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THE CITRIC ACID CYCLE

OUTLINE:
➢ INTRODUCTION
➢ THE ENTRY OF PYRUVATE INTO MITOCHONDRIA
➢ THE PYRUVATE DEHYDROGENASE COMPLEX AND PRODUCTION OF
ACTIVATED ACETATE
➢ FATE OF ACTIVATED ACETATE
➢ REACTIONS OF THE CITRIC ACID CYCLE AND ITS STAGES
➢ ESSENCE OF TCA SEEMING COMPLEXITY EXPLAINED
➢ REGLATIONS OF THE CITRIC ACID CYCLE
➢ IS T
...
A AND ONCOGENESIS RELATED ?
➢ TOTAL EXPENDITURE OF ACETATE CARBON IN TCA AND ITS EXCEPTION:
THE GLYOXYLATE SHUNT OF TCA

Eukaryotic cells and bacteria which are “aerobes” oxidize their organic fuel to CO2 &
H2O
...
This
aerobic phase of catabolism is called “Respiration
Cellular respiration occurs in three phases:
1
...
The acetyl groups are then fed into the TCA cycle; which enzymatically oxidize
them to CO2, and the energy produced at this stage is conserved in the
reduced(Hydrogen containing) electron carriers: NADH &FADH2
3
...
in the course of the electron
transfer to O2, large amount of energy is released and are stored as ATP
...
A specific carrier present in mitochondrial membrane
transports pyruvate across mitochondrial membrane via the pyruvate-H+ symport
mechanism
...

Pyruvate product of glycolysisis oxidized to acetylCoA and CO2 by the “Pyruvate
Dehydrogenase (PDH) Complex”; a cluster of multiple copies of 3 enzymes, located in
the mitochondria of eukaryotes and cytosol of prokaryotes
...


The oxidative decarboxylation activity of PDH complex requires the sequential action
of its three constituent enzymes and five coenzymes
...


Pyruvate dehydrogenase, designated E1

2
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Dihyrolipoyl dehydrogenase, designated E3
The functional coenzymes of the PDH complex are:
1
...
Flavin adenine dinucleotide (FAD)
3
...
Nicotinamide adenine dinucleotide (NAD)
5
...

Be careful to notice that we can infer that certain vitamins are crucial constituents or
precursors of these coenzymes: Thiamine (in TPP), Riboflavin (in FAD), Niacin (in
NAD), Pantothenate (in CoA-SH)
Also, it is worthy to note that CoA-SH has a reactive thiol (-SH) group that is critical to
its role as an “acyl carrier” in a number of metabolic reactions, when acylgroup is
covalently attached to the thiol group, a thioester with a very high acyl group transfer
potential is formed
...

NAD & FAD are electron carriers, while TPP performs the decarboxylase activity of the
PDH complex
...
E2 has 3 distinct domain:
1
...
The central E1 and E3 binding domain
3
...

These domains are separated by oligopeptide linkers
...


The C1 (carboxylic group) of pyruvate is cleaved off and released as CO2 and the
C2 (keto group) becomes attached to TPP to form an “hydroxyethyl” group
...


2
...
Thus
the acetyl moiety becomes esterified to one of the lipoyl –SH groups
...
The lipoyl-acetyl ester is transesterified to CoA-SH to form acetylC0A
4
...

Here, the reduced lipoyl moiety is oxidized into a cyclic di-sulphide by reacting
with FAD, reducing the FAD to FADH2 in the presence of E3 enzyme
...
The reduced FADH2 is oxidized to FAD by reaction with NAD+, thus reducing
NAD+ to NADH
...
Thus the
intermediate of this multiple sequence never leave the PDH complex surface and
concentration of the substrate of E2 is high
...
Thiamine deficiency is known as Beriberi, common in
alcoholics and population feeding on polished rice
...


FATE OF ACETYLCoASH
AcetylCoA once formed has both anabolic and catabolic fate
...


REACTIONS OF THE CITRIC ACID CYCLE
Now having formed the acetylCoA from pyruvate, the acetylCoA undergoes oxidation
and chemical transformation, carried out by “the citric acid cycle (TCA)”
...

Four of the eight steps of TCA are oxidations in which energy liberated is conserved in
form of reduced coenzyme NADH & FADH2
...

Mitochondrion is the intracellular organelle which contains all the enzymes of TCA
cycle, also last stage of respiration includes electron transfer and ATP synthesis
...
In this reaction, the –CH3 end of acetylCoA is linked to the C2 carbonyl
group of the oxaloacetate
...
Hydrolysis of
the thiol (-SH) of CoA-SH is highly energetic, and uses the free energy to drive this
step1 reaction to spontaneity
...

Aconitase is a mutase enzyme that function through the reversible addition of water
to the doube bond of the Cis-aconitase
...

Naturally it is non-toxic, but in the body, it is metabolized to fluoroacetyl-CoA, which
gives rise to fluorocitrate after condensing with oxaloacetate
...
This is a form of metabolic lethal synthesis
...
This reaction involves a carbonyl intermediate
(oxalosuccinate)
...
in
this reaction, NAD+ is the electron acceptor and CoA-SH functions as the carrier of the
succinyl group
...

STEP 5:
Conversion of SuccinylCoA to Succinate: succinylCoA has a thioester
bond, and the energy released by breaking this bond is used to power the synthesis of
phosphoanhydrides(ATP or GTP) at substrate level, known as substrate level
phosphorylation, and succinate is formed
...


STEP 6:
Oxidation of Succinate to Fumarate: the succinate formed from
succinylCoA is oxidized to fumarate by the flavoprotein enzyme “Succinate
dehydrogenase” which contains a molecule of covalently bound FAD
...


The enzyme “fumarase” is highly stereospecific, i
...

STEP 8 :
oxidation of Malate to Oxaloacetate:
NAD-linked “L-malate
dehydrogenase” enzyme catalyzes the oxidation of L-malate to oxaloacetate
...
Energy released in this oxidation is thoroughly conserved in the
reduction of NAD+, FAD and also in the production/synthesis of ATP or GTP
...


REASON FOR THE CITRIC ACID CYCLE COMPLEXITY
The 8 cyclic steps of TCA seem cumbersome and biologically uneconomical, this is
necessarily the situation because the role of TCA is not confined to the oxidation of
acetate
...
4 & 5-carbon end products of
many catabolic processes fits into the TCA cycle as fuels, e
...

Also, intermediates may also under some conditions be siphoned off the TCA cycle, to
be used as precursors in other biosynthetic pathways for example:
1
...
g αketoglutarate and oxaloacetate are transaminated to glutamate and aspartate
respectively- both which are also useful for nucleotide biosynthesis
...
The use of TCA intermediates for gluconeogenesis
3
...

Thus, TCA cycle is an “Amphibolic Pathway” i
...


We also need learn that as the intermediates of TCA are removedfor other
biosynthetic activities, they loss is often comensated and replenished by “Anaplerotic
reactions”
...

In mammals, the most important anaplerotic reaction is the carboxylation of pyruvate
to form oxaloacetate b y the enzyme “Pyruvate carboxylate”
...
This reaction requires energy which is often supplied by ATP
...
e until there is enough acetylCoA for
other cycles activity, pyruvate will not suspend acetylCoA formation for carboxylation
into oxaloacetate
...

All known anaplerotic reactions are summarized as shown below:

It is also worthy to note that activity of the pyruvate carboxylase enzyme requires the
vitamin “Biotin”- functioning as a prosthetic group of the enzyme
...
The carboxylation of pyruvate which occurs in a two step
reaction exemplifies the activity of biotin:
a
...
Then the carboxyl group is transferred to pyruvate to form oxaloacetate
Biotin is an essential vitamin required in diet which could also be supplemented by
enterobacteria synthesis
...


REGULATION OF THE CITRIC ACID CYCLE
The flow of electron from pyruvate into and through the TCA is under tight regulation
at two crucial junctions:
i
...


Entry of acetylCoA into the TCA cycle (citrate synthase reaction)

The cycle is also regulated at isoitrate dehydrohenase and α-ketoglutarate
dehydrogenase reactions
To explain in comprehensive terms:
The PDH complex of mammals is strongly inhibited by aceylCoA, NADH, and ATP- the
products of the reaction catalyzed by the complex
...


On the other hand, accumulation of CoA-SH, NAD+ and AMP “activates” PDH complex
reaction
...
PDH complex is inhibited as “Protein kinase” covalently phosphorylates the
E1 of PDH complex, not forgetting the fact that protein kinase is activated by ATP
prescence: in summary, ATP accumulation activates protein kinase and activated
protein kinase inhibits the E1 of PDH complex
...


Also, metabolites and intermediate flow in the TCA cycle is regulated by factors as :
a
...
Inhibition by accumulating products
c
...
e acetylCoA &
oxaloacetate in step1 varies with the step condition and limits the rate of citrate
formation
...
This mechanism is also slightly
seen in malate dehydrogenase activity
...

Lastly, citrate is an important allosteric inhibitor of phosphofructokinase-1 in the
glycolytic pathway
...
C
...
For example,
it is found that mutations in genes coding for the “Fumarase” enzyme is closely
associated with tumors of the smooth muscle and the kidney, also mutations in genes
coding for “Succinate dehydrogenase” enzyme is closely associated with tumors of the
adrenal gland
...
Thus studies suggest
that some TCA enzymes genes (as exemplified in fumarase and succinate
dehydrogenase) also functions as tumor suppressor genes
...
In a normal
cell, α-ketoglutarate & Fe3+ are important cofactor for the activity of a family of

“Histone demethylase” which help place tight regulation on the expression of genes
...


TOTAL EXPENDITURE OF ACETATE CARBON IN T
...
A AND ITS EXCEPTION:
THE GLYOXYLATE SHUNT OF T
...
A
The carbon of acetate siphoned into the citric acid are oxidized and lost as CO and are
therefore not available for gluconeogenesis
...

Some organisms (other than vertebrates) have a pathway that help salvage acetate into
gluconeogenesis: this pathway is known as the “Glyoxylate cycle”
...


Step1: Acetate condenses with oxaloacetate to form citrate; just as in TCA cycle,
and the citrate to isocitrate
...
Step2: Then the isocitrate is (not oxidized by isocitrate dehydrogenase but)
cleaved by the enzyme “Isocitrate lyase” to form Succinate and Glyoxylate
...
Step3: The glyoxylate is then condensed with another molecule of acetylCoA to
yield Malate by the enzyme “Malate synthase”
The malate is subsequently oxidized to oxaloacetate to start another cycle or can be
converted to phosphoenolpyruvate by the enzyme “phosphoenolpyruvatecarboxykinase”; and thus to glucose by gluconeogenesis
...
Also in plants, the glyoxylate cycle helps developing seed to
make glucose prior to the ability to make glucose by photosynthesis

In conclusion, it is imperative that we learn the co-ordinate regulation between the
glyoxylate cycle and the citric acid cycle
...
Fatty acid catabolism into acetylCoA (in the glyoxysome)
b
...
The citric acid cycle (in the mitochondria)
d
...
Isocitrate dehydrogenase is regulated by covalent modification:
through a specific Protein kinase which phosphorylates and inactivates the
dehydrogenase and shunts isocitrate (the substrate) into the glyoxylate cycle
...


Other intermediates of glycolysis and TCA cycle that activate Isocitrate dehydrogenase
are allosteric inhibitors of isocitrate lyase
...
All pieced together in a simplified easily digestible
form for all and sundry
...


Lehningher principles of biochemistry, 6th edition, David Nelson & Micheal Cox

2
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Mallikarjuna Rao
3
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Murray, Granner, Mayer,
Rodwell
4
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MN Chatterjea, Rana shinde
5
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David Metzler
6
...
Donald & Judith Voet,
Charlotte Pratt

THANK YOU FOR YOUR TIME & ATTENTION…
SEND YOUR QUESTIONS, OBSERVATIONS AND ADVICE TO US AT:
successbiochemacademy@gmail
...
com
+2348096136004



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