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

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Metabolism and Energy Overview

Proteins Carbo hydrates Amino Acids Simple Sugars Lipids

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 (CO 2 , H 2 O) E.A. DENNIS 2010

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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.

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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, non polar, hydrophobic.

Triacylglycerol

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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

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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)

12 9 18 1

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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

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What are Essential Fatty Acids?

Linoleic acid Arachidonic acid Diet Linolenic acid EPA • 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 E.A. DENNIS 2010

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Key Enzyme: Acetyl-CoA Carboxylase

• Acetyl-CoA Carboxylase is a key enzyme • Converts acetyl-CoA into malonyl-CoA – The CO 2 later is released – 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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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

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FA Synthesis: Step 2

Step 2: Set up malonyl-ACP Acetyl-CoA-ACP Transacylase HCO 3 Acetyl-CoA Carboxylase Malonyl-CoA-ACP Transacylase

This step will iterate many times, adding carbons to the growing FA backbone. E.A. DENNIS 2010

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FA Synthesis: Step 3

Step 3: Condense them, giving Acetoacetyl-ACP and CO 2 condensing enzyme CO 2 ACP

The condensing enzyme is also known as b-ketoacyl-ACP synthase . It is part of the FA synthase complex E.A. DENNIS 2010

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FA Synthesis: Step 4

Step 4: Use NADPH to reduce the

b

-carbonyl to a hydroxyl group H + + NADPH NADP +

b

-ketoacyl-ACP reductase

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FA Synthesis: Step 5

Step 5: Remove the hydroxyl group as H 2 O leaving a double bond

b

-hydroxylacyl-ACP dehydratase

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FA Synthesis: Step 6

Step 6: Use another NADPH to reduce the double bond H + + NADPH NADP + Enoyl-ACP reductase

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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 palmitoyl ACP • Palmitate is released.

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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 2 O 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

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[2a] Acetyl-CoA enters cycle

Acetyl-CoA Carboxylase

Acetyl-CoA [1]

Acetyl-CoA-ACP transacylase

Acetyl-ACP

Fatty acid synthase cycle

Release from FA synthase complex Malonyl-CoA

Malonyl-CoA-ACP transacylase

[2b]

Initiation

Malonyl-ACP [3]

b-ketoacyl ACP synthase

Acetoacetyl-ACP

Elongation b-ketoacyl-ACP reductase

[4]

b

-hydroxybutyryl ACP

thioesterase

Palmitoyl-ACP Butyryl-ACP [6]

enoyl-ACP reductase b-hydroxyacyl-ACP dehydratase

[5] Palmitate 2-trans-butenoyl-ACP

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The FA Synthase Enzyme

• FA synthase is an enzyme complex • It includes

all

the FA synthesis enzymes

except

carboxylase for acetyl-CoA • 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, 2 nd ed. Reprinted with permission of John Wiley & Sons, Inc. Figures: Nelson DL, Cox MM (2005), Lehninger Principles of Biochemistry, 4 th ed. W.H. Freeman & Co. E.A. DENNIS 2010

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Reported Tuberculosis in the US 1982-2008 AIDS increase

50% decrease

Year

Tuberculosis

• • • •

Incidence

diseases : one of the leading causes of death due to infectious – Often follows HIV infection

Symptoms

: pulmonary infection – cough, sputum, pleural effusions • see picture below left – urogenital & brain affects also seen

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 Tuberculosis infection

Source: CDC E.A. DENNIS 2010

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Treating Tuberculosis

Figure: Draper, Nat. Med. 6, 977-8 (2000).

Mycolic acid; R 1 and R 2 long-chain are aliphatic hydrocarbons • • •

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

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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 Regulation occurs primarily at acetyl CoA carboxylase , the rate limiting step

• 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

dependent PKA].

, which signal immediate energy needs, phos phorylate and slow the enzyme [via AMP -dependent protein kinase and also via CMP E.A. DENNIS 2010

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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

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