Download Triacylglycerol and Phospholipid Biosynthesis

Triacylglycerol and Phospholipid Biosynthesis
March 28, 2003
Bryant Miles
I. Regulation of Fatty acid Metabolism
It is energetically wasteful to have fatty acid synthesis and β-oxidation occurring at the same time. Hence
these 2 metabolic pathways are reciprocally regulated. This coordinated regulation is also related to the
regulation of glycolysis, and the regulation of the citric acid cycle. The end product of β-oxidation is
acetyl CoA, the end product of glycolysis is pyruvate which can be converted into acetyl CoA by pyruvate
dehydrogenase. Acetyl CoA is activated in the form of malonyl CoA for fatty acid biosynthesis. Acetyl
CoA is oxidized into CO2 in the citric acid cycle. In short acetyl CoA is the common metabolite of all of
these metabolic pathways.
Allosteric Regulation
When fatty acid biosynthesis is turn on, the concentration of malonyl CoA rises. High concentrations of
malonyl CoA prevent the degradation of fatty acids by inhibiting carnitine acyl transferase I. The
inhibition of carnitine acyl transferase I stops the transport of fatty acids across the inner mitochondrial
membrane. When the transport is stopped, β-oxidation stops.
Citrate activates acetyl CoA carboxylase.
Palmitoyl-CoA, stearoyl CoA and arachidyl CoA all inhibit acetyl CoA carboxylase.
Hormonal Regulation
We have already discussed the effects of phosphorylation of acetyl CoA carboxylase(ACC). ACC
contains 8-12 residues that may be phosphorylated by a variety of protein kinases. These protein kinases
are under hormonal control. We are already familiar with the hormone glucagon which is the signal that
the blood glucose concentration is low. Glucagon binds to cell receptors activating an intracellular
phosphorylation cascade which activates protein kinases that phosphorylate ACC. The phosphorylated
ACC enzyme favors the inactive protameric form of the enzyme. Only a high concentration of citrate can
activate the phosphorylated form of the enzyme. Conversely, a small concentration of the acyl-CoA is
required to keep the equilibrium in favor of the inactive protamers.
The phosphorylated ACC enzyme can be reactivated by a specific phosphatase which dephosphorylates
the enzyme.
Glucagon binds to the adipose cell receptors triggering a phosphorylation cascade that activates the
lipases to produce fatty acids from triacylglycerols stored in the tissue. These fatty acids are carried by
serum albumin to the tissues that need them.
Thus glucagon simultaneously stops fatty acid biosynthesis and activates fatty acid transport from the
adipose tissue.
The effects of glucagon are counteracted by the hormone insulin. Insulin is the hormone that signals high
blood glucose concentrations. Insulin activates the phosphodiesterase that hydrolyzes cAMP terminating
the glucagon phosphorylation cascade. Insulin starts its own phosphorylation cascade that activates a
variety of phosphatases. One of these phosphatases dephosphorylates the phosphorylated lipase in the
adipose tissue which stops the liberation of fatty acids. Another insulin activated phosphatase
dephosphorylates ACC and thereby activating it. So insulin simultaneously turns on fatty acid
biosynthesis and stops fatty acid transport from the adipose tissue.
II. Desaturation of Fatty Acids.
The addition of cis double bonds in eukaryotes does not occur until the fatty acid has reached full length.
The desaturation occurs in the endoplasmic reticulum. There are two common naturally occurring
monosaturated fatty acids palmitoleic (16:∆9) acid and oleic acid(18:∆9). Oleic acid is produced by the
dehydrogenation of stearic acid. This conversion is catalyzed by stearoyl CoA desaturase which uses
stearoyl CoA as the substrate.
O
C
S
CoA
S
CoA
Stearoyl CoA Desaturase
O
C
Stearoyl CoA desaturase contains a nonheme iron center, NADH and an oxygen binding site. Two other
proteins are also required, cytochrome b5 and cytochrome b5 reductase, which is a flavoprotein. All three
of these proteins are associated with the endoplasmic reticulum membrane. Cytochrome b5 reductase
transfers electrons one at a time from NADH through FAD to cytochrome b5. Cytochrome b5 transfers an
electron to reduce the nonheme iron from the ferric to the ferrous state. The ferrous iron coordinates to an
O2 molecule. At this iron center the cis double bond at the 9,10 position of the substrate is formed. O2 is
the terminal electron acceptor in this fatty acid desaturation cycle. 2 molecules of water are produced per
oleoyl CoA which means that four electrons were transferred in the overall process. 2 electrons came
from the NADH and the other 2 electrons form stearoyl CoA.
Mammals lack the enzymes to introduce cis double bonds at carbons beyond C9 in the fatty acid chain.
Hence mammals cannot synthesize linoleate (18:2∆9,12) and linolenate (18:3∆9,12,15). Plants however have
the ability to desaturated oleoyl CoA at the 12 position to produce linoleate or at both the 12 and 15
positions to generate linolenate. These two polyunsaturated fatty acids must come from the diet and are
hence called essential fatty acids.
Mammals can synthesize arachidonic acid, plants cannot. Mammals take linoleic acid (obtained from the
diet) and converted it into arachidonic acid (20:4∆5,8,11,14). The biosynthesis occurs in the endoplasmic
reticulum. Arachidonic acid is an important precursor for leukotrienes, proacyclins, thromboxanes and
prostaglandins.
O
C
O-
CoASH + ATP
Acyl CoA synthetase
O
AMP + PPi
C
CoA
S
Desaturase
S
C
Malonyl CoA
CoA
O
Elongation
CO2 + CoA
S
C
CoA
O
Desaturase
O
CoA
C
S
H2O
CoA
O
C
O-
III. Eicosanoids
Eiconsanoids are 20 carbon fatty acids that are produced by the breakdown of selected phospholipids.
Hormonal signals activate phospholipase A2 which cleaves the fatty acids from the C2 position of
phospholipids which is position occupied by unsaturated fatty acids. One of these unsaturated fatty acids
is arachidonic acid whose biosynthesis we just discussed. The hormonal signal also activates
phospholipase C which produces diacylglycerols which in turn are substrates for diacylglycerol lipase
which cleaves fatty acids from the C2 position.
Eicosanoids are local hormones.
•
•
•
•
•
•
Eicosonaids have short lifetimes (between 30-200 seconds).
Eicosanoids exert their effects at very low concentrations (10-14M)
Eicosanoids act at sites near their biosynthesis.
Eicosanoids include thromboxanes (Tx), leukotrienes, prostaglandins(PGF), ect.
Eicosanoids are synthesized from arachidonic acid in the endoplasmic reticulum.
The first step of eicosanoid synthesis is the simultaneous epoxidation and cyclization catalyzed by
a cyclooxygenase (COX).
A variety if stimuli activated the release of arachidonate and the conversion into eicosanoids including
histamines, hormones, proteases and proteins such as serum albumin. Thromboxane A2 is produced by
platelets to stimulated platelet aggregation. One of the post important roles of eicosanoids is involved in
tissue injury, inflammation and pain sensitivity. When tissue is damaged, special inflammatory cells,
monocytes and neutrophils, invade the injured tissue and interact with the smooth muscle cells and
fibroblasts. This interaction stimulates arachidonate release and eicosanoid production. Examples of
tissue injury which stimulates eicosanoid biosynthesis include heart attacks, rheumatoid arthritis and
ulcers.
Aspirin reduces pain sensitivity by irreversibly inhibiting COX activity by transferring an acetyl group
from acetylsalicylic acid to an active site serine residue which prevents the first step of eicosanoid
formation. Thus aspirin prevents the formation of a great number of eicosanoids which produces a
number of side effects including reducing the ability of platelets to aggregate and form blood clots and
inhibiting the secretion of mucin in the stomach which protects the gastric wall.
Ibuprofen (Motrin) and Acetaminophen (Tylenol) are nonsteroidal anti-inflammatory,
anti-fever, pain relievers. Ibuprofen and acetaminophen are reversible, competitive
inhibitors for the arachidonate binding site.
COOH
Ibuprofen (Motrin)
There are two isozymes of COX called COX-1 and COX-2. Motrin and Tylenol
reversibly inhibit both of them. Aspirin irreversibly inactivates both of them. COX-1 is
expressed in many tissues. COX-1 is important for the secretion of mucin, regulating
gastric acid secretion, maintaining renal blood flow, and platelet aggregation. COX-2 is
not expressed under normal conditions. COX-2 expression is induced by inflammatory
mediators such as interleukin-1. COX-2 produces the eicosanoids that promote
inflammation, fever and pain.
O
H2N
S
O
O
N
N
O
CF3
O
H3 C
S
H3C
O
Vioxx
Celebrex
There is a new generation of pain killers which are
COX-2 inhibitors. These inhibitors block pain, fever
and swelling but do not interrupt mucin formation in
the stomach or interfere with platelet aggregation by
specifically targeting the COX-2 isozyme. Celebrex
and Vioxx are COX-2 inhibitors Celebrex and Vioxx
are weak competitive inhibitors of COX-1, but a tight
binding competitive inhibitors of COX-2.
IV. Phosphatidic Acid Biosynthesis
Triacylglycerols and glycerophospholipids have glycerol as a backbone. The biosynthesis of these
compounds proceeds through the formation of phosphatidic acid. Phosphatidic biosynthesis occurs in the
endoplasmic reticulum and the outermitochondrial membrane. The pathway is shown below.
H2C
HO
OH
H
C
H2C
ATP
OH
Glycerokinase
+
NADH + H
NAD
Pi
OH
H2C
O
+
H2C
O
C
H2C
O
P
O
HO
Glycerol 3P
Dehydrogenase
-
OH
C
H
O
H2C
O
P
O
OR1
C
O
-
O
OR1
SCoA
C
SCoA
Glycerol 3-phosphate
Acyltransferase
DHAP
Acyltransferase
CoA
CoA
+
H2C
O
O
O
HO
P
O
O
R1
O
C
H2C
C
NADP+
NADPH + H
O
O
-
AcylDHAP
Reductase
-
O
R2
C
H2C
O
C
C
H
O
H2C
O
P
R
1
O
-
O
SCoA
-
1-Acylglycerol 3P
Acyltransferase
CoA
O
O
R2
C
O
H2C
O
C
C
H
O
H2C
O
P
O-
Phosphatidic Acid
R1
O
-
The starting point for phosphatidic acid biosynthesis begins with either glycerol 3-phosphate or DHAP.
Dihydroxtacetone phosphate can be reduced by glycerol -3-phosphate dehydrogenase to form glycerol 3P. Eukaryotes can acylate DHAP by DHAP acyltransferase to form 1-Acyldihydroxyacetone phosphate
which can be reduced by acyl-DHAP reductase to form 1-Acylglycerol-3-phosphate.
Glycerol 3-phosphate acyltransferase is specific for saturated acyl-CoAs.
The eukaryotic DHAP acyltransferase is also specific for saturated acyl-CoAs.
V. Triacylglycerol Biosynthesis
Triacylglycerol biosynthesis occurs in the intestinal mucosa cells, the adipose tissue and the liver.
Triacylglycerols are used primarily for energy storage. For some animals triacylglycerols also provide
insulation for survival in cold climates. In the liver and adipose tissue, triacylglycerols are synthesized by
diacylglycerol acyltransferase. This enzyme is found on the cytoplasmic face of the endoplasmic
reticulum. A different route for triacylglycerol biosynthesis occurs in the intestinal mucosa cells. In the
small intestine triacylglycerols are degraded into 2-monoacylglycerols and free fatty acids. The intestinal
mucosa cells absorb the monoacylglycerols and fatty acids. A monoacylglycerol acyltransferase produces
diacylglycerol which then goes on to form triacylglycerols as shown below.
O
O
R2
C
H2C
O
C
C
H
O
H2C
O
P
O
R1
O-
O-
H2O
Phosphatidic acid Phosphatase
O
CoAS
H2 C
O
R2
C
O
C
H2 C
C
Pi
R1
O
CoA
O
OH
H
OH
R2
C
H2 C
O
C
H
O
Monoacylglycerol
acyltransferase
H2 C
C
OH
R1
O
CoAS
C
R
3
Diacylglycerol Acyltransferase
CoA
O
O
R2
C
O
H2 C
O
C
C
H
O
H2 C
O
C
R1
R3
VI. Glycerophospholipid Biosynthesis
Phosphatidic acid can be converted into phospholipids. Phospholipids are the main components of
biological membranes.
Phosphatidyl Choline and Phosphatidyl Ethanolamine Biosynthesis
The first step in the biosynthesis of these 2 glycerophospholipids is the activation of choline or
ethanolamine. The activation requires 2 steps. The first step is phosphorylation with ATP to produce
phosphoethanolamine or phosphocholine.
CH3
HO
C
H2
C
H2
NH3
HO
ATP
C
H2
C
H2
N
CH3
ATP
Ethanolamine Kinase
Choline Kinase
ADP
ADP
O
O
-
O
The second step involves
cytidylyltransferase to generate
cytosine diphosphate ethanolamine
or cytosine diphosphate choline.
This reaction produces
pyrophosphate which of course is
hydrolyzed by pyrophosphatase
which makes this activation
irreversible.
CH3
P
O
C
H2
C
H2
-
NH3
O
CH3
P
O-
O
O
C
H2
C
H2
N
-
CH3
CH3
CTP
CTP
CTP: Phosphoethanolamine
cytidylyltransferase
O
CTP: Phosphocholine
cytidylyltransferase
PPi
O
PPi
R2
C
Cytidine
C
C
H
O
H2C
O
P
R1
O-
O
O
O
O
O
Cytidine
O
H2C
P
O
P
O-
O
C
H2
C
H2
O
O-
CH3
NH3
O
P
O
P
O
C
H2
OO-
CDP-Ethanolamine
C
H2
N
O-
CH3
H2O
CH3
Phosphatidic acid Phosphatase
CDP-Choline
Pi
O
O
CMP
O
C
R2
H2C
O
C
C
H
O
H2C
O
P
O
O
R1
H2 C
O
R2
O
C
H2
C
H2
NH3
C
O
C
H2 C
O-
Phosphatidylethanolamine
Phosphatidylcholine
O
O
R2
C
O
H 2C
O
C
C
H
O
H 2C
O
P
O
O
C
H2
C
H2
C
H2
NH3
CH
C
Phosphatidylethanolamine
Transferase
HO
R2
C
O
C
H
O
H2 C
O
P
O-
OH
R1
CH3
O
C
H2
C
H2
N
CH3
CH3
Phosphatidic acid is
dephosphorylated by phosphatidic
acid phosphatase to form
diacylglycerol.
Phosphatidylserine
Phosphatidylserine is synthesized in mammals by
phosphatidylethanolamine transferase. In this enzyme the hydroxyl group
of serine attacks the phosphate group of phosphatidylethanolamine
eliminating ethanolamine to form phosphatidyl serine.
C
H2
C
H2
NH3
O
C
O-
H
R1
O
O-
O
C
P
C
H2C
H O
O
O
C
The diacylglycerol then reacts with CDP-ethanolamine or CDP-choline to
form phosphatidylethanolamine or phosphatidylcholine.
The enzymes that catalyze these two reactions have incredibly long names:
CDPethanolamine: 1,2-diacylglycerol phosphoethanolamine transferase
and CDPcholine: 1,2-diacylglycerol phosphocholine transferase.
-
HO
H2 C
O
C
O
R1
NH3
O
R2
CMP
O
H2C
R1
O
O
C
H2
CH
NH3
C
O-
Phosphatidylinositol, Phosphatidylglycerol and Cardiolipin Biosynthesis.
In order to synthesize these phospholipids, the phosphatidic
acid must be activated in order to be condensed with an
alcohol to produce the corresponding phospholipids.
Phosphatidic acid is activated in the same manner as
ethanolamine and choline were activated. CDP:phosphatidate
cytidylyltransferase which reacts CTP with phosphatidate to
form cytidine diphosphodiacylglycerol. This activation
reaction produces pyrophosphate which of course is
hydrolyzed by pyrophosphatase which makes this activation
irreversible.
O
O
R2
C
H2C
O
C
C
H
O
H2C
O
P
O
R1
O-
O
CTP
PPi
NH2
O
O
R2
C
H2C
O
O
C
C
H
O
H2C
O
P
O-
N
R1
O
O
P
N
O
O
OH
H
OH
OH
H
H
O
NH 2
NH2
O
O
O
R2
O
C
H 2C
O
C
C
H
O
H 2C
O
O
R2
O
P
O
-
N
P
O
O
C
C
H
O
H 2C
O
P
O
O
N
R1
O
N
P
O
-
O
O
O
O
-
H
H
OH
OH
H
H
OH
H 2C
H
OH
H
OH
H
OH
HO
OH
OH
OH
H 2C
O
C
O
O
-
H
OH
O
N
R1
C
H
O
H 2C
O
P
O
CMP
Glycerophosphate phosphatidyltransferase
O-
CMP
-
OH
O
O
O
R2
C
O
OH
H 2C
O
C
C
H
O
H 2C
O
P
O
R1
R2
H2 C
O
C
H
O
H2 C
O
P
O-
O
H
H 2C
C
O
C
C
R1
O
OH
O
O
C
H2
O
O-
OH
OH
Phosphatidylglycerophosphate
Phosphatase
OH
O-
P
Pi
OH
O
O
C
H 2C
O
C
C
H
O
H 2C
O
P
O-
O
H
H 2C
C
O
NH 2
R1
O
O
R2
C
O
H2 C
O
C
C
H
O
H2 C
O
P
O-
O
O
P
N
OH
OH
N
R1
C
H2
O
O
Cardiolipid Synthase
O
OH
H
H
OH
H
OH
CMP
O
O
R2
C
H 2C
O
O
C
R1
C
H
O
H 2C
O
P
O-
O
H
H2 C
C
O
R1
C
H2
OH
Cardiolipin
O
C
O
CH 2
O
H
C
P
O
CH 2
O-
O
O
C
R2