Fatty Acid Oxidation

A 16-C fatty acid with numbering conventions is shown.

Most naturally occurring fatty acids have an even number of carbon atoms & unsaturated fatty acids are in the cis configuration

The pathway for catabolism of fatty acids is referred to as the -oxidation pathway, because oxidation occurs at the -carbon (C-3).

C

O

O1

23

4

fatty acid with a cis-9 double bond

Triacylglycerols (triglycerides) are the most abundant dietary lipids.

Each triacylglycerol has a glycerol backbone to which are esterified 3 fatty acids

Most triacylglycerols are “mixed.” The 3 fatty acids differ in chain length & number of double bonds.

g ly c e ro l fa tty a c id tr ia c y lg ly c e ro l

H 2 C

HC

H 2 C

OH

OH

OH

H 2 C

HC

H 2 C

O

O

O

C R

O

C

C R

OR

O

HO C R

O

Lipases hydrolyze triacylglycerols, yielding glycerol and three fatty acids

g ly c e ro l fa tty a c id tr ia c y lg ly c e ro l

H 2 C

HC

H 2 C

OH

OH

OH

H 2 C

HC

H 2 C

O

O

O

C R

O

C

C R

OR

O

HO C R

O

Free fatty acids are transported in the blood bound to albumin, a plasma protein produced by the liver.

Several proteins have been identified that facilitate transport of long chain fatty acids into cells

C

O

O1

23

4

fatty acid with a cis-9 double bond

Fatty acid activation:

Acyl-CoA Synthases (Thiokinases) of ER & outer mitochondrial membranes catalyze activation of long chain fatty acids, esterifying them to coenzyme A.

This process is ATP-dependent, & occurs in 2 steps.

There are different Acyl-CoA Synthases for fatty acids of different chain lengths. 

Acyl-CoA Synthases

Exergonic PPi (P~P) hydrolysis, is catalyzed by Pyrophosphatase

2 ~P bonds of ATP are cleaved.

The acyl-CoA product includes one "~" thioester linkage.

Fatty acid activation

N

NN

N

NH2

O

OHOH

HH

H

CH2

H

OPOPOP O

O

O O

O O

O

N

NN

N

NH2

O

OHOH

HH

H

CH2

H

OPOC

O

O

R

O

SCR

O

CoA

R C

O

O

PPi

CoA SH

AMP

2 Pi

fatty acid

ATP

acyl- adenylate

acyl-CoA

Summary of fatty aid activation:

fatty acid + ATP + HS-CoA acyl-CoA + AMP + 2 Pi

Fatty acyl-CoA formed outside can pass through the outer mitochondrial membrane, but cannot penetrate the inner membrane.

-Oxidationpathway inmatrix

Fatty acyl-CoA formed in cytosol by enzymesof outer mitochondrial membrane & ER

Mitochondrion

Fatty acid-oxidation is considered to occur in the mitochondrial matrix.

Fatty acids must enter the matrix to be oxidized.

Carnitine Palmitoyl Transferases catalyze transfer of a fatty acid between the thiol of Coenzyme A and the hydroxyl on carnitine.

H3C N CH2 CH CH2

CH3

CH3

OH

COO+

R

C

SCoA

O+

H3C N CH2 CH CH2

CH3

CH3

O

COO+

C

R

O

+ HSCoA

carnitine

fatty acyl carnitine

Carnitine Palmitoyl Transferase

Transfer of the fatty acid across the inner mitochondrial membrane involves carnitine. 

Carnitine-mediated transfer of the fatty acyl into the mitochondrial matrix is a 3-step process:

1. Carnitine Palmitoyl Transferase I, an enzyme on the cytosolic surface of the outer mitochondrial membrane, transfers a fatty acid from CoA to the OH on carnitine.

2. An antiporter in the inner mitochondrial membrane mediates exchange of carnitine for acylcarnitine.

cytosol mitochondrial matrix O O

R-C-SCoA HO-carnitine HO-carnitine R-C-SCoA HSCoA R-C-O-carnitine R-C-O-carnitine HSCoA

O O

1 2

3

3. Carnitine Palmitoyl Transferase II, an enzyme within the matrix, transfers the fatty acid from carnitine to CoA. (Carnitine exits the matrix in step 2.)

The fatty acid is now esterified to CoA in the matrix.

cytosol mitochondrial matrix O O

R-C-SCoA HO-carnitine HO-carnitine R-C-SCoA HSCoA R-C-O-carnitine R-C-O-carnitine HSCoA

O O

1 2

3

Control of fatty acid oxidation is exerted mainly at the step of fatty acid entry into mitochondria.

Malonyl-CoA (which is also a precursor for fatty acid synthesis) inhibits Carnitine Palmitoyl Transferase I.

Malonyl-CoA thus inhibits fatty acid oxidation by preventing its transport into mitochondria.

H3C C SCoA

O

CH2 C SCoA

O

OOC

acetyl-CoA

malonyl-CoA

bond between carbon atoms 2 & 3.

There are different Acyl-CoA Dehydrogenases for short (4-6 C), medium (6-10 C), long and very long (12-18 C) chain fatty acids.

H3C(CH2)nCCCSCoA

H

H

H

HO

123

H3C(CH2)nCCCSCoA

H

HO

H3C(CH2)nCCH2CSCoA

OH

O

H2O

FADH2

FAD

H

H3C(CH2)nCCH2CSCoA

OO

H+ + NADH

NAD+

CH3CSCoA

O

H3C(CH2)nCSCoA +

O

HSCoA

fatty acyl-CoA

trans-2-enoyl-CoA

Acyl-CoA Dehydrogenase

-Oxidation Pathway:

Step 1. Acyl-CoA Dehydrogenase catalyzes oxidation of the fatty acid of acyl-CoA to produce a double

FAD is the prosthetic group that functions as eacceptor for Acyl-CoA Dehydrogenase.

The reaction is stereospecific, yielding a trans double bond in enoyl-CoA.

H3C(CH2)nCCCSCoA

H

H

H

HO

123

H3C(CH2)nCCCSCoA

H

HO

H3C(CH2)nCCH2CSCoA

OH

O

H2O

FADH2

FAD

H

H3C(CH2)nCCH2CSCoA

OO

H+ + NADH

NAD+

CH3CSCoA

O

H3C(CH2)nCSCoA +

O

HSCoA

fatty acyl-CoA

trans-2-enoyl-CoA

Acyl-CoA Dehydrogenase

Step 2.

Enoyl-CoA Hydratase catalyzes stereospecific hydration of the trans double bond produced in the 1st step, yielding L-hydroxyacyl-Coenzyme A.

H3C (CH2)n C C C SCoA

H

H

H

H O

123

H3C (CH2)n C C C SCoA

H

H O

H3C (CH2)n C CH2 C SCoA

OH

O

H2O

FADH2

FAD

H

H3C (CH2)n C CH2 C SCoA

OO

H+ + NADH

NAD+

CH3 C SCoA

O

H3C (CH2)n C SCoA +

O

HSCoA

fatty acyl-CoA

trans-2-enoyl-CoA

3-L-hydroxyacyl-CoA

Acyl-CoA Dehydrogenase

Enoyl-CoA Hydratase

H3C (CH2)n C C C SCoA

H

H

H

H O

123

H3C (CH2)n C C C SCoA

H

H O

H3C (CH2)n C CH2 C SCoA

OH

O

H2O

FADH2

FAD

H

H3C (CH2)n C CH2 C SCoA

OO

H+ + NADH

NAD+

CH3 C SCoA

O

H3C (CH2)n C SCoA +

O

HSCoA

3-L-hydroxyacyl-CoA

-ketoacyl-CoA

fatty acyl-CoA acetyl-CoA (2 C shorter)

Hydroxyacyl-CoA Dehydrogenase

-Ketothiolase

Step 3.

Hydroxyacyl-CoA Dehydrogenase catalyzes oxidation of the hydroxyl in the position (C3) to a ketone.

NAD+ is the electron acceptor.

Acetyl-CoA is released, leaving the fatty acyl in thioester linkage to the CoA -fatty acyl-CoA (2 C less).

H3C (CH2)n C CH2 C SCoA

OO

CH3 C SCoA

O

H3C (CH2)n C SCoA +

O

HSCoA-ketoacyl-CoA

fatty acyl-CoA acetyl-CoA (2 C shorter)

-Ketothiolase

Step 4.

-Ketothiolase catalyzes thiolytic cleavage.

Thiol sulfur of CoA attacks the -keto carbon

Summary of one round of the -oxidation pathway:

fatty acyl-CoA + FAD + NAD+ + HS-CoA

fatty acyl-CoA (2 C less) + FADH2 + NADH + H+

+ acetyl-CoA

The -oxidation pathway is cyclic.

The product, 2 carbons shorter, is the input to another round of the pathway.

If, as is usually the case, the fatty acid contains an even number of C atoms, in the final reaction cycle butyryl-CoA is converted to 2 molecules of acetyl-CoA. 

FADH2 & NADH produced during fatty acid oxidation are reoxidized by transfer of electrons to respiratory chain.Transfer of electrons in the respiratory chain leads to production of ATP

Acetyl-CoA can enter Krebs cycle, yielding additional NADH, FADH2, and ATP.

Fatty acid oxidation is a major source of cell ATP.

The reactions presented accomplish catabolism of a fatty acid with an even number of C atoms & no double bonds.

Additional enzymes deal with catabolism of fatty acids with an odd number of C atoms or with double bonds.

The final round of -oxidation of a fatty acid with an odd number of C atoms yields acetyl-CoA & propionyl-CoA.

Propionyl-CoA is converted to the Krebs cycle intermediate succinyl-CoA, by a pathway involving vitamin B12.

Most double bonds of naturally occurring fatty acids have the cis configuration.

They are not correct substrates for Enoyl-CoA hydratase, which acts only on trans compounds.

Additional enzymes, isomerase and reductase , are required for oxidation of unsaturated fatty acids.

-Oxidation of very long-chain fatty acids also occurs within peroxisomes.

Within the peroxisome, FADH2 generated by fatty acid oxidation is reoxidized producing hydrogen peroxide:

FADH2 + O2 FAD + H2O2

The peroxisomal enzyme Catalase degrades H2O2:

2 H2O2 2 H2O + O2

These reactions produce no ATP.

Once fatty acids are reduced in length within the peroxisomes they may shift to the mitochondria to be catabolized to acetylCoA.

This impedes entry of acetyl-CoA into Krebs cycle.

Acetyl-CoA in liver mitochondria is converted then to ketone bodies, acetoacetate & -hydroxybutyrate.

Glucose-6-phosphatase glucose-6-P glucose

Gluconeogenesis Glycolysis

pyruvate fatty acids

acetyl CoA ketone bodies cholesterol oxaloacetate citrate

Krebs Cycle

During fasting or carbohydrate starvation, oxaloacetate is depleted in liver due to gluconeogenesis.

H3C CH2C C

O O

SCoA

H3C C

O

SCoA

HSCoA

H2C C

H2C C

OH O

SCoA

CH3

C

O

O

H3C C

O

SCoA + H3C C

O

SCoA

HSCoA

O CH2C C

O O

CH3 H3C C

O

SCoA+

acetyl-CoA acetyl-CoA

acetoacetyl-CoA

acetyl-CoA

HMG-CoA

acetoacetate acetyl-CoA

Thiolase

HMG-CoA Synthase

HMG-CoA Lyase

Ketone body synthesis:

-Ketothiolase. The final step of the -oxidation pathway runs backward.

HMG-CoA Synthase catalyzes condensation with a 3rd acetate (from acetyl-CoA).

HMG-CoA Lyase cleaves HMG-CoA to yield acetoacetate & acetyl-CoA.

Ketone bodies are transported in the blood to other cells, where they are converted back to acetyl-CoA for catabolism in Krebs cycle, to generate ATP.

Ketone bodies thus function as an alternative fuel.

Ketoacidosis is caused by excess of ketone bodies.

-H ydroxybutyrate D ehydrogenase

C H 3

C

C H 2

C O O

O

C H 3

C H

C H 2

C O O

H O

acetoacetate D - -hydroxybutyrate

H + N A D H N A D +

-Hydroxybutyrate Dehydrogenase catalyzes reversible interconversion of the ketone bodies acetoacetate & -hydroxybutyrate.

Fatty Acid Synthesis

ATP-dependent carboxylation provides energy input.

The CO2 is lost later during condensation with the

growing fatty acid.

H 3C C SC o A

O

C H 2 C SC o A

O

O O C

acetyl-C oA

m alonyl-C oA

The input to fatty acid synthesis is acetyl-CoA, which is carboxylated to malonyl-CoA.

As with other carboxylation reactions, the enzyme prosthetic group is biotin. 

ATP-dependent carboxylation of the biotin, carried out at one active site 1 , is followed by transfer of the carboxyl group to acetyl-CoA at a second active site 2 .

Acetyl-CoA Carboxylase catalyzes the 2-step reaction by which acetyl-CoA is carboxylated to form malonyl-CoA.

ll

Enzyme-biotin HCO3

- + ATP

ADP + Pi

Enzyme-biotin-CO2-

O

CH3-C-SCoA acetyl-CoA O

-O2C-CH2-C-SCoA malonyl-CoA

ll

Enzyme-biotin

1

2

The overall reaction may be summarized as:

HCO3 + ATP + acetyl-CoA ADP + Pi + malonyl-CoA

ll

Enzyme-biotin HCO3

- + ATP

ADP + Pi

Enzyme-biotin-CO2-

O

CH3-C-SCoA acetyl-CoA O

-O2C-CH2-C-SCoA malonyl-CoA

ll

Enzyme-biotin

1

2

Biotin is linked to the enzyme by an amide bond between the terminal carboxyl of the biotin side chain and the -amino group of a lysine residue.

CHCH

H2CS

CH

NHC

N

O

(CH2)4 C NH (CH2)4 CH

CO

NH

O

CO

O

Carboxybiotin lysine residue

Fatty acid synthesis from acetyl-CoA & malonyl-CoA occurs by a series of reactions that are:

catalyzed by individual domains of a very large polypeptide that includes an ACP domain. This multienzyme complex is called Fatty Acid Synthase

NADPH serves as electron donor in the two reactions involving substrate reduction.

The NADPH is produced mainly by the Pentose Phosphate Pathway.

Fatty AcidSynthase prosthetic groups:

the thiol of the side-chain of a cysteine residue.

the thiol of phosphopantetheine, equivalent in structure to part of coenzyme A. 

N

N N

N

NH2

O

OHO

HH

H

CH2

H

OPOPOH2C

O

O O

O

P

O

O O

C

C

C

NH

CH2

CH2

C

NH

CH3H3C

HHO

O

CH2

CH2

SH

O

-mercaptoethylamine

pantothenate

ADP-3'- phosphate

Coenzyme A

phosphopantetheine

H3N+ C COO

CH2

SH

H

cysteine

Phosphopantetheine (Pant) is covalently linked via a phosphate ester to a serine OH of the acyl carrier protein (ACP) of Fatty Acid Synthase. 

OPOH2C

O

OC

C

C

NH

CH2

CH2

C

NH

CH3H3C

HHO

O

CH2

CH2

SH

O

CH2 CH

NH

C O

-mercaptoethylamine

pantothenate

serine residue

phosphopantetheine of acyl carrier protein

phosphate

The condensation reaction (step 3) involves decarboxylation of the malonyl, followed by attack of the acetyl (or acyl).

Pant

SH

Cys

SH

Pant

SH

Cys

S

C

CH3

O

Pant

S

Cys

S

C

CH3

OC

CH2

COO

O

Pant

S

Cys

SH

C

CH2

C

O

CH3

O

acetyl-S-CoA HS-CoA malonyl-S-CoA HS-CoA CO2

1 2 3

1 Malonyl/acetyl-CoA-ACP Transacylase 2 Malonyl/acetyl-CoA-ACP Transacylase

3 Condensing Enzyme (-Ketoacyl Synthase)

Acetyl-CoA-ACP Transacylase

Malonyl-CoA-ACP Transacylase

4. The -ketone is reduced to an alcohol by e transfer from NADPH.

5. Dehydration yields a trans double bond.

6. Reduction by NADPH yields a saturated chain.

Pant

S

Cys

SH

C

CH2

C

O

CH3

O

Pant

S

Cys

SH

C

CH2

HC

O

CH3

Pant

S

Cys

SH

Pant

S

Cys

SH

NADPH NADP+NADPH NADP+

C

CH

HC

O

CH3

C

CH2

CH2

O

CH3

OH

H2O

4 5 6

4 -Ketoacyl-ACP Reductase

5 -Hydroxyacyl-ACP Dehydratase 6 Enoyl-ACP Reductase

Following transfer of the growing fatty acid from phosphopantetheine to cysteine sulfhydryl, the cycle begins again, with another malonyl-CoA.

Pant

S

Cys

SH

C

CH2

CH2

O

CH3

Pant

SH

Cys

S

C

CH2

O

CH2

CH3

Pant

S

Cys

S

C

CH2

O

CH2

CH3

C

CH2

COO

O

Malonyl-S-CoA HS-CoA

7 2

7 Condensing Enzyme 2 Malonyl/acetyl-CoA-ACP Transacylase (repeat). Malonyl-CoA-ACP Transacylase (repeat)

Product release:

When the fatty acid is 16 carbon atoms long, a Thioesterase catalyzes hydrolysis of the thioester linking the fatty acid to phosphopantetheine.

The 16-C saturated fatty acid palmitate is the final product of the Fatty Acid Synthase complex.

Palmitate, a 16-C saturated fatty acid, is the final product of the Fatty Acid Synthase reactions.

Summary (ignoring H+ & water):

acetyl-CoA + 7 malonyl-CoA + 14 NADPH palmitate + 7 CO2 + 14 NADP+ + 8 CoA

Accounting for ATP-dependent synthesis of malonate: 8 acetyl-CoA + 14 NADPH + 7 ATP palmitate + 14 NADP+ + 8 CoA + 7 ADP + 7 Pi

Fatty acid synthesis occurs in the cytosol. Acetyl-CoA generated in mitochondria is transported to the cytosol via a shuttle mechanism involving citrate.

-Oxidation & Fatty Acid SynthesisCompared

Oxidation Pathway Fatty Acid Synthesis

pathway location mitochondrial matrix cytosol

acyl carriers (thiols)

Coenzyme-A phosphopantetheine (ACP) & cysteine

e acceptors/donor FAD & NAD+ NADPH

-OH intermediate L D

2-C product/donor acetyl-CoA malonyl-CoA

(& acetyl-CoA)

Elongation beyond the 16-C length of the palmitate product of Fatty Acid Synthase occurs in mitochondria and endoplasmic reticulum (ER).

Fatty acid elongation within mitochondria involves the -oxidation pathway running in reverse, but NADPH serves as electron donor for the final reduction step.

Fatty acids esterified to CoA are substrates for the ER elongation machinery, which uses malonyl-CoA as donor of 2-carbon units.

The reaction sequence is similar to Fatty Acid Synthase but individual steps are catalyzed by separate proteins.

A family of enzymes designated Fatty Acid Elongases catalyze the initial condensation step for elongation of saturated or polyunsaturated fatty acids.

Desaturases introduce double bonds at specific positions in a fatty acid chain.

Mammalian cells are unable to produce double bonds at certain locations, e.g., 12.

Thus some polyunsaturated fatty acids are dietary essentials, e.g., linoleic acid, 18:2 cis 9,12 (18 C atoms long, with cis double bonds at carbons 9-10 & 12-13).

C

O

OH

910

oleate 18:1 cis 9

Formation of a double bond in a fatty acid involves the following endoplasmic reticulum membrane proteins in mammalian cells:

NADH-cyt b5 Reductase, a flavoprotein with FAD as prosthetic group.

Cytochrome b5

Desaturase

C

O

OH

910

oleate 18:1 cis 9

The overall reaction for desaturation of stearate (18:0) to form oleate (18:1 cis 9) is:

stearate + NADH + H+ + O2 oleate + NAD+ + 2H2O

There is a 4-electron reduction of O2 2 H2O as a fatty acid is oxidized to form a double bond.

2e pass from NADH to the desaturase via the FAD-containing reductase & cytochrome b5, the order of electron transfer being: NADH FAD cyt b5 desaturase

2e are extracted from the fatty acid as the double bond is formed.

Control of fatty acid synthesis is exerted mainly at acetyl-CoA carboxylase step

• Citrate & Insulin activate the enzyme

• Acyl-CoA & Glucagon & Epinephrine inhibite the enzyme

Thank you for your attention