Pyruvate Dehydrogenase Complex & Krebs Cycle

There are multiple videos

explain what happens to Pyruvate under aerobic cell conditions, aerobic metabolism, discussed in glycolysis, glucose → pyruvate.

If the cell does NOT have mitochondria, it is called anaerobic metabolism.

then, we will identify the enzymes and cofactors required in the conversion of pyruvate to acetylCoA

here is the pyruvate dehydrogenase complex, where the pyruvate on the left side is oxidized → acetyl-CoA and Co2 on the right side.

-this occurs via oxidative decarboxylation.

(pyruvate undergoes oxidative decarboxylation).

-since this pyruvate comes from glycolysis, there are two molecules of pyruvate entering this process, it is NECESSARY to remember to MULTIPLY BY 2.

you will find the pyruvate dehydrogenase complex located in the outer membrane of the mitochondria.

whereas Kreb’s Cycle and B-oxidation of fatty acids occurs in the mitochondrial matrix.

• mito- = thread (Greek mitos)

• chondrion = granule or grain

• matrix = womb / source / container (Latin mater = mother)

So mitochondrial matrix means: the inner compartment inside the mitochondrion where important metabolic reactions occur.

Here is an illustration of the mitochondria, the mitochondria is denominated as a semiautonomous organelle due to DNA replication and other important biological processes.

in this illustration, you can see the mitochondria inner and outer membrane, which have contrasting permeability characteristics.

The outer membrane is non-specifically permeable to all lower weight molecular solutes, whereas the inner membrane is impermeable to except through specific transporter.

then the matrix in the middle, is the soluble part where Kreb’s cycle and oxidative phosphorylation take place.

pyruvate dehydrogenase complex and the three enzymes are required are pyruvate dehydrogenase compound, denominated E1 with the coenzyme, TPP or thiamine pyrophosphate.

The Pyruvate Dehydrogenase Complex (PDC) is a large enzyme system that converts pyruvate into acetyl-CoA, which then enters the citric acid cycle (TCA cycle) in the mitochondrial matrix.

1. pyruvate dehydrogenase: Enzyme 1: E1 with coenzyme thiamine pyrophosphate (TPP).

2. dihydrolipyl transacetylase: Enzyme 2: E2 with coenzyme CoA and Lipoamide.

3. Dihydrolipoyl Dehydrogenase (E3) use NAD+ and FAD as coenzymes

WHAT DOES THIS MEAN?

1. TPP (Thiamine Pyrophosphate) Etymology

cofactor of pyruvate dehydrogenase (e1)

• thiamine = vitamin B₁

• pyrophosphate = two phosphate groups linked together

TPP = activated form of vitamin B₁.

What it does : TPP helps remove carbon dioxide (CO₂) from molecules.

In the pyruvate dehydrogenase complex it helps:

Pyruvate→ decarboxylation

So TPP stabilizes the reactive carbon intermediate after CO₂ is removed.

Simple role: TPP helps break carbon–carbon bonds.

2. Lipoamide Etymology

cofactor of dihydrolipyl transacetylase (e2)

• lipo- = fat

• amide = chemical bond involving nitrogen

It carries the acetyl group from one enzyme active site to another.

It also participates in redox reactions (accepting and donating electrons).

Simple role

Lipoamide transfers the acetyl group during the reaction.

3. CoA (Coenzyme A) Etymology

cofactor of dihydrolipyl transacetylase (e2)

• coenzyme = enzyme helper

• A originally meant acetylation

Simple role CoA is the carrier of acetyl groups.

4. FAD (Flavin Adenine Dinucleotide) Etymology

cofactor of Dihydrolipoyl Dehydrogenase (E3)

• flavin = yellow compound derived from riboflavin

• adenine = nitrogen base in nucleotides

• dinucleotide = two nucleotide units

What it does: FAD accepts electrons during oxidation reactions.

FAD+2e−+2H+→FADH2

It helps transfer electrons to the next molecule in the chain.

Simple role: FAD is an electron carrier.

5. NAD⁺ (Nicotinamide Adenine Dinucleotide) Etymology

cofactor of Dihydrolipoyl Dehydrogenase (E3)

• nicotinamide = derived from niacin (vitamin B₃)

• adenine dinucleotide = nucleotide structure

What it does NAD⁺ accepts high-energy electrons.

NAD++2e−+H+→NADH

NADH then carries these electrons to the electron transport chain, which produces ATP.

Simple role: NAD⁺ is another electron carrier used to make energy.

Dihydrolipoyl Dehydrogenase (E3) use NAD+ and FAD as coenzymes

the delta G for the complex as a whole is -8.3 Kcal/mol, it is an exergonic and irreversible process.

1. What the Pyruvate Dehydrogenase Complex does

After glycolysis:

Glucose→Pyruvate

Pyruvate must be converted into acetyl-CoA to enter the TCA cycle.

The reaction:

Pyruvate+CoA+NAD+→Acetyl-CoA+CO2+NADH

This reaction occurs in the mitochondrial matrix and requires three enzymes working together.

Step 1 — E1 (Pyruvate Dehydrogenase)

Coenzyme: TPP (Thiamine pyrophosphate)

Function: E1 removes CO₂ from pyruvate.

Pyruvate→ Hydroxyethyl-TPP + CO2

This step is called decarboxylation.

TPP stabilizes the reactive intermediate.

Vitamin connection:

TPP comes from vitamin B₁ (thiamine).

Step 2 — E2 (Dihydrolipoyl Transacetylase)

Coenzymes:

• Lipoamide

• CoA

Function: The hydroxyethyl group is transferred to lipoamide, forming an acetyl group.

Then the acetyl group is transferred to CoA.

Result: Acetyl-CoA

This is the molecule that enters the TCA cycle.

Step 3 — E3 (Dihydrolipoyl Dehydrogenase)

Coenzymes:

• FAD

• NAD⁺

Function: E3 restores (reoxidizes) the lipoamide so the cycle can continue.

Electron flow:

Reduced lipoamide→FAD→NAD+

This produces: NADH

NADH later generates ATP through the electron transport chain

here are shown all three enzymes, previously mentioned, forming a multi-enzymatic complex.

all three enzymes are strategically located since the product of each reaction is the substrate to the following enzyme. This allows a quick turnover

again, this is another illustration of pyruvate dehydrogenase complex shows how pyruvate enters on the left and the net product is acetyl-CoA on the top and NADH per pyruvate molecule that it is oxidized, and you can see the NADH on the right side.

Step 1 — Enzyme 1 (E1): Pyruvate Dehydrogenase

Cofactor: TPP (Thiamine pyrophosphate)

What happens:

• Pyruvate enters the enzyme.

• CO₂ is removed (decarboxylation).

• The remaining 2-carbon fragment attaches to TPP.

Intermediate formed: Hydroxyethyl-TPP

So:

Pyruvate→Hydroxyethyl-TPP+CO2​

1. Etymology of the name Pyruvate

So the enzyme acts on pyruvate.

Dehydrogenase

Breakdown of the word:

• de- = remove

• hydrogen = H atoms (or electrons)

• -ase = enzyme

dehydrogenase = an enzyme that removes hydrogen (electrons) from a molecule.

Why it’s called a dehydrogenase

Since the enzyme removes hydrogen/electrons from pyruvate and transfers them to NAD⁺:

NAD+→NADH\text{NAD}^+ \rightarrow \text{NADH}NAD+→NADH

the enzyme is classified as a dehydrogenase.

4. Important detail

Even though we call it pyruvate dehydrogenase, the reaction actually involves three enzymes working together (E1, E2, E3).

But the name comes from the first step, where pyruvate begins the reaction.

Step 2 — Enzyme 2 (E2): Dihydrolipoyl Transacetylase

dihydro: 2 hydrogen, “Two hydrogens added” or reduced form of a molecule.

In this enzyme, it refers to reduced lipoamide, which has two hydrogen atoms attached after it accepts electrons

Lipoyl comes from lipoic acid, a cofactor attached to the enzyme.

Trans-: “to move across” or “transfer.”

Acetyl refers to a 2-carbon group:

This is the same acetyl group that forms acetyl-CoA.

ase: enzyme

Cofactors:

• Lipoamide

• Coenzyme A (CoA)

1. Start: Oxidized lipoamide

Lipoamide initially exists in the oxidized form, which has a disulfide bond (-S-S-).

This is the starting form attached to E2.

Structure conceptually:

oxidized lipoamide
S—S (disulfide bond)

2. Transfer from TPP (cofactor of e1) → lipoamide (cofactor of e2)

From E1, the hydroxyethyl group attached to TPP, becomes oxidized to acetyl, and the acetyl is transferred to lipoamide.

During the hydroxyethyl transfer to Lipoamide:

• the hydroxyethyl group becomes oxidized → acetyl

• the lipoamide was in oxidized form, lipoamide disulfide bond is reduced (the hydroxyethyl group was oxidized to acetyl, the lipoamide was reduced to acetyl-lipoamide).

Result: acetyl-lipoamide (reduced) (NOT FULLY REDUCED yet).

Meaning:

• the acetyl group is now attached to lipoamide

• the disulfide bond has opened

So now we have: acetyl-lipoamide

3. Transfer of acetyl group to CoA

Next step: The acetyl group is transferred from lipoamide to CoASH.

(CoASH = Coenzyme A with a free sulfhydryl group).

Reaction:

acetyl-lipoamide + CoASH → acetyl-CoA + reduced lipoamide

Result:

• acetyl-CoA is produced

• lipoamide is now reduced (two -SH groups)

So now we have: FULLY reduced lipoamide

4. Regeneration of oxidized lipoamide

The enzyme complex must reset the lipoamide so the cycle can continue.

This happens in E3.

Steps:

1. Reduced lipoamide transfers electrons to FAD

2. FAD becomes FADH₂

3. FADH₂ transfers electrons to NAD⁺

4. NAD⁺ becomes NADH

During this process:

reduced lipoamide → oxidized lipoamide

Now the system is ready for another cycle.

This form is ready to accept the 2-carbon fragment from E1.

Result: Acetyl-CoA

This is the molecule that enters the Krebs cycle.

Step 3 — Enzyme 3 (E3): Dihydrolipoyl Dehydrogenase

Cofactors:

• FAD

• NAD⁺

What happens:

1. Lipoamide became reduced during step 2.

2. E3 re-oxidizes lipoamide so the enzyme can work again.

3. Electrons move:

Reduced lipoamide→ FAD→NAD+

This produces:

• FADH₂

• then NADH

Final product:

NADH

NADH carries electrons to the electron transport chain to make ATP.

The Pyruvate Dehydrogenase Complex (PDC) is a large enzyme system that converts pyruvate into acetyl-CoA, which then enters the citric acid cycle (TCA cycle) in the mitochondrial matrix.

what happens to acetyl-CoA once it is formed?

answer: acetyl-CoA can follow different directions depending on the metabolic needs of the cell.

1. One pathway is to go into the TCA cycle: where acetyl-CoA is combined with oxaloacetate to produce Citrate (acetyl-CoA + oxaloacetate= citrate).

2. another pathway leads to the formation of ketone bodies during starvation (acetyl-CoA can become ketone bodies during starvation)

3. or when blood glucose is high, acetyl-CoA can go into cholesterol, fatty acid synthesis, which is a fed state.

regulation

many reversible reactions need regulation for inhibition, and we will talk about the ones involved in pyruvate dehydrogenase complex

regulation of E1

Enzyme 1 (E1) is inhibited by high concentration of ATP, thus, we can say that ADP will cause the opposite and accelerate when there is low concentration of ATP. why?

Enzyme 1 (E1) of the pyruvate dehydrogenase complex converts:

Pyruvate→ Acetyl-CoA

This step allows pyruvate to enter the citric acid cycle, which produces ATP.

So activating this enzyme leads to more ATP production

regulation of E2

Enzyme 2 (E2) is inhibited by high concentration of its product, which is acetyl-CoA, by feedback inhibition as well.

1. What Enzyme 2 (E2) does

E2 in the pyruvate dehydrogenase complex is:

Dihydrolipoyl transacetylase

Its job is to transfer the acetyl group to CoA, producing: Acetyl-CoA

So E2 is the step that actually creates acetyl-CoA.

and the last enzyme, E3, is inhibited by its product NADH (why?)

What Enzyme 3 (E3) does

E3 is called:

Dihydrolipoyl dehydrogenase

Its job is to transfer electrons to NAD⁺, producing NADH.

Reaction:

NAD++2e−+H+→NADH

So E3 helps generate NADH, which carries high-energy electrons to the electron transport chain.

liver control over PDC with insulin, cAMP, glucagon and glucose.

another control seen in pyruvate dehydrogenase complex is in the liver, the first enzyme is phosphorylated and inactivated by the cyclic AMP (cAMP) cascade.

1. this cAMP cascade can be controlled by stress hormone glucagon since it is in the liver.

2. the presence of glucagon, during starvation will help preserve glucose and stop the pyruvate dehydrogenase complex, preventing formation of acetyl-CoA due to irreversibility.

3. then, in a fed state, insulin will be predominant and cyclic AMP will be activated by causing dependent phosphate.

as a summary, in this illustration, we see that E1 E2 and E3 are controlled by their products, and in the liver, is controlled by phosphorylation during starvation conditions

there are eight reactions or carbon versions in Kreb’s cycle, shown here.

oxidations are only four, in which three are made by NAD and one by FAD.

Here you can see the NAD (bottom right, 2 NAD+ AND 2NADH), another one here (bottom), and the last one on top (top left of the bottom circle), on the bottom left you can see the FAD (2 FAD, 2 FADH)

remember, this is once per cycle (he is referring to the big circle).

glucose is not the only source of acetyl-CoA.

Glucose is not the only source of acetyl-CoA .

as depicted here, the oxidation of many fields, like

1. fatty acids

2. ketone bodies

3. amino acids

will generate acetyl-CoA.

on top are the ones I just mentioned, and producing acetyl-CoA that will be into the kreb cycle

ATP not directly formed

ATP is not directly formed in Kreb’s cycle, but the reduction of NAD and FAD into NADH and FADH2 will carry the electrons needed in oxidation phosphorylation to produce ATP

1. What the Krebs cycle actually produces

When acetyl-CoA enters the Krebs (TCA) cycle, most of the energy is captured in reduced electron carriers:

Products per acetyl-CoA:

• 3 NADH

• 1 FADH₂

• 1 GTP (≈ ATP)

• CO₂

So technically one ATP-equivalent (GTP) is produced, but the major energy yield comes later.

2. What NADH and FADH₂ are

NADH and FADH₂ are electron carriers.

During the Krebs cycle:

• NAD⁺ accepts electrons → becomes NADH

• FAD accepts electrons → becomes FADH₂

These electrons store high-energy chemical energy.

Think of them like charged batteries.

3. Where ATP is actually made

The electrons carried by NADH and FADH₂ go to the electron transport chain in the inner mitochondrial membrane.

There, the electrons move through a series of proteins.

As they move:

• protons (H⁺) are pumped across the membrane

• this creates a proton gradient

4. Oxidative phosphorylation

The proton gradient powers the enzyme ATP synthase.

Protons flow back through ATP synthase and this drives:

ADP+Pi→ATP

This process is called oxidative phosphorylation.

only high energy compound formed during Kreb’s cycle

the only high energy compound formed during Kreb’s cycle is GTP from GDP. This will then GDP is interconverted to ATP by a nucleoside, diphosphate kinase.

why do fatty acids produce Acetyl-CoA?

1. Fatty acids → acetyl-CoA

Fatty acids are broken down by a process called β-oxidation in the mitochondrial matrix.

What happens

The long fatty acid chain is cut into 2-carbon pieces repeatedly.

Each cycle of β-oxidation removes:

2 carbons→acetyl-CoA

Example:

A 16-carbon fatty acid (palmitate):

16C→8 acetyl-CoA

So fatty acids produce many acetyl-CoA molecules, which is why fats are a very energy-rich fuel.

why do ketone bodies produce acetyl-CoA?

2. Ketone bodies → acetyl-CoA

Ketone bodies are produced in the liver during fasting or starvation.

Main ketone bodies:

1. acetoacetate

2. β-hydroxybutyrate

Other tissues (brain, muscle) convert these ketone bodies back into acetyl-CoA.

Example pathway: Acetoacetate→ Acetoacetyl-CoA

Then: Acetoacetyl-CoA→2 acetyl-CoA

These acetyl-CoA molecules then enter the TCA cycle to produce ATP.

why do amino acids produce acetyl-CoA?

3. Amino acids → acetyl-CoA

Some amino acids are called ketogenic amino acids because their breakdown produces acetyl-CoA.

Examples of ketogenic amino acids:

• Leucine

• Lysine

Other amino acids are partially ketogenic, such as:

• isoleucine

• phenylalanine

• tyrosine

• tryptophan

When these amino acids are metabolized, their carbon skeletons are converted into acetyl-CoA or acetoacetate.

SECOND VIDEO

pyruvate dehydrogenase complex and krebs cycle part 2

we are continuing this topic, we discussed the pyruvate dehydrogenase complex, which is a multienzymatic, converting pyruvate → acetyl-CoA

-the localization is in the outer membrane of the mitochondria

as a quick recap, we talk about Kreb’s and beta oxidation of fatty acids occur in the mitochondrial membrane and how the mitochondria is a semiautonomous organelle.

here is an illustration of the mitochondria with its different spaces.

we mentioned the different fields that produce acetyl-CoA, such as fatty acids, ketone bodies, and amino acids.

Kreb’s cycle illustration shows oxidation, are only four, in which three are made by NAD and one by FAD.

Remember that this GTP is interconverted to ATP.

ATP is not directly formed in Kreb cycle, but the oxidation of NAD and FAD into NADH and FADH2 will carry the electrons needed in the oxidative phosphorylation process to produce ATP.

When talking of energy production, you will find different sources with different values.

but for this lecture, we will assume that one NADH will form three ATP.

1 NADH= 3 ATP

3 NADH= 9 ATP.

Since there are three NADH in a single Kreb cycle, the total ATPs formed are 9.

for each FADH2, the ATP produced is 2.

1 FADH2= 2 ATP.

in addition, and as mentioned in the last diagram, remember that 1 GTP is converted to ATP by nucleoside diphosphate kinase. ( GTP+ADP→GDP+ATP).

What GTP is

GTP is very similar to ATP.

Both contain:

• a nucleotide

• three phosphate groups

• stored chemical energy

The only difference is the base:

Molecule	Base
ATP	Adenine
GTP	Guanine

Both can store and transfer energy.

3. What nucleoside diphosphate kinase does

The enzyme nucleoside diphosphate kinase transfers a phosphate group.

Reaction:

GTP+ADP→GDP+ATP

What happens:

• GTP donates one phosphate

• ADP receives that phosphate

• ATP is formed

the kreb’s cycle occurs under aerobic conditions (because oxygen is the final electron acceptor in oxidative phosphorylation). The reason is that NADH and FADH2 provides the electrons needed in the oxidative phosphorylation system, in the final phase, oxygen will accept that electron to be reduced and form water.

if oxygen is not present, as such in anaerobic conditions, there is no driving force to pull the electron through the oxidative phosphorylation system

anabolic aspect of the Kreb’s cycle

-the Krebs cycle is not only for energy (catabolism), it also supplies building blocks for biosynthesis (anabolism). This is why the cycle is often called an amphibolic (anabolic and catabolic) pathway.

the anabolic aspect of the kreb’s cycle means that the metabolites generated can also be precursors to other molecules like succinyl-CoA, can form heme or purines, alpha ketoglutarate can form glutamate, citrate to fatty acids, oxaloacetate to aspartate and then to amino acids.

as you can see, in this cycle, an H of the products.

anaplerotic reactions

Anaplerotic

• ana- = again / back

• plērōsis = filling or replenishing

Anaplerotic reactions = reactions that refill or replenish the Krebs cycle.

we mentioned that the molecules of the Kreb’s cycle can be used in other places, but if that happens, if they are not replaced back, the Kreb’s cycle can collapse.

Thus, in order to replace the lost compounds, we have the anaplerotic reactions to form the lost metabolites.

example of anaplerotic reactions are:

1. pyruvate to Oxaloacetate

2. Propionyl to Succinyl-CoA

3. Aspartate to Oxaloacetate

4. Glutamate to a-ketoglutarate

Remember: anaplerotic reactions = “refilling reactions.”

When Krebs cycle molecules are taken out to make other things (amino acids, heme, fatty acids, etc.), the cell must replace them so the cycle can keep producing energy.

1. Pyruvate → Oxaloacetate

Reaction: Pyruvate+CO2+ATP → Oxaloacetate

Enzyme: Pyruvate carboxylase

Meaning:

• Pyruvate (from glycolysis) is converted into oxaloacetate

• Oxaloacetate is the molecule that starts the Krebs cycle

This reaction refills oxaloacetate when levels are low.

2. Propionyl-CoA → Succinyl-CoA

Propionyl-CoA comes from breakdown of:

• odd-chain fatty acids

• certain amino acids

It is converted into:

Propionyl-CoA → Succinyl-CoA

Succinyl-CoA is a Krebs cycle intermediate, so this reaction adds new material into the cycle

3. Aspartate → Oxaloacetate

Aspartate is an amino acid.

It can be converted into oxaloacetate through a reaction called transamination.

Aspartate → Oxaloacetate

This again replenishes oxaloacetate in the Krebs cycle.

*both pyruvate and aspartate can form Oxaloacetate

4. Glutamate → α-ketoglutarate

Glutamate is another amino acid.

It can lose an amino group through deamination:

Glutamate→α-ketoglutarate

α-ketoglutarate is a Krebs cycle intermediate, so this reaction refills the cycle.

5. Why these reactions are important

The Krebs cycle constantly loses intermediates because they are used to make other molecules.

Examples:

• succinyl-CoA → heme

• oxaloacetate → aspartate

• citrate → fatty acids

If these molecules leave and are not replaced, the cycle slows down.

Anaplerotic reactions replace them so the cycle keeps running

most important anaplerotic reaction

from all the anaplerotic reactions mentioned, the most important is the one that goes from pyruvate to oxaloacetate because oxaloacetate is the substrate needed in the first reaction, the coenzyme used by pyruvate carboxylase in this step is biotanimite along with ATP.

pyruvate →> oxaloacetate

enzyme (pyruvate carboxylase, The enzyme adds a carbon dioxide (CO₂) to pyruvate.)

This process is called carboxylation (adding CO₂).

Oxaloacetate

• oxa- → refers to oxalic acid (a 2-carbon acid)

• acetate → a 2-carbon acetyl-related compound

So oxaloacetate is a 4-carbon molecule formed from oxalo- and acetyl-type fragments.

Chemically it is a four-carbon dicarboxylic acid used in metabolism.

first step of the krebs cycle

going step by step into the Kreb’s cycle, we start with the substrate of acetyl-CoA and oxaloacetate, the enzyme is called citrate synthase. through our condensation reaction, we will produce citrate. This is an exergonic reaction with a delta G of -7.5 Kcal/mole. This is an irreversible reaction or controlled reaction.

Acetyl-CoA + Oxaloacetate + H2​O→ Citrate+ CoA-SH

Enzyme: Citrate synthase

Acetyl-CoA = Coenzyme A carrying a 2-carbon acetyl group, It is the main fuel molecule entering the Krebs cycle.

Oxaloacetate = a 4-carbon molecule related to oxalo and acetate groups

A 2-carbon acetyl group joins a 4-carbon oxaloacetate to form a 6-carbon citrate.

3. Water (H₂O)

Water participates in the reaction because the enzyme performs a hydrolysis step.

Water helps break the thioester bond between acetyl and CoA.

This releases CoA-SH.

4. CoA-SH Etymology

SH

• sulfhydryl group

• thiol group

When acetyl leaves, CoA regains its free sulfhydryl (-SH).

So: CoA-SH = free coenzyme A

Citrate

Derived from citric acid

• citrus fruits contain citric acid

• first isolated from lemons

---

Citrate is a 6-carbon molecule formed when:

2C+4C=6C2C + 4C = 6C2C+4C=6C

Acetyl group + oxaloacetate → citrate.

Citrate is the first molecule of the Krebs cycle.

But citrate has a hydroxyl group in the wrong position, so it must be rearranged before oxidation can occur in the cycle.

the second step is a reversible one. Here we have a conversion of citrate → iso-citrate by aconitase enzyme.

2. Aconitase Etymology

• aconitic acid → a molecule related to citrate that contains a double bond

• -ase → enzyme

Aconitase = the enzyme that converts citrate through the aconitate intermediate.

3. Cis-aconitate Etymology

• aconitate → derived from aconitic acid, a compound with a carbon–carbon double bond

• cis → Latin “on the same side”

Cis-aconitate is a temporary intermediate formed when water is removed from citrate.

4. Isocitrate Etymology

• iso- = equal / rearranged

• citrate = citric acid derivative

Isocitrate = a rearranged form of citrate.

5. What actually happens in Step 2

The enzyme aconitase performs two reactions:

Step A — Dehydration

Water is removed:

Citrate→cis-aconitate+H2O\text{Citrate} \rightarrow \text{cis-aconitate} + H_2OCitrate→cis-aconitate+H2​O

This creates a double bond.

Step B — Rehydration

Water is added back, but in a different orientation:

cis-aconitate+H2O→Isocitrate\text{cis-aconitate} + H_2O \rightarrow \text{Isocitrate}cis-aconitate+H2​O→Isocitrate

This moves the –OH group to a different carbon.

6. Why this step is important

Citrate cannot easily undergo oxidation.

Rearranging it to isocitrate puts the hydroxyl group in the correct position for the next step:

Isocitrate→α-ketoglutarate

This step produces NADH and CO₂.

The aconitase enzyme has an iron sulfur center forming an inorganic molecule, this molecule is bound to cysteine.

This kinds of molecules are seen in redox reactions as explained in the lecture of oxidative phosphorylation.

this case is an exception where it supports substrate binding. The importance of knowing this fact is knowing there are external compounds that can affect this binding.

1. What the iron–sulfur center is

Aconitase contains an iron–sulfur cluster called a [4Fe–4S] cluster.

This cluster is made of:

• 4 iron atoms (Fe)

• 4 sulfur atoms (S)

These atoms form an inorganic metal cluster inside the enzyme.

2. Why cysteine is involved

The cluster is held in place by cysteine residues from the protein.

Cysteine contains a sulfur atom (-SH group).

The sulfur of cysteine binds to the iron atoms in the cluster.

So the enzyme structure looks conceptually like:

Cysteine-S → Fe
Cysteine-S → Fe
Cysteine-S → Fe

These cysteine residues anchor the iron–sulfur cluster inside the enzyme.

3. Iron–sulfur clusters in many enzymes

Iron–sulfur clusters are common in redox enzymes, especially in:

• electron transport chain proteins

• oxidative phosphorylation enzymes

In those systems, Fe–S clusters transfer electrons.

Example: Fe2+↔Fe3+

This allows them to participate in oxidation–reduction reactions.

4. Why aconitase is special

In aconitase, the Fe–S cluster is not mainly transferring electrons.

Instead, it helps bind the citrate molecule and orient it properly.

So here the cluster functions as a structural catalytic center, helping the enzyme:

• bind citrate

• remove water

• add water back to form isocitrate.

5. Why external compounds can affect this

Because the enzyme relies on the iron–sulfur cluster, substances that bind iron or sulfur can inactivate the enzyme.

A classic example is fluoroacetate poisoning.

Fluoroacetate becomes fluorocitrate, which inhibits aconitase.

This blocks the Krebs cycle, stopping cellular energy production.

6. Why this matters biologically

If aconitase stops working:

• citrate cannot become isocitrate

• the Krebs cycle stops

• ATP production drops dramatically.

This is why some toxins targeting this enzyme are very dangerous.

the third step is known as the rate limiting step of the cycle. It is very spontaneous and irreversible reaction.

Step 3 of the Krebs cycle (isocitrate → α-ketoglutarate) is called the rate-limiting step and is considered essentially irreversible because of the large release of energy and the loss of CO₂, which drives the reaction strongly forward.

isocitrate → oxalosuccinate → α-ketoglutarate
with NAD⁺ becoming NADH and CO₂ being released.

Big picture

This step is catalyzed by:

isocitrate dehydrogenase

Overall reaction:

Isocitrate+NAD+→α-ketoglutarate+CO2+NADH

1. Start with isocitrate

Isocitrate is a 6-carbon molecule.

It has an alcohol group (-OH) on the carbon that will be oxidized.

That alcohol is important, because alcohols can be oxidized into carbonyls.

2. NAD⁺ becomes NADH

The enzyme removes 2 hydrogens / electrons from isocitrate.

So:

• isocitrate is oxidized

• NAD⁺ is reduced to NADH

Reaction idea:

Isocitrate→Oxalosuccinate

while

NAD+→NADH

So NADH is formed because it accepts the electrons taken from isocitrate

3. Oxalosuccinate forms

This first oxidation converts the alcohol on isocitrate into a ketone.

That gives the intermediate:

oxalosuccinate

Etymology

• oxalo- = oxidized / oxalo-type acid relation

• succinate = related to succinic acid

Meaning:

It is a more oxidized 6-carbon intermediate between isocitrate and α-ketoglutarate.

This intermediate is usually not isolated because it quickly moves to the next step.

4. Oxalosuccinate gives up CO₂

Now oxalosuccinate is a beta-keto acid–type intermediate, and those are unstable.

So it undergoes decarboxylation:

Oxalosuccinate→α-ketoglutarate+CO2​

This means:

• one carbon leaves as CO₂

• the molecule goes from 6 carbons to 5 carbons

5. α-Ketoglutarate forms

After losing CO₂, the remaining 5-carbon molecule is:

α-ketoglutarate

Etymology

• alpha (α) = ketone is on the carbon next to the carboxyl group

• keto- = ketone group

• glutarate = 5-carbon dicarboxylic acid backbone

So α-ketoglutarate is a 5-carbon keto acid.

the fourth step is where alpha ketolglutarate is converted to succinyl CoA by a multisymatic complex

alpha ketoglutarate, CO2, NADH, Succinyl-CoA

Here we have an oxidative decarboxylation and the addition of coenzyme A.

The delta G is -7.2 Kcal/mole.

This is a controls step as well.

The Reaction (Step 4 of the Krebs Cycle)

α-ketoglutarate + CoA + NAD+→Succinyl-CoA+CO2+NADH

Enzyme complex: α-ketoglutarate dehydrogenase complex

Step-by-step puzzle

1. Start: α-ketoglutarate (5 carbons)

α-ketoglutarate is a 5-carbon molecule produced in Step 3.

Etymology:

• alpha (α) → ketone is on the first carbon next to the carboxyl group

• keto → ketone group (C=O)

• glutarate → a 5-carbon dicarboxylic acid

So it is a 5-carbon keto acid.

2. Oxidation occurs

α-ketoglutarate loses electrons.

Those electrons are transferred to NAD⁺.

NAD+→NADH

So:

• α-ketoglutarate is oxidized

• NAD⁺ is reduced to NADH

3. Decarboxylation occurs

A carboxyl group (-COO⁻) is removed.

This releases: CO2

4. Coenzyme A attaches

After CO₂ leaves, the remaining 4-carbon succinyl group attaches to Coenzyme A (CoA-SH).

This forms: Succinyl-CoA

Succinyl-CoA contains a high-energy thioester bond, which will be used in the next step to produce GTP (ATP equivalent).

Why the reaction releases energy

The ΔG is:

−7.2 kcal/mol-7.2 \text{ kcal/mol}−7.2 kcal/mol

This means the reaction releases energy because:

1. oxidation releases energy

2. CO₂ leaves the system

3. formation of a high-energy thioester bond

This makes the reaction essentially irreversible.

Why this is a control step

The enzyme α-ketoglutarate dehydrogenase is regulated by the cell’s energy state.

Inhibited by

• ATP

• NADH

• Succinyl-CoA (product inhibition)

Activated by

• Ca²⁺ (especially in muscle)

So the cycle slows when the cell already has enough energy.

Similar to the pyruvate dehydrogenase complex, the alpha ketoglutarate complex uses 3 enzymes and 5 coenzymes.

the enzyme 1 or E1 is the alpha ketoglutarate dehydrogenase component with the coenzyme TPP or thiamine pyrophosphate.

the enzyme 2 or E2, then transsuccinylase, with two coenzymes with are CoA and lipoamide

the enzyme 3 or E3, is dihydrolipoyl dehydrogenase, which uses NAD and FAD as coenzymes

1. Enzyme 1 (E1): α-ketoglutarate dehydrogenase

Cofactor: TPP (Thiamine pyrophosphate)

What happens

• α-ketoglutarate (5 carbons) binds the enzyme.

• CO₂ is removed (decarboxylation).

• The remaining 4-carbon fragment attaches to TPP.

Intermediate: Succinyl-TPP

So the molecule goes from:

5C→4C+CO2

2. Enzyme 2 (E2): Dihydrolipoyl transsuccinylase

Cofactors:

• Lipoamide

• Coenzyme A (CoA)

What happens

1. The succinyl group is transferred from TPP to lipoamide.

2. Lipoamide carries the group like a swinging arm.

3. The succinyl group is transferred to CoA.

Result: Succinyl-CoA

This molecule contains a high-energy thioester bond.

3. Enzyme 3 (E3): Dihydrolipoyl dehydrogenase

Cofactors:

• FAD

• NAD⁺

What happens

During step 2, lipoamide becomes reduced.

E3 restores it so the cycle can continue.

Steps:

1. Reduced lipoamide transfers electrons → FAD

2. FAD becomes FADH₂

3. FADH₂ transfers electrons → NAD⁺

4. NAD⁺ becomes NADH

Now the enzyme is reset

The 5 Cofactors (same as pyruvate dehydrogenase)

A common mnemonic:

TLCFN

Cofactor	Function
TPP	removes CO₂
Lipoamide	carries the succinyl group
CoA	forms succinyl-CoA
FAD	accepts electrons
NAD⁺	final electron carrier

the fifth step is a reversible reaction in which succinyl coa is converted to succinate, and GTP is formed by substrate level phosphorylation.

this GTP is then converted to ATP by the nucleoside triphosphate kinase.

succinyl Coa, GDP + Pi, GTP, succinate

The Reaction (Step 5)

Succinyl-CoA +GDP +Pi → Succinate + GTP + CoA−SH

Enzyme: Succinyl-CoA synthetase (also called succinate thiokinase)

This reaction uses the energy of the thioester bond in succinyl-CoA to make GTP.

1. Succinyl-CoA Etymology

Succinyl

• from succinic acid

• Latin succinum = amber
  (succinic acid was first isolated from amber)

-yl

• chemical suffix meaning derived group

So succinyl = the acyl group derived from succinic acid.

CoA

Coenzyme A

• co- = helper

• enzyme = assists enzymes

• A originally referred to acetyl transfer

CoA carries acyl groups using a sulfhydryl (-SH).

Succinyl-CoA = a 4-carbon succinyl group attached to coenzyme A

It contains a high-energy thioester bond.

Use

The energy of that bond is used to produce GTP.

2. GDP Etymology

G = guanine (nitrogenous base)

D = di (two)

P = phosphate

So: GDP = guanosine diphosphate

Structure:

• guanine base

• ribose sugar

• 2 phosphates

Use

GDP receives a phosphate to become GTP

3. Pi Meaning

Pi = inorganic phosphate

Etymology:

• P = phosphate

• i = inorganic

Use

Pi provides the phosphate that converts GDP → GTP.

4. GTP Etymology

G = guanine
T = tri (three)
P = phosphate

GTP = guanosine triphosphate

Use

GTP is an energy molecule, similar to ATP.

It can:

• power some cellular reactions

• or be converted into ATP.

5. Succinate Etymology

Succinate

From succinic acid (amber acid).

Succinate is a 4-carbon Krebs cycle intermediate.

It continues to the next step of the cycle.

6. Substrate-level phosphorylation Etymology

substrate

the molecule being acted upon in the reaction.

phosphorylation

• phospho = phosphate

• -ylation = adding a group

A phosphate group is directly transferred to GDP to make GTP.

This is different from oxidative phosphorylation.

6. Substrate-level phosphorylation Etymology

substrate the molecule being acted upon in the reaction.

phosphorylation

• phospho = phosphate

• -ylation = adding a group

Meaning A phosphate group is directly transferred to GDP to make GTP.

This is different from oxidative phosphorylation.

7. Nucleoside triphosphate kinase Etymology

nucleoside: base + sugar

triphosphate: three phosphates

kinase: enzyme that transfers a phosphate

Reaction GTP+ADP→GDP+ATP

Meaning: The enzyme transfers the phosphate from GTP to ADP, producing ATP

Putting the whole puzzle together

Step-by-step:

1. Succinyl-CoA has a high-energy thioester bond.

2. Breaking that bond releases energy.

3. That energy adds Pi to GDP, forming GTP.

4. CoA is released.

5. The remaining molecule becomes succinate.

6. GTP can then transfer its phosphate to ADP → ATP.

the sixth step is where succinate is converted to fumarate by the enzyme called succinate dehydrogenase

this is the only step where FAD will be oxidized and produce FADH2.

the enzyme is bound in the inner membrane of the mitochondria.

the reason is this enzyme participates in kreb’s cycle but also in oxidative phosphorylation.

succinate, FAD, FADH2, succinate dehydrogenase, fumarate

You are describing Step 6 of the Krebs (TCA) cycle, where succinate is oxidized to fumarate and FAD is reduced to FADH₂. This step is unique because the enzyme is also part of the electron transport chain (Complex II).

Let’s break down the etymology, meaning, and role of each term, and then put the biochemical puzzle together.

The Reaction (Step 6)

Succinate+FAD→Fumarate+FADH2

Enzyme: Succinate dehydrogenase

1. Succinate Etymology

Succinate comes from succinic acid.

• Latin succinum = amber

• Succinic acid was first isolated from amber resin

The suffix -ate means the ion or salt of an acid.

Meaning

Succinate is a 4-carbon molecule in the Krebs cycle.

Structure conceptually:

HOOC–CH₂–CH₂–COO⁻

Use Succinate is the substrate that gets oxidized in Step 6.

During the reaction, it loses two hydrogens and becomes fumarate.

2. FAD Etymology

FAD = Flavin Adenine Dinucleotide

Breakdown: Flavin

• from Latin flavus = yellow

• flavin molecules are yellow pigments

Adenine

a nitrogenous base in nucleotides

Dinucleotide

two nucleotides linked together.

Meaning

FAD is a coenzyme that accepts electrons and hydrogen atoms.

Use In this step: FAD→FADH2

It captures 2 hydrogens and 2 electrons from succinate.

3. FADH₂ Etymology

Same root as FAD, but with H₂ meaning two hydrogens added.

Meaning

FADH₂ = the reduced form of FAD

It now carries high-energy electrons.

Use FADH₂ transfers those electrons to the electron transport chain, producing ATP.

4. Succinate Dehydrogenase Etymology

Succinate the substrate molecule.

Dehydrogenase

• de- = remove

• hydrogen

• -ase = enzyme

Meaning: An enzyme that removes hydrogen from succinate.

Meaning: This enzyme removes two hydrogens from succinate, transferring them to FAD.

Use It catalyzes: Succinate→Fumarate

while producing FADH₂

5. Fumarate Etymology

Fumarate comes from fumaric acid.

• Latin fumus = smoke

Fumaric acid was first isolated from fumitory plants.

Meaning Fumarate is another 4-carbon Krebs cycle intermediate.

Structure: HOOC–CH=CH–COO⁻

Notice the double bond between the carbons.

Use Fumarate continues into the next step of the Krebs cycle: Fumarate→Malate

Why this step is special

1. Oxidation occurs

Succinate loses hydrogen atoms.

Succinate→Fumarate

2. FAD is reduced

FAD accepts the electrons and hydrogens: FAD→FADH2

3. Enzyme location

Succinate dehydrogenase is embedded in the inner mitochondrial membrane.

This is unique because all other Krebs cycle enzymes are in the mitochondrial matrix.

4. Link to oxidative phosphorylation

Succinate dehydrogenase is also:

Complex II of the electron transport chain.

So this enzyme participates in both:

Pathway	Role
Krebs cycle	oxidizes succinate
Electron transport chain	transfers electrons to ubiquinone

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Putting the puzzle together

Step-by-step:

1. Succinate loses two hydrogens.

2. The molecule forms a double bond, becoming fumarate.

3. FAD accepts the hydrogens, becoming FADH₂.

4. The electrons from FADH₂ enter the electron transport chain.

step 7 will convert fumarate into malate, by using fumarase enzyme

this hydration reaction can be a reversible reaction. the malate is known as the L-malate.

Malate comes from malic acid.

• Latin malum = apple

Malic acid was first found in apple juice.

Suffix -ate

the ionized form of malic acid.

Malate is a 4-carbon molecule with a hydroxyl group (-OH).

Structure conceptually:

HOOC–CH₂–CHOH–COO⁻

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Use Malate continues into the final step of the Krebs cycle, where it will be oxidized to:

Oxaloacetate

producing NADH.

The enzyme fumarase adds water in a very specific orientation.

This produces only the L-isomer of malate.

Etymology:

L

refers to Levo configuration, describing the spatial orientation of the molecule.

Only L-malate can be used in the next Krebs cycle reaction.

What actually happens in the reaction

The double bond in fumarate is broken.

Water adds across the bond:

• OH group attaches to one carbon

• H attaches to the other carbon

Result: Fumarate+H2O→L-malate

Putting the puzzle together

Step-by-step:

1. Fumarate has a carbon-carbon double bond.

2. Fumarase adds water across that double bond.

3. The double bond becomes a single bond.

4. A hydroxyl group forms, producing L-malate.

the final step (step 8), is the conversion of the L-malate into oxaloacetate by malate dehydrogenase.

it is reversible as well.

The Reaction (Step 8)

L-malate + NAD+→Oxaloacetate + NADH+H

Enzyme: Malate dehydrogenase

Type of reaction: Oxidation (dehydrogenation)

This reaction is reversible, although under normal conditions it proceeds forward because oxaloacetate is quickly used in the next step of the cycle.

Step-by-step:

1. L-malate contains a hydroxyl group (-OH).

2. The enzyme malate dehydrogenase removes hydrogens.

3. Those electrons go to NAD⁺, forming NADH + H⁺.

4. The alcohol group becomes a carbonyl (C=O).

5. The molecule becomes oxaloacetate.

control points in Kreb cycle

to summarize, there are three control points in the Kreb cycle.

1. the first one is the first step of the cycle, where the enzyme is citrate synthase is controlled by the level of succinyl-CoA, where a high amount will inhibit in a competitively manner.

the second one, pacemaker, is isocitrate dehydrogenase. (step 3)

this enzyme catalyzes the committed step, the enzyme can be inhibited allostetically, by high concentration of ATP and ADH.

vice versa, if ADP and NADH are high, then the enzyme is activated.

Why this step is called the “pacemaker”

It is considered the main regulatory step of the Krebs cycle because it determines how fast the cycle runs.

When this enzyme is active → the cycle runs faster.
When it is inhibited → the cycle slows.

the last control point is in the step where isocitrate is converted to alpha ketoglutarate by isocitrate dehydrogenase.

remember that this step is requires a multienzymatic complex

steps 1 and 3 are the control points in the Kreb’s cycle.

as we discussed before, the multi-enzymatic complex is regulated by three enzymes, where enzyme one, alpha ketoglutarate dehydrogenase complex with a cofactor TPP is inhibited allosterically by high concentration of ATP activated by ADP.

enzyme 2, transuccinylase with cofactors as coenzyme A lipoamide is inihibited allosterically by high concentrations of succinyl-CoA

enzyme 3, dihydrolipoyl dehydrogenase with cofactor of FAD and NAD is inhibited by allosterically by high concentration of NADH

a part of the inhibition of the kreb’s cycle we just mentioned, there is a compound called fluoroacetate (poison 1080) inhibits aconitase at the level of Fe4S4

if you recall in the step two of the Kreb’s cycle, the enzyme is aconitase. this enzyme is inhibited at the level of cofactor with the iron sulfur center. this inhibitions is not direct. what happens is that fluoroacetate is used as a substrate instead of acetate, which is converted to fluorocitrate, which is a very electronegative atom.

the floor of fluorocitrate binds tightly to aconitase, this is a very tight bond, so the enzyme is unable to work in the Kreb’s cycle.

remember, that the Kreb’s cycle is important in the central nervous system, and if there is a high concentration of this compound, it will lead to the death of the organism it is exposed to.

lastly, we have arsenites, which react with cofactor lipoamide.

we talk about alpha ketoglutarate dehydrogenase complex

arsenates will bind and inactivate the coenzyme lipoamide needed, the enzyme that it will affect is transuccinylase

THIRD VIDEO.

krebs cycle = citric acid cycle= TCA cycle

in the previous video, we discussed the pyruvate dehydrogenase complex mechanism and important cofactors.

it is important to remember that one product of the PDC reaction was acetyl-CoA

the acetyl-CoA produced during the PDC reaction will be used during the KREBS cycle

this happens because pyruvate cannot directly enter the Krebs cycle.

the PDC mechanism will ensure that pyruvate is converted to acetyl-CoA, the key substrate for energy production in the krebs cycle.

specifically, in the first step of the krebs cycle, acetyl-CoA combines with oxaloacetate to form citrate, which will start the cycle.

for a little bit more background on the kreb’s cycle, it is also known as the tri-carboxylic acid cycle, or the citric acid cycle.

it is a central metabolic pathway, that occurs in the mitochondrial matrix. as mentioned previously, it’s major key role is to generate energy by oxidizing acetyl-CoA.

this acetyl-CoA can be obtained from multiple sources like carbohydrates, fats, proteins, and, as previously discussed, the PDC.

once oxidized, acetyl-CoA will be converted to carbon dioxide, and with this cycle, electrons are transferred into the carrier molecules.

moving onto the steps of the kreb’s cycle.

the first step of the kreb’s cycle begins with acetyl-CoA

the acetyl-CoA, which has two carbons, combines with oxaloacetate, which has four carbons, by the action of citrate synthase, to form citrate, a six carbon intermediate.

what is happening during this reaction is the methyl group of the acetyl-CoA reacts with the carbonyl group of the oxaloacetate, this is known as an aldol condensation followed by a hydrolysis of carbon dioxide.

the second step of the krebs cycle is the conversion of citrate to isocitrate. this conversion is done by the aconitase enzyme.

what is happening during this reaction is: the citrate will undergo a dehydration to form a cis aconitase, which is then hydrated into isocitrate.

this ensures that the molecule is more reactive for oxidation.

during the third step of the cycle, isocitrate is oxidized into alpha ketoglutarate via the isocitrate dehydrogenase.

what is happening here is the hydroxyl group of isocitrate is oxidized to a carbonyl group forming oxalosuccinate.

then oxalosuccinate undergoes decarboxylation which will release CO2 and form alpha-ketoglutarate.

during the fourth step of the cycle, alpha ketoglutarate will be oxidized to form succinyl CoA

what happens here is a decarboxylation or removal of a carbon group of alpha ketoglutarate, which will form succinyl.

then this succinyl is attached or oxidized to coenzyme A via the alpha ketoglutarate enzyme which will form succinyl CoA.

during the fifth step of the krebs cycle, succinyl CoA will converted into succinate.

what is happening in this reaction is that the succinyl CoA synthase will hydrolyze the high energy thioester bond in succinyl CoA.

This hydrolysis releases energy which helps with the ATP or GTP production depending on the tissue.

this step is highly significant given that it is the only one that directly will form ATP or GTP.

during the sixth step of the cycle, succinate will be oxidized to fumarate via the succinate dehydrogenase

what is happening here is unlike other steps of the Krebs cycle, the succinate dehydrogenase enzyme is located in the inner mitochondrial membrane

this allows the enzyme to have two functions,

1) oxidation of succinate, which means succinate will lose two hydrogen atoms and form fumarate

2) to work as part of the electron transport chain, the two removed hydrogen groups from succinate will be transferred to FAD to produce FADH2. then FADH2 will donate its electron directly to the electron transport chain.

during the seventh step in the cycle, fumarate will be hydrated into malate via the fumerase enzyme.

what is happening in this step is that the fumarase will add a hydroxyl group and a hydrogen atom across the double bond in fumarate.

this step is mainly in preparation for the final oxidation step.

during the eighth and last step of the cycle, malate will be oxidized into oxaloacetate via the malate dehydrogenase.

what is happening here is that the hydroxyl group is malate is oxidized to a carbonyl group, during this oxidation, a pair of electrons REMOVED from the hydroxyl group of malate to form oxaloacetate.

then, the removed electrons are transferred to NAD reducing it to NADH.

this will finalize one cycle of the kreb cycle, specially producing an overall of two molecules of CO2, three molecules of NADH, one molecule of FADH2 and one molecule of ATP.

it’s very important to also keep in mind that 2 acetyl-CoA molecules are produces from one glucose molecule, which means the kreb cycle will run twice per glucose. This will double the product amount.

Here, we include a table with a summary of all the steps of the krebs cycle and important details to remember.

moving onto the regulation of the kreb’s cycle, it will occur at key steps of the involving enzymes that will catalyze irreversible reactions.

these checkpoints ensure that the cycle functions efficiently based on the the cell’s energy status and substrate availability.

specifically, we will have enzyme regulation step one, which will be inhibited by high levels of ATP, NADH or citrate.

activation on the other hand will be due to high levels of ADP, oxaloacetate or acetyl-CoA

regulation will also occur on step 3, specifically, inhibition will occur again at high levels of ATP or NADH, while activation occurs by the presence of ADP or calcium ions.

more regulation can occur in step four, which the reaction of the alpha ketoglutarate dehydrogenase is inhibited by high levels of ATP, high NADH levels, and high succinyl CoA levels.

and activation will occur by high levels of of calcium ions, other regulatory mechanisms like allosteric regulation, feedback inhibition, and energy charge of the cells will inhibit or activate this cycle.

all of these are important to prevent overprodcution of ATP to ensure intermediate of other pathways and to maintain metabolic flexiblity.

and finally we include the table with disorders and diseases related to dysfunction of the kreb cycle.

cancer

The cancer row in the table is highlighting that mutations in certain Krebs cycle enzymes can contribute to tumor growth. These enzymes are IDH, SDH, and FH, which are normally part of the TCA (Krebs) cycle in mitochondria.

Let’s unpack it step-by-step.

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1. The enzymes involved

The table lists three enzymes:

IDH

Isocitrate Dehydrogenase

Reaction in the Krebs cycle:

Isocitrate→α-ketoglutarate+CO2+NADH\text{Isocitrate} \rightarrow \alpha\text{-ketoglutarate} + CO_2 + NADHIsocitrate→α-ketoglutarate+CO2​+NADH

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SDH

Succinate Dehydrogenase

Reaction:

Succinate→Fumarate\text{Succinate} \rightarrow \text{Fumarate}Succinate→Fumarate

This enzyme is also Complex II of the electron transport chain.

---

FH

Fumarate Hydratase

Reaction:

Fumarate→Malate\text{Fumarate} \rightarrow \text{Malate}Fumarate→Malate

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2. What happens when these enzymes mutate

Normally these enzymes help generate energy.

But mutations can cause metabolic rewiring that favors tumor growth.

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Mutated IDH

Mutated IDH produces an abnormal metabolite:

2-hydroxyglutarate (2-HG)

This molecule interferes with:

• DNA methylation

• gene regulation

• cell differentiation

This can push cells toward cancerous behavior.

IDH mutations are seen in:

• gliomas

• acute myeloid leukemia (AML)

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Mutated SDH

If succinate dehydrogenase is defective:

Succinate accumulates.

High succinate can activate hypoxia signaling pathways even when oxygen is present.

This stimulates:

• angiogenesis

• tumor growth.

SDH mutations are associated with:

• paragangliomas

• pheochromocytomas.

---

Mutated FH

If fumarate hydratase is defective:

Fumarate accumulates.

Excess fumarate can alter:

• cellular signaling

• oxidative stress pathways

• gene regulation.

FH mutations are linked to:

• renal cancers

• hereditary leiomyomatosis.

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3. Why altered metabolism helps cancer

Cancer cells often change their metabolism to support:

• rapid cell growth

• biosynthesis

• survival in low oxygen environments.

These metabolic changes are sometimes called oncometabolite pathways.

Examples of oncometabolites:

• 2-hydroxyglutarate

• succinate

• fumarate

These molecules alter gene expression and promote tumor growth.

---

4. Clinical approach (from the table)

Because these mutations are specific, doctors can use targeted therapies.

Example:

IDH inhibitors

These drugs block the abnormal enzyme activity and reduce production of 2-hydroxyglutarate.

---

Simple summary

Enzyme mutated	What accumulates	Effect
IDH	2-hydroxyglutarate	alters gene regulation
SDH	succinate	activates hypoxia signaling
FH	fumarate	alters cell signaling

All of these changes can promote tumor growth.

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✅ Key idea

The table shows that mutations in Krebs cycle enzymes can create abnormal metabolites that disrupt gene regulation and cellular metabolism, helping drive cancer development.

TCA mitochondrial disorders

The “Mitochondrial Disorders” row in the table is explaining diseases where the mitochondria do not function properly, which disrupts the Krebs cycle and ATP production. Since mitochondria are the main site of cellular energy production, problems with them can affect many organs.

Let’s break down what the table means.

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1. What mitochondrial disorders are

Mitochondrial disorders are diseases caused by defects in:

• mitochondrial DNA (mtDNA), or

• nuclear genes that encode mitochondrial proteins.

Because mitochondria produce ATP, these disorders mainly affect tissues that need a lot of energy.

Examples of high-energy tissues:

• brain

• muscles

• heart

• liver

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2. Krebs cycle connection (Defective ATP production)

The table says:

“Defective ATP production.”

This happens because mitochondria contain:

• the Krebs (TCA) cycle

• the electron transport chain

• oxidative phosphorylation

If mitochondria malfunction:

1. The Krebs cycle cannot efficiently produce NADH and FADH₂.

2. The electron transport chain cannot produce ATP efficiently.

3. Cells become energy deficient.

Result:

Low ATP → poor cellular function.

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3. Symptoms of mitochondrial disorders

Because the body lacks energy, symptoms often involve:

Neurologic problems

• seizures

• developmental delay

• stroke-like episodes

Muscle problems

• muscle weakness

• exercise intolerance

Organ dysfunction

• cardiomyopathy

• liver disease

• vision problems

---

4. Why mitochondria are especially vulnerable

Mitochondria have their own DNA:

mtDNA

Characteristics:

• inherited from the mother

• more susceptible to mutations

• codes for proteins involved in energy production.

Mutations in mitochondrial genes disrupt ATP synthesis.

---

5. Clinical approach (from the table)

The table lists treatments aimed at supporting mitochondrial function, not curing the genetic defect.

Ketogenic diet

Provides fat-derived ketone bodies as an alternative energy source for the brain.

Ketones can enter the Krebs cycle as acetyl-CoA.

---

CoQ10 (Coenzyme Q10)

CoQ10 is part of the electron transport chain.

It helps transport electrons between:

• Complex I / II

• Complex III.

Supplementation can improve mitochondrial electron transport.

---

Antioxidants

Defective mitochondria produce excess reactive oxygen species (ROS).

Antioxidants help reduce oxidative damage.

Examples:

• vitamin C

• vitamin E

• alpha-lipoic acid.

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6. Why this relates to the Krebs cycle

The Krebs cycle feeds electrons into the electron transport chain.

It produces:

• NADH

• FADH₂

If mitochondria are damaged:

• these molecules cannot generate ATP efficiently

• the Krebs cycle slows

• energy production collapses.

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Big picture

Problem	Result
mitochondrial dysfunction	low ATP
impaired Krebs cycle	less NADH / FADH₂
defective electron transport	poor oxidative phosphorylation
cells lack energy	tissue damage

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✅ Key idea

The table is showing that mitochondrial disorders disrupt the Krebs cycle and oxidative phosphorylation, leading to defective ATP production, which particularly harms energy-demanding tissues like the brain and muscles.

The Neurodegenerative Diseases row in the table explains how problems in the TCA (Krebs) cycle and mitochondrial metabolism can contribute to diseases where neurons gradually die or lose function.

Let’s connect the puzzle step-by-step.

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1. What neurodegenerative diseases are

Neurodegenerative diseases are disorders where neurons progressively deteriorate.

Common examples include:

• Alzheimer’s disease

• Parkinson’s disease

• Huntington’s disease

• ALS (amyotrophic lateral sclerosis)

These diseases mainly affect the brain and nervous system.

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2. Why neurons depend heavily on the TCA cycle

Neurons require huge amounts of ATP because they constantly maintain electrical signaling.

Energy is required for:

• Na⁺/K⁺ ATPase pumps

• neurotransmitter release

• axonal transport

• synaptic activity.

Most of this ATP comes from mitochondrial metabolism, specifically:

1. TCA (Krebs) cycle

2. Electron transport chain

3. Oxidative phosphorylation

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3. Role of the TCA cycle in neuronal energy

The TCA cycle generates:

• NADH

• FADH₂

These molecules deliver high-energy electrons to the electron transport chain.

This process produces the majority of ATP in neurons.

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4. What happens if the TCA cycle is dysfunctional

If the TCA cycle is impaired:

• less NADH and FADH₂ are produced

• electron transport slows

• ATP production decreases

This causes neuronal energy deficits, which is exactly what the table states.

Since neurons have extremely high energy demands, they are very sensitive to ATP shortages.

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5. Mitochondrial dysfunction in neurodegenerative diseases

Many neurodegenerative diseases involve mitochondrial damage.

Examples:

Parkinson’s disease

Defects in mitochondrial Complex I lead to reduced ATP and increased oxidative stress.

Alzheimer’s disease

Impaired mitochondrial metabolism and TCA cycle enzymes lead to energy deficits in neurons.

Huntington’s disease

TCA cycle dysfunction and mitochondrial damage contribute to neuronal death.

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6. Oxidative stress also increases

When mitochondria malfunction:

• reactive oxygen species (ROS) accumulate

• oxidative damage occurs in neurons.

This further damages:

• mitochondrial DNA

• proteins

• membranes.

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7. Clinical approach (from the table)

The table lists:

“Mitochondria-targeted therapies.”

These treatments aim to improve mitochondrial function and energy metabolism.

Examples include:

• antioxidants (reduce oxidative stress)

• mitochondrial cofactors (e.g., CoQ10)

• metabolic therapies that improve energy production.

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Big picture

Problem	Effect
TCA cycle dysfunction	less NADH/FADH₂
ETC slows	less ATP
neurons lack energy	neuronal damage
mitochondrial ROS	oxidative stress

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✅ Key idea

Neurodegenerative diseases are linked to TCA cycle and mitochondrial dysfunction, which reduces ATP production and increases oxidative stress, leading to neuronal energy deficits and progressive neuron loss.

TCA cycle diabetes

The Diabetes row in the table is explaining how diabetes affects the TCA (Krebs) cycle and cellular energy metabolism, and how some treatments help restore mitochondrial function.

Let’s connect the biochemistry puzzle.

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1. What diabetes is (metabolically)

Diabetes mellitus is a condition where:

• insulin is deficient (Type 1), or

• cells are resistant to insulin (Type 2).

Insulin normally allows cells to take up glucose and use it for energy.

When insulin signaling is abnormal:

• glucose metabolism is altered

• energy metabolism shifts toward fat metabolism.

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2. How diabetes affects the TCA cycle

The table says:

“TCA cycle imbalance affects glucose metabolism.”

This happens through several mechanisms.

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Reduced glucose utilization

Normally:

Glucose → glycolysis → pyruvate → acetyl-CoA → TCA cycle

In diabetes:

• cells cannot use glucose efficiently

• less pyruvate enters mitochondria

• the TCA cycle becomes imbalanced.

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Increased fatty acid metabolism

Because glucose cannot be used well, the body relies more on fatty acid oxidation.

Fat breakdown produces large amounts of:

acetyl-CoA

If too much acetyl-CoA accumulates:

• the TCA cycle becomes overloaded

• excess acetyl-CoA is diverted into ketone body production.

This is why uncontrolled diabetes can lead to:

diabetic ketoacidosis (DKA).

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Mitochondrial stress

High glucose and fatty acids can also cause:

• mitochondrial dysfunction

• increased reactive oxygen species (ROS).

This damages the electron transport chain and worsens ATP production efficiency.

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3. Why the TCA cycle imbalance matters

If the TCA cycle and mitochondria are stressed:

• ATP production becomes inefficient

• oxidative stress increases

• cells become metabolically dysfunctional.

This contributes to long-term diabetic complications such as:

• neuropathy

• nephropathy

• retinopathy

• cardiovascular disease.

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4. Clinical approach mentioned in the table

The table lists:

Metformin (enhances mitochondrial function).

Metformin is one of the most common treatments for Type 2 diabetes.

It works by:

• reducing glucose production in the liver

• improving insulin sensitivity

• modifying mitochondrial metabolism

• reducing excess glucose entering metabolic pathways.

This helps restore metabolic balance, including effects on the TCA cycle.

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5. Big picture connection

Normal metabolism	Diabetes metabolism
Glucose → TCA cycle → ATP	Glucose not used efficiently
Balanced acetyl-CoA supply	Excess acetyl-CoA from fat
Efficient ATP production	mitochondrial stress

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✅ Key idea

In diabetes, impaired glucose metabolism and increased fatty acid oxidation disrupt the TCA cycle and mitochondrial energy balance, contributing to metabolic stress and diabetic complications.

TCA cycle ischemia/hypoxia

The Ischemia / Hypoxia row in the table explains how lack of oxygen slows the TCA (Krebs) cycle and ATP production, which can damage tissues.

Let’s connect the metabolic puzzle step-by-step.

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1. What ischemia and hypoxia mean Ischemia

From Greek:

• isch- = to hold back

• haima = blood

Meaning: reduced blood flow to tissue.

Example:

• heart attack (myocardial infarction)

• stroke.

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Hypoxia

From Greek:

• hypo = low

• oxia = oxygen

Meaning: low oxygen levels in tissues.

Ischemia often causes hypoxia because blood carries oxygen.

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2. Why oxygen matters for the TCA cycle

The TCA cycle itself does not directly use oxygen, but it depends on oxygen indirectly.

Why?

Because the TCA cycle produces:

• NADH

• FADH₂

These molecules deliver electrons to the electron transport chain (ETC).

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3. Oxygen is the final electron acceptor

In the electron transport chain:

O2+electrons+H+→H2OO_2 + electrons + H^+ → H_2OO2​+electrons+H+→H2​O

Oxygen accepts electrons at Complex IV.

This allows:

• NADH → NAD⁺

• FADH₂ → FAD

These oxidized cofactors (NAD⁺ and FAD) are required for the TCA cycle to continue.

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4. What happens during hypoxia

If oxygen is low:

1. Electron transport chain stops

2. NADH cannot be oxidized to NAD⁺

3. NAD⁺ becomes depleted

Without NAD⁺, several TCA reactions stop:

• Isocitrate dehydrogenase

• α-ketoglutarate dehydrogenase

• Malate dehydrogenase

Result:

➡ TCA cycle slows or stops

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5. Energy crisis in the cell

If the TCA cycle stops:

• less NADH

• less oxidative phosphorylation

• ATP production collapses

Cells switch to anaerobic glycolysis, which produces:

lactate

This causes lactic acidosis.

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6. Why ischemia damages tissues

High-energy tissues are especially vulnerable:

• brain

• heart

• kidneys.

Without ATP:

• ion pumps fail

• calcium accumulates

• cells swell

• cell death occurs.

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7. Clinical approaches in the table Hyperbaric oxygen

Patient breathes high-pressure oxygen.

This increases oxygen delivery to tissues and restores:

• electron transport

• ATP production.

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Ischemic preconditioning

This is a protective strategy where tissues are exposed to short, controlled episodes of low oxygen.

This activates protective pathways that make tissues more resistant to severe ischemia later.

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Big picture connection

Step	What happens
Oxygen decreases	ETC stops
NADH accumulates	NAD⁺ decreases
TCA cycle slows	ATP production drops
Cells rely on glycolysis	lactate accumulates
Energy failure	tissue damage

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✅ Simple key idea

In ischemia or hypoxia, lack of oxygen stops the electron transport chain, preventing regeneration of NAD⁺ and FAD, which causes the TCA cycle to slow and ATP production to collapse, leading to cellular injury.