CITRIC ACID CYCLE
SHARI BABU
DEPT. OF PHYSIOLOGICAL SCIENCES
SCHOOL OF MEDICINE, UNZA
shari.babu@unza.zm
Biomedical Importance
•
The citric acid cycle (Krebs cycle, tricarboxylic acid cycle) is the second stage of cellular
respiration.
•
It is a series of reactions in mitochondria that oxidize acetyl residues (as acetyl-CoA).
•
The acetyl groups are fed into the citric acid cycle, which enzymatically oxidizes them
to CO2. The energy released by oxidation is conserved in the reduced electron carriers
NADH and FADH2.
•
It is the final common pathway for the aerobic oxidation of carbohydrate, lipid, and
protein because glucose, fatty acids, and most amino acids are metabolized to acetylCoA or intermediates of the cycle.
•
It also produces important precurors that form building blocks of many other
molecules.
Cellular Aerobic Respiration
Catabolism of carbohydrates, lipids and amino acids generate
ACETYL-CoA
FATTY
ACID
PYRUVATE
KETONE
BODIES
KETOGENIC
AMINO
ACIDS
ACETYL-CoA
Transport and Production of Acetyl-CoA
•
Pyruvate generated in the cytoplasm by glycolysis must be transported
across the inner mitochondrial membrane via a pyruvate/H+ symport.
•
This transport uses some of the energy stored in the mitochondrial inner
membrane electrical potential gradient.
•
Pyruvate derived from glucose and other sugars by glycolysis, is oxidized to
acetyl-CoA and CO2 by the pyruvate dehydrogenase (PDH) complex,
located in the mitochondria of eukaryotic cells and in the cytosol of
prokaryotes.
The Pyruvate Dehydrogenase Complex Consists of Three
Distinct Enzymes
•
The PDH complex contains three enzymes—pyruvate dehydrogenase (E1),
dihydrolipoamide acetyltransferase (E2), and dihydrolipoamide
dehydrogenase (E3).
The Pyruvate Dehydrogenase Complex Requires Five
Coenzymes
•
They are thiamine pyrophosphate (TPP), flavin adenine dinucleotide
(FAD), coenzyme A, nicotinamide adenine dinucleotide (NAD), and
lipoate.
•
The active site of E1 has bound TPP, E2 is the point of connection for the
prosthetic group lipoate, and E3 has bound FAD.
SB1
SB2
SB3
CoASH
ACETYL-CoA
PYRUVATE
TPP
Acetyl-Lipoamide
E1
CO2
Hydroxyethyl
TPP
E2
Lipoamide
Reduced
Lipoamide
Oxidized
E3
FAD
FADH2
NADH + H+
E1 – Pyruvate dehydrogenase
E2 – Dihydrolipoamide acetyltransferase
E3 – Dihydrolipoamide dehydrogenase
NAD+
Slide 7
SB1
In the first step, E1 (pyruvate dehydrogenase) catalyzes oxidative decarboxylation of pyruvate. One carbon atom is removed from pyruvate as CO2.
The resulting 2-carbon molecule, hydroethyl is bound to TPP.
Shari Babu, 19/07/2021
SB2
Next step is catalyzed by E2 (dihydrolipoamide acetyltransferase), which transfers the hydroethyl group from TPP to the oxidized form of lipoamide.
The acetyl group is then transferred to free coenzyme A to form acetyl-CoA and reduced lipoamide.
Shari Babu, 19/07/2021
SB3
Finally, FAD-dependent E3 (dihydrolipoamide dehydrogenase) reoxidizes the lipoyl group E2. The electrons are accepted by FAD to form FADH2,
which is then donated to NAD+ to form NADH
Shari Babu, 19/07/2021
•
The overall reaction catalyzed by the pyruvate dehydrogenase complex is
an oxidative decarboxylation, an irreversible oxidation process in which
the carboxyl group is removed from pyruvate as a molecule of CO2 and the
two remaining carbons become the acetyl group of acetyl-CoA.
•
Deficiencies of thiamine can cause serious central nervous system
problems.
•
This is because brain cells are unable to produce sufficient ATP (via the TCA
cycle) if the PDH complex is inactive.
•
Beriberi, a disease that results from thiamine deficiency, is characterized
by loss of neural function.
•
Wernicke-Korsakoff, an encephalopathy-psychosis syndrome due to
thiamine deficiency, may be seen with alcohol abuse.
•
An elevated level of pyruvate in the blood is often an indicator of defects in
pyruvate oxidation due to one of these causes.
Regulation of acetyl-CoA production by the PDH complex
•
When AMP, CoA, and NAD+ accumulate, they allosterically activate the
pyruvate dehydrogenase complex.
•
The PDH complex is inhibited by reversible phosphorylation of E1.
• A specific protein kinase called PDH kinase, phosphorylates and
inactivates E1.
• PDH kinase is activated by ATP, acetyl-CoA and NADH.
• PDH kinase is inhibited by pyruvate.
•
A specific phosphatase called PDH phosphatase, removes the
phosphoryl group by hydrolysis and thereby activates E1.
• Calcium is a strong activator of PDH phosphatase
Pyruvate dehydrogenase deficiency:
• A deficiency in the E1 component of the PDH complex is the most common
biochemical cause of congenital lactic acidosis.
• This enzyme deficiency results in accumulation of pyruvate, causing it to be
converted to lactic acid.
• This causes problems for the brain, which relies on the TCA cycle for most of its
energy and is particularly sensitive to acidosis.
• Symptoms are variable and include neurodegeneration, muscle spasticity and, in the
neonatal onset form, early death.
• The E1 defect is X-linked it affects both males and females and is classified as Xlinked dominant.
• There is no proven treatment for PDH deficiency: however, dietary restriction of
carbohydrate and supplementation with TPP may reduce symptoms in select
patients.
Reactions of the Citric Acid Cycle
Acetyl-CoA
CoASH
Citrate
Oxaloacetate
NADH + H+
NAD+
Isocitrate
Malate
KERBS
CYCLE
NAD+
NADH + H+
α-Ketoglutarate
Fumarate
NAD+
FADH2
FAD
+ CO2
NADH + H+
Succinate
GTP/ATP
Succinyl
-CoA
GDP/ADP
+ CoASH
+ CO2
The Citric Acid Cycle Has Eight Steps
1.
•
Formation of Citrate: The first reaction of the cycle is the condensation of
acetyl-CoA with oxaloacetate to form citrate, catalyzed by citrate
synthase:
The CoA liberated in this reaction is recycled to participate in the oxidative
decarboxylation of another molecule of pyruvate by the PDH complex.
2.
•
Formation of Isocitrate via cis-Aconitate: Citrate is isomerized to
isocitrate by the enzyme aconitase.
Fluoroacetate is an inhibitor of TCA cycle.
• Fluoroacetate is first converted to fluorocitrate by citrate synthase.
• Fluorocitrate is a potent inhibitor of aconitase.
SB4
Slide 14
SB4
Fluoroacetate is found in a range of plant species. Some flourinated compounds such as in anticancer agents, pesticides, and industrial chemicals can
be metabolized to fluoroacetate.
Shari Babu, 19/07/2021
3.
•
Oxidation of Isocitrate to α-Ketoglutarate and CO2: In the next step,
isocitrate dehydrogenase catalyzes oxidative decarboxylation of isocitrate
to form α-ketoglutarate.
This is one of the rate-limiting steps of the TCA cycle. The enzyme is
allosterically activated by ADP and Ca , and is inhibited by ATP and NADH.
2+
4.
Oxidation of α-Ketoglutarate to Succinyl-CoA and CO2: The next step is
another oxidative decarboxylation, in which α-ketoglutarate is converted
to succinyl-CoA and CO2 by the action of the α-ketoglutarate
dehydrogenase complex.
• α-Ketoglutarate dehydrogenase is nearly identical to PDH in terms of
reactions catalysed and its structural features.
• TPP, lipoid acid, CoASH, FAD and NAD+ are required.
• α-Ketoglutarate dehydrogenase complex is inhibited by its products, NADH
and succinyl CoA, and activated by Ca .
2+
5.
Conversion of Succinyl-CoA to Succinate: Succinate thiokinase (succinyl
CoA synthetase) cleaves the high-energy thioester bond of succinyl CoA.
This reaction is coupled to phosphorylation of guanosine diphosphate
(GDP) to guanosine triphosphate (GTP).
•
GTP formed is used for mitochondrial synthesis of proteins, RNA and DNA.
•
The GTP formed can donate its phosphoryl group to ADP to form ATP, in a
reversible reaction catalyzed by nucleoside diphosphate kinase.
6.
Oxidation of Succinate to Fumarate: The succinate formed from succinylCoA is oxidized to fumarate by the flavoprotein succinate dehydrogenase:
• In eukaryotes, succinate dehydrogenase is tightly bound to the inner
mitochondrial membrane, and is the only enzyme of the citric acid cycle that
is membrane-bound.
• Succinate dehydrogenase is activated by ATP and succinate.
• Malonate, an analog of succinate, is a strong competitive inhibitor of
succinate dehydrogenase and the enzyme is also inhibited by oxaloacetate.
7.
Hydration of Fumarate to Malate: The reversible hydration of fumarate
to L-malate is catalyzed by fumarase.
8.
Oxidation of Malate to Oxaloacetate: In the last reaction of the citric acid
cycle, NAD+-linked L-malate dehydrogenase catalyzes the oxidation of Lmalate to oxaloacetate:
•
Products of one turn of the citric acid cycle. Three NADH, one FADH2, one
GTP (or ATP), and two CO2 are released in oxidative decarboxylation
reactions.
•
The three NADH, and one FADH2 enters into the electron transport chain
and drives ATP synthesis via oxidative phosphorylation.
•
The cycle generates the equivalent of 10 ATPs from one acetyl-CoA
(3 NADH = 7.5 ATPs, 1 FADH2 = 1.5 ATPs, 1 ATP (GTP) directly)
Arsenic Poisoning
• Arsenic poisoning” is primarily due to inhibition of enzymes that require
lipoic acid as a coenzyme, including E2 of the PDH complex and αketoglutarate dehydrogenase.
• Arsenic forms a stable complex with the thiol (–SH) groups of lipoic acid,
making it unavailable to serve as a coenzyme.
• When it binds to lipoic acid in the PDH complex, pyruvate (and consequently
lactate) accumulates.
• Like pyruvate dehydrogenase deficiency, this particularly affects the brain,
causing neurologic disturbances and death.
Citric Acid Cycle Source Of Biosynthetic Intermediates
•
α-ketoglutarate simple transamination is converted to glutamate, and in
nervous tissues converted to the neurotransmitter, γ-aminobutyrate
(GABA).
•
Oxaloacetate is transaminated to aspartate, the precursor of asparagine as
well as purine and pyrimidine bases.
•
Oxaloacetate is converted to phosphoenolpyruvate, key intermediate of
gluconeogenesis.
•
Succinyl-CoA is a central intermediate in the synthesis of the porphyrin ring
of heme groups, which serve as oxygen carriers (in hemoglobin and
myoglobin) and electron carriers (in cytochromes).
•
Citrate exported into cytosol is converted to oxaloacetate and acetyl-CoA,
precursor for fatty acid and cholesterol synthesis.
GLUCOSE
AcetylCoA
Oxaloac
etate
ASPARTATE
FATTY ACID
SYNTHESIS &
CHOLESTEROL
Citrate
Malate
Isocitrate
GLUTAMATE
Fumarat
e
α-KG
GABA
Succinat
e
Succinyl
-CoA
HEME