Biochemistry-Ii: Dr. Jhinuk Chatterjee Department of Biotechnology
Biochemistry-Ii: Dr. Jhinuk Chatterjee Department of Biotechnology
Biochemistry-Ii: Dr. Jhinuk Chatterjee Department of Biotechnology
TCA cycle
https://doi.org/10.1042/EBC20190041
BIOCHEMISTRY-II
• Pyruvate, the product of glycolysis, is converted to acetyl-CoA, the starting
material for the citric acid cycle, by the pyruvate dehydrogenase complex.
• The PDH complex is in the mitochondria of eukaryotic cells and in the cytosol
of prokaryotes
• Composed of multiple copies of three enzymes: pyruvate dehydrogenase, E1
(with its bound cofactor TPP); dihydrolipoyl transacetylase, E2 (with its
covalently bound lipoyl group); and dihydrolipoyl dehydrogenase, E3 (with its
cofactors FAD and NAD).
• E1 catalyzes first the decarboxylation of pyruvate, producing hydroxyethyl-TPP,
and then the oxidation of the hydroxyethyl group to an acetyl group. The
electrons from this oxidation reduce the disulfide of lipoate bound to E2, and
the acetyl group is transferred into thioester linkage with one SH group of
reduced lipoate.
• E2 catalyzes the transfer of the acetyl group to coenzyme A, forming acetyl-
CoA.
• E3 catalyzes the regeneration of the disulfide (oxidized) form of lipoate;
electrons pass first to FAD, then to NAD.
• The long lipoyllysine arm swings from the active site of E1 to E2 to E3,
tethering the intermediates to the enzyme complex to allow substrate
channeling. Principles of Biochemistry-Lehninger
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NATURE COMMUNICATIONS
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Reaction mechanism and cryo-EM reconstruction of E. coli PDHc.
a Scheme of the reactions catalyzed by the individual components of
PDHc.
b Overall reaction catalyzed by PDHc.
c High-resolution cryo-EM reconstruction of the inner E2p core in
alignment with the low-resolution reconstruction of the outer shell of
the complex. Catalytic cores of E2p are colored magenta, lipoyl
domains cyan, and the outer shell components (E1p/E3) yellow.
d Ball and-stick representation of the dihydrolipoyllysine moiety.
e Scheme of the domain organization of E2p.
F A simplified scheme of the covalent swinging-arm substrate
channeling mechanism in the catalytic cycle of PDHc. Only one LD is
shown and only one copy of each enzyme is shown, but the LDs can
reach multiple copies of each enzyme in the multienzyme complex.
PSBD can bind to either E1p or E3. E1p—pyruvate dehydrogenase,
E2p—dihydrolipoyl transacetylase, E3—dihydrolipoyl dehydrogenase,
LD—lipoyl domain, PSBD—peripheral subunit-binding domain.
NATURE COMMUNICATIONS
BIOCHEMISTRY-II
• The PDH complex of mammals is strongly inhibited by ATP
and by acetyl-CoA and NADH, the products of the reaction
catalyzed by the complex.
• The allosteric inhibition of pyruvate oxidation is greatly
enhanced when long-chain fatty acids are available.
• AMP, CoA, and NAD, all of which accumulate when too little
acetate flows into the citric acid cycle, allosterically activate
the PDH complex.
• Thus, this enzyme activity is turned off when ample fuel is
available in the form of fatty acids and acetyl-CoA and when
the cell’s [ATP]/[ADP] and [NADH]/[NAD] ratios are high,
and it is turned on again when energy demands are high
and the cell requires greater flux of acetyl-CoA into the citric
acid cycle.
Principles of Biochemistry-Lehninger
BIOCHEMISTRY-II
• In mammals, these allosteric regulatory mechanisms are
complemented by a second level of regulation: covalent protein
modification.
• The PDH complex is inhibited by reversible phosphorylation of a
specific Ser residue on one of the two subunits of E1.
• In addition to the enzymes E1, E2, and E3, the mammalian PDH
complex contains two regulatory proteins whose sole purpose is
to regulate the activity of the complex.
• A specific protein kinase phosphorylates and thereby inactivates
E1, and a specific phosphoprotein phosphatase removes the
phosphoryl group by hydrolysis and thereby activates E1.
• The kinase is allosterically activated by ATP: when [ATP] is high
(reflecting a sufficient supply of energy), the PDH complex is
inactivated by phosphorylation of E1.
• When [ATP] declines, kinase activity decreases and phosphatase
action removes the phosphoryl groups from E1, activating the
complex.
Principles of Biochemistry-Lehninger
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https://doi.org/10.1042/EBC20190041
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https://doi.org/10.1042/EBC20190041
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Important steps of TCA
Step 1 – The combination of 2-carbon acetyl CoA and 4-carbon
oxaloacetate forms 6-carbon citrate. Here, citrate can either
move into the cytosol to initiate fatty acid synthesis or is destined
to carry through the oxidation steps involved in the TCA cycle.
This reaction is irreversible and highly regulated with a highly
negative G.
Step 3 – This step is highly regulated and allows the commitment
of citric acid to the TCA cycle instead of fatty acid synthesis. This
step is activated and stimulated by NAD+ and ADP but inhibited
by NADH and ATP. Here, the first NADH molecules are formed
along with CO2.
Step 5 – This reaction is coupled to the phosphorylation of GDP
to GTP, making it the only reaction in the TCA cycle to generate a
high energy phosphate. Following the formation of GTP, this can
either be converted to ATP or used in protein synthesis.
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Anaplerosis and cataplerosis
The NADH that is generated from the TCA cycle is used by the cell
in the form of ATP. Even though the TCA cycle forms over 70% of
the ATP that is used by a cell, it also provides the intermediate
building blocks for other processes. As a result of this, the
subsequent intermediates within the TCA cycle can be used up
excessively and these must be replenished through the process of
anaplerosis.
https://doi.org/10.1042/EBC20190041
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https://doi.org/10.1042/EBC20190041
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Principles of Biochemistry-Lehninger
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• The citric acid cycle is amphibolic, serving in both catabolism and
anabolism; cycle intermediates can be drawn off and used as the
starting material for a variety of biosynthetic products.
• Besides its role in the oxidative catabolism of carbohydrates, fatty
acids, and amino acids, the cycle provides precursors for many
biosynthetic pathways, through reactions that served the same
purpose in anaerobic ancestors.
• alpha–ketoglutarate and oxaloacetate can, serve as precursors of the
amino acids aspartate and glutamate by simple transamination.
• Through aspartate and glutamate, the carbons of oxaloacetate and –
ketoglutarate are then used to build other amino acids, as well as
purine and pyrimidine nucleotides.
• Oxaloacetate is converted to glucose in 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).
Principles of Biochemistry-Lehninger
Dr. Jhinuk Chatterjee
Department of Biotechnology
jhinukchatterjee@pes.edu
+91 80 2672 1983 Extn 345