Transcript Fatty Acid Oxid - Univerzita Karlova v Praze

Fatty Acid Oxidation

4
b
O

3
2
C
1
O
fatty acid with a cis-9
double bond
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 b-oxidation pathway, because oxidation occurs
at the b-carbon (C-3).
O
H2C
OH
HC
OH
O
HO
H2C
C
OH
glycerol
fatty acid
H2C
O
C
O
R
HC
O
C
O
R
H2C
O
C
R
R
triacylglycerol
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.
O
H2C
OH
HC
OH
O
HO
H2C
C
OH
glycerol
fatty acid
H2C
O
C
O
R
HC
O
C
O
R
H2C
O
C
R
R
triacylglycerol
Lipases hydrolyze triacylglycerols, yielding glycerol and
three fatty acids

4
b
O

3
2
C
1
O
fatty acid with a cis-9
double bond
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
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.
NH2
Fatty acid activation
O
fatty acid
O

O
O
P
O
R
O
O
O
N
P

O
O
CH2

H
2 Pi
R
O
C
H
OH
H
OH
P
N
N
O
CH2
O
CoA
SH
H
O
H
H
OH
H
OH
N
acyladenylate
AMP
O
R
NH2
N
O
O
N
ATP
H
PPi
O
N
O
O
P

C
N
C
S
CoA
acyl-CoA
Summary of fatty aid activation:
fatty acid + ATP + HS-CoA  acyl-CoA + AMP + 2 Pi
Mitochondrion
Fatty acid b-oxidation
is considered to occur
in the mitochondrial
matrix.
Fatty acids must enter
the matrix to be
oxidized.
b-Oxidation
pathway in
matrix
Fatty acyl-CoA formed in cytosol by enzymes
of outer mitochondrial membrane & ER
Fatty acyl-CoA formed outside can pass through the
outer mitochondrial membrane, but cannot penetrate the
inner membrane.
CH3
H3C
Transfer of the
fatty acid
across the inner
mitochondrial
membrane
involves
carnitine.
+
N
CH3
CH2
OH
R
CH CH2 COO +
C
carnitine
O
SCoA
Carnitine Palmitoyl
Transferase
R
C
CH3
H3C
+
N
CH3
O
O
CH2
CH CH2 COO
+ HSCoA
fatty acyl carnitine
Carnitine Palmitoyl Transferases catalyze transfer of a
fatty acid between the thiol of Coenzyme A and the
hydroxyl on carnitine.
cytosol
mitochondrial matrix
O
O
R-C-SCoA HO-carnitine
1
HO-carnitine R-C-SCoA
3
2
HSCoA R-C-O-carnitine
O
R-C-O-carnitine HSCoA
O
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
1
HO-carnitine R-C-SCoA
3
2
HSCoA R-C-O-carnitine
O
R-C-O-carnitine HSCoA
O
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.
O
H3C
C
SCoA
acetyl-CoA
O

OOC
CH2
C
SCoA
malonyl-CoA
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.
H H O
b-Oxidation
3
2
1
H3C (CH2)n C C C SCoA
Pathway:
b
fatty acyl-CoA
Step 1. Acyl-CoA
H H
FAD
Dehydrogenase
Acyl-CoA Dehydrogenase
FADH2
catalyzes oxidation
H O
of the fatty acid of
H3C (CH2)n C C C SCoA
acyl-CoA to
trans-2-enoyl-CoA
H
produce a double
bond between carbon atomsH22O& 3.
H
O
There are different Acyl-CoA Dehydrogenases
for short
(4-6 C), medium (6-10H3C),
long
and very long
(12-18 C)
C (CH
SCoA
2)n C CH2 C
chain fatty acids.
OH
H+ + NADH
NAD+
O
O
H
3
H3C (CH2)n C
b
H
FAD
H
O
2
C
H
C
1
SCoA
fatty acyl-CoA
Acyl-CoA Dehydrogenase
FADH2
H
O
H3C (CH2)n C
C
C
H
SCoA
trans-2-enoyl-CoA
FAD His2Othe prosthetic group that functions as e acceptor
H Dehydrogenase.
O
for Acyl-CoA
H
SCoA
(CH2)n C isCH
The
stereospecific,
3C reaction
2 C
bond in enoyl-CoA.
OH
H+ + NADH
NAD+
O
O
yielding a trans double
H
Step 2.
Enoyl-CoA
Hydratase
catalyzes
stereospecific
hydration of the
trans double bond
produced in the
1st step, yielding
L-hydroxyacylCoenzyme A.
3
H3C (CH2)n C
b
H
FAD
H
O
2
C
H
C
1
fatty acyl-CoA
Acyl-CoA Dehydrogenase
FADH2
H
O
H3C (CH2)n C
C
C
H
H2O
SCoA
trans-2-enoyl-CoA
Enoyl-CoA Hydratase
H
O
H3C (CH2)n C CH2 C
OH
H+ + NADH
SCoA
SCoA
3-L-hydroxyacyl-CoA
H
H2O
H
O
H3C (CH2)n C CH2 C
Step 3.
Hydroxyacyl-CoA
Dehydrogenase
catalyzes oxidation
of the hydroxyl in
the b position (C3)
to a ketone.
NAD+ is the
electron acceptor.
+
OH
NAD
H+ + NADH
SCoA
3-L-hydroxyacyl-CoA
Hydroxyacyl-CoA
Dehydrogenase
O
O
H3C (CH2)n C CH2 C
SCoA
b-ketoacyl-CoA
b-Ketothiolase
HSCoA
O
O
H3C (CH2)n C
SCoA + CH3 C
fatty acyl-CoA
(2 C shorter)
SCoA
acetyl-CoA
O
Step 4.
b-Ketothiolase
catalyzes thiolytic
cleavage.
Thiol sulfur of
CoA attacks the bketo carbon
O
H3C (CH2)n C CH2 C
SCoA
b-ketoacyl-CoA
HSCoA
O
O
H3C (CH2)n C
SCoA + CH3 C
fatty acyl-CoA
(2 C shorter)
SCoA
acetyl-CoA
b-Ketothiolase
Acetyl-CoA is released, leaving the fatty acyl in thioester
linkage to the CoA -fatty acyl-CoA (2 C less).
Summary of one round of the b-oxidation pathway:
fatty acyl-CoA + FAD + NAD+ + HS-CoA 
fatty acyl-CoA (2 C 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 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 b-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.
b-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.
Glucose-6-phosphatase
glucose-6-P
glucose
Gluconeogenesis
Glycolysis
pyruvate
fatty acids
During fasting
acetyl CoA
ketone bodies
or carbohydrate
cholesterol
starvation,
oxaloacetate
citrate
oxaloacetate is
depleted in
Krebs Cycle
liver due to
gluconeogenesis.
This impedes entry of acetyl-CoA into Krebs cycle.
Acetyl-CoA in liver mitochondria is converted then to
ketone bodies, acetoacetate & b-hydroxybutyrate.
Ketone body
synthesis:
b-Ketothiolase. The
final step of the boxidation 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.
O
H3C
O
C
acetyl-CoA
SCoA + H3C
Thiolase
HSCoA
O
H3C
O
H3C
C
SCoA
acetyl-CoA
O
H2
C
C
SCoA
HMG-CoA Synthase
O
O
C
SCoA
acetoacetyl-CoA
acetyl-CoA HSCoA

C
C
OH
H2
C
C
O
H2
C
CH3
C
SCoA
HMG-CoA
HMG-CoA Lyase
O

O
C
O
H2
C
acetoacetate
C
O
CH3 + H3C
C
SCoA
acetyl-CoA
b-Hydroxybutyrate Dehydrogenase
b-Hydroxybutyrate
CH3
+
H 
Dehydrogenase
C O NADH
catalyzes reversible
interconversion of
CH2
the ketone bodies
COO
acetoacetate &
acetoacetate
b-hydroxybutyrate.
CH3
+
NAD HO
CH
CH2
COO
D-b-hydroxybutyrate
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.
Fatty Acid Synthesis
O
H3C
C
SCoA
acetyl-CoA
The input to fatty acid
synthesis is acetyl-CoA,
which is carboxylated to
malonyl-CoA.
O

OOC
CH2
C
SCoA
malonyl-CoA
ATP-dependent carboxylation provides energy input.
The CO2 is lost later during condensation with the
growing fatty acid.
Acetyl-CoA
Carboxylase
catalyzes the
2-step reaction
by which
acetyl-CoA is
carboxylated
to form
malonyl-CoA.
Enzyme-biotin
HCO3 + ATP
1
ADP + Pi
Enzyme-biotin-CO2
O
ll
CH3-C-SCoA
acetyl-CoA
2
Enzyme-biotin
O
-
ll
O2C-CH2-C-SCoA
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 .
Enzyme-biotin
HCO3 + ATP
1
ADP + Pi
Enzyme-biotin-CO2
O
ll
CH3-C-SCoA
acetyl-CoA
2
Enzyme-biotin
O
-
ll
O2C-CH2-C-SCoA
malonyl-CoA
The overall reaction may be summarized as:
HCO3 + ATP + acetyl-CoA  ADP + Pi + malonyl-CoA
O
O
C
C
O
N
NH
CH CH
CH
H2C
S
Carboxybiotin
O
O
(CH2)4 C
NH
C
(CH2)4 CH
lysine NH
residue
Biotin is linked to the enzyme by an amide bond between
the terminal carboxyl of the biotin side chain and the
e-amino group of a 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.
H
H3N+ C
SH
COO
CH 2
CH2
CH 2
SH
NH
Fatty Acid
cysteine
Synthase
prosthetic groups:
 the thiol of the sidechain of a cysteine
residue.
 the thiol of
phosphopantetheine,
equivalent in structure
to part of coenzyme A.
Coenzyme A
C
b-mercaptoethylamine
O
CH 2
CH 2
pantothenate
NH
C
NH 2
O
ADP-3'phosphate
HO
C
H
H3C
C
CH 3 O
H2C
O
P
N
N
O
O
O
P
N
N
O
CH 2
O
O
H
H
O
H
OH
H
phosphopantetheine

O
P
O
O
SH
phosphopantetheine
of acyl carrier protein
CH2
Phosphopantetheine
(Pant) is covalently
linked via a phosphate
ester to a serine OH of
the acyl carrier protein
(ACP) of Fatty Acid
Synthase.
CH2
b-mercaptoethylamine
NH
C
O
CH2
CH2
pantothenate
NH
C
O
HO
C
H
H 3C
C
CH3 O
H2C
O
P
NH
O
O
phosphate
CH2
CH
C
serine
residue
O
acetyl-S-CoA HS-CoA
Pant
SH
Cys
SH
Pant
1
SH
CO2
malonyl-S-CoA HS-CoA
Cys
Pant
2
S
C
CH3
O
Cys
S
S
C
O C
CH2
1 Malonyl/acetyl-CoA-ACP
Transacylase COO
Acetyl-CoA-ACP Transacylase
2 Malonyl/acetyl-CoA-ACP
Transacylase
Malonyl-CoA-ACP Transacylase
3 Condensing Enzyme (b-Ketoacyl Synthase)
CH3
3
O
Pant
Cys
S
SH
C
O
CH2
C
O
CH3
The condensation reaction (step 3) involves
decarboxylation of the malonyl, followed by attack of
the acetyl (or acyl).
NADPH NADP+
Pant
Cys
S
SH
C
O
O
CH3
Pant
Cys
S
SH
C
O
HC
5
Pant
Cys
S
SH
C
CH
CH2
CH2
C
4
NADPH NADP+
H2O
OH
CH3
HC
CH3
O
6
Pant
Cys
S
SH
C
O
CH2
CH2
CH3
4 b-Ketoacyl-ACP Reductase
5 b-Hydroxyacyl-ACP Dehydratase
6 Enoyl-ACP Reductase
4. The b-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.
Malonyl-S-CoA HS-CoA
Pant
Cys
S
SH
C
O
7
Pant
Cys
SH
S
C
2
O
Pant
Cys
S
S
C
O
C
CH2
CH2
CH2
CH2
CH2
CH2
COO
CH2
CH3
CH3
O
CH3
7 Condensing Enzyme
Malonyl-CoA-ACP Transacylase
(repeat)(repeat).
2 Malonyl/acetyl-CoA-ACP
Transacylase
Following transfer of the growing fatty acid from
phosphopantetheine to cysteine sulfhydryl, the cycle
begins again, with another malonyl-CoA.
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.
b-Oxidation & Fatty Acid Synthesis
Compared
b Oxidation Pathway Fatty Acid Synthesis
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)
pathway location
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
b-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.
10 9
O
C
OH
oleate 18:1 cis 9
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).
10 9
O
C
OH
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
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