الله الرحمن الرحيم مسب - JUdoctors · 27-9-2012  · that was added to make Malonyl CoA from the carboxylation of Acetyl CoA, is now released. The Ketoacyl CoA,

Biochemistry Sheet 27 Fatty Acid Synthesis Dr. Faisal Khatib P

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بسم ميحرلا نمحرلا هللا

On Thursday, we discussed the synthesis of fatty acids and its regulation. We also went on

to talk about the synthesis of Triacylglycerol (TAG).

Last time, we started talking about the synthesis of fatty acids, so I will shortly recap before

explaining the topics discussed in this lecture.

The Requirements of fatty acid synthesis include the following:

1) Carbon Source: Acetyl CoA

2) Reducing Power: NADPH

3) Energy Input: ATP

The process of fatty acid synthesis starts off with the Carboxylation of Acetyl CoA to form

Malonyl CoA by a biotin containing enzyme called Acetyl CoA Carboxylase.

Notice that the pink CO2 was the one added during carboxylation of Acetyl CoA to Malonyl CoA.

All remaining steps of fatty acid synthesis are catalyzed by a multifunctional enzyme

complex called Fatty Acid Synthase. This enzyme is a dimer composed of two identical

chains, each of which has seven catalytic activities.

- One of the activities of this enzyme is the Condensing Enzyme portion that has a

reactive –SH group.

- One Domain is linked to Phosphopantetheine also with a reactive –SH group. This

domain carries Acyl intermediates such as Acetyl and Malonyl groups during

catalysis, it is thus known as the Acyl Carrier Protein (ACP).

The Acyl Carrier Protein carries an acetyl group and then transfers to the condensing

enzyme of Fatty Acid Synthase. Following the carboxylation of another acetyl group to

Malonyl CoA by Acetyl CoA Carboxylase as discussed earlier, this Malonyl CoA is transferred

to the Acyl carrier protein of Fatty Acid Synthase, which already has an acetyl group linked

to its condensing enzyme portion.

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Now that both the Acetyl CoA and Malonyl CoA are attached to different portions of the

same Fatty Acid Synthase Enzyme complex, condensation takes place by combining the

acetyl group with only 2 of the 3 carbons of Malonyl CoA, hence producing Ketoacyl ACP. In

other words, the CO2 that was added to make Malonyl CoA from the carboxylation of Acetyl

CoA, is now released.

The Ketoacyl CoA, which is a 4 Carbon unit attached to the Acyl Carrier Protein in Fatty Acid

Synthase, undergoes 3 reactions after condensation, finally resulting in a butyric acid; a 4

carbon fatty Acyl group.

1) Reduction by NADPH.

2) Dehydration by removal of water.

3) Another reduction by NADPH.

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This butyric acid is now transferred to the condensing enzyme of Fatty Acid Synthase.

Consequently, the Acyl Carrier Protein is now empty and so it takes on a new Malonyl CoA

group and the whole process of condensation takes place again; this time resulting in a 6

Carbon fatty Acyl group with a ketone group at carbon Beta (Carbon # 3).

This cycle of condensation, reduction, dehydration and lastly reduction is carried out

repeatedly until the fatty Acyl Palmitate (16 Carbon fatty acid) is formed.

Why does fatty acid Synthase put an end to the synthesis cycle at 16 Carbons?

This is because this enzyme has a specificity to produce a 16 Carbon fatty acid using a cycle

of the same steps. The production of a fatty acid, longer than 16 Carbons by Fatty Acid

Synthase, is NOT possible.

Following the production of Palmitate, hydrolysis of the high energy bond between the fatty

acid and ACP, takes place in order to release it.

Biochemistry Sheet 27 Fatty Acid Synthesis Dr. Faisal Khatib P

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Net reaction of Palmitate synthesis

*How many cycles of condensation? 7 cycles

Why? Since 16/2=8 – 1 because the first cycle results in the formation of a 4 Carbon fatty

Acyl group, after that 2 Carbons are added with every cycle.

*How many Malonyl CoA are used throughout the synthesis of Palmitate?

7 Malonyl CoA groups, since in the first cycle, condensation of acetyl CoA and Malonyl CoA

takes place, followed by the addition of 2 Carbons of the Malonyl group with every cycle.

*How many Acetyl CoA were used directly in the synthesis of Palmitate?

Only 1, as acetyl CoA is used in the first cycle only and it forms the methyl omega carbon.

Acetyl CoA carboxylated to Malonyl CoA are not directly utilized in the synthesis.

*How many NADPH were used?

14, since 2 NADPH are used in every cycle and we have 7 cycles.

Note by the doctor: All these numbers can be deduced by studying the reactions above, in

the exam he might give you an example of the synthesis of a 12 Carbon fatty acid for

example and ask you about the net information such as the ones provided above, so it is

important to know how to calculate the number of cycles and number of substrates used.

What is the source of Acetyl CoA for fatty synthesis?

*It is Pyruvate Dehydrogenase, from the metabolism of carbohydrates or

degradation of amino acids that are converted to pyruvate. When there is

excess carbohydrate or protein intake, the acetyl CoA is made available for fatty

acid synthesis.

*The Doctor emphasized the point that the source of Acetyl CoA for fatty acid

synthesis is NOT Beta Oxidation of fatty acids, since synthesis and degradation

cannot take place at the same time in the same location. Otherwise, it would be

a waste of energy. Synthesis and degradation can take place in different places

at the same time like glycolysis in muscle tissue and Gluconeogenesis in the

liver, but this is a special case. Thus, the synthesis and oxidation of fatty acids

should be regulated.

Biochemistry Sheet 27 Fatty Acid Synthesis Dr. Faisal Khatib P

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Production of Cytosolic Acetyl CoA for Fatty Acid synthesis:

Fatty Acid synthesis takes place in the cytosol, while Acetyl CoA production, from Pyruvate

Dehydrogenase, takes place in the mitochondria. Furthermore, the Inner Mitochondrial

membrane is impermeable to Acetyl CoA like it is to Acyl CoA, as demonstrated when we

took beta oxidation of fatty acids.

So the question here is, how does Acetyl CoA reach the cytosol for fatty acid synthesis?

First, Acetyl CoA condenses with Oxaloacetate to form Citrate, which is also the first step in

the Citric Acid Cycle. If the cell is in a high energy state, Citrate is not used in the Krebs Cycle

and thus leaves the mitochondria by means of a Citrate Carrier. The action of this Citrate

carrier depends on the concentration of Citrate in the mitochondria, if high enough; it

transports Citrate to the Cytosol where it is cleaved back to Acetyl CoA and Oxaloacetate.

*This is not the reverse of the condensation reaction (Acetyl CoA + OAA -> Citrate) that took

place in the mitochondria, although it might appear so chemically. This is because the

reformation or condensation to form Citrate in the CYTOSOL would require ATP. In other

words, the cleavage reaction of Citrate in the cytosol would require energy to reverse it to

the formation of citrate, like that in the mitochondria. Thus the irreversible exergonic

cleavage of Citrate in the cytosol to give Acetyl CoA, is not the opposite of the first reaction

of the Krebs Cycle.

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To validate this point even further, we have been accustomed to the fact that reverse

reactions usually utilize the same enzyme, this cleavage reaction in the cytosol uses a

different enzyme from the one used in the production of Citrate in the mitochondria.

The Names of the enzymes are:

- Citrate Synthase in the mitochondria.

- ATP Citrate Lyase; the enzyme responsible for the synthesis of Cytosolic Acetyl CoA

needed for fatty acid synthesis.

Another explanation for the abovementioned reaction , would be that the process of

linking Acetyl group to Coenzyme A while cleaving the acetate by lyase enzyme requires

ATP (for making the high energy bond) , but cleaving the citrate to acetic acid and OAA

doesn’t require any ATP , so energy is required ,because the reaction is actually not

reversible and you need to make it reversible .

Extra Notes:

- Lyases differ from other enzymes in that they require only one substrate for the

reaction in one direction, but two substrates for the reverse reaction.

- ATP Citrate Lyase is activated by Insulin.

After Acetyl CoA was made available in the cytosol for fatty acid synthesis, Oxaloacetate

should return to the Mitochondria, however it does not enter as such, as it is reduced to

Malate. This is the opposite of the last reaction of the Krebs cycle.

Theoretically speaking, Malate can enter the mitochondria, however sometimes according

to the Doctor; it can’t enter unless it undergoes oxidative decarboxylation to pyruvate. COO

is removed as CO2 and NADP is reduced to NADPH. This is similar to oxidative

decarboxylation in the citric acid cycle.

The enzyme for this reaction is Malate Dehydrogenase or more commonly known as Malic

Enzyme.

If Malate can enter the mitochondria as such, why is it converted by Malic enzyme to

pyruvate by the process of oxidative decarboxylation? This is because, during oxidative

decarboxylation, NADP is reduced to NADPH, which is needed during fatty acid synthesis.

8 out of the 14 NADPH required during fatty acid synthesis of Palmitate are produced in this

way when 8 Acetyl CoA leave the mitochondria.

After this, in the mitochondria, pyruvate is carboxylated to Oxaloacetate (requiring an ATP

molecule ), thus closing the cycle.

Biochemistry Sheet 27 Fatty Acid Synthesis Dr. Faisal Khatib P

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The figure on the right shows the reaction

explained on the previous page.

The Doctor made a comment about a

mistake in one of the old editions of

Lippincott.

The mistake was that during the

reduction of OAA to Malate, NADH is

oxidized to NAD+ and not the opposite.

Should you own one of the earlier

editions of Lippincott, correct this

mistake.

To summarize the net of what happens is:

1) The exit of Acetyl CoA from the mitochondria to the cytosol.

2) The production of NADPH by preventing Malate from returning to the mitochondria

before decarboxylating it to pyruvate.

3) 2 ATP consumption per Acetyl CoA, which is energetically costly, but this occurs during

high energetic state of the cell.

*Pyruvate Carboxylase catalyzes the carboxylation of pyruvate to OAA. The Doctor

mentioned that this is an Anaplerotic reaction; which are ‘fill up’ reactions that form

intermediates of metabolic pathways such as the TCA cycle.

Now that we’ve finished both the synthesis and degradation of fatty acids, let’s take a look at

the regulation of these two processes.

Regulation of Fatty Acid Metabolism

The 2 processes should be separated, as no synthesis should take place during degradation

and vice versa. Otherwise, energy would be lost since the degradation does not produce

ATP while the synthesis requires ATP for Fatty Acid Synthase and transporting Acetyl CoA.

Thus, these two processes should be strictly coordinated and regulated.

There are mechanisms for the regulation of oxidation and others for the regulation of

synthesis. Let’s start with the regulation of synthesis.

Biochemistry Sheet 27 Fatty Acid Synthesis Dr. Faisal Khatib P

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Regulation of Synthesis of Fatty Acids

The limiting or committed step in fatty acid synthesis is carboxylation of Acetyl CoA

catalyzed by Acetyl CoA Carboxylase.

There are 3 methods for regulating this step and enzyme:

1) Allosteric Regulation (seconds)

2) By phosphorylation mechanism (minutes)

3) Amount of enzymes (long term)

1) Allosteric Regulation of the enzyme Acetyl CoA Carboxylase:

This takes place within a very short period of time (seconds).

*Active Form: Polymer converts Acetyl CoA to Malonyl CoA

Inactive Form: Dimer

*Activator: Citrate

Inhibitor: Long Chain Fatty Acyl CoA

The significance of stimulation of this enzyme by Citrate is that Citrate is made available to

the pathway of fatty acid synthesis when the cell is in high energy state and here is

abundance of building blocks. Thus, when the concentration level of Citrate is high during

fed state, Acetyl CoA Carboxylase is activated into its polymer form and fatty acid synthesis

is stimulation.

On the other hand, Fatty Acyl CoA or Palmitoyl CoA to be more specific is a long chain fatty

acid or Fatty Acyl CoA group (LCFA). This works as an inhibitor by feedback inhibition of

the very early committed step.

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2) Covalent Modification and Regulation by Phosphorylation of the enzyme Acetyl CoA

Carboxylase:

This requires more time than the Allosteric regulation (minutes).

cAMP – dependant protein Kinase converts the active form to inactive form by phosphorylation.

Active Form: Dephosphorylated

Inactive Form: Phosphorylated

This is stimulated by Glucagon and Epinephrine, when there is low blood glucose, the levels

of these hormones rise and thus, fatty acid synthesis is stopped.

Remember the rule of thumb: phosphorylation corresponds to conserving

Glucose, so fatty acid synthesis is stopped.

How does the enzyme return to its active form (Dephosphorylated and polymerized)?

The enzyme Phosphatase dephosphorylates it, stimulated by Insulin when blood glucose

level is high, so fatty acid synthesis resumes.

An Insulinoma is a tumor of the insulin producing cells. Upon formation, it causes the

person to gain weight very quickly, as insulin is continuously produced by the tumor cells,

so Acetyl CoA Decarboxylase is always activated and performing fatty acid synthesis, thus

keeping blood glucose level low and creating the hungry sensation. This leads to obesity at a

very high rate.

Biochemistry Sheet 27 Fatty Acid Synthesis Dr. Faisal Khatib P

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3) Regulation of Fatty Acid Synthesis not only Acetyl CoA Dehydrogenase, according to the

amount of enzymes:

The last method of regulation of fatty acid synthesis involves the amount of enzymes

present in the body. If the fed state persists, in other words whenever the person feels a

little hungry he eats, the number of enzymes involved in fatty acid synthesis increase to

accommodate for the high calorific intake.

Enzymes including Fatty Acid Synthase, Malic Enzyme, Glucose-6-Phosphate

Dehydrogenase, among others increase in number in the body. The number of enzymes, that

produce NADPH needed for fatty acid synthesis, also increase.

Regulation of Oxidation of Fatty Acids

There are also 3 mechanisms regulating the oxidation of fatty acids, they mostly involve the

substrate presence or absence.

1) Supply of Fatty Acids

2) Availability of NAD+

3) Entry into the mitochondria

1) If the fatty acid level increases, then their activation into Fatty Acyl CoA increases,

followed by increased transport of Fatty Acyl group by Carnitine shuttle into the

mitochondria, then Fatty Acyl CoA is oxidized by the 3 steps we studied earlier.

When Glucagon stimulates Lipase, fatty acids are mobilized from adipose tissue. When there

is a high concentration of fatty acids in the blood, the rate of oxidation increases.

(Proportional)

2) The availability of NAD+

Oxidation of fatty acids needs NAD+. NAD+ is either present oxidized or reduced in cells. If all

the NAD+ is reduced totally to NADH, then NAD+ is not available for fatty acid oxidation. This

is an indirect way of regulating fatty acid oxidation. NADH is rapidly reoxidized by the

electron transport chain (ETC) in order to make NAD+ available.

Fatty acid available corresponds to higher oxidation. NAD+ available, also a substrate whose

availability participates in the regulation of fatty acid oxidation.

3) Malonyl CoA acts as an inhibitor for the entrance of Fatty Acyl to the mitochondria by

inhibiting its attachment to Carnitine shuttle. Malonyl CoA is an intermediate during

synthesis of fatty acids, thus it inhibits fatty acid oxidation by blocking transfer of fatty acyl

to mitochondria. Even though fatty Acyl is available, it does not enter the mitochondria and

does not undergo oxidation. This is direct regulation between synthesis and oxidation, as

one of the intermediates of synthesis is inhibiting the oxidation.

Biochemistry Sheet 27 Fatty Acid Synthesis Dr. Faisal Khatib P

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*The Doctor then read an article about the discovery of the Acetyl CoA Decarboxylase

enzyme to reduce fatty acid synthesis, and its importance in discovering drugs for managing

obesity, which is now a major problem in developed and even developing countries. He also

mentioned that most drugs inhibit enzymes and do not stimulate them.

We will now move to the topic of fatty acid modification which includes both elongation and

desaturation (introducing double bonds).

Elongation of Fatty Acids

Fatty acid synthesis involves reaching a fatty acid of up to 16 Carbons, so the question is

how do we obtain fatty acids which are longer than 16 Carbons?

- Elongation does not take place in the cytoplasm, it taken place in the endoplasmic

reticulum; it involves a similar sequence of reactions (method and cofactors) but with

different enzymes. This is because the specificity of fatty acid Synthase enzyme stops at 16

Carbons. The Endoplasmic Reticulum is a membranous structure so the products of fatty

acid synthesis enter it directly for elongation.

- Elongation can take place in the mitochondria. The reactions involved are similar to those

of Beta oxidation but are opposite. However the last reaction that reduces FAD to FADH2

instead oxidizes NADPH to NADP, because synthesis requires NADPH and because FADH2 is

fixed and does not leave the enzyme. The enzyme involved in this step is also different as

demonstrated in the figure.

If elongation takes place in the mitochondria, fatty acids with less than 16 Carbons are used.

Those are obtained from the food we eat ex. 6 Carbon fatty acid. In order to store these

short fatty acids in the body, they must be elongated in the mitochondria. They do not go to

fatty acid Synthase. This involves similar sequence of steps but with modification.

Biochemistry Sheet 27 Fatty Acid Synthesis Dr. Faisal Khatib P

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Desaturation of Fatty Acids

This involves the introduction of double bonds to fatty acids in the endoplasmic reticulum.

Let’s start with the synthesis of monounsaturated fatty acids.

Taking Oleic Acid (18 Carbons) and Palmitoleic Acid (16 Carbons) as examples because they

both have a single double bond at carbon number 9.

How are monounsaturated fatty acids produced?

Example: The conversion of Stearoyl CoA (18:0) to Oleoyl CoA (18:1), or Palmitoyl (16:0)

CoA converted Palmitoleoyl CoA (16:1)

Rule of thumb: No double bonds can be introduced beyond Carbon number 9

in human cells. We can’t synthesize fatty acids with double bonds at carbon

number 12 and 15 for example. Oleic and Palmitoleic Acids can be synthesized

because the double bond is at Carbon number 9. Fatty Acids, with double

bonds on carbons higher than 9, must be ingested from dietary sources. Thus

the synthesis of monounsaturated fatty acids is possible in humans, because

they can be at Carbon 9.

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This is done by Hydroxylation or introducing a hydroxyl group followed by dehydration

(removal of H2O) to produce a double bond. This is done by enzyme Δ9 Desaturase,

Cytochrome b5 which requires NADPH and O2.

We are oxidizing the fatty acid by adding hydroxyl group, and NADPH is required despite its

being a reductant. This is because oxygen (O2) is used as a source for the oxygen atom in

hydroxyl group, thus NADPH reduces the other oxygen to prevent free radical formation.

Cytochrome b5 transfers 2 electrons to the other oxygen atom in order to reduce it, while

the other oxygen atom is introduced into fatty acid.

Formation and Modification of Polyunsaturated Fatty Acids (PUFA)

We can’t synthesize PUFA’s but we can modify them by elongation and desaturation. This is

done by addition of double bonds at Carbon 4, 5, or 6 but not after Carbon number 9.

- Linoleic Acid (18:2) has 2 double bonds at Carbon 9 and 12. If we want to add a double

bond, the double bond is introduced at Carbon number 6 not 7 or 5 because the difference

between double bonds must be 3 carbons. Desaturase enzyme adds double bonds at 6,9,12.

- If elongated, it becomes a 20 carbon fatty acid, each double bond pushed by 2 C’s.

Thus it is now (20:3 Δ 8, 11, 14). The omega classification is still the same ω6.

- Another desaturation at carbon 5 takes place now, not at 6 since 8-3=5.

The fatty acid becomes (20:4 Δ 5, 8, 11, 14) which is Arachidonic acid.

Note: The Omega classification does not change upon modification, because modification

does not take place from the omega side. Thus, fatty acids are divided into omega

classification families such as the ω 3 family, the ω 6 family, and the ω 9 family.

Biochemistry Sheet 27 Fatty Acid Synthesis Dr. Faisal Khatib P

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The last topic for this lecture is the biosynthesis of triacylglycerol:

Triacylglycerol (TAG) is composed of a glycerol esterified to 3 fatty acids.

Triacylglycerol and phosphoacylglycerol

are very similar in structure, but the latter

has phosphoric acid esterified to the third

carbon of glycerol instead of a third fatty

acid.

Owing to the huge similarity in their

structures, their pathways of synthesis

should logically share many common

steps.

Phosphatidic acid is a common intermediate for the synthesis of both, despite the fact that

TAG has no phosphate group.

Biosynthesis of Triacylglycerol requires:

- Adding Fatty Acids in their active form: Acyl CoA

- Glycerol phosphate

The active form of Acyl CoA is used because Fatty acids can’t be added as such, they must be

activated so that breaking the high energy thioester bond between Acyl and CoA releases

energy needed to bind DAG and Acyl.

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- Hydrolysis of TAG produces Diacylglycerol (DAG), ΔG= –ve exergonic.

- DAG + FA ΔG=+ve endergonic

Fatty acid as Acyl CoA has a high energy bond which when broken releases a lot of energy

needed to transfer the fatty acid to Diacylglycerol. This is similar to the synthesis of

acetylcholine.

DAG + Acyl CoA gives TAG which has ΔG= -ve, that’s why the active form is needed.

Now let’s look at this in a step wise manner;

1) Acyl CoA is transferred to Glycerol 3-phosphate, whereby the fatty acid is transferred to

carbon number 1, hence producing a derivative of Phosphatidic acid called

Lysophosphatidic acid.

*Lysophosphatidic Acid differs from Phosphatidic Acid in that it does not have a fatty acid at

carbon number 2.

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2) Next would be the transfer of another Acyl group to carbon number 2 of lysophosphatidic

Acid, thus producing Phosphatidic acid; a common intermediate between TAG and

phosphoacylglycerol. This Phosphatidic Acid has 2 fatty acids and a phosphoric acid as

mentioned earlier.

3) The next step in TAG synthesis would be the removal of phosphate or phosphoric acid by

an enzyme called Phosphatase (not phosphorylase). In this manner, Diacylglycerol is

produced.

4) Then addition of Acyl group to DAG gives Triacylglycerol.

Note: The enzyme used in all these steps except the removal of phosphate group is Acyl

Transferase. Step number 3 uses enzyme Phosphatidate Phosphatase.

How is glycerol 3-phosphate produced/obtained?

By the phosphorylation of glycerol using an enzyme called Glycerol Kinase.

Note: This does not take place in adipose tissue; there is another method by which glycerol

3-phosphate is obtained.

*Dihydroxyacetone (DHAP); an intermediate of glycolysis, is used to make glycerol

phosphate by using NADH. We took this in the mobilization of TAG.

Enzyme used: glycerol 3-phosphate Dehydrogenase.

This concludes this very long lecture, good

luck everyone!

Done By: Zeina Hani Kalaji