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Transcript
Lecture 31
– Quiz on Wed (today): fatty acid synthesis
Final on Monday morning 8-10AM
Glycosylphosphatidylinositol (GPI)
•
•
•
Anchor proteins to the exterior of the eukaryotic membrane.
Alternative to transmembrane polypeptides
Proteins destined to be anchored to the surface of the
membrane are synthesized with membrane spanning Cterminal sequences which are removed after GPI addition.
Figure 11–20 Plasma membrane proteins have a variety of functions.
Figure 9.19 Passive transport of solute molecules through a permeable
membrane
Figure 9.20 Three types of membrane transport
Figure 9.23 Glucose permease of erythrocyte membrane
Passive transport system - intracellular [glucose] =< plasma
[glucose] concentration.
Also transports epimers of glucose - mannose, galactose at slower
rates (20%)
Figure 9.23 Transport of sodium and potassium ions by Na+-K+
ATPase transporter (pump)
Three Na+ ions are transported out of the cell for every two K+ that
move inside
Lipases
•
•
How are lipids accessed for energy production?
Know the differences between the triacylglycerol lipase
and phospholipase A2 mechanisms.
•
Triacylglycerol lipase uses a catalytic triad similar to Ser
proteases (Asp, His, Ser)
Phospholipase A2 uses a catalytic triad but substitutes water
for Ser.
•
Page 911
Figure 25-3a Substrate binding to phospholipase A2. (a)
A hypothetical model of phospholipase A2 in complex
with a micelle of lysophosphatidylethanolamine.
Page 912
Figure 25-4a The X-ray structure of porcine
phospholipase A2 (lavender) in complex with the
tetrahedral intermediate mimic MJ33.
Figure 25-4bThe catalytic
mechanism of
phospholipase A2.
Page 912
What other
mechanism does this
look like?
What are the
differences?
Fatty acid binding proteins
•
•
•
Fatty acids form complexes with intestinal fatty acidbinding protein (I-FABP) which makes them more soluble.
Chylomicrons-transport exogenous (dietary) triacylglycerols
and chloestorl packaged into lipoprotein molecules from the
intestine to the tissues.
Chylomicrons are released into the bloodstream via transport
proteins named for their density.
•
VLDL (very low density lipoproteins), LDL (low density
lipoproteins) - transport endogenous (internally produced)
triacylglycerols and cholesterols from the liver to tissues
“Bad”
•
HDL (high density lipoproteins), - transport endogenous
cholesterol from the tissues to liver - “Good”
Page 439
Table 12-6 Characteristics of the Major Classes of
Lipoproteins in Human Plasma.
Page 442
Lipoprotein lipase
•
•
Hydrolyzes triacylglycerol components fo chylomicrons and
VLDL to free fatty acids and glycerol.
Fatty acids taken up by tissues.
•
Glycerol is returned to liver or kidneys to convert to DHAP
Fatty acid oxidation
•
•
•
•
Once fatty acids are taken into the cell they undergo a series
of oxidations to yield energy.
In eukaryotes, occurs in the matrix of mitochondria (same
place as TCA cycle)
Triglycerides found in fat cells (adipocytes) or in cytoplasm.
Was found by Knoop back in the day (1904) through the
following experiment that the oxidation of the carbon atom 
to the carboxyl-group is involved in fatty acid breakdown.
Page 914
Figure 25-8 Franz Knoop’s classic experiment
indicating that fatty acids are metabolically oxidized at
their -carbon atom.
Beta Oxidation of Fatty Acids
• Process by which fatty acids are degraded by
removal of 2-C units
• -oxidation occurs in the mitochondria matrix
• The 2-C units are released as acetyl-CoA, not
free acetate
• The process begins with oxidation of the
carbon that is "beta" to the carboxyl carbon, so
the process is called"beta-oxidation"
Fatty acid oxidation
•
•
•
•
•
Triglycerides are broken down into free fatty acids in the
cytoplasm.
Beta-oxidation takes place in the mitochondrial matrix.
Fatty acids must be imported into the matrix
Requires activation of fatty acids in the cytosol (fatty acids
converted to acyl-CoA form)
Activated fatty acids (acyl-CoA form) are imported into the
mitochondrion.
Fatty acids must first be activated by
formation of acyl-CoA
• Acyl-CoA synthetase condenses fatty acids with CoA, with
simultaneous hydrolysis of ATP to AMP and PPi
• Formation of a CoA ester is expensive energetically
• Reaction just barely breaks even with ATP hydrolysis Go’ATP
hydroysis = -32.3 kJ/mol, Go’ Acyl-CoA synthesis +31.5 kJ/mol.
• But subsequent hydrolysis of PPi drives the reaction strongly
forward (Go’ –33.6 kJ/mol)
Fatty acid + CoA + ATP
acyl-CoA + AMP + PPi
Import of acyl-CoA into mitochondria
•
-oxidation occurs in the mitochondria, requires import of
long chain acyl-CoAs
• Acyl-CoAs are converted to acyl-carnitines by carnitine
acyltransferases.
• A translocator then imports Acyl carnitine into the matrix
while simultaneously exporting free carnitine to the
cytosol
• Acyl-carnitine is then converted back to acyl-CoA in the
matrix
Page 915
Figure 25-10 Acylation of carnitine
catalyzed by carnitine palmitoyltransferase.
Page 916
Figure 25-11 Transport of fatty acids into
the mitochondrion.
Import of acyl-CoA into mitochondria
• The acyl group of cytosolic acyl-CoA is transferred to
carnitine-releases CoA to cytosol.
• Acyl-carnitine is transported into the matrix by the
carnitine carrier protein
• The acyl group is transferred to a CoA molecule from the
mitochondrion.
• Carnitine is returned to the cytosol.
Deficiencies of carnitine or carnitine
transferase or translocator activity are
related to disease state
• Symptons include muscle cramping during exercise, severe
weakness and death.
• Affects muscles, kidney, and heart tissues.
• Muscle weakness related to importance of fatty acids as
long term energy source
• People with this disease supplement diet with medium chain
fatty acids that do not require carnitine shuttle to enter
mitochondria.
Activation of fatty acids for -oxidation
• Activation of fatty acid to acyl-CoA form by acyl-CoA synthetaserequires CoASH and ATP (converted to AMP + PPi ) in the cytosol.
• Acyl-CoA is converted to acyl-carnitine by carnitine acyltransferase
(carnitine palmitoyl transferase I) in the cytosol for transport into the
mitochondrion.
• Acyl-carnitine is transported across the membrane by the carnitine
carrier protein.
• Acyl-carnitine is converted to acyl-CoA by carnitine palmitoyl
transferase II in the mitochondrial matrix.
• The fatty acyl-CoA is ready for the reactions of the oxidation pathway
-oxidation
•
The first 3 steps resemble the citric acid reactions that convert succinate to
oxaloacetate.
- OC-CH -CH -CO 2
2
2
2
FAD
Succinate
Succinate dehydrogenase
FADH2
H2O
H
- OC-C=C-CO 2
2
H
Fumarate
Fumarase
OH
- OC-CH -C-CO 2
2
2
NAD+
Malate
Malate dehydrogenase
NADH + H+
O
- OC-CH -C-CO 2
2
2
Oxaloacetate
1.
2.
Page 917
3.
4.
Formation of a trans  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 -ketoacylCoA.
C-C bond cleavage by -ketoacyl-CoA
thiolase (KT)
-oxidation
• Strategy: create a carbonyl group
on the -C
• First 3 reactions do that; fourth
cleaves the "-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-C 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
• Mitochondria have four acyl-CoA dehydrogenases
• Specificities for short (C4 to C6), medium (C6 to C10), long
(C8-C12), very long (C12 to C18) chain fatty acyl-CoAs.
• Reoxidized via the Electron Transport Chain.
Figure 25-13 Ribbon diagram of the active site region in
a subunit of medium-chain acyl-CoA dehydrogenase
from pig liver mitochondria in complex with octanoylCoA.
Page 917
FAD = green
Octonoyl=blue
CoA =white
Glu376 -red
General base
Acyl-CoA Dehydrogenase
Net: 2 ATP/2 e- transferred
1.
2.
Page 917
3.
4.
Formation of a trans  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 -ketoacylCoA.
C-C bond cleavage by -ketoacyl-CoA
thiolase (KT)
Enoyl-CoA Hydratase
• aka crotonases
• Adds water across the double bond
• Uses substrates with trans-2-and
cis 2 double bonds (impt in boxidation of unsaturated FAs)
• With trans-2 substrate forms Lisomer, with cis 2 substrate forms
D-isomer.
• Normal reaction converts transenoyl-CoA to L--hydroxyacyl-CoA
Enoyl hydratase
1.
2.
Page 917
3.
4.
Formation of a trans  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 -ketoacylCoA.
C-C bond cleavage by -ketoacyl-CoA
thiolase (KT)
Hydroxyacyl-CoA
Dehydrogenase
• Oxidizes the Hydroxyl 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  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 hydroxyacyl-CoA by 3-L-hydroxyacyl-CoA
dehydrogense (HAD) to form -ketoacylCoA.
C-C bond cleavage by -ketoacyl-CoA
thiolase (KT)
Thiolase
• Nucleophillic sulfhydryl
group of CoA-SH attacks
the -carbonyl carbon of
the 3-keto-acyl-CoA.
• Results in the cleavage of
the C-C 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 ketoacyl-CoA thiolase.
1.
2.
Page 917
3.
4.
Formation of a trans  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 hydroxyacyl-CoA by 3-L-hydroxyacyl-CoA
dehydrogense (HAD) to form -ketoacylCoA.
C-C bond cleavage by -ketoacyl-CoA
thiolase (KT)
-oxidation
• Each round of -oxidation produces 1 NADH, 1 FADH2 and 1 acetylCoA.
 -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 (9 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.
-oxidation of unsaturated fatty acids
•
•
•
•
•
-oxidation occurs normally for 3
rounds until a cis-3-enoyl-CoA is
formed.
Acyl-CoA dehydrogenase can not add
double bond between the  and
carbons.
Enoyl-CoA isomerase converts this to
trans- 2 enoyl-CoA
Now the -oxidation can continue on
w/ the hydration of the trans-2-enoylCoA
Odd numbered double bonds handled
by isomerase
-oxidation of fatty acids with even
numbered double bonds
-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.
• -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).
One of only 2
known C-metal
bonds in biology.
Page 923
Co is coordinated
by the corrin ring’s
4 pyrrole N atoms,
a N from the
dimehylbenzimad
azole (DMB), and
C5’ from the
deoxyribose unit.
Page 923
Figure 25-20 The rearrangement catalyzed by
methylmalonyl-CoA mutase.
Methylmalonyl-CoA mutase
•
•
•
Unusual  barrel enzyme. Most  barrel enzymes have active
sites at the C-terminus, but the methlymalonyl-CoA mutase AdoCbl
group is located at the N-terminus.
The Co atom is coordinated by His610 instead of the N from the 5,6
dimehylbenzimadazole (DMB)
Has a narrow tunnel through the center of the barrel for the substrate
and provides the only access to the active site, protecting the free
radical intermediates that are produced from the side reactions.
Figure 25-22a
X-Ray structure of P.
shermanii methylmalonyl-CoA mutase in complex with
2-carboxypropyl-CoA and AdoCbl. (a) The catalytically
active  subunit.
2-carboxypropyl-CoA
Page 925
N-term
AdoCb
C-term
Page 925
Figure 25-22b
The
arrangement of AdoCbl and 2carboxypropyl-CoA molecules in
the  barrel of P. shermanii
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  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 -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 -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.
Ketone Bodies
• A special source of fuel and energy for certain tissues
• Produced when acetyl-CoA levels exceed the capacity of
the TCA cycle (depends on OAA levels)
• Under starvation conditions no carbos to produced
anpleorotic intermediates
• Some of the acetyl-CoA produced by fatty acid oxidation in
liver mitochondria is converted to acetone, acetoacetate
and -hydroxybutyrate
• These are called "ketone bodies"
• Source of fuel for brain, heart and muscle
• Major energy source for brain during starvation
• They are transportable forms of fatty acids!
Ketone bodies
• Acetyl-CoA can be
converted through
ketogenesis to
acetoacetate, D-hydroxybutyrate, and
acetone.
• Acetoacetate and D-hydroxybutyrate
are carried in the
bloodstream to other
tissues where they are
converted to acetylCoA.
Ketone bodies
• Acetoacetate and D-hydroxybutyrate are carried
in the bloodstream to other
tissues where they are
converted to acetyl-CoA.
• Catalyzed by three enzymes:
-hydroxybutyrate
dehydrogenase, 3ketoacyl-CoA transferase,
thiolase
Formation of
ketone bodies
Re-utilization
of
ketone bodies
Ketone Bodies and Diabetes
• Lack of insulin related to uncontrolled fat breakdown in
adipose tissues
• Excess -oxidation of fatty acids results in ketone body
formation.
• Can often smell acetone on the breath of diabetics.
• High levels of ketone bodies leads to condition known as
diabetic ketoacidosis.
• Because ketone bodies are acids, accumulation can lower
blood pH.