The Fatty Acid Biosynthesis Enzyme FabI Plays a Key Role in the
LM1 Fatty Acid Biosynthesis
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LIPID M A P S Lipid M etabolom ics Tutorial Fatty A cid B iosynthesis ProfessorEdw ard A . D ennis D epartm ent of C hem istry and Biochem istry D epartm ent of Pharm acology, School ofM edicine U niversityof C alifornia, San D iego C opyright/attribution notice: You are free to copy, distribute, adaptand transm itthis tutorial or individual slides (w ithout alteration) foracadem ic,non-profit and non-com m ercial purposes. Attribution: Edw ard A.D ennis (2010)“LIPID M APS Lipid M etabolom ics Tutorial”w w w .lipidm aps.org E.A.DENNIS 2010 ©
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Transcript of LM1 Fatty Acid Biosynthesis
LIPID MAPS Lipid Metabolomics Tutorial Fatty Acid Biosynthesis
Professor Edward A. DennisDepartment of Chemistry and Biochemistry
Department of Pharmacology, School of MedicineUniversity 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
• 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)
Lipids
FattyAcids
Carbo-hydrates
Simple Sugars
Acetyl CoA
Pyruvate
Proteins
AminoAcids
Energy(CO2, H2O)
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What is a “Fatty Acid”?
Palmitic acid
O
OH
Ester group
Fatty acid: a carboxylic acid with a long hydrocarbon chain. Usually, they have an even number of carbons. Reactive and toxic.
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.
O
O
HHOOH
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What is a “Triglyceride”?
Glycerol: common name for 1,2,3-trihydroxy-propane.
Triglyceride: a glycerol molecule with three esterfied fatty acid side chains. Also known more correctly as a “triacylglycerol”. Stable, non-polar, hydrophobic. Triacylglycerol
Glycerol
HOHHO
OH
O
O
HOR1 O
O
R2
O
R3
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O
OH
Common Saturated Fatty AcidsSaturated FA’s have no double bonds
16 Carbons = Palmitic Acid (Palmitate)
18 Carbons = Stearic Acid (Stearate)
O
OH
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OH
O
Common Unsaturated Fatty AcidsUnsaturated 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 Carbons, 2 double bonds at c9 and c12 = Linoleic Acid (Linoleate)
1918
OH
O
1918 12
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More Unsaturated Fatty Acids
18 Carbons, 3 cis double bonds at 9, 12 & 15 = a-Linolenic Acid (a-Linolenate)
20 Carbons, 4 cis double bonds at 5,8,11 & 14 Arachidonic Acid (Arachidonate)
(5Z,8Z,11Z,14Z-Eicosatetraenoic Acid)
OH
O
1918 1215
O
OH
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What are Essential Fatty Acids?• Two “Essential” FA’s
cannot be synthesized by humans– Linoleic acid– Linolenic acid
• Used in the biosynthesis of polyunsaturated fatty acid
• Must come from diet
Linoleicacid
Linolenicacid
Arachidonicacid EPA
Diet
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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, rate-limiting enzyme in FA synthesis
“E” above is the enzyme acetyl-CoA carboxylase, which is conjugated to biotin.
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CCH2
O
S-CoAC
-O
O
CH3C
O
S-CoA
E-Biotin-CO2-
E-Biotin
HCO3- +ATP
ADP + Pi Acetyl-CoA
Malonyl-CoA
Carboxybiotinyl-enzyme
Biotinyl-enzyme
+ E-Biotin
CH3C
O
S-CoA
Acetyl-CoA
CH3C
O
S-ACP
Acetyl-ACP
Acetyl-CoA-ACPTransacylase
FA Synthesis: Step 1Step 1: Set up acetyl-ACP
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.
Acetyl-CoA-ACPTransacylase
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FA Synthesis: Step 2Step 2: Set up malonyl-ACP
This step will iterate many times, adding carbons to the growing FA backbone.
CH3C
O
S-CoAC
H3C
O
S-ACP
CCH2
O
S-CoAC
-O
O
CCH2
O
S-ACPC
-O
O
Acetyl-CoA Carboxylase
Acetyl-CoA-ACPTransacylase
Malonyl-CoA-ACPTransacylase
HCO3-
Malonyl-ACPMalonyl-CoA
Acetyl-CoA Acetyl-ACP
HCO3-
Malonyl-CoA-ACPTransacylase
Acetyl-CoA-ACPTransacylase
Acetyl-CoACarboxylase
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FA Synthesis: Step 3Step 3: Condense them, giving Acetoacetyl-ACP and CO2
The condensing enzyme is also known as b-ketoacyl-ACP synthase. It is part of the FA synthase complex
CH3C
O
S-ACP
CCH2
O
S-ACPC
-O
O
CCH2
O
S-ACPC
H3C
O
condensing enzyme
Acetoacetyl-ACP
Malonyl-ACP
Acetyl-ACP
CO2 ACPCO2 ACP
condensing enzyme
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FA Synthesis: Step 4Step 4: Use NADPH to reduce the b-carbonyl to a hydroxyl group
CCH2
O
S-ACPC
H3C
O
Acetoacetyl-ACP
CCH2
O
S-ACPC
H3C
OHH
b-hydroxybutyryl-ACP
b-ketoacyl-ACP reductase
H+ + NADPH
NADP+
b-ketoacyl-ACPreductase
H+ + NADPH
NADP+
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FA Synthesis: Step 5Step 5: Remove the hydroxyl group as H2O leaving a double bond
CCH2
O
S-ACPC
H3C
OHH
b-hydroxybutyryl-ACP
CC
O
S-ACPC
H3C
H
H
ab-trans-butenoyl-ACP
b-hydroxyacyl-ACP dehydrataseb-hydroxylacyl-ACP dehydratase
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FA Synthesis: Step 6Step 6: Use another NADPH to reduce the double bond
CC
O
S-ACPC
H3C
H
H
ab-trans-butenoyl-ACP
CCH2
O
S-ACP
CH2
H3C
enoyl-ACP reductase
H+ + NADPH
NADP+
Butyryl-ACP
H+ + NADPH
NADP +
Enoyl-ACPreductase
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FA Synthesis: Repeat CycleRepeat from step 2 using the new 4-carbon butyryl-ACP in place of acetyl-ACP
• 6 iterations makes Palmitoyl-ACP.
• Finally, the enzyme thioesterasecleaves the ACP from palmitoyl-ACP
• Palmitate is released.
O
S-ACP
CCH2
O
S-ACP
CH2
H3C
Butyryl-ACP
O
O-
Palmitoyl-ACP
Palmitate
thioesterase
recycle reactions 2-6six more times
H2O
recycle reactions 2-6 six more times
thioesterase
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Fatty Acid Synthesis SummaryOne 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 H2O 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
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CH3C
O
S-CoA CCH2
O
S-CoAC
-O
O
CCH2
O
S-ACPC
-O
OC
H3C
O
S-ACP
CCH2
O
S-ACPC
H3C
O
CCH2
O
S-ACPC
H3C
OHH
CC
O
S-ACPC
H3C
H
H
CCH2
O
S-ACP
CH2
H3C
O
S-ACP
O
O-
Acetyl-CoACarboxylase
b-ketoacyl-ACP synthase
Acetyl-CoA-ACP transacylase Malonyl-CoA-ACP transacylase
b-ketoacyl-ACP reductase
b-hydroxyacyl-ACP dehydratasethioesterase
Initiation
Elongation
Acetyl-CoA
Acetyl-ACP Malonyl-ACP
Malonyl-CoA
Acetoacetyl-ACP
b-hydroxybutyryl-ACP
2-trans-butenoyl-ACP
Butyryl-ACPPalmitoyl-ACP
Palmitate
Release fromFA synthase complex
enoyl-ACP reductase
Fatty acidsynthase cycle
Acetyl-CoA enters cycle
[1]
[5]
[4]
[3]
[6]
[2a]
[2b]
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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
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Figure: Voet, D, Voet JG, Pratt CW (2002), Fundamentals of Biochemistry: Life 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.
Tuberculosis• 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
• Mechanism: Mycobacteriumtuberculosis 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 Tuberculosis infectionSource: CDC
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Reported Tuberculosis in the US 1982-2008
Year
Tube
rcul
osis
cas
es (t
hous
ands
)
AIDS increase 50% decrease
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 (“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
Figure: Draper, Nat. Med. 6, 977-8 (2000).
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Mycolic acid; R1 and R2 are long-chain aliphatic hydrocarbons
Bacteria vs. Mammals
FA Synthesis in Bacteria
• Acyl carrier protein (ACP) is a separate molecule, as are each of the six enzymes.
• “Acetyl CoA carboxylase”is two separate enzymes, plus a biotin cofactor joined to a third enzyme.
FA Synthesis in Mammals
• ACP is part of the FA synthase complex, which is one large protein present as a dimer.
• Acetyl-CoA carboxylase is one enzyme conjugated to a biotin cofactor.
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Regulation of FA Synthesis
Feedback Mechanisms• Citrate, which builds up
when acetyl-CoA is plentiful, accelerates FA synthesis.
• Palmitoyl-CoA weakly inhibits FA synthesis.
Hormonal Mechanisms• Insulin, which signals a resting,
energy rich state, dephosphoryl-ates and accelerates the enzyme.
• Glucagon, epinephrine and norepinephrine, which signal immediate energy needs, phos-phorylate and slow the enzyme [via AMP -dependent protein kinase and also via CMP-dependent PKA].
Regulation occurs primarily at acetyl-CoA carboxylase, the rate limiting step
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AcknowledgementThis 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. DennisSeptember, 2010La Jolla, California
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