Fatty Foods Have Very High Energy Content

1
Introduction to Lipid Metabolism
Lipids are fats, cholesterol, steriods, bile
salts, fatty acids, etc. Lipids play roles
in energy storage compose cell
membranes hormones and signaling molecules
Fatty Foods Have Very High Energy
Content and Fats are the Primary Form in Which
Metabolic Energy is Stored
Food Stuff Specific Enthalpy (DH, kJ
g-1) Carbohydrates 16 Fats 37 Proteins 17
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Lipid Metabolism The Structure of Lipids
Fatty Acids Carboxylic acids with long carbon
chains. May be saturated (no double bonds)
or unsaturated (with double
bonds) Chemical and physical properties depend
on degree of saturation stereochemistry at
double bond sites Rarely occur free in
nature occur as esters of glycerol
laurate C12H23O2
glycerol
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Some Common Fatty Acids
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Properties of Some Common Fatty Acids
Increasing chain length tends to increase the
melting point. The presence of double bonds
tends to greatly decrease the melting point.
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A General Triacylglycerol or Fat
Glycerol
Triacylglycerol
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Fatty Acids and Fats Form Micelles in Aqueous
Solution
The formation of a micelle minimizes the amount
of hydrophobic surface area that must be solvated
by (polar) water. This is known as the
hydrophobic effect. It is also the driving
force behind protein folding. It is primarily an
entropic phenomenon.
Polar head group (e.g., glycerol
esters) Non-polar interior (long carbon chains)
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Triacylglyerols are First Processed by Pancreatic
Lipase
Promotes ester hydrolysis at the 1 and 3
positions.
lipase
2-acylglycerol 2 fatty acids
1,2,3-triacylglycerol
Because fats occur in aqueous solution as
micelles, lipase must function at a lipid-water
interface. Lipase forms a complex with
colipase. The complex exhibits an amphiphilic
surface, facilitating chemistry at the
lipid-water interface.
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The Active LipaseColipase Complex Forms at The
Lipid-Water Interface
A lid (yellow) prevents access to the lipase
active site in the inactive conformation. The
lid reorganizes at the lipid-water interface and
allows access to the active site. Colipase
(magenta) binds the non-catalytic C-terminal
domain of lipase. Its loops present
hydrophobic residues. A surface of 50 Å is
created. This phenomenon is called interfacial
activation.
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Ester Hydrolysis by Lipase Proceeds Similarly to
Peptide Hydrolysis by Chymotrypsin. In
particular, lipase exhibits a catalytic triad.
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Phospholipids
Membrane components. Signaling molecules.
A representative phospholipid. Phosphatidylcholine
with stearoyl and oleyl side chains
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Phospholipids Are Degraded by Phospholipases
Phospholipase A2 promotes hydrolysis at the 2
position.
phospholipase
2,3-acyl-1-phosphocholine glycerol
3-acyl-1-phosophocholine glycerol (lysophospholip
id)
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Overview of Lipid Metabolism Lipids are absorbed
by the intestinal mucosa (site of lipase
activity). Action of lipase releases fatty acids
? form micelles aid in further breakdown and
absorption. Intestinal Mucosa packaged into
triacyl-glycerol containing lipoprotein particles
called chylomicrons. These are sent into the
lymph for entry into the bloodstream and delivery
to muscle and adipose (fat) tissue. Capilaries
in adipose and muscle tissue lipoprotein lipase
hydrolyzes triacyl-glycerols into glycerol and
fatty acids. Fatty acids taken up by tissue ?
oxidation entry into TCA cycle. Glycerol sent
to liver ? Converted to DHAP entry into
glycolysis.
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Intestinal Fatty Acid Binding Protein
Strands A, B, C, D, E, F form a gapped b-sheet
which is roughly orthogonal to the sheet formed
from strands G, H, I, J. In yellow, a bound
palmitate molecule occupies the gap between
strands D and E. Carboxy function coordinated
by R, Q side chains and two H2O molecules.
Hydrophobic tail in contact with mostly
aromatic hydrophobic residues.
I-FABP provides for the solubility of fatty acids
absorbed by the intestines.
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The Blood Protein Serum Albumin Binds and
Transports Fatty Acids
Uncomplexed fatty acids would exhibit
solubilitiies of 10-6 M in complex with serum
albumin, fatty acid concentrations in blood
approach 2 mM.
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The Fate of Glycerol Produced in Lipid
Metabolism The glycerol liberated in lipid
metabolism is directed to the liver or kidneys
where it enters glycolysis as DHAP.
TIM
DHAP
GAP
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The Oxidation of Fatty Acids and the Knoop
Experiment (1904) Fatty Acids are Oxidized at
their b-Carbons
The isolation of acetyl-CoA in the 1950s, and
elucidation of fatty acid (and glucose and TCA)
metabolic pathways confirmed Knoops deduction.
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Fatty Acid Oxidation Requires a Priming Step
Acyl-CoA Synthetases (or Thiokinases)
Recall that inorganic pyrophosphatase is
ubiquitous, and that the degradation of PPi is
highly energetically favourable.
18O labeled fatty acids result in 18O labeled
acyl-CoA and 18O labeled AMP species. Evidence
for an acyl-adenylate mixed anhydride
intermediate that is attacked by CoASH.
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Fatty-acyl-CoA Species Do Not Cross the
Mitochondrial Inner Membrane
Rather, carnitine acyl-transferases liberate CoA
in the cytoplasm and consume CoA in the
mitochondrial matrix.
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Entry of Fatty Acids into the Mitochondrial
Matrix Another Shuttle System
Free carnitine is transported from the matrix to
the cytosol as acyl-carnitines are transported
from the cytosol to the matrix. Carnitine
acyl-transferases remove CoASH in the cytosol,
attach CoASH in the matrix.
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The b-Oxidation Pathway

• Formation of a trans-a,b double bond through
  dehydrogenation
• by the flavoenzyme acyl-CoA dehydrogenase (AD).
• Hydration of the double bond by enoyl-CoA
  hydratase (EH) to
• form a 3-L-hydroxyacyl-CoA.
• NAD-dependent dehydrogenation of the
  b-hydroxyacyl-CoA
• to form the corresponding b-ketoacyl-CoA by
• 3-L-hydroxyacyl-CoA dehydrogenase (HAD).

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The b-Oxidation Pathway

• Formation of a trans-a,b double bond through
  dehydrogenation
• by the flavoenzyme acyl-CoA dehydrogenase (AD).
• Hydration of the double bond by enoyl-CoA
  hydratase (EH) to
• form a 3-L-hydroxyacyl-CoA
• NAD-dependent dehydrogenation of the
  b-hydroxyacyl-CoA
• to form the corresponding b-ketoacyl-CoA by
• 3-L-hydroxyacyl-CoA dehydrogenase (HAD).
• The three reactions above are reminiscent of the
• succinate ? fumarate ? malate ? oxaloacetate
• transformations of the citric acid cycle.
• Ca-Cb cleavage in a thiolysis reaction with CoA
  as catalyzed by
• b-ketoacyl-CoA thiolase (KT) to form acetyl-CoA
  and a new
• acyl-CoA with two fewer carbons, which then
  becomes a
• substrate for the AD, in (1).

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The b-Oxidation Pathway Acyl-CoA dehydrogenase
(AD)
The first transformation in fatty acid oxidation
Dehydrogenation across the a-b bond to the
trans-D2-enoyl-CoA product. The reaction
produces FADH2 which ultimately enters oxidative
phosphorylation. This transformation is similar
to the succinate dehydrogenase reaction in the
TCA cycle.
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Crystal Structure of Medium Chain Acyl-CoA
Dehydrogenase (AD) in Complex with Octanoyl-CoA
Implications for Mechanism
CoA
Octanoyl- group
FAD
In red, the side chain of Glu 376, which is
thought to function as a general base in the
abstraction of the a proton in the
dehydrogenation reaction.
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The b-Oxidation Pathway Enoyl-CoA hydratase (EH)
The second transformation in fatty acid
oxidation Hydration of the trans-D2-enoyl-CoA
reactant to the 3-L-hydroxyacyl-CoA
product. This transformation is similar to the
fumarase reaction in the TCA cycle.
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The b-Oxidation Pathway HAD
The third transformation in fatty acid oxidation
Dehydrogenation of the 3-L-hydroxyacyl-CoA
reactant to the b-ketoacyl-CoA product. Carried
out by 3-L-hydroxyacyl-CoA dehydrogenase
(HAD). The reaction produces NADH which enters
oxidative phosphorylation. This transformation
is similar to the malate dehydrogenase reaction
in the TCA cycle.
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The b-Oxidation Pathway KT
The fourth transformation in fatty acid
oxidation Capture of the b-ketone function by
CoASH and elimination of acetyl-CoA. Carried out
by b-ketoacyl thiolase (KT). The fatty acyl-CoA
product is now two carbon atoms shorter, and
these have been converted into Acetyl-CoA.
Acetyl-CoA may now enter the citric acid
cycle. The acyl-CoA product may now return to
reaction (1) at AD for subsequent oxidation.
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Mechanism of b-ketoacyl-CoA Thiolase (KT)
Stabilization of a carbanion at the CoA center.
Recall citrate synthase.
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Fatty Acid Oxidation is Highly Exergonic (I)
Each round of b-oxidation 1 FADH2 (AD) 1 NADH
(HAD) 1 acetyl-CoA (1) Each acetyl-CoA 3
NADH 1 FADH2 1 GTP
FADH2 and NADH enter oxidative
phosphorylation FADH2 and NADH enter
oxidative phosphorylation
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Fatty Acid Oxidation is Highly Exergonic (II)
Example The oxidation of one molecule of
palmitate (C16) 7 rounds of b-oxidation ? 7
NADH, 7 FADH2, 8 acetyl-CoA 8 acetyl-CoA ? 24
NADH, 8 FADH2, 8 GTP Totals 31 NADH, 15 FADH2,
8 GTP ATP Equivalents NADH ? 2.5 ATP FADH2
? 1.5 ATP GTP ? 1 ATP
31 NADH 77.5 ATP 15 FADH2 22.5 ATP 8 GTP 8
ATP Total 108 ATP
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Fatty Acid Biosynthesis
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Acetyl-CoA Carboxylase (ACC)
Catalyzes the first committed step of fatty acid
biosynthesis Biotin containing enzyme. Biotin
activates bicarbonate ion. Carboxylate is added
to acetyl-CoA Reaction chemistry similar to that
of pyruvate carboxylase (gluconeogenesis).
Ebiotin ATP
EbiotinCO2- ADP
Ebiotin malonyl-CoA
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Acetyl-CoA Carboxylase is Hormonally Regulated
Becomes phosphorylated (inactivation) in a
cAMP dependent manner. Responsive to
glucagon, adrenaline, noradrenaline (inactivati
on) insulin (activation) Polymerizes on
dephosphorylation monomer ? inactive polymer ?
active
Also responsive to citrate (activation)
palmitoyl-CoA (deactivation negative feedback).
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Acyl-Carrier Protien (ACP)
Acyl-carrier protein is the site of fatty acid
elongation. The growing acyl chain is esterified
at the phosphopantetheine group. Very similar
chemistry to CoA.
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The Logic of Fatty Acid Synthesis (I)

• Acetyl group transfer from acetyl-CoA to ACP.
• (malonyl/acetyl-CoA-ACP transacylase (MAT).
• 2. Loading of b-ketoacyl-ACP synthase (KS)
• Acetyl group transfer from ACP to KS cysteine
  residue.
• (1.) Malonyl-ACP formation.
• Transfer of the malonyl group from malonyl-CoA
  to ACP (MAT)
• (2.) Coupling of the acetyl group on KS to the
  b-carbon of the malonyl group on ACP.
• Concommittant decarboxylation of malonyl group.
• Formation of acetoacetyl-ACP.
• (Or, more generally, formation of a b-ketone and
  addition of a two carbon unit.)

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The Logic of Fatty Acid Synthesis (II)
3. Reduction of b-carbonyl on acetoacetyl ACP to
the alcohol. b-ketoacyl-ACP reductase
(KR) 4. Elimination of water by
b-hydroxyacyl-ACP-dehydrase (DH). Yields the
a,b-trans-alkene. 5. Reduction of the
a,b-transalkene by enoyl-ACP-reductase
(ER) Yields the saturated product. 6. Repetition
of reactions 2 through 5 six more times to
generate palmitoyl-ACP. 7. Release of palmitate
group by palmitoyl thioesterase (TE).
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Acetyl-CoA and Malonyl-CoA Chemistry in Fatty
Acid Biosynthesis
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The Condensation Reaction Between Malonyl-ACP and
the Growing Fatty Acid Chain
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The Reductive Reactions of Fatty Acid Biosynthesis
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Repetition of Steps, and the Release of Product
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The Logic of Fatty Acid Synthesis (I)

• Acetyl group transfer from acetyl-CoA to ACP.
• (malonyl/acetyl-CoA-ACP transacylase (MAT).
• 2. Loading of b-ketoacyl-ACP synthase (KS)
• Acetyl group transfer from ACP to KS cysteine
  residue.
• (1.) Malonyl-ACP formation.
• Transfer of the malonyl group from malonyl-CoA
  to ACP (MAT)
• (2.) Coupling of the acetyl group on KS to the
  b-carbon of the malonyl group on ACP.
• Concommittant decarboxylation of malonyl group.
• Formation of acetoacetyl-ACP.
• (Or, more generally, formation of a b-ketone and
  addition of a two carbon unit.)

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The Logic of Fatty Acid Synthesis (II)
3. Reduction of b-carbonyl on acetoacetyl ACP to
the alcohol. b-ketoacyl-ACP reductase
(KR) 4. Elimination of water by
b-hydroxyacyl-ACP-dehydrase (DH). Yields the
a,b-trans-alkene. 5. Reduction of the
a,b-transalkene by enoyl-ACP-reductase
(ER) Yields the saturated product. 6. Repetition
of reactions 2 through 5 six more times to
generate palmitoyl-ACP. 7. Release of palmitate
group by palmitoyl thioesterase (TE).
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The material following is supplemental.
43
Hypothetical Model of Phospholipase A2 Binding to
Phospholipid Micelle
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Apparent Mechanism of Phospholipase A2 A
catalytic dyad.
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Lipid Metabolism Involves the Hydrolysis of Fatty
Acid Esters of Glycerol
3 H2O
3
Ester Hydrolysis is Similar to Peptide Hydrolysis

H2O
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Lipid Metabolism A Short Detour into Peptide
Hydrolysis and Protease Chemistry
The hydrolysis of a peptide bond addition of
water across a peptide bond yields an acid and
an amine Biologically this reaction is
accomplished by proteases. e.g. chymotrypsin tr
ypsin elastase HIV-1 protease

Serine proteases possess Catalytic triads
Aspartic protease
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Chymotrypsin Uses an Internal Serine Side Chain
as its Nucleophile
Scissile bond The bond to be cleaved is called
the scissile bond.
S195
But, serine side chains present an alcohol
function ? and, like water, alcohol functions
are poor nucleophiles.
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Chymotrypsin Exhbits a Hydrogen Bond Network That
Dramatically Enhances the Nucleophilicity of S195
H57, D102, and S195 comprise the catalytic
triad.
Alkoxide anions are very potent nucleophiles.
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Catalytic Triad From the Crystal Structure of
Chymotrypsin
His57
2.6 Å
3.1 Å
Asp102
Ser195 nucleophile
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Chymotrypsin has Evolved a Means of Activating
The S195 Nucleophile But
The hydrolysis involves a tetrahedral
intermediate exhibiting an oxyanion. The
interior of proteins are typically hydrophobic.
How shall an anion be accommodated in a
hydrophobic environment?
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An Oxyanion Hole is Available Which Presents
Hydrogen Bond Donors to the Developing Negative
Charge in the Tetrahedral Intermediate
Oxyanion hole
Scissile bond
Nucleophilic g-O of S195
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Resolution of the Tetrahedral Intermediate
Cleavage of the Scissile Bond Release of the
Cleaved N-terminal Fragment Formation of an
Acyl-enzyme Intermediate
53
Resolution of the Acyl-enzyme Intermediate is
Facilitated by Diffusion of Water into the Active
Site
Notice how H57 and D102 can also enhance the
nucleophilicity of active site water. Think of
this as a catalytic dyad.
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The Full Reaction Pathway for Peptide Bond
Hydrolysis by Chymotrypsin
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Ester Hydrolysis by Lipase Proceeds Similarly to
Peptide Hydrolysis by Chymotrypsin
2 H2O
In particular, lipase exhibits a catalytic triad.
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Phospholipids
Membrane components. Signaling molecules.
A representative phospholipid. Phosphatidylcholine
with stearoyl and oleyl side chains
57
Phospholipids Are Degraded by Phospholipases
Phospholipase A2 promotes hydrolysis at the 2
position.
phospholipase
2,3-acyl-1-phosphocholine glycerol
3-acyl-1-phosophocholine glycerol (lysophospholip
id)
58
Phospholipase A2 and Other Phospholipase
Activities
59
Hypothetical Model of Phospholipase A2 Binding to
Phospholipid Micelle
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Phospholipase A2 MJ33 Transition State Analog
Structure
61
Apparent Mechanism of Phospholipase
A2 Recall the activation of water in
the resolution of the acyl-enzyme intermediate in
peptide hydrolysis. Catalytic Dyad