Transcript Introduction to Carbohydrates

UNIT II:
Intermediary Metabolism
Tricarboxylic Acid Cycle
Figure 9.1. The tricarboxylic acid cycle shown as a part of the central
pathways of energy metabolism.
Overview
• 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 CHO’s, aa’s
& 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
aa’s, & it provides building blocks for synthesis of some aa’s &
heme.
• Intermediates of TCA cycle can also be synthesized by catabolism
of some aa’s.
• This cycle should not be viewed as a closed circle, but instead as
a traffic circle with cpds entering & leaving as required.
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 can’t
be formed from acetyl CoA via gluconeogenesis
Figure 9.2
Oxidative decarboxylation of
pyruvate.
•
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 enz’s: 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
Figure 9.3
Mechanism of action of the pyruvate dehydrogenase complex. TPP =
thiamine pyrophosphate; L = lipoic acid.
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 cAMPindependent protein kinase phosphorylates and,
thereby, inhibits E1, whereas phosphoprotein
phosphatase activates E1
• 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 conc’s 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
Figure 9.4
Regulation of pyruvate
dehydrogenase complex.
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
- A 3rd form causes episodic ataxia (an inability to
coordinate voluntary muscles) that is induced by a CHOrich 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
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 enz’s that require lipoic acid as a cofactor,
including pyruvate dehydrogenase, α-ketoglutarate
dehydrogenase, and branched-chain α-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
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)
Figure 9.5
Formation of a-ketoglutarate from acetyl
CoA and oxaloacetate.
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
E. Oxidative decarboxylation of α-KG
- Conversion of α-KG to succinyl CoA is catalyzed
by the α-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
- The equil of reaction is far in direction of succinyl
CoA, a high-energy thioester similar to acetyl
CoA.
- α-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: α-KG is also produced by oxidative
deamination or transamination the aa glu
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 aa’s
(e.g., ile, val)
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 aa’s,
phe & tyr.
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.
Figure 9.7. Formation of oxaloacetate from malate.
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 e’s are transferred during one turn
of the cycle: 3 pairs of e’s 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:
Figure 9.8. Number of ATP molecules produced from the
oxidation of one molecule of acetyl CoA (using both
substrate-level and oxidative phosphorylation).
IV. 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, & α-KG dehydrogenase complex
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
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
[ADP][Pi]/[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
Figure 9.9. A. production of reduced coenzymes,
ATP, and CO2 in TCA cycle. B. inhibitors and
activators of the cycle.
Summary
• 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
• 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 α-KG, producing CO2 and NADH. The
enz is inhibited by ATP & NADH, & is activated by ADP &
Ca2+.
• α-KG is oxidatively decarboxylated to succinyl CoA by αKG dehydrogenase complex, producing CO2 & NADH.
The enz is very similar to pyruvate dehydrogenase and
uses the same coenzymes.
• α-KG dehydrogenase complex is activated by Ca2+, and
inhibited by ATP, GTP, NADH, & succinyl CoA.
• 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
Figure 9.10
Key concept map for
tricarboxylic acid cycle.