leads to formation
of 3 ATP, whereas oxidation of FADH2 yields 2
ATP - Total yield of ATP from oxidation of one acetyl
CoA is
28Figure 9.8. Number of ATP molecules produced from
the oxidation of one molecule of acetyl CoA
(using both substrate-level and oxidative
phosphorylation).
29IV. Regulation of the TCA cycle
- A. Regulation by activation and inhibition of
enzyme activity - In contrast to glycolysis which is regulated
primarily by PFK, the TCA cycle is controlled by
regulation of several enz activities. The most
important of these are citrate synthase,
isocitrate dehydrogenase, a-KG dehydrogenase
complex
30- B. Regulation by availability of ADP
- 1. Effect of elevated ADP
- - Energy consumption as a result of muscular
contraction, biosynthetic reactions or other
processes result in hydrolysis of ATP to ADP
Pi. - Resulting increase in conc of ADP accelerates
rate of reactions that use ADP to generate ATP,
most important of which is oxphos - Production of ATP increases until it matches rate
of ATP consumption by energy-requiring reactions
31- 2. Effect of low ADP
- If ADP (or Pi) is present in limiting conc,
formation of ATP by oxphos decreases as a result
of the lack of phosphate acceptor (ADP) or
inorganic phosphate (Pi) - The rate of oxphos is proportional to
ADPPi/ATP this is known as respiratory
control of energy production - Oxidation of NADH FADH2 by ETC also stops if
ADP is limiting. This is because the processes of
oxidation phospho are tightly coupled occur
simultaneously - As NADH FADH2 accumulate, their oxidized forms
become depleted causing oxidation of acetyl CoA
by the TCA cycle to be inhibited as a result of a
lack of oxidized coenzymes
32Figure 9.9. A. production of reduced coenzymes,
ATP, and CO2 in TCA cycle. B. inhibitors and
activators of the cycle.
33Summary
- Pyruvate is oxidatively decarboxylated by
pyruvate dehydrogenase complex producing acetyl
CoA, which is the major fuel for TCA cycle - This enz complex requires five coenzymes
thiamine pyrophosphate, lipoic acid, FAD, NAD,
and coenzyme-A (which contains the vitamin
pantothenic acid) - The reaction is activated by NAD, coenzyme-A,
and pyruvate, and inhibited by ATP, acetyl CoA,
NADH, and Ca2. - Pyruvate dehydrogenase deficiency is the most
common biochemical cause of congenital lactic
acidosis. Because the deficiency deprives the
brain of acetyl CoA, the CNS is particularly
affected, with profound psychomotor retardation
death occurring in most patients. The deficiency
is X-linked dominant - Arsenic poisoning causes inactivation of pyruvate
dehydrogenase by binding to lipoic acid
34- Citrate is synthesized fro OAA and acetyl CoA by
citrate synthase. This enz is allosterically
activated by ADP, and inhibited by ATP, NADH,
succinyl CoA, and fatty acyl CoA derivatives - Citrate is isomerized to isocitrate by aconitase.
Isocitrate is oxidized decarboxylated by
isocitrate dehydrogenase to a-KG, producing CO2
and NADH. The enz is inhibited by ATP NADH,
is activated by ADP Ca2. - a-KG is oxidatively decarboxylated to succinyl
CoA by a-KG dehydrogenase complex, producing CO2
NADH. The enz is very similar to pyruvate
dehydrogenase and uses the same coenzymes. - a-KG dehydrogenase complex is activated by Ca2,
and inhibited by ATP, GTP, NADH, succinyl CoA.
35- Succinyl CoA is cleaved by succinate thiokinase
( succinyl CoA synthetase), producing succinate
and GTP. This is an e.g. of substrate-level
phospho. - Succinate is oxidized to fumarate by succinate
dehydrogenase, producing FADH2. this enz is
inhibited by OAA. - Fumarate is hydrated to malate by fumarase (
fumarate hydratase), and malate is oxidized to
OAA by malate dehydrogenase, producing NADH. - Three NADH, one FADH2, and one GTP (whose
terminal phosphate can be transferred to ADP by
nucleoside diphosphate kinase, ? ATP) are
produced by one round of TCA cycle. - Oxidation of NADHs and FADH2 by ETC yields 11
ATPs, making 12 the total of ATPs produced
36Figure 9.10 Key concept map for tricarboxylic
acid cycle.