Fatty Acid Synthesis - General

Question 1

What are the three steps of fatty acid synthesis?

Answer 1

1. Acetyl CoA, the building block for fatty acids, is shuttled from the mitochondria to the cytosol where fatty acid synthesis occurs.
2. Acetyl CoA is activated to form malonyl CoA.
3. Palmitate is synthesized in a five-step elongation cycle.

Question 2

What problem does acetyl CoA pose in regards to the mitochondrial membrane? How is this problem solved (4 steps)?

Answer 2

The mitochondrial membrane is not permeable to acetyl CoA.

Steps:

1. Acetyl CoA reacts with oxaloacetate to form citrate by the enzyme citrate synthase. The citrate can enter the CAC; but if the energy needs of the cell have been met, the citrate can be shuttled out of the mitochondria through a transport protein in the mitochondrial membrane.
2. Citrate in the cytosol is acted upon by ATP-citrate lyase, cleaving citrate into acetyl CoA and oxaloacetate. Acetyl CoA has now been brought into the cytosol at the expense of an ATP.
3. The oxaloacetate is reduced to malate using NADH. Malate is then converted to pyruvate by malic enzyme which produces a molecule of NADPH.
4. Pyruvate is able to be transported back into the mitochondria and converted by a carboxylation reaction to oxaloacetate catalyzed by pyruvate carboxylase, which can combine with more acetyl CoA to form citrate.

Question 3

What is the active form of acetyl CoA? What enzyme catalyzes this change? What are the steps?

Answer 3

Malonyl CoA, a combined form of acetyl CoA and bicarbonate. This is catalyzed by acetyl CoA carboxylase and occurs in two steps:

1. Biotin in aC-carboxylase is carboxylated and coupled with ATP hydrolysis which drives the reaction forward.
   
   • Biotin-enzyme + ATP + HCO₃- → CO₂-biotin + ADP + P_i + H+
2. The carboxyl group is transferred from biotin to acetyl CoA to form malonyl CoA, a 3-carbon molecule.
   
   • ​CO2-biotin + acetyl CoA → malonyl CoA + biotin-enzyme

Question 4

How do odd-chain fatty acids differ in assembly?

Answer 4

Odd-chained fatty acids are synthesized by beginning with propionyl CoA, a 3-carbon molecule, rather than acetyl CoA.

Question 5

What is the main difference between the fatty acid synthases between the ones in bacteria and plants, the ones in yeast, and the ones in vertebrates?

Answer 5

• In bacteria and plants, the fatty acid synthase complex is made up of individual proteins that each contain a separate catalytic activity.
• In yeast, all of the catalytic activities are present in two polypeptides.
• In vertebrates, all of the enzyme activities are contained within one large polypeptide.

Question 6

How many carbons does each cycle in the synthesis of palmitate add?

Answer 6

2 carbons.

Question 7

How many steps are required to synthesize palmitate? What enzyme system catalyzes these steps?

Answer 7

Four steps are required. A large enzyme system called fatty acid synthase performs the task of synthesizing fatty acids.

Question 8

What is the important structural component of the fatty acid synthase complex and how does this aid the enzyme?

Answer 8

The important component of the fatty acid synthase complex is the acyl carrier protein (ACP). ACP has a phosphopantetheine prosthetic group linked to it (also a major component of Coenzyme A). The phosphopantetheine group has a free sulfhydryl group, and it is to this group that the intermediates of fatty acid synthesis are linked.

ACP thus acts as an arm, which moves the fatty acid intermediate from one enzyme to the other as it synthesizes palmitate.

Question 9

What is the first step of palmitate synthesis? What enzyme is involved? Describe the reactions.

Answer 9

Condensation reaction catalyzed by β-ketoacyl synthase

The acetyl group from acetyl ACP reacts with malonyl ACP to form acetoacetyl ACP (4-carbons), with the release of CO₂ and the ACP from acetyl ACP. There is a substantial release of energy which drives this reaction forward and makes it energetically favorable.

Question 10

What is the acetoacetyl group linked to in the first reaction of the pathway? What is the role of this group?

Answer 10

The acetoacetyl group is physically linked to ACP, as all intermediates in this pathway are.

The ACP “arm” moves the intermediates from one enzyme to the next.

Question 11

What is the second step of palmitate synthesis? What enzyme is involved? Describe the reactions. What must occur to fully reduce the carbon?

Answer 11

Reduction step catalyzed by β-ketoacyl reductase

• Acetoacetyl ACP is reduced to 3-hydroxybutyryl ACP using NADPH as the reducing agent.
  
  • The keto group is now a hydroxyl group as a result of the reduction.

In order to fully reduce the carbon, the oxygen has to be gotten rid of.

Question 12

What is the third step of palmitate synthesis? What enzyme is involved? Describe the reactions.

Answer 12

​Dehydration step catalyzed by 3-hydroxyacyl dehydratase

• In this step, a water molecule is removed (thus a dehydration), forming crotonyl ACP.
  
  • This removes the oxygen group and leaves a double bond.

Question 13

What is the fourth step of palmitate synthesis? What enzyme is involved? Describe the reactions. What is the final product of these 4 reactions? How many more rounds must occur before palmitate is formed?

Answer 13

Reduction step catalyzed by enoyl reductase.

• The double bond in crotonyl ACP is reduced through the consumption of another molecule of NADPH, to form butyryl ACP

​At this point, a 4-carbon molecule has been formed. Therefore, 6 more rounds have to occur before palmitate is formed.

Question 14

What must happen to acetyl CoA and malonyl CoA before fatty acid synthesis can occur? What enzymes catalyze this?

Answer 14

Acetyl CoA and malonyl CoA are both linked to ACP via the sulfhydryl group to form acetyl ACP and malonyl ACP, respectively. The enzymes that catalyze these reactions are acetyl transacylase and malonyl transacylase, respectively.

Question 15

If you take butyryl ACP and replace acetyl CoA with it at the top of the pathway, how will the pathway occur?

Answer 15

Butyryl ACP undergoes condensation with another malonyl ACP, to generate a 6-carbon long acyl molecule linked to ACP.

The next keto group is then completely reduced by the four reactions mentioned above and so on, until a 16-carbon long molecule is formed that has all carbons fully reduced except carbon 1 which is a carboxyl group and linked to ACP by a thioester bond. This molecule is palmitoyl-ACP.

Question 16

Once a complete fatty acid has been synthesized, what must occur to free it? What enzyme catalyzes this and by what mechanism?

Answer 16

In order to complete the synthesis of a free fatty acid, palmitate has to be released from ACP. This is accomplished by the action of an enzyme called thioesterase, which hydrolytically (using H₂O) cleaves the link between the carboxyl group of the fatty acid and the sulfhydryl group of ACP

Question 17

How many NADPH molecules are required for the synthesis of one palmitate? What are the two major sources of NADPH for the synthesis of fatty acids? How many molecules do each of these methods contribute to the NADPH needed?

Answer 17

14.

1. Pentose Phosphate Pathway
2. When acetyl CoA was transported from the mitochondrial matrix to the cytosol so that it could be used as a substrate for fatty acid synthesis, a molecule of NADPH is also produced in the same cellular compartment. This is accomplished through the reduction of oxaloacetate to malate using NADH, followed by the oxidative decarboxylation of malate to pyruvate, which generates a molecule of NADPH.

Since eight molecules of acetyl CoA are required to make palmitate, 8 molecules of NADPH required for palmitate synthesis come through the transport of acetyl CoA into the cytosol. The other 6 molecules required come from the Pentose Phosphate Pathway.

Question 18

How is fatty acid metabolism altered in tumor cells? What does this difference allow therapeutically?

Answer 18

Because they grow very fast, their need for fatty acids is much greater and thus they have higher rates of fatty acid synthesis. This offers opportunities for therapeutic intervention to inhibit cancer cell growth. Inhibitors of β-ketoacyl ACP synthase, which catalyzes the first condensation step of fatty acid synthesis, inhibit the growth of some tumor cells and induce cell death.

Question 19

How are fatty acids elongated and desaturated? Where is this located?

Answer 19

Longer fatty acids are made by adding 2-carbon units to the carboxyl end of both saturated and unsaturated fatty acids, using malonyl CoA as the substrate and reactions almost analogous with those carried out by the fatty acid synthase complex. The specialized enzymes required to do these processes are located on the cytoplasmic side of the ER membrane.

Question 20

What enzyme converts stearate to oleate (contains a double bond) and what does it use?

Answer 20

An oxidase that uses molecular oxygen (O₂) and NADH.

Question 21

Which two essential fatty acids are must be provided by our diet? What are these also called? What issue in mammals do they solve?

Answer 21

Linoleate (18:2 cis-∆⁹, ∆¹²) and linolenate (18:3 cis-∆⁹, ∆¹², ∆¹⁵). These are referred to as omega fatty acids. Mammals lack the ability to introduce double bonds beyond carbon 9 and we need unsaturated fatty acids that have double bonds after carbon 9 that must be found in our diet.

Question 22

What do omega fatty acids serve as a precursor for? Give the example given in the notes.

Answer 22

They act as precursors for other unsaturated fatty acids, some of which are important hormones. One of these is arachidonate, a fatty acid that has four double bonds.

Question 23

What is arachidonate synthesized from? What does it serve as a precursor for and what do these molecules do?

Answer 23

It can be synthesized from linoleate. Arachidonate is the precursor for a group of signalling molecules called eicosanoids.

• Eicosanoids are local hormones, acting on cells in close proximity
• Functions: inflammation, blood flow, ion transport, synaptic transmission, sleep, and many more.

Question 24

How does Aspirin work on a cellular level?

Answer 24

Aspirin promotes the addition of an acetyl group to an amino acid in the active site of the enzyme that catalyzes the conversion of arachidonic acid to prostaglandin H₂. This inactivates the enzyme, which reduces the synthesis not only of PGH₂ but of all the other eicosinoids downstream of PGH₂ including other prostaglandins, prostacyclins, and thromboxanes.

Question 25

Which enzyme is the key regulator of fatty acid metabolism? When is it inhibited and when is it activated?

Answer 25

Acetyl CoA carboxylase (acetyl CoA → malonyl CoA)

• Activated: when there is a large amount of glucose and a large flux through glycolysis (carbohydrates plentiful and fatty acids low)
• Inhibited: energy levels are low and there is a need for fatty acid breakdown

Question 26

What protein inhibits the activity of acetyl CoA carboxylase and how does it do this? When is this protein active and when is it inactivated?

Answer 26

AMP-activated protein kinase (AMPK) phosphorylates acetyl CoA carboxylase which inhibits the enzyme.

• Activated: low energy conditions (AMP levels are high).
• Inactivated: energy state of the cell is high (ATP inhibits AMPK)

Question 27

What protein reverses the inhibition of acetyl CoA carboxylase? How?

Answer 27

Ever-present protein phosphatase 2A, which dephosphorylates acetyl CoA carboxylase putting it in a more active state

Question 28

What molecule activates acetyl CoA carboxylase? How does it do this? What molecule reverses this effect?

Answer 28

Citrate (represents high energy state).

• The inactive form of acetyl CoA carboxylase is a dimer of identical subunits.
• Citrate induces polymerization of the dimers into long, active filaments, through assistance by a protein called MIG12.
• Citrate can partially reverse the inhibition caused by phosphorylation of the enzyme.

Palmitoyl CoA, a fatty acyl CoA, induces depolymerisation of acetyl CoA carboxylase into inactive dimers.

Question 29

What 3 hormones regulate acetyl CoA carboxylase activity?

Answer 29

Stimulation

• Insulin: causes the activity of acetyl CoA carboxylase to increase by both inhibiting AMPK and stimulating phosphatase 2A.The net effect is to shift most of the acetyl CoA carboxylase into the active form.

Inhibition

• Glucagon and Epinephrine: mechanism is unclear but it uses AMPK.​