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Fatty Acid Oxidation

Copyright 1999-2007 by Joyce J. Diwan.

All rights reserved.

Molecular Biochemistry II

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

Most naturally occurring fatty acids have an even number

ofcarbon atoms.

The pathway for catabolism of fatty acids is referred to

as the b-oxidation pathway, because oxidation occursat the b-carbon (C-3).

C

O

O

12

34

b

fatty acid with a cis-9

double bond

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Triacylglycerols (triglycerides) are the most abundant

dietary lipids. They are the form in which we store reduced

C for energy.

Each triacylglycerol has a glycerolbackbone to which are

esterified 3 fatty acids

Most triacylglycerols are mixed. The 3 fatty acids differ

in chain length & number of double bonds.

glycerol fatty acid triacylglycerol

H2C

HC

H2C

OH

OH

OH

H2C

HC

H2C

O

O

O

C R

O

C

C R

OR

O

HO C R

O

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Lipid digestion, absorption, transport will be covered

separately.

Lipases hydrolyze triacylglycerols, releasing 1 fatty acid

at a time, yielding diacylglycerols, & eventually glycerol.

glycerol fatty acid triacylglycerol

H2C

HC

H2C

OH

OH

OH

H2C

HC

H2C

O

O

O

C R

O

C

C R

OR

O

HO C R

O

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glycerol glycerol-3-P dihydroxyacetone-P

CH2

CH

CH2

OH

HO

O PO3

CH2

CH

CH2

OH

HO

OH

CH2

C

CH2

OH

O PO3

O

ATP ADPH

++

NAD

+

NADH

1 2

Glycerol, arising from hydrolysis of triacylglycerols, is

converted to the Glycolysis intermediate

dihydroxyacetone phosphate, by reactions catalyzed by:1 Glycerol Kinase

2 Glycerol Phosphate Dehydrogenase.

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Free fatty acids, which in solution have detergent

properties, are transported in the blood bound to

albumin, a serum protein produced by the liver.Several proteins have been identified that facilitate

transport of long chain fatty acidsinto cells, including

the plasma membrane protein CD36.

C

O

O

12

34

b

fatty acid with a cis-9

double bond

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Fatty acid activation:

Fatty acids must be esterified toCoenzyme A before theycan undergo oxidative degradation, be utilized for

synthesis of complex lipids, or be attached to proteins as

lipid anchors.

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.

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Acyl-CoA

Synthases

Exergonic PPi(P~P) hydrolysis,

catalyzed by

Pyrophosphatase,

makes the coupled

reaction

spontaneous.

2 ~P bonds of ATP

are cleaved.The acyl-CoA

product includes

one "~" thioester

linkage.

Fatty acid activation

N

N

N

N

NH2

O

OHOH

HH

H

CH2

H

OPOPOPOO

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

2Pi

fatty acid

ATP

acyl-adenylate

acyl-CoA

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Summary of fatty aid activation:

fatty acid + ATP

acyladenylate + PPiPPi 2 Pi

acyladenylate + HS-CoA acyl-CoA + AMPOverall:

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

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For most steps of the pathway there are multiple enzymes

specific for particular fatty acid chain lengths.

b-Oxidationpathway in

matrix

Fatty acyl-CoA formed in cytosol by enzymes

of outer mitochondrial membrane & ER

Mitochondrion

b-Oxidationpathway:

Fatty acids are

degraded in the

mitochondrial matrix

via the b-Oxidation

Pathway.

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Fatty acyl-CoA formed outside can pass through the

outer mitochondrial membrane (which has large VDAC

channels), but cannot penetrate the inner membrane.

b-Oxidationpathway in

matrix

Fatty acyl-CoA formed in cytosol by enzymes

of outer mitochondrial membrane & ER

MitochondrionMany of the constituent

enzymes are soluble

proteins located in themitochondrial matrix.

But enzymes specific

for very long chainfatty acids are

associated with the

inner membrane,

facing the matrix.

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Carnitine Palmitoyl Transferases catalyzes 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 PalmitoylTransferase

Transfer of the fatty

acid moiety acrossthe mitochondrial

inner membrane

involves carnitine.

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Carnitine-mediated transfer of the fatty acyl moiety 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

12

3

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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 OR-C-SCoA HO-carnitine HO-carnitine R-C-SCoA

HSCoA R-C-O-carnitine R-C-O-carnitine HSCoAO O

12

3

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Control of fatty acid oxidation is exerted mainly at the

step offatty acid entry into mitochondria.

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

Malonyl-CoA is produced from acetyl-CoA by the

enzyme Acetyl-CoA Carboxylase.

H3C C SCoA

O

CH2 C SCoA

O

OOC

acetyl-CoA

malonyl-CoA

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Activated Kinase, leading to decreased malonyl-CoA.

The decrease in malonyl-CoA concentration leads to

increased activity ofCarnitine Palmitoyl Transferase I.

Increased fatty acid oxidation then generates acetyl-CoA,

for entry into Krebs cycle with associated ATP production.

AMP-Activated Kinase,

a sensor of cellular energy

levels, is allosterically

activated by AMP, which

is high in concentration

when [ATP] is low.

Acetyl-CoA Carboxylaseis inhibited when

phosphorylated by AMP-

H3C C SCoA

O

CH2 C SCoA

O

OOC

acetyl-CoA

malonyl-CoA

ATP + HCO3

ADP + Pi

Acetyl-CoACarboxylase(inhibited by

AMP-ActivatedKinase)

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AMP-Activated Kinase functions under a variety of

conditions that lead to depletion of cellular ATP

(reflected as increased AMP), including:

glucose deprivation, exercise, hypoxia & ischaemia.

AMP-Activated Kinase regulates various metabolic

pathways to:

promote catabolism leading to ATP synthesis

(e.g., stimulation of fatty acid oxidation)

inhibit energy-utilizing anabolic pathways(e.g., fatty acid synthesis).

AMP-Activated Kinase in the hypothalamus of the

brain is involved also in regulation of food intake.

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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.

Very Long Chain Acyl-CoA Dehydrogenase is bound to

the inner mitochondrial membrane. The others are soluble

enzymes located in the mitochondrial matrix.

H3C (CH2)n C C C SCoA

H

H

H

H O

123

b


H

H OFADH2

FAD

fatty acyl-CoA

trans-

2

-enoyl-CoA

Acyl-CoA Dehydrogenase

b-OxidationPathway:

Step 1. Acyl-CoA

Dehydrogenase

catalyzes oxidation

of the fatty acid

moietyofacyl-CoA

to producea double

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FAD is the prosthetic group that functions as e acceptor

for Acyl-CoA Dehydrogenase. Proposed mechanism:

A Glu side-chain carboxyl extracts a proton from the

-carbon of the substrate, facilitating transfer of 2 e

with H+ (a hydride) from the b position to FAD.

The reduced FAD accepts a 2nd

H+

, yielding FADH2.


H

H

H

H O

123

b


H

H OFADH2

FAD

fatty acyl-CoA

trans-2

-enoyl-CoA


H3N+ C COO

CH2

CH2

C

H

O

O

glutamate

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The carbonyl O of the thioester substrate is hydrogenbonded to the 2'-OH of the ribityl moiety of FAD, giving

this part of FAD a role in positioning the substrate and

increasing acidity of the substrate -proton.


H

H

H

H O

123

b


H

H OFADH2

FAD

fatty acyl-CoA

trans-2-enoyl-CoA


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The carbonyl O of the thioester substrate is hydrogenbonded to the 2'-OH of the ribitol moiety of FAD, giving

the sugar alcohol a role in positioning the substrate and

increasing acidity of the substrate -proton.

C

CCH

C

C

HC

NC

CN

NC

NHC

H3C

H3C

O

O

CH2

HC

HC

HC

H2C

OH

O P O P O

O

O-

O

O-

Ribose

OH

OH

Adenine

C

CCH

C

C

HC

NC

C

HN

NH

C

NHC

H3C

H3C

O

O

CH2

HC

HC

HC

H2C

OH

O P O P O

O

O-

O

O-

Ribose

OH

OH

Adenine

FAD FADH2

2 e

+ 2 H+

dimethylisoalloxazine

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The reactive Glu and FAD are on opposite sides of thesubstrate at the active site.

Thus the reaction is stereospecific, yielding a trans

double bond in enoyl-CoA.


H

H

H

H O

123

b


H

H OFADH2

FAD

fatty acyl-CoA

trans-2-enoyl-CoA


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FADH2is reoxidized by transfer of 2 electrons toan electron transfer flavoprotein (ETF), which in

turn passes the electrons to coenzyme Q of the

respiratory chain.

Matrix

H+

+NADH NAD++2H+ 2H++O2 H2O

2e I Q III IV

+ +

4H+ 4H+ 2H+

Intermembrane Space

cytc

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Step 2.

Enoyl-CoA

Hydratase

catalyzes

stereospecifichydration of the

trans double bond

produced in the

1st step, yieldingL-hydroxyacyl-

Coenzyme A.


H

H

H

H O

123

b


H

H O

H3C (CH2)n C CH2 C SCoA

OH

O

H2O

FADH2

FAD

H

fatty acyl-CoA

trans-2-enoyl-CoA

3-L-hydroxyacyl-CoA


Enoyl-CoA Hydratase

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OH

OH


OO

H++NADH

NAD+

CH3 C SCoA

O

H3C (CH2)n C SCoA +

O

HSCoA

3-L-hydroxyacyl-CoA

b-ketoacyl-CoA

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

Hydroxyacyl-CoA

Dehydrogenase

b-Ketothiolase

Step 3.

Hydroxyacyl-CoA

Dehydrogenase

catalyzes oxidation

of the hydroxyl in

the b position (C3)

to a ketone.NAD+ is the

electron acceptor.

H

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A cysteine S attacks the b-keto C.

Acetyl-CoA is released, leaving the fatty acyl moiety in

thioester linkage to the cysteine thiol.

The thiol of HSCoA displaces the cysteine thiol, yielding

fatty acyl-CoA (2 C less).


OO

CH3 C SCoA

O

H3C (CH2)n C SCoA +

OHSCoA

b-ketoacyl-CoA

fatty acyl-CoA acetyl-CoA

(2 C shorter)

b-Ketothiolase

H3N+ C COO

CH2

SH

H

cysteine

Step 4.

b-Ketothiolasecatalyzes thiolytic

cleavage.

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A membrane-bound trifunctional protein complex

with two subunit types expresses the enzyme

activities for steps 2-4 of the b-oxidation pathway forlong chain fatty acids.

Equivalent enzymes for shorter chain fatty acids are

soluble proteins of the mitochondrial matrix.

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Summary of one round of the b-oxidation pathway:

fatty acyl-CoA + FAD + NAD+ + HS-CoAfatty acyl-CoA (2C less) + FADH2 + NADH + H

+

+ acetyl-CoA

The b-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 copies of acetyl-CoA.

ADP P ATP

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NADH produced during fatty acid oxidation is reoxidized

by transfer of2e to respiratory chain complex I.Transfer of 2e from complex I to oxygen causes sufficientproton ejection to yield approximately 2.5 ATP.

Recall that 4H+ enter the matrix per ATP synthesized,

taking into account transmembrane flux of ADP, ATP & Pi.

Matrix

H+


2e

I Q III IV

+ +

4H+ 4H+ 2H+

Intermembrane Space

cytc 3H+

F

Fo

ADP+Pi ATP

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FADH2 of Acyl-CoA Dehydrogenase is reoxidized by

transfer of 2e via ETF to CoQ of the respiratory chain.

H+ ejection from the matrix that accompanies transfer of

2e from coenzyme Q to oxygen, leads to production ofapproximately 1.5 ATP.

Matrix

H+


2

e

I Q III IV

+ +

4H+ 4H+ 2H+

Intermembrane Space

cytc 3H+

F

Fo

ADP+Pi ATP

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Acetyl-CoA can enter Krebs cycle, yielding

additional NADH, FADH2, and ATP.

Fatty acid oxidation is a major source of cell ATP.

M i ADP + P ATP

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Catabolism oftwo 6-C glucose through Glycolysis, Krebs,

& ox phos yields about 60 ~P bonds ofATP (30/glucose).

Compare energy yield oxidizing a 12-C fatty acid. Assume:

1.5 ATP produced per FADH2 reoxidized in the

respiratory chain (via coenzyme Q).

2.5 ATP produced per NADH reoxidized in the

respiratory chain.

Matrix

H+


2e

I Q III IV

+ +

4H+ 4H+ 2H+

Intermembrane Space

cytc 3H+

F

Fo

ADP+Pi ATP

Problem(See web

handout,

tutorial)

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How many "high energy" (~) bonds are utilized in activating the fatty acid, by

esterifying it to coenzyme A? ()________

How many times is the b-oxidation pathway repeated during oxidation of a 12-C

fatty acid? _________

How many each of NADH______, FADH2______, and Acetyl CoA______ are

produced, per 12-carbon fatty acid, in the b-oxidation pathway?

Oxidation of each acetyl CoA in Krebs cycle yields 3 NADH and one FADH2

(from succinate), resulting in additional production of _______NADH and

_______FADH2.

Thus the yield is a total of _______NADH and _______FADH2.

In the respiratory chain, approx. 2.5 ~ bonds of ATP are produced per NADH and

1.5 ~ bonds of ATP per FADH2 (electrons entering the respiratory chain via

coenzyme Q). Thus from reoxidation of NADH and FADH2

a total of _______

~ bonds of ATP are produced per 12-C fatty acid.

Add to this the ~P bonds of GTP produced in Krebs Cycle (one GTP per acetyl-

CoA) for a total of _______ ~P bonds produced.

Summing input and output yields a total of _______ ~P bonds per 12-C fatty acid

oxidized. Does fat yield more energy than carbohydrate? _______

2

5

5 5 6

18

623 11

74

80

78

YES

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Human genetic diseases have been identified that

involve mutations in:

the plasma membrane fatty acid transporter CD36

Carnitine Palmitoyltransferases I & II (required for

transfer of fatty acids into mitochondria)

Acyl-CoA Dehydrogenases for various chain lengthsof fatty acids

Hydroxyacyl-CoA Dehydrogenases for medium &

short chain length fatty acids

Medium Chain b-Ketothiolase

the trifunctional protein complex

Electron Transfer Flavoprotein (ETF).

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Human genetic diseases:

Symptoms vary depending on the specific genetic

defect but may include: hypoglycemia and failure to increase ketone body

production during fasting

fatty degeneration of the liver

heart and/or skeletal muscle defects

maternal complications of pregnancy

sudden infant death (SIDS).

Hereditary deficiency ofMedium Chain Acyl-CoA

Dehydrogenase (MCAD), the most common genetic

disease relating to fatty acid catabolism, has been

linked to SIDS.

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The reactions presented accomplish catabolism of a

fatty acid with an even number of Catoms &

no double bonds.

Additional enzymes deal with catabolism of fatty

acids with an odd number ofC atoms or with double

bonds. The final round ofb-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 (to be presented later).

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Most double bonds of naturally occurring fatty acids

have the cis configuration.

As C atoms are removed two at a time, a double bond

may end up in the wrong position or wrong

configuration to be the correct substrate for Enoyl-

CoA Hydratase.

The reactions that allow unsaturated fatty acids to befully catabolized by the b-oxidation pathway aresummarized in the textbook.

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b-Oxidationof very long-chain fatty acids also occurswithin peroxisomes.

FAD is e acceptor for peroxisomal Acyl-CoA Oxidase,

which catalyzes the 1

st

oxidative step of the pathway.

Single membrane

Enzymes, some of which produce H2O2, &

always including Catalase, that degrades H2O2.

Crystalline inclusion

often present

Peroxisome

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Within the peroxisome, FADH2 generated by fatty acid

oxidation is reoxidized producing hydrogen peroxide:FADH2 + O2 FAD + H2O2

The peroxisomal enzyme Catalase degrades H2O2:

2H2O2

2H2O + O2These reactions produce no ATP.

Once fatty acids are reduced in length within the

peroxisomes they may shift to the mitochondria to becatabolized all the way to CO2.

Carnitine is involved in transfer of fatty acids into and

out of peroxisomes.

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Serious genetic diseases are associated with defects in or

deficiency of enzymes of the peroxisomalb-oxidationsystem.

Peroxisomes also contain enzymes for an essential-oxidation pathway that degrades fatty acids havingmethyl branches, such as phytanic acid, a breakdown

product of chlorophyll.

Gl 6 h h

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This impedes entry of acetyl-CoA into Krebs cycle.

Acetyl-CoA in liver mitochondria is converted then to

ketone bodies, acetoacetate & b-hydroxybutyrate.

Glucose-6-phosphatase

glucose-6-P glucose

Gluconeogenesis Glycolysis

pyruvatefatty acids

acetyl CoA ketone bodies

cholesterol

oxaloacetate citrate

Krebs Cycle

During fasting

or carbohydrate

starvation,oxaloacetate is

depleted in

liver due to

gluconeogenesis.

O OK t b d

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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-CoAacetyl-CoA

acetoacetyl-CoA

acetyl-CoA

HMG-CoA

acetoacetate acetyl-CoA

Thiolase

HMG-CoA Synthase

HMG-CoA Lyase

Ketone body

synthesis:

b-Ketothiolase. Thefinal step of the b-oxidation pathway

runs backward.

HMG-CoA

Synthase catalyzes

condensation with a

3rd acetate moiety

(from acetyl-CoA).

HMG-CoA Lyase

cleaves HMG-CoA to

yield acetoacetate &

acetyl-CoA.

b-Hydroxybutyrate Dehydrogenase

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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.

While ketone bodies thus function as an alternative fuel,

amino acids must be degraded to supply input to

gluconeogenesis when hypoglycemia occurs, since acetate

b-Hydroxybutyrate Dehydrogenase

CH3

C

CH2

COO

O

CH3

CH

CH2

COO

HO

acetoacetate D-b-hydroxybutyrate

H+

NADH NAD+

b-HydroxybutyrateDehydrogenase

catalyzes reversible

interconversion of

the ketone bodies

acetoacetate &b-hydroxybutyrate.

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