Transcript BIO 322_Rec_4part1_Spring 2013

FATTY ACID CATABOLISM
Lehninger Ch. 17
BIO 322 Recitation 4 / Spring 2013
Role and oxidation of FA
2
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

Electrons from FA to ETC
Acetyl CoA may be oxidized completely to
In liver, acetyl CoA
ketone bodies



in TCA
Brain and other tissues use as water soluble fuel when
glucose is not available.
In higher plants, acetyl CoA is used for biosynthesis,
secondary for fuel purposes.
All use 4 step process called, β oxidation, where FA is
converted to acetyl CoA.
Triacylglycerols
3
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
Suitable storage fuels
Long alkyl chains - Complete Oxidation is 38 kJ/g


Insoluble in water, aggregate in lipit droplets



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more than twice of the carbs and proteins.
Do not raise the osmolarity of the cytosol.
Inert, no undesired chemical rxn, stored in large quantities.
Ingested TG should be emulsified before digested by water
soluble enzymes of the intestine or mobilized via protein
attachment.
C-C , C1-coenzyme A, allows oxidation of the fatty acyl
groups at C3 or beta position, hence the name beta
oxidation
Three Stages
4
The chemical steps of FA oxidation in mitochondria
1.
Oxidation of long chain FA into 2C fragments in
the form of acetyl CoA. (β oxidation) (CH 17)
2.
Acetyl CoA – TCA cycle (CH 16)
3.
ETC (CH 19)
Digestion, Mobilization and Transport of Fats
5
•
Fatty acid fuels 3 sources: Diet, Stored as lipid droplets, Synthesis
•
In industrialized countries ~ 40% daily energy from TG < %30 is recommended.
•
Provide more than half of the required energy for liver, heart and resting skelatal muscle.
•
Sole source of energy in hibernating animals and migrating birds
Dietary Fats are Absorbed in the Small Intestine
1) Solubilization of TG into finely dispersed
microscopic micelles via bile salts.

Bile Salts

synthesized from cholestrol in liver, stored in
gall bladder, released into SI after ingestion
of fatty meal.

Biological Detergents, amphipathic

Convert dietary fats into mixed micelles of
bile salts and TG – accesible to water soluble
lipases in SI
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2.
Lipase converts TG into
•
Diacylglycerols
•
Monoglycerols
•
Free FA
•
Glycerol
3.
Diffuse into epithelial cells lining the intestinal surface
4.
They are reconverted into TG and packed into lipoprotein aggregates called
chylomicrons together with cholestrol and specific proteins.

Apoliproteins (Apo means detached, lipid free form) – lipid binding proteins for
transport of TG and other lipid derivatives between organs

VLDL to VHDL (Hydrophobic core, hydrophilic protein side chains and lipid head
groups at the surface)
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5. Chylomicrons from SI to
lymphatic system, then to blood
 carried to muscle and adipose
tissue via apoCII.
6. Within the capillaries of the
tissues, ApoCII activated
lipoprotein lipase hydrolyses TG
into FA and glycerol
7. Taken up by cells
8. Muscle FA for energy. In adipose
FA reesterified for storage as TG.
 Chylomicron remnants back to liver. The left over TG by this route may be used
for energy or for production of ketone bodies.
 More FA than needed, (liver) packed as VLDL – blood to adipose tissue -storage.
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Hormones Trigger Mobilization of Stored Triacylglycerols
9

Neutral lipids are stored in adipocytes, adrenal cortex,
ovary, testes – as lipid droplets : Sterol esters and TG in
the core surrounded by phospholipids
 Coated with Perilipins – restrict acces to the lipid
droplet, prevent lipid mobilization.




Hormonal signal for energy (Epi, or glucagon)
Adenylyl cyclase in the plasma memb of adipocyte 
cAMP  cAMP dependent kinase (PKA)
phosphorylates perilipin A, phospho-perilipin activated
hormone senstive lipase in the cytosol so that
hydrolysis of TG into FFA and glycerol can begin.
PKA can also phosphorylate hormone sensitive lipase
and duplicate, triplicate its activity. 50X increase via
perilipin phosphorylation
Hydrolysis of FA FFA pass from adipocytes into blood

Bind to protein serum albumin ( 10 FA per protein)
and carried to the tissue for fuel
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95% of bio available
energy resides in three long
chain FAs and 5% in
glycerol
Glycerol released by lipase
action is phosphorylated by
glycerol kinase
Glycerol 3-phosphate is
oxidized into
dihydroxyacetone phosphate
Glycolytic enzyme triose phosphate
isomerase converts DHAP into
glyceraldehyde 3-phosphate, which
is oxidized in glycolysis.
Fatty Acids are Activated and Transported into Mitochondria
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•
Enzymes of FA oxidation are located in
mitochondrial matrix
•
Chain length 12 or fewer enter mitoch. w/o any
help of a membrane transporter.
•
14 or more carbons containing FA, that
constitutes most of the FFA from diet cannot
pass mitochondrial membrane,
via 3 enzymatic reactions called carnitine
shuttle.
•
1.
Rxn by a family of isozymes (different isozymes specific for short,
intermediate, long carbon chains) present in the outer mitoch. membrane,
acyl-CoA synthetases
•
Thioester linkage between carboxyl of FA to thiol of CoA, coupled to ATP
cleavage
12
• Fatty acyl-CoAs are high energy
compounds – hydrolysis to free FA +
acetyl CoA ∆G’º= -31kj/mol
•Formation of FA-CoA is made
favorable by the hydrolysis of two high
energy bonds in ATP. Pyrophosphate
hydrolized by inorganic phosphatase
•
• Together with ATP hydrolysis that the
overall reaction is pulled towards fatty
acyl-CoA formation.
Fatty acyl CoA ester formed in cytosolic side
of the outer mitochondrial membrane
•
•
Transported either into mitochondrion, oxidized
or may be used for membrane lipid synthesis.
If destined to mitochondria
•
Fatty acyl attached to the hydroxyl group of
carnitine to form fatty acyl-carnitine  The
second reaction of shuttle
•
13
•
•
•
•
•
Fatty acyl CoA into fatty
acyl carnitine via outer
mitochondrial membrane
enzyme carnitine acyl
transferase I.
Either acyl-CoA passes outer membrane and is converted to the carnitine
ester in intermembrane space or carnitine ester is formed on the cytosolic
face of the outer membrane, then moved across the outer membrane to
intermembrane space – current evidence does not reveal which.
Passage into innermemb. space is done via large pores called porin proteins.
Fatty acyl-carnitine ester enter the matrix via faciliated diffusion through acyl
carnitine/carnitine transporter of the inner mitochondrial membrane.
At the final, 3rd step, carnitine acyl transferase II regenarates fatty acyl CoA in
the matrix, the relased carnitine back to intermemb via same transporter.
Linkage of CoA and fatty acyl CoA pools in the cytosol (Biosynthesis) and
mitochondria (Energy).
Oxidation of Fatty Acids
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

Mitochondrial Oxidation of FA – three stages
First stage – beta oxidation – oxidative
removal of two carbon units in the form of
acetyl CoA starting from carboxyl end of
fatty acyl chain.


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Ex. 16 carbon palmitic acid yields 8 two
carbon acetyl CoA molecules.
Formation of each acetyl CoA requires
removal of 4 hydrogen atoms via
dehydrogenases.
Second stage – acetyl CoA into TCA (also in
mitochondrial matrix)
Third stage -First two stages produced NADH
and FADH2 donate electrons to ETC.
The β oxidation of Saturated Fatty Acids
Has Four Basic Steps
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
First step of fatty acid oxidation – 4 enzymes (Saturated)
1) Dehydrogenation of fatty-acyl CoA produces a double bond
between α and β carbons (C-2,C-3)  trans-∆2-enoyl-CoA
(delta for position of the double bond, FA nomenclature – HW)
 new double bond in trans config, whereas naturally occuring
unsaturated fatty acid in cis config.

Acyl-CoA dehydrogenase (three isozymes) : VLCAD – 12 to
18 C, MCAD – 4 to 14 C, SCAD – 4 to 9 C

All three are flavoproteins with FAD, electrons removed from
fatty acyl CoA are transferred to FAD and reduced
dehydrogenase donates these electrons to electron carrier of
ETC flavoproteins (ETF).

Analogous to succinate dehydrogenase rxn, both enzymes
are bound to inner membrane of mitochondria, a double
bond is introduced into a carboxylic acid α and β carbons ,
FAD is the electron acceptor, electrons to ETC, 1.5 ATP per
electron pair.
2. Water added to the double bond of trans enoyl CoA to
form L stereoisomer of beta-hydroxyacyl-CoA,
catalyzed by enoyl-CoA hydratase -- Analogous to
fumerase reaction in citric acid cycle.
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3. L- beta hydroxyacyl-CoA is dehydrogenated to form
beta-ketoacyl-CoA, by the action of beta-hdroxyacyl
CoA dehydrogenase
 NAD is the electron acceptor, specific for L stereoisomer
 NADH formed donates electrons to NADH dehydrogenase
of ETC, analogous to the malate dehyrogenase of citric
acid cycle.
4. Beta-ketoacyl-CoA with free CoA to split off the
carboxy terminal 2C fragment of original FA as acetyl
CoA by thiolase (acyl-CoA acetyltransferase)
 Other product is the coenzyme A thioester of FA, now
shortened by 2C. (also called Thiolysis, similar to hydrolysis)
 Last 3 steps, fatty acyl chains of 12 or more C are
catalyzed by a multienzyme complex in inner mitoch.
memb. Trifunctional protein (TFP)
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
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TFP is heterooctamer of α4β4 subunits.
α subunit: 2 activities enoyl-CoA hydratase
and β-hydroxyacyl-CoA dehydrogenase
β subunit: thiolase activity
Allows substrate channeling
After TFP shortenes the fatty acyl chain into
12 or fewer carbons, further oxidation is
catalyzed by 4 soluble enzymes in the
matrix.
Acetyl-CoA Can Be Further Oxidized in the TCA
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
FADH2 : 1.5 ATP

NADH: 2.5 ATP

In TCA 3NADH, 1 FADH2 , ATP=10 ATP

Beta oxidation + TCA
Oxidation of Unsaturated Fatty Acids
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saturated: the SFA’s of a lipid have no
double bonds between carbons in chain
polyunsaturated: more than one double
bond in the chain
Saturated – single bonds in the carbon
chain – typical 4 steps in oxidation
Most of the FA in triacyglycerols and
phospholipids are unsaturated – one or
more double bonds – cis configuration
cannot be acted upon by enoyl-CoA
hydratase (acts on trans double bond)
2 new enzymes: an isomerase for
monounsaturated fatty acid and an
isomerase together with a reductase for
polyunsaturated fatty acids
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Odd number FA
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FA Oxidation is Tightly Regulated
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
In liver, fatty acyl-CoA in cytosol
 1) β oxidation in mitochondria


2) conversion into triacylglycerols and phospholipids in cytosol
Dependent on long chain fatty acyl-CoA transfer into mitoch via
carnitine shuttle – rate limiting step of fatty acid oxidation –
important point of regulation. Once enter the shuttle  committed
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
When Malonyl-CoA – first intermediate of LCFA sythesis from acetyl-CoA is high

When excess glucose cannot be oxidized or stored as glycogen is converted into FA in
cytosol for storage as triacylglycerols

Malonyl CoA inhibits carnitine acytransferase I to stop producing energy from fatty
acid oxidation.

NADH/NAD ratio is high β -hydroxyacyl-CoA dehydrogenease is inhibited

High concentrations of acetyl-CoA inhibits thiolase

AMPK also phophorylates ACC (Acetyl-CoA carboxylase) – allows β oxidation
Peroxisomes Also Carry Out β Oxidation
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
Mitochondria – major site of fatty acid oxidation in animal cells.
Peroxisomes - β oxidation in plant cell (similar but not identical)
Intermediates for β acid oxidation of FA are coenzyme A
derivaties
1.
Dehydrogenation
2.
Addition of water to the resulting double bond
3.
Oxidation of b-hydroxyacyl CoA to a ketone
4.
Thiolytic clevage by coenzyme A


The difference in the chemistry of the first step
In peroxisomes - Flavoprotein acyl-CoA oxidase that introduces the
double bond, passes electrons direct to oxygen, producing
hydrogen peroxide (H2O2).



Damaging oxidant cleaved to water and oxygen via catalase
Mitoch – first step electrons – through ETC to O2 for ATP, H2O
Peroxisome – first step electrons – to catalase rxn, which is
exothermic, dissipates heat not conserved as ATP.
The ω Oxidation of FA Occurs in the ER
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
Omega oxidation – minor pathway in mammals,
generally active because of a mutation or carnitine
deficiency-- defect in β oxidation

Takes place in ER, where carbon most distant to
carboxyl group is oxidized.

First step introduces hydroxyl group on the omega
carbon, molecular oxygen is used that involves
cytochrome p450 and electron donor NADPH,
catalyzed by mixed fuction oxidases.

Alcohol dehydrogenase – oxidizes hydroxyl group
into an aldehyde

Aldehyde dehydrogenase – oxidizes aldehyde into
carboxylic acid, producing a FA with carboxylgroup
each end.

Can be attached coenzyme A – mitoch. – beta ox.

Yields dicarboxylic acids such as succinic acid that
can enter TCA cycle.
Phytanic Acid Undergoes α
Oxidation in Peroxisomes
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The presence of methyl group on
β carbon of a FA make β
oxidation impossible
These branched FA are
catabolized in peroxisomes of
animal cells
Example: Phytanic acid,



phytanoyl-CoA is hydroxylated on its
alpha carbon that involves the use of
molecular oxygen
decarboxlated to form an aldehyde
one C shorther
Oxidized to carboxylic acid, that can
be further oxided by beta oxidation
Ketone Bodies
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
Acetyl-CoA formed in the liver

TCA

Ketone bodies (matrix of mitoch.) export to other
tissues

Acetone in smaller quantities, is exhaled.

Acetoacetate and d-β-hydroxybutyrate


To other extrahepatic tissues  converted to acetyl-CoA for TCA
cycle provide energy for heart, muscle and renal cortex.
The production and export of ketone bodies to
extrahepatic tissues allows continued oxidation of FA
in liver when acetyl-CoA is not being oxidized in TCA
cycle.
Ketone Bodies, Formed in Liver, Are Exported to
Other Organs as Fuel
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1) Formation of acetoacetate: Enzymatic
condensation of two molecules of acetyl-CoA
catalyzed by thiolase, reversal of last step of β
oxidation.
2) Acetoacetyl-CoA then condenses with acetyl-CoA
to form beta-hydroxy-β-methylglutaryl-CoA
(HMG-CoA).
3) HMG-CoA is cleaved into acetoacetate and
acetyl-CoA.
4) Acetoacetate reduced to d-β-hydroxybutyrate by
mitochondrial d-β dehydroxybutyrate
dehydrogenease. Specific for d isomer.
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

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

Extrahepatic tissues, d-β-hydroxybutyrate
oxidized to acetoacetate by d-β hydroxybutyrate
dehydrogenase
The acetoacetate is activated to its CoA ester by
transfer of CoA from succiniyl CoA of TCA cycle
by β-ketoacyl-CoA transferase.
Acetoacetyl-CoA is cleaved into 2 acetyl-CoAs by
thiolase.
Used as fuel in extrahepatic tissues, because liver
lacks (or little) β-ketoacyl-CoA transferase.
When TCA intermediates are siphoned off for
glucose synthesis by gluconeogenesis, cycle slows,
so acetyl-CoA oxidation.
Ketone Body Formation and Export from The Liver
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

Liver contains only a limited
amount of CoA, most of it bound
to acetyl-CoA, β oxidation slows
to supply free coenzyme.
Production and export of ketone
bodies frees CoA, allowing
continued FA oxidation.