1 | P a g e

Tricarboxylic Acid Cycle

Overview (Also called KREBS CYCLE , or CITRIC ACID CYCLE )

Occur totally in mitochondria .

TCA cycle is an aerobic pathway , bcz O2 is required as the final

electron accepter.

Supplies intermediates for a number of important synthetic reactions,

and provides building blocks for synthesis of some amino acids and heme.

Reactions of TCA cycle

1. condensation of oxaloacetate and acetyl coA to form

citrate.

-catalyzed by citrate synthase.

-citrate composed of 6 carbons, with 3 carboxyl groups,

and a hydroxyl group on the middle carbon, so it is a

symmetrical molecule.

-in the following steps we'll need to decarboxylise the

middle carbon (removing CO2), but we cannot do so while

there is a hydroxyl group on the same carbon , so that

we have to transfer the hydroxyl group to the adjacent

carbon by forming "isocitrate".

2. isomerization of citrate

-citrate is isomarized to isocitrate by aconitase.

-this enzyme catalyzes the dehydration-rehydration

reaction (removing and adding H2O) to form isocitrate.

-there's an intermediate in this reaction called aconitase

so that we named the enzyme so.

-isocitrate is not symmetrical , so that decarboxylation

can occur now.

2 | P a g e

3. oxidation and decarboxylation of isocitrate

-this reaction occurs by two steps :

a) oxidation of the hydroxyl group to ketone group

since there is a ketone group on the middle carbon,

then CO2 is ready now to be removed.

b)decarboxylation of isocitrate to α-katoglutarate.

-catalyzed by isocitrate dehydrogenase (according to the

type of the reaction and the name of the substrate)

-the enzyme was not named " decarboxylase" bcz it's an

oxidation reduction reaction, it did not decarboxylated

simply .

-irreversible step.

-produces the first NADH and the first CO2 in the cycle.

α-katoglutarate resembles an amino acid called Glutamate , the

difference between them is that there is a ketone group on α-

carbon of α-katoglutarate , replaced by an amino group in

Glutamate , also called (α-aminoglutarate).

α-katoglutarate has 5 carbons , when we remove CH2 we'll get

oxaloacetate.

4. oxidative decarboxylation of α-katoglutarate

-The conversion of α-katoglutarate to succinyl CoA is

catalyzed by the α-katoglutarate dehydrogenase complex (an

aggregation of three enzymes), which works in a similar way

to "Pyruvate dehydrogenase complex" that was used for the

conversion of pyruvate to acetyl coA, but here it's not

regulated by phospholyration dephosphorylation reactions.

3 | P a g e

-this complex catalyzes the reaction by three steps:

decaboxylation : removal of CO2.

Oxidation of the aldehyde group to carboxyl group,

which causes the reduction of NAD+ to NADH.

Transfer of the resultant acetyl group to coA.

This reaction requires 5 cofactors : flavin , riboflavin

,niacin, lipoic acid , coA

4 vitamins are needed: thiamine , riboflavin ,niacin ,

coA (synthesized from pantothenic acid (vitamin B5)

and cysteine (alfa amino acid ))

-The coenzymes required are: thiamine pyrophosphate

(TPP) , lipoic acid, FAD, NAD+, and coA.

5. cleavage of succinyl coA

-catalyzed by succinate thiokinase.

-succinyl coA contain a high energy bond (thioester bond)

, so that we can utilize the energy that is produced from

the cleavage of that bond to form GTP from GDP+Pi

-GTP and ATP are energetically interconvertible by the

nucleside diphosphate kinase reaction:

GTP + ADP GDP + ATP

-the generation of GTP by succinate thiokinase is an

example of substrate-level phosphorylation .

-the reaction releases the second CO2 and produces the

second NADH of the cycle.

4 | P a g e

6. oxidation of succinate

-Succinate is oxidized to Fumarate by succinate

dehydrogenase, as two hydrogens transferred from two

adjacent carbons to FAD FADH2 .

- Usually when we transfer 2H from an adjacent carbons,

the accepter will be FAD not NAD . while NAD accept

them from hydroxyl groups.

- succinate dehydrogenase is part of electron transport

chain "complex II" .

7. hydration of fumarate

Adding of water to fumarate produces malate in a freely

reversible reaction , catalyzed by fumarase.

8. oxidation of malate

- Malate is oxidized to oxaloacetate by malate

dehydrogenase , which produces the third NADH .

-this reaction is highly favorable in the opposite direction

, but what pushes it in the forward direction is that

oxaloacetate is used in the following reaction which makes

the cycle go on and on.

-oxaloacetate and malate "maarGa 3leina b-Ge99et el-

shuttel"

Aspartate-malate shuttle : for transferring NADH from

cytoplasm to mitochondria.

A SIMPLE WAY TO REMEMBER THE INTERMEDIATES

OF THE CYCLE

CIA Sends Soldiers From My Office

5 | P a g e

NET REACTION

Acetyl-CoA + 3 NAD+ + FAD + GDP + Pi + 2 H2O→

CoA-SH + 3 NADH + 3 H+ + FADH2 + GTP + 2 CO2

In short : 2 carbon atoms enter the cycle.

Two molecules of CO2 are released.

Substrate level phosphorylation (succinyl coA Succinate)

Four reduced coenzyme molecules per acetyl coA oxidized to CO2.

ENERGY PRODUCED BY THE TCA CYCLE

TCA cycle is a source of biosynthetic precursors , many of

its intermediates used in different pathways :

Citrate fatty acids , sterols.

α-katoglutarate +ammonia Glutamate other amino acids

(Glutamine) purins (Adenine , Guanine) .

succinyl coA pophyrins , heme, chlorophyll

oxaloacetate Asparate, other amino acids, purins,

pyrimidines.

In case of using the intermediates in pathways other than TCA

cycle , the rate of the cycle will decrease.

(the cycle will go on but at a very much slower rate).

6 | P a g e

In that case , to retain the normal rate we have to supply the

cycle with any intermediate (other than acetyl coA ).

-A way by which we can replenish the intermediates is by

converting pyruvate to oxaloacetate.

(carboxylation of pyruvate , catalyzed by pyruvate

carboxylase).

-pyruvate (3 carbons), oxaloacetate (4 carbons) , CO2 can be

added in the form of bicarbonate (CO2 + H2O H2CO3 ).

-this reaction needs energy, since it is a carboxylation reaction.

-converting 0xaloacetate to pyruvate is a decarboxylation

reaction , so that it did not require ATP .

-carbozylation reactions need a vitamin called BIOTIN , a

vitamin from the B vitamin groups , it is a carrier for the

activated carboxyl group (CO2 is a gas , so it require a carrier

so as to be added to pyruvate).

Anaplerotic reactions "fill up rxns" : replenishing the cycle

with different intermediates.

REGULATION OF THE TCA CYCLE

• It is the final common pathway for the aerobic oxidation of fuel

molecules

• It is an important source of building blocks for a number of

biomolecules.

• Entry into the cycle and the rate of the cycle itself are controlled at

several stages.

7 | P a g e

pyruvate= pyruvic acid = حمض الحصرم

-Bacteria can convert Acetyl coA back to pyruvate.

Regulation should be stated on the level of irreversible steps which is here

"pyruvate dehydrogenase complex" , which is a large complex composed of

about 60 subunits , two of them are "pyruvate dehydrogenase kinase" and

"pyruvate dehydrogenase phospatase" which carry on the regulation process by

phosphorylation\dephosphorylation rxns.

-The dephosphorylated form of the complex is the active form.

-Phsphatase works to remove the phosphate group from the complex

, hence , transforming it to the active form (glucose will be converted

to pyruvate).

-kinase works to phosphorylate the complex , converting it to the

inactive form (the rxn will be switched off).

-those regulatory proteins (phosphatase, kinase) are part of the

complex , which increases the efficiency of the regulation process.

High concentrations of reaction products inhibit the complex.

NADH, Acetyl coA inhibit the dehosphorylated form (the active

form).

8 | P a g e

Increasing the NADH/NAD+ , acetyl coA/coA , or ATP/ADP ratio

promots phosphorylation.

Promot the kinase activity phsphorylation will occur the complex

is switched off the reaction will be stopped.

Pyruvate , coA , NAD+ inhibit the kinase enzyme.

Pyruvate as well as ADP activate the dehydrogenase by inhibiting

the kinase.

The rise in mitochondrial Ca2+ activates the pyruvate dehydrogenase

complex by stimulating the phosphatase.

This is particularly important in skeletal muscle, where release of Ca2+

during contraction stimulates the PDH complex, and thereby energy

production.

Insulin also accelerates the conversion of pyruvate into acetyl coA by stimulating the phosphatase.

9 | P a g e

Phosphatase Deficiency

(i.e. pyruvate dehydrogenase phosphatase deficiency)

Pyruvate dehydrogenase is always phosphorylated and thus

inactive.

Glucose is processed to lactic acid lactic acidosis

Malfunctioning of many tissues, especially the central nervous

system (that's goes for 2 reasons) :

- Glucose is a source of energy

- Acetyl coA + choline Ach (neurotransmitter in the CNS)

In this case , acetyl coA will not be found in proper

amounts , hence , no production of Ach, this will affect the

function of the CNS .

REGULATION OF CYCLE ITSELF

Here, the regulation should be done at the level where CO2 are

released, (the release of CO2 indicates that the reaction is

almost irreversible), decarboxylation reactions is irreversible , so

they must be regulated.

Regulation at the level of isocitrate

dehydrogenase (IDH)

- Activated by : ADP, Ca+

- Inhibited by : ATP, NADH

*high concentrations of NADH indicates that the

energy level in the cell is high.

*in case of inhibiting the activity of isocitrate

dehdrogenase , the accumulated citrate will be

converted to fat.

11 | P a g e

*ATP inhibitor, ADP activator

when we have high concentrations of ATP , the

concentration of ADP will be decreased.

∑ATP+ADP+AMP = constant

Regulation at the level of α-katoglutarate

dehydrogenase

*As we said , it resembles to a high extent the

pyruvate dehydrogenase complex , the only

difference between them is that the later cannot

be phosphorylated.

- Activated by : Ca+

- Inhibited by : NADH, succinyl coA (its products)

, ATP.

Regulation at the level of citrate synthase

- Regulated only in bacteria not in humans.

- Regulates the process of condensation of

Acetyl CoA with Oxaloacitate to form Citrate.

11 | P a g e

Dietary deficiency of thiamine (vitamin B1)

Causes a disease called " Beriberi "

• A serious health problem in communities where rice is the

major food, "the processed rice is very poor in vitamins , one

of which is thimine".

-causes difficulty in walking. The patient may also suffer from

complications affecting the cardiovascular, nervous, muscular,

and gastrointestinal systems.

• In alcoholics who are severely malnourished

• Characterized by neurologic and cardiac symptoms

• Thiamine pyrophosphate is cofactor of : pyruvate

dehydrogenase , α-ketoglutarate dehydrogenase, and

transketolase.

• ↑ ↑ pyruvate and α-ketoglutarate in the blood.

The vitamin is missing the enzyme is not functioning the

concentrations of the substrates (pyruvate and α-

ketoglutarate) will increase in the blood.

• transketolase activity of red cells, easily measured, is

reliable diagnostic indicator of the disease.

Mercury or Arsenite (AsO33-) Poisoning

Arsenite = الزرنيخ

Both elements have a high affinity for neighboring

sulfhydryls, (dihydrolipoyl groups)

The binding of mercury or arsenite to the dihydrolipoyl

groups inhibits the complex

energy production central nervous system pathologies

Treatment for these poisons is by administration of

sulfhydryl reagents with adjacent sulfhydryl groups

12 | P a g e

(2,3-Dimercaptopropanol PDH (antidote) has high affinity

to bind Arsenite, the produced complex will be secreted in

urine)

"Dedicated to all those with the willpower to stay awake in Bacteriology, and all those who were kicked out of Virology for being 2 minutes late"

Special thanks to : Mokhlisa AL-Remawi <3 & Khalid Al-Jawhari

Done by : me