Lecture 32
– Last lecture!!
– Fatty acid biosynthesis
b-oxidation
• Strategy: create a carbonyl group
on the b-C
• First 3 reactions do that; fourth
cleaves the "b-keto ester" in a
reverse Claisen condensation
• Products: an acetyl-CoA and a fatty
acid two carbons shorter
Acyl-CoA Dehydrogenase
• Oxidation of the C-Cb bond
• Mechanism involves proton
abstraction, followed by
double bond formation and
hydride removal by FAD
• Electrons are passed to an
electron transfer
flavoprotein (ETF), and then
to the electron transport
chain.
Acyl-CoA Dehydrogenase
Net: 2 ATP/2 e- transferred
1.
2.
Page 917
3.
4.
Formation of a trans b double bond by
dehydrogenation by acyl-CoA
dehydrogenase (AD).
Hydration of the double bond by enoyl-CoA
hydratase (EH) to form 3-L-hydroxyacylCoA
NAD+-dependent dehydrogenation of bhydroxyacyl-CoA by 3-L-hydroxyacyl-CoA
dehydrogense (HAD) to form b-ketoacylCoA.
C-Cb bond cleavage by b-ketoacyl-CoA
thiolase (KT)
Enoyl-CoA Hydratase
• aka crotonases
• Adds water across the double bond
• Uses substrates with trans-D2-and
cis D2 double bonds (impt in boxidation of unsaturated FAs)
• With trans-D2 substrate forms Lisomer, with cis D2 substrate forms
D-isomer.
• Normal reaction converts transenoyl-CoA to L-b-hydroxyacyl-CoA
1.
2.
Page 917
3.
4.
Formation of a trans b double bond by
dehydrogenation by acyl-CoA
dehydrogenase (AD).
Hydration of the double bond by enoyl-CoA
hydratase (EH) to form 3-L-hydroxyacylCoA
NAD+-dependent dehydrogenation of bhydroxyacyl-CoA by 3-L-hydroxyacyl-CoA
dehydrogense (HAD) to form b-ketoacylCoA.
C-Cb bond cleavage by b-ketoacyl-CoA
thiolase (KT)
Hydroxyacyl-CoA
Dehydrogenase
• Oxidizes the bHydroxyl Group to keto
group
• This enzyme is
completely specific for
L-hydroxyacyl-CoA
• D-hydroxylacyl-isomers
are handled differently
• Produces one NADH
1.
2.
Page 917
3.
4.
Formation of a trans b double bond by
dehydrogenation by acyl-CoA
dehydrogenase (AD).
Hydration of the double bond by enoyl-CoA
hydratase (EH) to form 3-L-hydroxyacylCoA
NAD+-dependent dehydrogenation of bhydroxyacyl-CoA by 3-L-hydroxyacyl-CoA
dehydrogense (HAD) to form b-ketoacylCoA.
C-Cb bond cleavage by b-ketoacyl-CoA
thiolase (KT)
Thiolase
• Nucleophillic sulfhydryl
group of CoA-SH attacks
the b-carbonyl carbon of
the 3-keto-acyl-CoA.
• Results in the cleavage of
the C-Cb bond.
• Acetyl-CoA and an acylCoA (-) 2 carbons are
formed
1.
2.
3.
4.
5.
An active site thiol is added to the
substrate b-keto group.
C-C bond cleavage forms an
acetyl-CoA carbanion intermediate
(Claisen ester cleavage)
The acetyl-CoA intermediate is
protonated by an enzyme acid
group (acetyl-CoA released)
CoA binds to the enzyme-thioester
intermediate
Acyl-CoA is released.
Net reaction reduces fatty acid by 2C and
acyl-CoA group is free to pass through
the cyle again.
Page 919
Figure 25-15 Mechanism of action of bketoacyl-CoA thiolase.
1.
2.
Page 917
3.
4.
Formation of a trans b double bond by
dehydrogenation by acyl-CoA
dehydrogenase (AD).
Hydration of the double bond by enoyl-CoA
hydratase (EH) to form 3-L-hydroxyacylCoA
NAD+-dependent dehydrogenation of bhydroxyacyl-CoA by 3-L-hydroxyacyl-CoA
dehydrogense (HAD) to form b-ketoacylCoA.
C-Cb bond cleavage by b-ketoacyl-CoA
thiolase (KT)
b-oxidation
• Each round of b-oxidation produces 1 NADH, 1 FADH2 and 1 acetylCoA.
 b-oxidation of palmitate (C16:0) yields 129 molecules of ATP
• C 16:0-CoA + 7 FAD + 7 NAD+ + 7 H2O + 7 CoA  8 acetyl-CoA + 7
FADH2 + 7 NADH + 7 H+
• Acetyl-CoA = 8 GTP, 24 NADH, 8 FADH2
• Total = 31 NADH = 93 ATPs + 15 FADH2 = 30 ATPs
• 2 ATP equivalents (ATP  AMP + PPi, PPi  2 Pi) consumed during
activation of palmitate to acyl-CoA
• Net yield = 129 ATPs
Beta-oxidation of unsaturated fatty acids
•
•
Nearly all fatty acids of biological origin have cis double bonds between C9
and C10 (D9 or 9-double bond).
Additional double bonds occur at 3-carbon intervals (never conjugated).
Examples: oleic acid and linoleic acid.
In linoleic acid one of the double bonds is at an even-numbered carbon and the
other double bond is at an odd-numbered carbon atom.
4 additional enzymes are necessary to deal with these problems.
•
Need to make cis into trans double bonds
•
•
•
Page 920
Figure 25-17
Problems in
the oxidation of
unsaturated fatty
acids and their
solutions.
b-oxidation of unsaturated fatty acids
•
•
•
•
•
b-oxidation occurs normally for 3
rounds until a cis-D3-enoyl-CoA is
formed.
Acyl-CoA dehydrogenase can not add
double bond between the  and
bcarbons.
Enoyl-CoA isomerase converts this to
trans- D2 enoyl-CoA
Now the b-oxidation can continue on
w/ the hydration of the trans-D2-enoylCoA
Odd numbered double bonds handled
by isomerase
b-oxidation of fatty acids with even
numbered double bonds
b-oxidation of odd
chain fatty acids
• Odd chain fatty acids are less common
• Formed by some bacteria in the stomachs of
ruminants and the human colon.
• b-oxidation occurs pretty much as w/ even
chain fatty acids until the final thiolase
cleavage which results in a 3 carbon
acyl-CoA (propionyl-CoA)
• Special set of 3 enzymes are required to
further oxidize propionyl-CoA
• Final Product succinyl-CoA enters TCA cycle
Propionyl-CoA Carboxylase
•
•
•
1.
2.
The first reaction
Tetrameric enzyme that has a biotin prosthetic group
Reactions occur at 2 sites in the enzyme.
Carboxylation of biotin at the N1’ by bicarbonate ion (same as
pyruvate carboxylase). Driven by hydrolysis of ATP to ADP and Piactivates carboxyl group for transfer
Stereospecific transfer of the activated carboxyl group from
carboxybiotin to propionyl-CoA to form (S)-methylmalonyl-CoA.
Occurs via nucleophillic attack on the carboxybiotin by a carbanion at
C2 of propionyl-CoA
Page 922
Methylmalonyl-CoA Racemase
•
•
•
2nd reaction for odd
chain fatty acid
oxidation
Transforms (S)methylmalonyl-CoA to
(R)-methylmalonylCoA
Takes place through a
resonance stablized
carbanion intermediate
(p. 923)
Methylmalonyl-CoA mutase
•
•
•
1.
2.
3rd reaction of the pathway: converts (R)-methylmalonyl-CoA to
succinyl-CoA
Utilizes 5’-deoxyadenosylcobalamin (AdoCbl) - coenzyme B12.
AdoCbl has a reactive C-Co bond that is used for 2 types of reactions:
Rearrangements in which a hydrogen atom is directly transferred
between 2 adjacent C atoms.
Methyl group transfers between molecules.
H X
-C1-C2-
X H
-C1-C2-
Figure 25-21 Structure of 5¢deoxyadenosylcobalamin
(coenzyme B12).
Co is coordinated by the
corrin ring’s 4 pyrrole N
atoms, a N from the
dimethylbenzimadazole
(DMB), and C5’ from the
deoxyribose unit.
Page 923
One of only 2 known C-metal
bonds in biology.
Page 923
Figure 25-20 The rearrangement catalyzed by
methylmalonyl-CoA mutase.
Methylmalonyl-CoA mutase
•
•
•
•
•
•
Mechanism begins with homolytic cleavage of the C-Co(III) bond.
The AdoCbl is a free radical generator
C-Co(III) bond is weak and it is broken and the radical is stabilized
favoring the formation of the adenosyl radical.
Rearrangement to form succinyl-CoA from a cyclopropyloxy radical
Abstraction of a hydrogen atom from 5’deoxyadenosine to regenerate
the adenosyl radical
Release of succinyl-CoA
Page 926
Odd chain fatty acids
•
•
•
•
•
•
Transform odd chain length FAs to succinyl-CoA
3 enzymes
Propionyl-CoA carboxylase (biotin cofactor): activates bicarbonate
and transfers to propionyl-CoA to form S-methylmalonyl-CoA.
Methylmalonyl-CoA racemase: Transforms (S)-methylmalonyl-CoA
to (R)-methylmalonyl-CoA through a resonance-stabilized
intermediate.
Methylmalonyl-CoA mutase (B12 cofactor(AdoCbl)): Transforms
(R)-methylmalonyl-CoA to succinyl-CoA by generating a radical.
Succinyl-CoA enters TCA cycle
Combination of fatty acid activation,
transport into mitochondrial matrix
and b oxidation
• Resulting acetyl CoA
enters citric acid
cycle.
• Production of NADH,
FADH2, oxidized by
respiratory chain.
Fatty Acid Breakdown Summary
• Even numbered fatty acids are broken down into acetylCoA by 4 enzymes: acyl-CoA dehydrogenase (AD),
enoyl-CoA hydratase (EH), 3-L-hydroxyacyl-CoA
dehydrogenase (HAD) and b-ketoacyl-CoA thiolase
(KT).
• The breakdown of unsaturated fatty acids (cis double
bonds) requires 4 additional enzymes in mammals:
enoyl-CoA isomerase, 2,4 dienoyl-CoA reductase, 3,2enoyl-CoA isomerase, and 3,5-2,4-dienoyl-CoA
isomerase. In bacteria, they only need enoyl-CoA
isomerase and 2,4-dienoyl-CoA reductase.
• Have to convert cis double bonds to trans double
bonds.
• Unsaturated fatty acids b-oxidation results in the
production of acetyl-CoA.
Fatty Acid Breakdown Summary
• Odd numbered fatty acids are broken down into
propionyl-CoA.
• Propionyl-CoA is converted to S-Methylmalonyl-CoA by
propionyl-CoA carboxylase with ATP and CO2. Uses a
carboxybiotynyl cofactor for the mechanism.
• S-Methylmalonyl-CoA is converted to R-MethylmalonylCoA by methylmalonyl-CoA racemase.
• R-Methylmalonyl-CoA is converted to Succinyl-CoA by
methylmalonyl-CoA mutase. Uses a 5’deoxyadenosylcobalimin (AdoCbl aka coenzyme B12)
cofactor for the mechanism.
Fatty Acid Synthesis
• Fatty acid biosynthesis occurs through condensation
of C2 units (reverse of b-oxidation)
• Acetyl-CoA is the precursor molecule; converted to
malonyl-CoA
• In mammals fatty acid synthesis occurs primarily in
the liver and adipose tissues
• Also occurs in mammary glands during lactation.
• Fatty acid synthesis and degradation go by different
routes
• There are four major differences between fatty acid
breakdown and biosynthesis
The differences between fatty acid
biosynthesis and breakdown
• Intermediates in synthesis are linked to -SH
groups of acyl carrier proteins (as compared to SH groups of CoA)
• Synthesis in cytosol; breakdown in mitochondria
• Enzymes of synthesis are one polypeptide in
eukaryotes.
• Dissociated in bacteria
• Biosynthesis uses NADPH/NADP+; breakdown
uses NADH/NAD+
ACP vs. Coenzyme A
•Intermediates in synthesis are linked to -SH
groups of acyl carrier proteins (ACP) as compared
to -SH groups of CoA
Page 931
Figure 25-28 A comparison of fatty acid b oxidation and
fatty acid biosynthesis.
Fatty Acid Synthesis Occurs in the
Cytosol
• Must have source of acetyl-CoA
• Most acetyl-CoA in mitochondria
• Citrate-malate-pyruvate shuttle provides cytosolic
acetate units and reducing equivalents for fatty acid
synthesis
Citrate Lyase
Citrate synthase
Malate
dehydrogenase
Pyruvate
carboxylase
Malate Enzyme
Fatty Acid Synthesis
• Fatty acids are built from 2-C units derived
from acetyl-CoA
• Acetate units are activated for transfer to
growing FA chain by conversion to malonylCoA
• Decarboxylation of malonyl-CoA and reducing
power of NADPH drive chain growth
• Chain grows to 16-carbons (eight acetyl-CoAs)
• Other enzymes add double bonds and more
carbons
Acetyl-CoA Carboxylase
Acetyl-CoA + HCO3- + ATP  malonyl-CoA + ADP
• The "ACC enzyme" commits acetate to fatty
acid synthesis
• Carboxylation of acetyl-CoA to form malonylCoA is the irreversible, committed step in
fatty acid biosynthesis
Acetyl-CoA
Carboxylase
Regulation of Acetyl-CoA
Carboxylase (ACCase)
• ACCase forms long, active filamentous
polymers from inactive protomers
• Accumulation of palmitoyl-CoA (product)
leads to the formation of inactive
polymers
• Accumulation of citrate leads to the
formation of the active polymeric form
• Phosphorylation modulates citrate
activation and palmitoyl-CoA inhibition
Page 932
Figure 25-30 Association of acetyl-CoA carboxylase
protomers.
Regulation of Acetyl-CoA
Carboxylase (ACCase)
• Unphosphorylated ACCase
has low Km for citrate and is
active at low citrate
• Unphosphorylated ACCase
has high Ki for palmitoyl-CoA
and needs high palmitoyl-CoA
to inhibit
• Phosphorylated E has high
Km for citrate and needs high
citrate to activate
• Phosphorylated E has low Ki
for palmitoyl-CoA and is
inhibited at low palmitoyl-CoA
Page 933
Fatty acid biosynthesis
1.
Acetyl-CoA is converted by MAT to
Acetyl ACP
2.
Acetyl-ACP is attached to KS
(condensation reaction).
3.
Malonyl ACP is formed by MAT.
4.
Acetyl-group is coupled to beta
carbon of malonyl-ACP with release
of CO2 to form acetoacetyl-ACP(2b)
by KS.
5.
Reduction of acetoacetyl-ACP with
NADPH to form D-b-hydroxybutyrlACP by DH
6.
Dehydration of D-b-hydroxybutyrlACP by ER to form a,b-transbutenoyl-ACP
7.
Reduction of the double bond to
form butyryl-ACP
8.
Repeat until Palmitoyl-ACP (C16) is
formed.
9.
ACP is cleaved by TE releasing free
fatty acid.
Fatty Acid Synthesis
• Step 1: Loading – transferring acetyl- and malonylgroups from CoA to ACP
• Step 2: Condensation – transferring 2 carbon unit
from malonyl-ACP to acetyl-ACP to form 2 carbon
keto-acyl-ACP
• Step 3: Reduction – conversion of keto-acyl-ACP to
hydroxyacyl-ACP (uses NADPH)
• Step 4: Dehydration – Elimination of H2O to form
Enoyl-ACP
• Step 5: Reduction – Reduce double bond to form 4
carbon fully saturated acyl-ACP
Step 1: Loading Reactions
O
H3C
O
C S CoA
C
acetyl-CoA
acetyl-CoA:ACP
transacylase
O
HS-ACP
MAT
H
O
C
C S CoA
H
malonyl-CoA
HS-ACP
malonyl-CoA:ACP
transacylase
HS-CoA
HS-CoA
O
H3C
C S ACP
acetyl-ACP
O
C
O
H
O
C
C S ACP
H
malonyl-ACP
Step 2: Condensation Rxn
O
H3C C S ACP
acetyl-ACP
HS-Ketoacyl-ACP Synthase
b-ketoacyl-ACP synthase (KS)
HS-ACP
O
C
O
O
H
O
C
C S ACP
H
+
H3C C S
ketoacyl-ACP Synthase
malonyl-ACP
keto-ACP synthase
CO2
O
H
O
H3C C
C
C S ACP
H
acetoacetyl-ACP
Step 3: Reduction
O
H
O
H3C C
C
C S ACP
H
acetoacetyl-ACP
KR
NADPH + H+
Ketoacyl-ACP Reductase
NADP+
OH H
H3C C
C
H
H
O
C S ACP
b-hydroxybutyryl-ACP
Step 4: Dehydration
OH H
H3C C
C
H
H
O
C S ACP
b-hydroxyacyl-ACP
DH
b-hydroxyacyl-ACP
dehydrase
H20
H3C C
H
H
O
C
C S ACP
trans-enoyl-ACP
Step 5: Reduction
H3C C
H
O
C
C S ACP
trans-enoyl-ACP
H
NADPH + H+
ER
enoyl-ACP reductase
NADP+
H
H
O
H3C C
C
C S ACP
H
H
trans-enoyl-ACP
Step 6: next condensation
H H O
H3C C C C S ACP
H H
butyryl-ACP
HS-Ketoacyl-ACP Synthase
KS
HS-ACP
O
C
O
H H O
H
O
C
C S ACP
H
+
H3C C C C S KAS
H H
malonyl-ACP
keto-ACP synthase
CO2
H H O
H
O
H3C C C C
C
C S ACP
H H
H
ketoacyl-ACP
H O
Termination
of Fatty Acid
Synthesis
H3C C C S ACP
H
Palmitoyl-ACP
14
Thioesterase
HS-ACP
H O
H3C C C O
H
Palmitic Acid
14
ATP + HS-CoA
Acyl-CoA
synthetase
AMP + PPi
H O
H3C C C S CoA
H
14
Palmitoyl-CoA
Page 933
Fatty acid biosynthesis
1.
Acetyl-CoA is converted by MAT to
Acetyl ACP
2.
Acetyl-ACP is attached to KS
(condensation reaction).
3.
Malonyl ACP is formed by MAT.
4.
Acetyl-group is coupled to beta
carbon of malonyl-ACP with release
of CO2 to form acetoacetyl-ACP(2b)
by KS.
5.
Reduction of acetoacetyl-ACP with
NADPH to form D-b-hydroxybutyrlACP by DH
6.
Dehydration of D-b-hydroxybutyrlACP by ER to form a,b-transbutenoyl-ACP
7.
Reduction of the double bond to
form butyryl-ACP
8.
Repeat until Palmitoyl-ACP (C16) is
formed.
9.
ACP is cleaved by TE releasing free
fatty acid.
Organization of Fatty Acid
Synthesis Enzymes
• In bacteria and plants, the fatty acid synthesis
reactions are catalyzed individual soluble
enzymes.
• In animals, the fatty acid synthesis reactions are
all present on multifunctional polypeptide.
• The animal fatty acid synthase is a homodimer of
two identical 250 kD polypeptides.
Animal Fatty Acid Synthase
Regulation of FA Synthesis
• Allosteric modifiers, phosphorylation and
hormones
• Malonyl-CoA blocks the carnitine
acyltransferase and thus inhibits betaoxidation
• Citrate activates acetyl-CoA carboxylase
• Fatty acyl-CoAs inhibit acetyl-CoA
carboxylase
• Hormones regulate ACC
• Glucagon activates lipases/inhibits ACC
• Insulin inhibits lipases/activates ACC
Allosteric regulation
of fatty acid
synthesis occurs at
ACCase and the
carnitine
acyltransferase
Glucagon inhibits
fatty acid synthesis
while increasing
lipid breakdown and
fatty acid boxidation
Insulin prevents
action of glucagon
Regulation
• Pancreatic  and b cells directly sense the dietary and energy
state of the organism through [glucose] in the blood.
  cells respond to low blood glucose by secreting glucagon.
 b cells respond to the high blood glucose by secreting insulin.
• Both involved in glycogen metabolism.
• These hormones determine whether fatty acids will be oxidized
or synthesized.
• Target the flux-generating regulatory enzymes of fatty acid
synthesis (acetyl-CoA carboxylase).
• Short-term regulation
• ACCase inhibited by cAMP-dependent phosphorylation
(glucagon).
• Activated by insulin-dependent dephosphorylation.
Regulation
• ACCase inhibitied by palmitoyl-CoA.
• Activated by citrate.
• Long-term regulation: control the amount of enzyme present
over hours or days.
• Polyunsaturated fatty acids decreases the lipid biosynthesis
enzymes.
• Adipose tissue lipoprotein lipase-enzyme that inititates fats for
storage is increased by insulin and decreased by starvation.
• Starvation and/or regular exercise decreases blood glucosechanges hormone balance.
• Results in long-term changes in gene expression that increase
the levels of fatty acid oxidation enzymes and decrease those of
lipid biosynthesis.
Regulation
• Fatty acid oxidation regulated by concentrations of fatty acids in
blood.
• Controlled by hydrolysis rates of triacylglycerols in adipose
tissue by hormone-sensitive triacylgycerol lipase.
• Regulated by phosphorylation(active)/dephosphorylation (less
active) in response to cAMP.
• Epinephrine and norepinephrine act to increase adipose tissue
cAMP concentrations -> lead to protein kinase A
phosphorylation, increase phosphorylation of enzymes.
• Stimulates lipolysis in adipose tissue raising blood fatty acid
levels and activates b-oxidation in liver and muscles.
Regulation
• AMP-dependent protein kinase (AMPK) phosphorylates
ACCase (inactive) -inhibits fatty acid biosynthesis.
• AMPK measures energy levels of the cell. Activated by AMP
and inhibited by ATP.
• Insulin has opposite effect of glucagon and epinephrine:
stimulates glycogen and triacylglycerol formation.
• Decreases cAMP levels.
• Stimulates dephosphorylation of ACCase.
• Ratio of glucagon/insulin important for rate and direction
of fatty acid metabolism.
• Carnitine palmitoyltransferase I is inhibited by malonyl-CoA.
• Keeps new fatty acids from getting into the mitochondria.
Page 941