Transcript Fatty Acid Biosynthesis

LIPID MAPS Lipid Metabolomics Tutorial
Fatty Acid Biosynthesis
Professor Edward A. Dennis
Department of Chemistry and Biochemistry
Department of Pharmacology, School of Medicine
University of California, San Diego
Copyright/attribution notice: You are free to copy, distribute, adapt and transmit this tutorial or
individual slides (without alteration) for academic, non-profit and non-commercial purposes.
Attribution: Edward A. Dennis (2010) “LIPID MAPS Lipid Metabolomics Tutorial” www.lipidmaps.org
E.A. DENNIS 2010 ©
Metabolism and Energy Overview
•
Proteins
Carbohydrates
Lipids
•
•
Amino
Acids
Simple
Sugars
Fatty
Acids
•
Many biomolecules are
degraded to Acetyl CoA
Acetyl CoA provides
biologic energy
Excess acetyl CoA is
stored as Fatty Acids
(FA’s)
FA’s are assembled into
more complex lipids like
triglycerides (TG’s)
Pyruvate
Acetyl CoA
Energy
(CO2, H2O)
E.A. DENNIS 2010 ©
What is a “Fatty Acid”?
Palmitic acid
Fatty acid: a carboxylic acid with a long hydrocarbon chain. Usually,
they have an even number of carbons. Reactive and toxic.
Ester group
Fatty acid ester: a fatty acid in which the carboxylic acid
group has reacted with the alcohol group of another molecule
(often glycerol) to form a stable, less reactive ester bond.
E.A. DENNIS 2010 ©
What is a “Triglyceride”?
Glycerol: common name for
1,2,3-trihydroxy-propane.
Glycerol
Triglyceride: a glycerol
molecule with three esterfied
fatty acid side chains. Also
known more correctly as a
“triacylglycerol”. Stable, nonpolar, hydrophobic.
Triacylglycerol
E.A. DENNIS 2010 ©
Common Saturated Fatty Acids
Saturated FA’s have no double bonds
16 Carbons = Palmitic Acid (Palmitate)
18 Carbons = Stearic Acid (Stearate)
E.A. DENNIS 2010 ©
Common Unsaturated Fatty Acids
Unsaturated FA’s have at least one double bond,
usually in the Z (cis) conformation
18 Carbons, 1 double bond at c9 = Oleic Acid (Oleate)
18
9
1
18 Carbons, 2 double bonds at c9 and c12 = Linoleic Acid (Linoleate)
18
12
9
1
E.A. DENNIS 2010 ©
More Unsaturated Fatty Acids
18 Carbons, 3 cis double bonds at 9, 12 & 15 =
a-Linolenic Acid (a-Linolenate)
18
15
12
9
1
20 Carbons, 4 cis double bonds at 5,8,11 & 14
Arachidonic Acid (Arachidonate)
(5Z,8Z,11Z,14Z-Eicosatetraenoic Acid)
E.A. DENNIS 2010 ©
What are Essential Fatty Acids?
• Two “Essential” FA’s
cannot be synthesized
by humans
Diet
Linoleic
acid
Linolenic
acid
Arachidonic
acid
EPA
– Linoleic acid
– Linolenic acid
• Used in the
biosynthesis of
polyunsaturated fatty
acid
• Must come from diet
E.A. DENNIS 2010 ©
Key Enzyme: Acetyl-CoA Carboxylase
• Acetyl-CoA Carboxylase is
a key enzyme
• Converts acetyl-CoA into
malonyl-CoA
– The CO2 is released
later
– Biotin is a cofactor
• It is the “committed step”
in FA synthesis
• It is the regulated, ratelimiting enzyme in FA
synthesis
“E” above is the enzyme acetyl-CoA
carboxylase, which is conjugated to biotin.
E.A. DENNIS 2010 ©
FA Synthesis: Step 1
Step 1: Set up acetyl-ACP
Acetyl-CoA-ACP
Transacylase
ACP is “acyl carrier protein” and is
a part of a large enzyme complex.
It holds the reactants in place
while other enzymes catalyze the
subsequent reaction steps.
E.A. DENNIS 2010 ©
FA Synthesis: Step 2
Step 2: Set up malonyl-ACP
Acetyl-CoA-ACP
Transacylase
HCO3-
Acetyl-CoA
Carboxylase
Malonyl-CoA-ACP
Transacylase
This step will iterate many times, adding carbons to the growing FA backbone.
E.A. DENNIS 2010 ©
FA Synthesis: Step 3
Step 3: Condense them, giving Acetoacetyl-ACP and CO2
condensing enzyme
CO2
ACP
The condensing enzyme is also known as b-ketoacyl-ACP
synthase. It is part of the FA synthase complex
E.A. DENNIS 2010 ©
FA Synthesis: Step 4
Step 4: Use NADPH to reduce the b-carbonyl to a hydroxyl group
H+ + NADPH
b-ketoacyl-ACP
reductase
NADP+
E.A. DENNIS 2010 ©
FA Synthesis: Step 5
Step 5: Remove the hydroxyl group as H 2O leaving a double bond
b-hydroxylacyl-ACP
dehydratase
E.A. DENNIS 2010 ©
FA Synthesis: Step 6
Step 6: Use another NADPH to reduce the double bond
H+ + NADPH
Enoyl-ACP
reductase
NADP +
E.A. DENNIS 2010 ©
FA Synthesis: Repeat Cycle
Repeat from step 2 using the new 4-carbon butyryl-ACP in place of acetyl-ACP
recycle reactions 2-6
six more times
thioesterase
• 6 iterations
makes
Palmitoyl-ACP.
• Finally, the
enzyme
thioesterase
cleaves the ACP
from palmitoylACP
• Palmitate is
released.
E.A. DENNIS 2010 ©
Fatty Acid Synthesis Summary
One iteration:
Step 1: Set up acetyl-ACP
Step 2: Set up malonyl-ACP
Step 3: Condense them, giving Acetoacetyl ACP
Step 4: Use NADPH to reduce distal carbonyl to a hydroxyl group
Step 5: Remove the hydroxyl group as H 2O leaving a double bond
Step 6: Use another NADPH to reduce the double bond
REPEAT: From step 2 using the new 4-carbon butyryl-ACP in
place of acetyl-ACP in step 3
E.A. DENNIS 2010 ©
[2a]
Acetyl-CoA
Carboxylase
Acetyl-CoA enters cycle
Acetyl-CoA
[1]
Malonyl-CoA
Acetyl-CoA-ACP transacylase
Malonyl-CoA-ACP transacylase
[2b]
Initiation
Acetyl-ACP
Malonyl-ACP
Fatty acid
synthase cycle
[3]
b-ketoacylACP synthase
Release from
FA synthase complex
Acetoacetyl-ACP
[4]
b-ketoacyl-ACP reductase
Elongation
Palmitoyl-ACP
b-hydroxybutyrylACP
Butyryl-ACP
thioesterase
[6]
Palmitate
enoyl-ACP reductase
b-hydroxyacyl-ACP dehydratase
2-trans-butenoyl-ACP
[5]
E.A. DENNIS 2010 ©
The FA Synthase Enzyme
• FA synthase is an enzyme
complex
• It includes all the FA synthesis
enzymes except for acetyl-CoA
carboxylase
• Actually exists as a dimer of two
complete, anti-parallel complexes
-- like Ying and Yang
• Cytosolic
Figure: Voet, D, Voet JG, Pratt CW (2002),
Fundamentals of Biochemistry: Lif e at the
Molecular Level, 2nd ed. Reprinted with
permission of John Wiley & Sons, Inc.
Figures: Nelson DL, Cox MM (2005), Lehninger Principles of
Biochemistry, 4th ed. W.H. Freeman & Co.
E.A. DENNIS 2010 ©
Tuberculosis
Tuberculosis cases (thousands)
Reported Tuberculosis in the US 1982-2008
AIDS increase
50% decrease
• Incidence: one of the leading
causes of death due to infectious
diseases
– Often follows HIV infection
• Symptoms: pulmonary infection
– cough, sputum, pleural effusions
• see picture below left
– urogenital & brain affects also seen
Year
• Mechanism: Mycobacterium
tuberculosis infection
• Treatments:
– First-line combination:
• Pyrazinamide and Isoniazid
– stop mycobacterial FA synthase!
• Rifampin (RNA transcription inhibitor)
– Various second-line agents
– If needed, HIV treatment
Normal lung
Source: CDC
Tuberculosis infection
Treating Tuberculosis
• Mycobacteria
– make their outer membrane with
mycolic acids using FA synthases
• FAS-1 is a single, eukaryote-like
enzyme with multiple actions
• FAS-2 is a multi-unit, prokaryote-like
enzyme with multiple actions
• Pyrazinamide
Figure: Draper, Nat. Med. 6, 977-8 (2000).
Mycolic acid;
R1 and R2 are
long-chain
aliphatic
hydrocarbons
(“peer-ah-ZIN-a-mide”)
– Inhibits FAS-I
– Relatively specific for M. tuberculosis
– Arrests synthesis of both fatty acids
and mycolic acids
• Isoniazid (“eye-so-NYE-a-zid”)
– Inhibits FAS-II
– Stops synthesis of mycolic acids
E.A. DENNIS 2010 ©
Bacteria vs. Mammals
FA Synthesis in Bacteria
FA Synthesis in Mammals
• Acyl carrier protein (ACP)
is a separate molecule, as
are each of the six
enzymes.
• ACP is part of the FA
synthase complex, which
is one large protein
present as a dimer.
• “Acetyl CoA carboxylase”
is two separate enzymes,
plus a biotin cofactor
joined to a third enzyme.
• Acetyl-CoA carboxylase is
one enzyme conjugated to
a biotin cofactor.
E.A. DENNIS 2010 ©
Regulation of FA Synthesis
Regulation occurs primarily at acetylCoA carboxylase, the rate limiting step
Feedback Mechanisms
Hormonal Mechanisms
• Citrate, which builds up
when acetyl-CoA is
plentiful, accelerates FA
synthesis.
• Insulin, which signals a resting,
energy rich state, dephosphorylates and accelerates the enzyme.
• Palmitoyl-CoA weakly
inhibits FA synthesis.
• Glucagon, epinephrine and
norepinephrine, which signal
immediate energy needs, phosphorylate and slow the enzyme
[via AMP -dependent protein
kinase and also via CMPdependent PKA].
E.A. DENNIS 2010 ©
Acknowledgement
This tutorial is based on an evolving subset of lectures and
accompanying slides presented to medical students in the Cell
Biology and Biochemistry course at the School of Medicine of the
University of California, San Diego.
I wish to thank Dr. Bridget Quinn and Dr. Keith Cross for aid in
developing many of the original slides, Dr. Eoin Fahy for advice in
applying the LIPID MAPS nomenclature and structural drawing
conventions [Fahy et al (2005) J Lipid Res, 46, 839-61; Fahy et al
(2009) J Lipid Res, 50, S9-14] and Masada Disenhouse for help in
adopting to the tutorial format.
Edward A. Dennis
September, 2010
La Jolla, California
E.A. DENNIS 2010 ©