Ketone Body Formation; Fatty acid and Cholesterol ... - Ecu

Ketone Body
Formation; Fatty acid
and Cholesterol
Biosynthesis

• Most acetyl CoA from β-oxidation is
routed through the TCA cycle
• Excess is used to synthesize ketone bodies;
β-hydroxybutyrate, acetoacetate and
acetone
• Ketone bodies serve as fuel molecules
• Liver is the site of synthesis in mammals
(occurs in the mitochondrial matrix)

Ketone Body Synthesis:
O
H 3
C-C-S-CoA
Acetyl CoA
+
O
H 3
C-C-S-CoA
Acetyl CoA

O
H 3
C-C-S-CoA
Acetyl CoA
+
O
H 3
C-C-S-CoA
Acetyl CoA
HSCoA
thiolase
O
O
H 3
C-C-CH 2
-C-S-CoA
Acetoacetyl CoA

O
H 3
C-C-S-CoA
Acetyl CoA
+
O O
H C-C-CH -C-S-CoA
3 2
Acetoacetyl CoA

O
O
O
H 3
C-C-S-CoA
Acetyl CoA
+
H 3
C-C-CH 2
-C-S-CoA
Acetoacetyl CoA
HSCoA
OH
HMG CoA Synthase
O
-
OOC-CH 2
-C-CH 2
-C-S-CoA
CH 3
3-Hydroxy-3-Methylglutaryl CoA (HMG CoA)

OH O
-
OOC-CH -C-CH -C-S-CoA
2 2
CH 3
3-Hydroxy-3-Methylglutaryl CoA (HMG CoA)
HMG CoA Lyase
O
H 3
C-C-S-CoA
Acetyl CoA
O
-
OOC-CH 2
-C-CH 3
Acetoacetate

O
-
OOC-CH 2
-C-CH 3
Acetoacetate
NADH
β-Hydroxybutyrate
D’Hase
CO 2
O
CH 3
-C-CH 3
Acetone
NAD + OH
-
OOC-CH -CH-CH 2 3
β-Hydroxybutyrate

Ketone Body Breakdown:
OH
-
OOC-CH 2
-CH-CH 3
β-Hydroxybutyrate
*Liver enzyme catalyzes
a near equilibrium
reaction
β-Hydroxybutyrate
D’Hase
*Isozyme found in
other cells catalyzes
an irreversible
reaction
NAD +
NADH
O
-
OOC-CH -C-CH 2 3
Acetoacetate

O
-
OOC-CH 2
-C-CH 3
Acetoacetate
Succinyl CoA Transferase
+
O
-
OOC-CH 2
-CH 2
-C-S-CoA
Succinyl CoA
O
O
H 3
C-C-CH 2
-C-S-CoA
+
-
OOC-CH 2
-CH 2
-COO -
Succinate
Acetoacetyl CoA

O O
H C-C-CH -C-S-CoA
3 2
Acetoacetyl CoA
Thiolase
O
H 3
C-C-S-CoA
Acetyl CoA
O
H 3
C-C-S-CoA
Acetyl CoA

Fatty acid
Biosynthesis

• Occurs through the condensation of C2
units
• Occurs largely in adipocytes and liver
and to a lesser extent in specialized
tissues
• Occurs in three stages: 1) transport of
acetyl Co A to the cytosol; 2) formation
of malonyl CoA; 3) assembly of the
fatty acid chain

Citrate Transport System:
Acetyl CoA + OAA
Citrate
α-Kg
or
malate
Citrate:Dicarboxylic
acid Carrier
Cytosol
Matrix

Citrate Transport System:
Citrate
Acetyl CoA + OAA
Citrate
α-Kg
or
malate
Citrate:Dicarboxylic
acid Carrier
α-Kg
or malate
Cytosol
Matrix

Citrate Transport System:
citrate lyase
malate
d’hase
Cytosol
Citrate
ATP
ADP
OAA + Acetyl CoA
malate
Malic
enzyme
NADH
NAD+
NADP+
NADPH
CO 2
pyruvate
Citrate:Dicarboxylic
acid Carrier
pyruvate
translocase
pyruvate
Matrix

• Formation of Malonyl CoA by Acetyl CoA
carboxylase
• Loading Step: Transfer of
acetyl CoA and malonyl CoA
to ACP to form acetyl-ACP
and malonyl ACP
• Successive rounds of
condensation; reduction;
dehydration, reduction
}
Fatty Acid
Synthetase

O
H 3
C-C-S-CoA
acetyl-CoA
+ HCO 3
ATP
ADP, Pi
bicarbonate
Acetyl CoA Carboxylase
O
-
OOC-CH 2
-C-S-CoA
malonyl-CoA

Fatty Acid Synthetase Complex:
Bacteria, Plants
Seven activities in 7 separate proteins
Yeast
Seven activities in 2 separate proteins
Vertebrates
Seven activities in 1 large protein

acetyl-CoA
O
H 3
C-C-S-CoA
malonyl-CoA
O
-
OOC-CH 2
-C-S-CoA
Acetyl CoA:ACP
transacylase
HS-ACP
HS-CoA
HS-ACP
HS-CoA
Malonyl CoA:ACP
transacylase
O
H 3
C-C-S-ACP
acetyl-ACP
O
-
OOC-CH 2
-C-S-ACP
malonyl-ACP
Loading Step

O
H 3
C-C-S-ACP
acetyl-ACP
O
-
OOC-CH 2
-C-S-ACP
malonyl-ACP
ketoacyl-ACP synthase
Condensation

O
H 3
C-C-S-ACP
acetyl-ACP
O
-
OOC-CH 2
-C-S-ACP
malonyl-ACP
CO 2
HS-ACP
ketoacyl-ACP synthase
O O
H C-C-CH -C-S-ACP
3 2
acetoacetyl-ACP
Condensation

O
O
H 3
C-C-CH 2
-C-S-ACP
acetoacetyl-ACP
NADPH
NADP +
ketoacyl-ACP
reductase
OH
O
H 3
C-C-CH 2
-C-S-ACP
H
b-hydroxybutyryl-ACP
Reduction

OH O
H C-C-CH -C-S-ACP
3 2
H
b-hydroxybutyryl-ACP
b-hydroxyacyl-ACP
dehydrase
H 2
0
H O
H C-C=C-C-S-ACP
3
H
butenoyl-ACP
Dehydration

H O
H C-C=C-C-S-ACP
3
H
butenoyl-ACP
NADPH
NADP
+
enoyl-ACP
reductase
O
H 3
C-CH 2
-CH 2
-C-S-ACP
butyryl-ACP
Reduction

• Fatty Acid Synthesis is sometimes called
palmitate synthesis because palmitate is the
predominant product (in mammals)
• Rounds of synthesis continue until a C16
palmitoyl group is formed
• Thiolase then catalyzes the formation of
palmitate and ACP-SH
Palmitoyl-ACP
thiolase
Palmitate +
ACP-SH

Overall stoichiometry for
palmitate synthesis:
Acetyl-CoA + 7 Malonyl CoA + 14 NADPH, H +
palmitate + 7CoA + 7HCO 3
14 NADP +

Regulation:
• Key regulatory step: acetyl-CoA carboxylase
• Citrate allosterically activates.
• Palmitate is a feedback inhibitor
• Fatty acyl-CoAs allosterically inhibit
• Phosphorylation (stimulated by glucagon
and epinephrine) inactivates

Regulation:
• Fatty acid synthesis occurs in the
chloroplast (stroma) in plants
• The plant acetyl-CoA carboxylase is
not regulated by phosphorylation; it
is activated by increases in stromal
pH and magnesium

• Synthesis of unsaturated fatty acids requires the
activity of a number of desaturases
• Animal desauturases form double bonds as
much as 9 carbons removed from the carboxyl
end of a fatty acid
• Only plant desaturases form double bonds
positioned farther than 9 carbons from the
carboxyl end; thus fatty acids such as linoleate
(18:2 ∆9,12 ) must be acquired in the diet of animals
and are considered essential fatty acids

Linoleate
arachidonic acid (C20:4 ∆5,8,11,14 )
cyclooxygenase
lipoxygenase
leukotrienes
eicosanoids
prostaglandins (smooth muscle
contraction; pain; inflammation)
trigger allergic
responses

Linoleate
arachidonic acid (C20:4 ∆5,8,11,14 )
X
cyclooxygenase
lipoxygenase
Aspirin
eicosanoids
prostaglandins (smooth muscle
contraction; pain; inflammation)
leukotrienes
trigger allergic
responses

Cholesterol Biosynthesis
• Pathway substantially active only in liver cells
• All carbon atoms arise from acetyl-CoA
• Squalene, C30 linear hydrocarbon, is an
intermediate
• Squalene is formed from 5 carbon units
(isoprene)
H 3
C CH 2
H 2
C C C H

Stage 1: Acetyl Co-A to
Isopentenyl Pyrophosphate
2 Acetyl Co-A Acetoacetyl Co-A
thiolase
2 NADP +
HS-CoA
2
NADPH
HMG CoA
HMG CoA
synthase
OH
HMG CoA
reductase
-
OOC-CH 2
-C-CH 2
-CH 2
-OH
CH 3
Mevalonate

Stage 1: Acetyl Co-A to
Isopentenyl Pyrophosphate
2 Acetyl Co-A Acetoacetyl Co-A
thiolase
NADP +
HS-CoA
NADPH
HMG CoA
HMG CoA
synthase
OH
X
HMG CoA
reductase
-
OOC-CH 2
-C-CH 2
-CH 2
-OH
CH 3
Mevalonate
Lovostatin
(Mevacor);
Zocor

OH
-
OOC-CH 2
-C-CH 2
-CH 2
-OH
melvalonate kinase
CH 3
Mevalonate
ATP
ADP, Pi
OH
Phosphomevalonate
-
OOC-CH -C-CH -CH -OPO
=
kinase
2 2 2 3
CH 3
OH
Phosphomevalonate
-
OOC-CH 2
-C-CH 2
-CH 2
-OP 2
O 6
3-
ATP
ADP, Pi
CH 3
Pyrophosphomevalonate

Pyrophosphomevalonate
ATP
CH 3
CH =C-CH -CH -OP O 3-
2 2 2 2 6 Isopentenyl pyrophosphate
Decarboxylase
ADP, Pi
CO 2
isomerase
CH 3
CH -C=CH-CH -OP O 3-
3 2 2 6 Dimethylallyl pyrophosphate

Stage 2: Isopentenyl Pyrophosphate to
Squalene
Isopentenyl pyrophosphate +
Dimethylallyl pyrophosphate
prenyl transferase
condense head to tail
PPi
OP 2
O 6
3-
Geranyl
pyrophosphate
(C10)

OP 2
O 6
3-
Geranyl
pyrophosphate
(C10)
+
OP 2
O 6
3-
Isopentenyl
pyrophosphate
(C5)
prenyl transferase
OP 2
O 6
3-
PPi
Farnesyl
pyrophosphate
(C15)

2 PPi
OP 2
O 6
3-
NADPH
2 Farnesyl
pyrophosphates
condense head to head
NADP +
Reaction catalyzed
by Squalene
synthase
Product is Squalene (C30) !

Stage 3: Squalene to Cholesterol
• Numerous steps involving addition of oxygen (to
form squalene 2,3 epoxide) and closure of rings
• Lanosterol is the first intermediate containing all
four fused rings; it accumulates in large amounts
in cells actively synthesizing cholesterol
• Lanosterol is converted to cholesterol in 20
more reactions
• Squalene 2,3 epoxide is precursor to plant
sterols

Cholesterol metabolism is a source of a large
number of other cellular constituents:
• Isopentenyl pyrophosphate is precursor to fat
soluble vitamins (A, E, K), ubiquinone in
animal cells, plastoquinone and the phytol side
chain of chlorophyll in plant cells, certain oils
such as musk, lemon, eucalayptus
• Cholesterol is precursor for bile salts, vitamin
D, steroid hormones, mineralocorticoids, and
is also an important component of membranes.

Regulation of HMG-CoA Reductase:
• Phosphorylation by c-AMP dependent
protein kinase inactivates.
• Gene expression: Cholesterol levels
control the amout of mRNA. If [cholesterol]
is high, mRNA levels are reduced. If
[cholesterol] is low, mRNA is increased.
• Degradation of the enzyme: Half-life depends
upon the cholesterol level; high cholesterol
means a short half-life.

Lipoproteins: Good vs. Bad Cholesterol
Phospholipids, triacylglycerols and cholesterol circulate
in blood in the form of lipoproteins.
Classified according to the relative amounts of lipid
and protein in the complex (the more protein and less
lipid the denser the complex).
Thus, we have high-density lipoproteins (HDL);
low-density lipoproteins (LDL); intermediate-density
lipoproteins (IDL); very-low density lipoproteins (VLDL)
and chylomicrons.

All lipoproteins consist of a core of triacylglycerols or
cholesterol esters surrounded by a single phospholipid
layer into which is inserted a mixture of cholesterol and
proteins. The proteins serve as recognition sites for
lipoprotein receptors throughout the body.
HDL and VLDL are synthesized primarily in the ER of the
liver; chylomicrons are synthesized in the intestine.
LDL is synthesized from VLDL and is the major
circulatory complex for transport of cholesterol and
cholesterol esters from liver to other tissues.
Chylomicrons transport triacylglycerols mostly (and
some cholesterol esters) from intestines to other tissues.

VLDL released in to the bloodstream is converted to IDL
and LDL by the action of lipases which cleave
triacylglycerols.
The half-life of LDL is about 24 hours. It is removed from
circulation by endocytosis in coated vesicles and
degradation by lysosomal acid lipases.
HDL has a half-life of 5-6 days. Newly formed HDL contains
no cholesterol esters. These accumulate over time through
the action of enzymes that transfer cholesterol esters. HDL
functions to return cholesterol and cholesterol esters to the
liver and hence remove them from circulation. Hence, HDL
is the “good cholesterol”. LDL, because it is the major
circulating form of cholesterol is the “bad cholesterol”
correlated with increased risk of cardiovascular disease.

Receptor-Mediated Endocytosis
Discovered by Brown and Goldstein (1985 Nobel
Prize).
ApoB-100, the major protein in LDL particles, is
recognized by the LDL receptor.
Binding of LDL to the LDL receptor initiates
endocytosis which brings LDL and its receptors into
the cell inside an endosome.
Endosomes fuse with lysosomes, which contain
enzymes that hydrolyze cholesterol esters, releasing
cholesterol and fatty acids into the cytosol.

ApoB-100 is degraded to amino acids, but the LDL
receptor is recycled back to the cell surface.
Cholesterol entering the cell in this manner is used
to synthesize membranes, or is reesterified for
storage in cytosolic lipid droplets.
Defective LDL receptors result in the genetic disease
familial hypercholesterolemia. This is characterized
by the inability of cells to take up cholesterol; hence
blood cholesterol levels are extremely high.
The excess blood cholesterol accumulates and
contributes to the formation of atherosclerotic
plaques. Heart failure from atherosclerosis is the
leading cause of death in industrialized societies.

Coated pit
Clathrin
Receptor with ligand
Adaptor