Chapter 5

Metabolism of Lipids
The biochemistry and molecular biology department of CMU

Concept
Lipids are substances that are insoluble or immiscible in water, but soluble in organic solvents.

Fats (Triglyceride or triacylglycerole) Lipids

To store and supply energy To be important membrane components

Phospholipids Glycolipids Lipoids Cholesterol Cholesterol ester

Contents
Section 1 Fatty acids Section 2 Metabolism of Triglycerids

Section 3 Metabolism of Phospholipids

Section 4 Metabolism of Cholesterols

Section 5 Metabolism of Plasma Lipoproteins

Section 1 Fatty acids

1.1 Classification of fatty acids

Numerical Symbol
14:0 16:0 18:0 16:1 9 18:1 9

Common Name
Myristic acid Palmitic acid Stearic acid Palmitoleic acid Oleic acid

Comments
Saturated Saturated Saturated Unsaturated Unsaturated

18:2 9,12 18:3 9,12,15 20:4 5,8,11,14

Linoleic acid Linolenic acid Arachidonic acid

EFA EFA EFA

Essential Fatty Acids (EFA)

Linoleic, linolenic and arachidonic acids are called essential fatty acids, because they cannot be synthesized by the body and must be obtained through diet.

1.2 Important Derivatives of Arachidonic acids

Arachidonic acids (AA) in turn gives rise to biologically important substances known as the eicosanoids. Prostaglandins (PGs)
Thromboxanes (TXs)

Leukotrienes (LTs)

Section 2 Metabolism of Triglycerides

Triglyceride (TG) or triacylglycerol (TAG)

Glycerol

O O
1

CH2 O C R1 O

R2 C O C H
3

CH2 O C R3

Overview of triglycerides metabolism

Triglycerides (fats) Esterification Diet Lipogenesis Carbohydrate Amino acids Fatty acids -Oxidation Steroidogenesis Lipolysis Steroids

Acetyl-CoA Cholesterol CholesteroloKe genesis tog en es TAC is Ketone bodies 2CO2

 2.1 Degradation of TG
2.1.1 Fat catabolism (lipolysis)

2.1.2 -Oxidation of Fatty acids

2.1.3 Other Oxidations of Fatty acids

2.1.4 Ketone Bodies Formation and Utilization

 2.1.1 Fat catabolism (lipolysis)

Fat mobilization
The triacylglycerol stored in the adipocytes are hydrolyzed by lipases, to produce free fatty acids (FFA) and glycerol, which are released to the blood, this process is called fat mobilization.

The fatty acids thus released diffusively from the adipocyte into the blood, where they bind to the serum albumin.

Hormone sensitive lipase (HSL)

TG lipase is the rate-limiting enzyme in the TG degradation in adipose tissue. It is also named HSL because it is regulated by some hormones.

Effect of hormones on lipolysis

Lipolytic Hormones:

epinephrine
norepinephrine adrenocorticotropic hormone (ACTH) thyroid stimulating hormone (TSH) Glucagon etc.

Antilipolytic Hormones: insulin

glycerol metabolism
Place: liver, kidney, intestine
g ADP CH2OH CH2OH ATP 3-phlycerol deh osph yd r o HO C H a HO C H g en t e glycerol as e CH2O P kinase CH2OH NAD+ CH2OH Glycerol L-Glycerol 3-phosphate O C NADH+H+
Glycolysis Glyconeogenesis CHO H C OH CH2O P D-Glyceraldehyde 3-phosphate CH2O P Dihydroxyacetone triose phosphate phosphate isomerase

Note
In muscle cells and adipocytes, the activity of glycerol kinase is low, so these tissues cannot use glycerol as fuel.

 2.1.2 -Oxidation of Fatty acids

Fatty acids are one of the main energy materials of human and other mammalian. Fatty acid catabolism can be subdivided into 3 stages.

Stage 1 Activation of FAs

Acyl-CoA Synthetase (Thiokinase), which locates on the cytoplasm, catalyzes the activation of long chain fatty acids.
ATP + HSCoA O Fatty acid AMP + PPi O Mg2+ R C acyl-CoA S CoA synthetase acyl-CoA

O R C

Key points of FA activation

1. Irreversible 2. Consume 2 ~P 3. Site: cytosol

Stage 2 Transport of acyl CoA into the mitochondria ( rate-limiting step)

Carrier: carnitine

Rate-limiting enzyme
carnitine acyltransferase
CH3 OH + H3C N CH2 CH CH2 COO CH3 Carnitine R C CH3 + H3C N CH2 CH3 O CH CH2 COO

R C O

SCoA carnitine acyltransferase

O HSCoA

Fatty acyl carnitine

Stage 3: -oxidation of FAs

-oxidation means -C reaction.
Four steps in one round

step 1: Dehydrogenate
step 2: Hydration

step 3: Dehydrogenate
step 4: Thiolytic cleavage

Step 1. Dehydrogenate
H H3C (CH2)n C

H C H

O C SCoA Fatty acyl-CoA

H FAD FADH2

acyl-CoA dehydrogenase H H3C (CH2)n C H C O C SCoA

trans-2-enoyl-CoA

Step 2. Hydration
H H3C (CH2)n C H H2O C O C SCoA

Trans-2-enoyl-CoA enoyl-CoA Hydratase O C SCoA

OH H H3C (CH2)n C H C H

3-L-Hydroxyacyl-CoA

Step 3. Dehydrogenate
OH H H3 C (CH2)n C C H O C SCoA

H NAD+ NADH + H+ O H3 C (CH2)n C

3-L-Hydroxyacyl-CoA

hydroxyacyl-CoA dehydrogenase O CH2 C SCoA

-Ketoacyl-CoA

Step 4. Thiolytic cleavage

O H 3C (CH2)n C CH2 O C SCoA

-Ketoacyl-CoA HSCoA O H3 C (CH2)n C SCoA + CH3 -Ketothiolase O C SCoA

Fatty acyl-CoA (2C shorter)

Acetyl-CoA

- oxidation of fatty acids

The -oxidation pathway is cyclic

Summary
one cycle of the -oxidation:

fatty acyl-CoA + FAD + NAD+ + HS-CoA

fatty acyl-CoA (2 C less) + FADH2 +

NADH + H+ + acetyl-CoA

The product of the -oxidation is in the form of FADH2, NADH, acetyl CoA, only after Krebs cycle and oxidative phosphorylation, can ATP be produced.

Energy yield from one molecule of palmitic acid

palmitic acid -2 ~P activation 7 2 respiratory chain

8 acetyl CoA + 7 FADH2 + 7 NADH + 7 H+ palmitoyl-CoA 7 turns of -oxidation TAC respiratory chain 8 12 7 3

The net ATP production: 1312 = 129

 2.1.3 Other Oxidations of Fatty acids

1. Oxidation of unsaturated fatty acids 2. Peroxisomal fatty acid oxidation 3. Oxidation of propionyl-CoA

1. Oxidation of unsaturated fatty acid

Mitochondria Isomerase: cis trans Epimerase: D (-) L (+)

2. Peroxisomal fatty acid oxidation

Very long chain fatty acids
FAD Acyl-CoA oxidase

shorter chain fatty acids

-oxidation

3. Oxidation of propionyl-CoA
propionyl-CoA Carboxylase (biotin) Epimerase Mutase (VB12)
succinyl-CoA

 2.1.4 Ketone Bodies Formation and Utilization

Ketone bodies are water-soluble fuels normally exported by the liver but overproduced during fasting or in untreated diabetes mellitus, including acetoacetate, hydroxybutyrate, and acetone.

The formation of ketone bodies (Ketogenesis)

Location: hepatic mitochondria
Material: acetyl CoA Rate-limiting enzyme: HMG-CoA synthase

O CH3 C S CoA

CH3 2 Acetyl-CoA

O C S CoA

HSCoA CH3 thiolase

C CH2 C S CoA Acetoacetyl-CoA Acetyl-CoA HSCoA O

HMG-CoA synthase OH CH3 CH CH2 COO OOC CH2 -Hydroxy-butyrate NAD+ OH

C CH2 C S CoA

CH3 -Hydroxy- -methylglutaryl-CoA HMG-CoA HMG-CoA lyase O CH3 CO2 C CH2 COO Acetoacetate Acetyl-CoA

-hydroxybutyrate dehydrogenase NADH+H+ O CH3 C CH3 Acetone

Utilization of ketone bodies (ketolysis) at extrahepatic tissues

Succinyl-CoA transsulfurase

HSCoA ATP -

AMP PPi

Acetoacetate thiokinase

Lack of succinyl-CoA transsulfurase and Acetoacetate thiokinase in the liver.

Biological Significance
Ketone bodies replace glucose as the major source of energy for many tissues especially the brain, heart and muscles during times of prolonged starvation.

Normal physiological responses to carbohydrate shortages cause the liver to increase the production of ketone bodies from the acetyl-CoA generated from fatty acid oxidation.

Hepatocyte

Acetoacetate, -hydroxybutyrate, acetone Ketone body formation Fatty Acetyl-CoA acids -oxidation CoA

Citric Acid cycle

Ketone bodies exported as energy source for heart, skeletal muscle, kidney, and brain

oxaloacetate gluconeogenesis Glucose Glucose exported as fuel for tissues such as brain

Plasma concentrations of metabolic fuels (mmol/L) in the fed and starving states

Ketosis consists of ketonemia, ketonuria and smell of acetone in breath

Causes for ketosis

Severe diabetes mellitus

Starvation
Hyperemesis (vomiting) in early pregnancy

 2.2 Lipogenesis

 2.2.1 Synthesis of fatty acid

O C-S-CoA H3C
palmitic acid (C16:0)

palmitoylCoA

O C-S-CoA
H3C
stearic acid (C18:0) 9 oleic acid (C18:1 D9) 18 stearoylCoA

O C-S-CoA
1 oleoylCoA

H3C

1. Palmitic Acid Synthesis

Location: cytosol of liver, adipose tissue, kidney, brain and breast.
Precursor: acetyl CoA

Other materials: ATP, NADPH, CO2

Citrate-pyruvate cycle
mitochondrion Acetyl CoA citrate cytosol citrate Acetyl CoA

TCAC oxaloacetate malate

oxaloacetate NADH malate NADPH CO2 glucose

pyruvate

pyruvate

The sources of NADPH are as follows:

Pentose phosphate pathway

Malic enzyme

Cytoplasmic isocitrate dehydrogenase

Process of synthesis:
(1) Carboxylation of Acetyl CoA
(2) Repetitive steps catalyzed by fatty acid synthase

(1) Carboxylation of Acetyl CoA

O CH3 C SCoA + HCO3 acetyl-CoA ATP ADP + Pi biotin O OOC CH2 C SCoA malonyl-CoA

acetyl-CoA carboxylase

Malonyl-CoA serves as the donor of twocarbon unit.

Acetyl-CoA Carboxylase is the rate limiting enzyme of the fatty acid synthesis pathway. The mammalian enzyme is regulated, by phosphorylation allosteric regulation by local metabolites.

glucagon ATP

insulin ADP + Pi

acetyl-CoA + HCO3 + H+ malonyl-CoA acetyl-CoA carboxylase (biotin) long chain acyl-CoA citrate isocitrate

(2) Repetitive steps catalyzed by fatty acid synthase

Fatty acid synthesis from acetyl-CoA & malonyl-CoA occurs by a series of reactions that are: in bacteria catalyzed by seven separate enzymes. in mammals catalyzed by individual domains of a single large polypeptide.

Fatty acid synthase complex (multifunctional enzyme)

Acyl carrier protein (ACP) Acetyl-CoA-ACP transacetylase (AT) -Ketoacyl-ACP synthase (KS) Malonyl-CoA-ACP transferase (MT) -Ketoacyl-ACP reductase (KR) -Hydroacyl-ACP dehydratase (HD) Enoyl-ACP reductase (ER) Thioesterase (TE)

AT KS Cys HS HS PhP TE ACP KR ER

MT HD ER KR ACP TE PhP HS HS Cys KS HD MT AT

Functional division

Subunit division

ACP contains 4-phosphopantotheine.

HS CH3 C S

O OOC CH 2 C S CoA
MT

CH 3 C S CoA
ACP-HS KS-HS

O HS HS

AT

HS CoA

HS CoA O

OOC CH2 C S CH3 C S

O

O CH3 (CH2)14 C O
(After 7 rounds) H2O TE

HS CH3 CH2 CH2 C S

condensation KS
CO 2

O
O CH3 CH2 CH2 C S HS
AT

CH3 C CH2 C S HS O

reduction
KR NADPH + H+ NADP+

reduction
NADP+ ER

O
NADPH + H+

CH3 CH CH2 C S HS OH

CH3 CH CH C S HS

dehydration
HD H2O

The overall reaction of synthesis:

acetyl-CoA + 7 malonyl-CoA + 14 NADPH + 14H+

palmitate + 7 CO2 + 14 NADP+ + 8 HSCoA + 6H2O

Differences in the oxidation and synthesis of FAs

-oxidation Site Intermediates Enzymes Sequential units Co-enzymes Mitochondria Present as CoA derivatives Present as independent proteins Fatty acid synthesis Cytoplasm Covalently linked to SH group of ACP Multi-enzyme complex

2 carbon units split off 2 carbon units added, as 3 as acetyl CoA carbon malonyl CoA NAD+ and FAD are reduced NADPH used as reducing power

Routes of synthesis of other fatty acids

2. Elongation of palmitate
Elongation beyond the 16-C length of the palmitate occurs in mitochondria and endoplasmic reticulum (ER).

 Fatty acid elongation within mitochondria uses the acetyl-CoA as donor of 2-carbon units and NADPH serves as electron donor for the final reduction step. Fatty acids esterified to coenzyme A are substrates for the ER elongation machinery, which uses malonyl-CoA as donor of 2-carbon units.

3. The synthesis of unsaturated fatty acid

Formation of a double bond in a fatty acid involves several endoplasmic reticulum membrane proteins in mammalian cells

O 10 9 C OH

oleate 18:1 cis D9

Desaturases introduce double bonds at specific positions in a fatty acid chain.

 2.2.2
Synthesis of Triacylglycerol
Monoacylglycerol pathway (small intestine) Diacylglycerol pathway (liver, adipose tissue)

1. Monoacylglycerol pathway
O O CH2 HSCoA OH acyl CoA O CH2 O C R1

R2 C O C H CH2 OH

acyl CoA transferase

R2 C O C H CH2 OH

2-monoacylglycerol
HSCoA acyl CoA

1,2-diacylglycerol
O O CH2 O C R1 O

acyl CoA transferase

R2 C O C H

CH2 O C R3

triacylglycerol

2. Diacylglycerol pathway
glycolysis

Summary
Places: tissue small intestine, liver, adipose

Materials: Endogenous: glucoseamino acid glycerol Exogenous: free fatty acid and monoacylglycerol

Adipose tissue generate fat mainly from glucose

In adipose tissue, the acetyl CoA for the synthesis of fatty acid is mainly from glucose. The lack of glycerol kinase make the only source of glycerol 3-phosphate in adipose tissue is glucose.

Obesity results from an imbalance between energy input and output

Food
adipose tissue

Work or Growth

fatty acids & triacylglcerols ADP ATP

Obesity

Heat

CO2 + H2O

Section 3 Metabolism of Phospholipids

 Phospholipid refers to phosphorouscontaining lipids. Glycerophospholipids Phospholipids Sphingolipids

 3.1 Classification and Structure of Glycerophospholipids

Glycerophospholipids are lipids with a glycerol, fatty acids, a phosphate group and a nitrogenous base.

glycerol

fatty acids

nitrogenous base

Phosphatidylcholine

 glycerol O R2 C O CH2 O C CH2 H O

O C O P OH R1

fatty acyl group

fatty acyl group

Nitrogenous O X base

The basic structure of glycerophospholipid

In general, glycerophospholipids contain a saturated fatty acid at C-1 and an unsaturated fatty acid (usually arachidonic acid) at C-2.

The major function of phospholipids is to form biomembrane.

 Hydrophobic tail = fatty acids Polar head = nitrogenous base

Some common glycerophospholipid

Some common glycerophospholipid

(continue)

 3.2 Synthesis of Glycerophospholipid

Location: All tissue of body, especially liver & kidney Endoplasmic reticulum
Pathways: CDP-diacylglycerol pathway Diacylglycerol pathway

The system of synthesis

a. FA Glycerol from carbohydrate

b. poly unsaturated fatty acid from plant oil c. choline ethanolamine serine inositol d. ATP, CTP

from food or synthesis in body

e. Enzymes and cofactors

Diacylglycerol pathway
CO2 HO CH2 CH COOH NH2 HO CH2 CH2 NH2 3 SAM HO CH2 CH2 N(CH3)3

Ethanolamine
ATP ADP P O CH2 CH2 NH2

Choline
ATP ADP P O CH2 CH2 N(CH3)3

Serine

Phosphoethanolamine
CTP PPi CDP O CH2 CH2 NH2

Phosphocholine
CTP PPi CDP O CH2 CH2 N(CH3)3

CDP-ethanolamine
DG CO2 CMP

CDP-choline
DG CMP 3 SAM

Phosphatidyl serine

Phosphatidyl ethanolamine

Phosphatidyl choline

CDP-Diacylglycerol pathway
Dihydroxyacetone phosphate

Glycerol 3-phosphate

Phosphotidate CTP PPi CDP-diacylglycerol Inositol Phosphatidyl glycerol CMP

CMP Phosphatidyl inositol

Serine CMP

Diphosphatidyl glycerol (cardiolipin) Phosphatidyl serine

Phosphatidylcholine (Lecithin)

Phosphatidylethanolamine (Cephalin)

CDP-diacylglycerol

Phosphatidylserine

Phosphatidylglycerol

Diphosphatidyl glycerol (Cardiolipin)

Phosphatidylinositol

 3.3 Degradation of glycerophospholipids by phospholipase

A1 O A2 O R2 C O CH2 C CH2 H O C O C D O P OH O X R1

O CH2 HO C CH2 H O O C B1 O P OH Lysophospholipid-1 O X R1 R2 O C O B2 CH2 C CH2 H O OH O P OH Lysophospholipid-2 O X

Actions of phospholipases on lecithin

PLA1: fatty acid + lysolecithin
PLA2: fatty acid + acyl glycerophosphoryl choline PLC: 1,2 diacylglycerol + phosphoryl choline

PLD: phosphatidic acid + choline

Lysophospholipids, the products of Phospholipase A hydrolysis, are powerful detergents.

O O R2 C O CH2 C H O C O PLA2 CH2O P O phospholipid O X R1 H2O R2 O C O HO CH2 C H O O P O Lysophospholipid O X O C R1

CH2O

Section 4 Metabolism of Cholesterol

 4.1 Structure and function of

cholesterol
1. Function of cholesterol: (1) It is a constituent of all cell membranes. (2) It is necessary for the synthesis of all steroid hormones, bile salts and vitamin D.

2. Structure of cholesterol
All steroids have cyclopentano penhydro phenanthrene ring system.
H3C 21
18 CH3 12 19 CH3 1 2 3 11 9 10 5 20 27 CH3 15 22 23 24 25

CH3
26

17 13 14

D 16

A
4

B 8
6 7

HO

Cholesterol ester

O C R O

 4.2 Synthesis of cholesterol

Location: All tissue except brain and mature red blood cells. The major organ is liver (80%). Enzymes locate in cytosol and endoplasmic reticulum. Materials: Acetyl CoA, NADPH(H+), ATP

Acetyl-CoA is the direct and the only carbon source.

Acetyl-CoA HMG-CoA

Acetoacetyl-CoA

HMG CoA reductase is the rate-limiting enzyme

The total process of cholesterol de novo synthesis

Regulation of cholesterol synthesis

fasting

Glucagon

HMG CoA

HMG CoA reductase

MVA

cholesterol

after meal

insulin

thyroxine

bile acid

 4.3 Transformation and excretion of cholesterol

Bile acids

Steroid hormones Cholesterol

Vitamin D

1. Conversion of Cholesterol into bile acid

(1) Classification of bile acids The primary bile acids are synthesized in the liver from cholesterol. The 7hydroxylase is rate-limiting enzyme in the pathway for synthesis of the bile acids.

The secondary bile acids are products that the primary bile acids in the intestine are subjected to some further changes by the activity of the intestinal bacteria.

Classification of bile acids

Classification Free bile acids Conjugated bile acids

Cholic acid Primary bile acids Chenodeoxycholic acid

Glycocholic acid Glycochenodeoxycholic acid

Taurocholic acid

Taurochenodeoxycholic acid

Secondary bile acids

Deoxycholic acid
Lithocholic acid

Glycodeoxycholic acid
Glycolithocholic acid

Taurodeoxycholic acid
Taurolitho-cholic acid

(2) Strcture of bile acids

OH
12

COOH

COOH

HO

H cholic acid

OH

HO

OH H chenodeoxycholic acid OH CONHCH2CH2SO3H

OH

CONHCH2COOH

HO

OH

HO

OH

glycocholic acid

taurocholic acid

OH

COOH

COOH

HO

H deoxycholic acid

HO

H lithocholic acid

(3) Enterohepatic Cycle of bile acids

Conversion to bile salts, that are secreted into the intestine, is the only mechanism by which cholesterol is excreted. Most bile acids are reabsorbed in the ileum , returned to the liver by the portal vein, and re-secreted into the intestine. This is the enterohepatic cycle.

(4) Function of bile acids

Bile acids are amphipathic, with detergent properties. Emulsify fat and aid digestion of fats & fat-soluble vitamins in the intestine.

Increase solubility of cholesterol in bile.

2. Conversion of cholesterol into steroid hormones

Tissues: adrenal cortex, gonads

Steroid hormones: cortisol (glucocorticoid), corticosterone and aldosterone (mineralocorticoid), progesterone, testosterone, and estradiol

Steroids derived from cholesterol

3. Conversion into 7-dehydrocholesterol

cholesterol in skin 25-hydroxylase (microsome in the liver)

ultraviolet light 7-dehydrocholecalciferol (VD3) cholesterol

25-OH-D3

1 -hydroxylase (mitochondria in the kidney)

1,25-(OH)2-D3 active Vit D3

 4.4 Esterification of cholesterol

in cells
SHCoA acyl CoA acyl CoA cholesterol R C O acyl transferase cholesteryl ester (ACAT)
O

HO

cholesterol

in plasma

Section 5 Plasma lipoprotein

 5.1 blood lipid

Concept: All the lipids contained in plasma, including fat, phosphalipids, cholesterol, cholesterol ester and fatty acid. Blood lipid exist and transport in the form of lipoprotein.

blood lipids

ester lecithin phospholipids sphingolipids cephalin FFA

TG cholesterol

free

 5.2 Classification of plasma

lipoproteins
1. electrophoresis method: - Lipoprotein pre -Lipoprotein -Lipoprotein CM (chylomicron) fast

slow

2. Ultra centrifugation method

high density lipoprotein (HDL) high low density lipoprotein ( LDL) very low density lipoprotein ( VLDL) CM (chylomicron ) low

electron microscope

CM

LDL

VLDL

HDL

Origin

CM

Pre-

Separation of plasma lipoproteins by electrophoresis on agarose gel

 5.3 Structure

 5.4 Composition of lipoprotein

CM Density(g/ml) <1.006 VLDL 0.951.006 LDL 1.0061.063 HDL 1.0631.210

Protein
Phospholipids Cholesterol Cholesteryl esters TG

2
9 1 3 85

10
18 7 12 50

23
20 8 37 10

55
24 2 15 4

 5.5 Apolipoproteins

Functions of apolipoproteins
a . To combine and transport lipids.

b . To regulate lipoprotein metabolism. apo A II activates hepatic lipaseHL apo A I activates LCAT apo C II activates lipoprotein lipase LPL c. To recognize the lipoprotein receptors.

 5.6 Metabolism of plasma lipoprotein

1. CM
Chylomicrons are formed in the intestinal mucosal cells and secreted into the lacteals of lymphatic system.

structure of CM

Apolipoproteins

phospholipids

Cholesterol

Triacylglycerols and cholesteryl esters

Metabolic fate of CM

summary of CM
Site of formation: intestinal mucosal cells Function: transport exogenous TG key E: LPL in blood HL in liver apoC is the activator of LPL

apo E and apo B-48 will be recognized by the LRP receptor

2. VLDL
Very low density lipoproteins (VLDL) are synthesized in the liver and produce a turbidity in plasma.

Nascent VLDL

Metabolic fate of VLDL and production of LDL

Summary of VLDL
Formation site: liver

Function: VLDL carries endogenous triglycerides from liver to peripheral tissues for energy needs.
key E: LPL in blood HL in liver

3. LDL
Most of the LDL particles are derived from VLDL, but a small part is directly released from liver. They are cholesterol rich lipoprotein molecules containing only apo B-100.

LDL receptors
Cholesterol ester

protein

Cholesterol

LDL

Cholesteryl oleate

Amino acids LDL binding Internalization Lysosomal hydrolysis

Michael Brown and Joseph Goldstein were awarded Nobel prize in 1985 for their work on LDL receptors.

Summary of LDL
Formation site: from VLDL in blood Function: transport cholesterol from liver to the peripheral tissues. LDL concentration in blood has positive correlation with incidence of cardiovascular diseases.

Fates of cholesterol in the cells

1. Incorporated into cell membranes.

2. Metabolized to steroid hormones.

3. Re-esterified and stored. The reesterification is catalyzed by ACAT. 4. Expulsion of cholesterol from the cell, esterified by LCAT and transported by HDL and finally excreted through liver.

4. HDL
LDL variety is called bad cholesterol whereas HDL is known as good cholesterol .

Liver VLDL BAD LDL Cholesterol Deposit

Heart

Good Excretion

HDL

Forward and reverse cholesterol transport

Reverse cholesterol transport

Cholesterol from tissues reach liver, and is later excreted. This is called reverse cholesterol transport by HDL.

Metabolism of HDL in reverse cholesterol transport

CETP
Cholesterol ester transfer protein (CETP) transfer cholesterol ester in HDL to VLDL and LDL.

Summary of HDL
Formation site: liver and intestine Function: transport cholesterol from peripheral tissues to liver

summary of lipoprotein metabolism

 5.7 Hyperlipidemias
classification a b Lipoprotein CM LDL LDL, VLDL Blood lipids TAG CH CH CH TAG

IDL
VLDL VLDL, CM

CH TAG
TAG TAG CH

Molecular Biochemistry II

Fatty Acid Synthesis

Copyright 1999-2009 by Joyce J. Diwan. All rights reserved.

O H3C C SCoA

The input to fatty acid O synthesis is acetyl OOC CH2 C SCoA CoA, which is malonyl-CoA carboxylated to malonyl-CoA. ATP-dependent carboxylation provides energy input. The CO2 is lost later during condensation with the growing fatty acid.
The spontaneous decarboxylation drives the condensation reaction.

acetyl-CoA

Acetyl-CoA 1 Carboxylase ADP + Pi Enzyme-biotin-CO2 catalyzes the O 2-step ll 2 CH3-C-SCoA reaction by Enzyme-biotin acetyl-CoA which acetylO CoA is ll O2C-CH2-C-SCoA carboxylated malonyl-CoA to form malonylAs with other carboxylation reactions, the enzyme CoA. prosthetic group is biotin. ATP-dependent carboxylation of the biotin, carried out at one active site 1 , is followed by transfer of the carboxyl group to acetyl-CoA at a second active site 2 .

Enzyme-biotin HCO3 + ATP

Enzyme-biotin HCO3 + ATP ADP + Pi Enzyme-biotin-CO2 O CH3-C-SCoA acetyl-CoA

-

ll

2
Enzyme-biotin O
ll

O2C-CH2-C-SCoA malonyl-CoA

The overall reaction, which is spontaneous, may be summarized as: HCO3 + ATP + acetyl-CoA ADP + Pi + malonylCoA

O O C O N C NH CH CH H2C S CH O (CH2)4 C NH O C

(CH2)4 CH

Carboxybiotin

lysine NH residue

Biotin is linked to the enzyme by an amide bond between the terminal carboxyl of the biotin side chain and the e-amino group of a lysine residue. The combined biotin and lysine side chains act as a long flexible arm that allows the biotin ring to

Acetyl-CoA Carboxylase, which converts acetylCoA to malonyl-CoA, is the committed step of the fatty acid synthesis pathway. The mammalian enzyme is regulated, by

phosphorylation
allosteric control by local metabolites.

Conformational changes associated with regulation:

In the active conformation, Acetyl-CoA Carboxylase associates to form multimeric filamentous complexes. Transition to the inactive conformation is associated with dissociation to yield the monomeric form of the enzyme (protomer).

AMP functions as an energy sensor and regulator of metabolism. When ATP production does not keep up with needs, a higher portion of a cell's adenine nucleotide pool is in the form of AMP. AMP promotes catabolic pathways that lead to synthesis of ATP. AMP inhibits energy-utilizing synthetic pathways.

E.g., AMP regulates fatty acid synthesis and catabolism by controlling availability of malonylCoA.

O H3C C SCoA

AMP-Activated Kinase catalyzes phosphorylation of Acetyl-CoA Carboxylase. This causes inhibition of ATP-utilizing of malonylCoA production.

acetyl-CoA ATP + HCO3 Acetyl-CoA Carboxylase (inhibited by AMP-Activated Kinase)

SCoA

ADP + Pi

O OOC CH2 C

malonyl-CoA

Fatty acid synthesis is diminished by lack of the substrate malonyl-CoA. As discussed earlier, fatty acid oxidation is stimulated due to decreased inhibition by malonyl-CoA of transfer of fatty acids into mitochondria.

O H3C C SCoA

acetyl-CoA ATP + HCO3 Acetyl-CoA Carboxylase (inhibited by AMP-Activated Kinase)

ADP + Pi

A cAMP cascade, O activated by glucagon OOC CH2 C SCoA & epinephrine when malonyl-CoA blood glucose is low, may alsophosphorylation of Acetyl-CoA Carboxylase result in via cAMP-Dependent Protein Kinase.

With Acetyl-CoA Carboxylase inhibited, acetyl-CoA remains available for synthesis of ketone bodies, the alternative metabolic fuel used when blood glucose is low.

Phosphorylated protomer of Acetyl-CoA Carboxylase (inactive) Citrate

Dephosphorylated, e.g., by insulinactivated Protein Phosphatase

Palmitoyl-CoA
Phosphorylated, e.g., via AMP-activated Kinase when cellular stress or exercise depletes ATP.

Dephosphorylated Polymer of Acetyl-CoA Carboxylase (active) Regulation of Acetyl-CoA Carboxylase

The antagonistic effect of insulin, produced when blood glucose is high, is attributed to activation of Protein Phosphatase.

Phosphorylated protomer of Acetyl-CoA Carboxylase (inactive) Citrate

Dephosphorylated, e.g., by insulinactivated Protein Phosphatase

Palmitoyl-CoA
Phosphorylated, e.g., via AMP-activated Kinase when cellular stress or exercise depletes ATP.

Regulation of Acetyl-CoA Carboxylase by local metabolites:

Dephosphorylated Polymer of Acetyl-CoA Carboxylase (active) Regulation of Acetyl-CoA Carboxylase

Palmitoyl-CoA (product of Fatty Acid Synthase) promotes the inactive conformation, diminishing production of malonyl-CoA, the precursor of fatty acid synthesis.

Glucose-6-phosphatase glucose-6-P glucose Gluconeogenesis Glycolysis pyruvate fatty acids acetyl CoA ketone bodies cholesterol citrate

Citrate oxaloacetate allosterically activates AcetylKrebs Cycle CoA Carboxylase. [Citrate] is high when there is adequate acetyl-CoA entering Krebs Cycle.

Excess acetyl-CoA is then converted via malonylCoA to fatty acids for storage.

Fatty acid synthesis from acetyl-CoA & malonylCoA occurs by a series of reactions that are: in bacteria catalyzed by 6 different enzymes plus a separate acyl carrier protein (ACP)

in mammals catalyzed by individual domains of a very large polypeptide that includes an ACP domain.
Evolution of the mammalian Fatty Acid Synthase apparently has involved gene fusion. NADPH serves as electron donor in the two reactions involving substrate reduction. The NADPH is produced mainly by the Pentose Phosphate Pathway.

H H3N+ C CH2 SH COO

SH CH2 CH2 NH C CH2 CH2 NH C HO H3C C C H2C O H CH3 O O P O O O

Coenzyme A
-mercaptoethylamine

Fatty Acid cysteine Synthase prosthetic groups: the thiol of the sidechain of a cysteine residue of Condensing Enzyme domain. the thiol of phosphopantethein e, equivalent in structure to part of

pantothenate
NH2 N

ADP-3'phosphate
O P O O

N CH2 H H O

N O H H OH O

phosphopantetheine

P O

SH

Phosphopantetheine (Pant) is covalently linked via a phosphate ester to a serine OH of the acyl carrier protein domain of Fatty Acid Synthase. The long flexible arm of phosphopantetheine helps its thiol to move from one active site to another within the complex.

CH2 CH2 NH C CH2 CH2 NH C HO H3C C C H2C O H CH3 O O P O

phosphopantetheine of acyl carrier protein -mercaptoethylamine

pantothenate

NH O CH2 CH C

serine residue
O

phosphate

Enoyl ACP N- Condensing Malonyl/acetyl-CoA Dehydratase Reductase -Ketoacyl (Pant) Thioesterase -C Enzyme (Cys) Transacylase (Ser) Reductase

Order of domains in primary structure of mammalian Fatty Acid Synthase

As each of the substrates acetyl-CoA & malonylCoA bind to the complex, the initial attacking group is the oxygen of a serine hydroxyl group of the Malonyl/acetyl-CoA Transacylase enzyme domain.
Each acetyl or malonyl moiety is transiently in ester linkage to this serine hydroxyl, before being transferred into thioester linkage with the phosphopantetheine thiol of the acyl carrier protein (ACP) domain. Acetate is subsequently transferred to a cysteine thiol of the Condensing Enzyme domain.

acetyl-S-CoA HS-CoA Pant SH Cys SH

malonyl-S-CoA HS-CoA Cys S C CH3 O Pant Cys S O C CH3 O

CO2 Pant Cys SH O

Pant

SH

S C CH2

S C CH2 C CH3 O

1 Malonyl/acetyl-CoA-ACP Transacylase COO 2 Malonyl/acetyl-CoA-ACP Transacylase 3 Condensing Enzyme (-Ketoacyl Synthase)

The condensation reaction (step 3) involves decarboxylation of the malonyl moiety, followed by attack of the resultant carbanion on the carbonyl carbon of the acetyl (or acyl) moiety.

NADPH NADP+ Pant S C CH2 C CH3 O O Cys SH Pant Cys SH O

H2O Pant

NADPH NADP+ Cys SH O Pant Cys SH O

S C CH2 HC CH3

S C CH

S C CH2 CH2 CH3

OH

HC CH3

4 -Ketoacyl-ACP Reductase 5 -Hydroxyacyl-ACP Dehydratase 6 Enoyl-ACP Reductase

4. The -ketone is reduced to an alcohol by e transfer from NADPH. 5. Dehydration yields a trans double bond. 6. Reduction by NADPH yields a saturated chain.

Malonyl-S-CoA HS-CoA Pant S C CH2 CH2 CH3 O Cys SH Pant Cys S C CH2 CH2 CH3 O Pant Cys S O C CH2 CH2 CH3 O

SH

S C CH2 COO

7 Condensing Enzyme 2 Malonyl/acetyl-CoA-ACP Transacylase (repeat).

Following transfer of the growing fatty acid from phosphopantetheine to the Condensing Enzyme's cysteine sulfhydryl, the cycle begins again, with another malonyl-CoA.

Product release: When the fatty acid is 16 carbon atoms long, a Thioesterase domain catalyzes hydrolysis of the thioester linking the fatty acid to phosphopantetheine. The 16-C saturated fatty acid palmitate is the final product of the Fatty Acid Synthase complex.

Enoyl ACP N- Condensing Malonyl/acetyl-CoA Dehydratase Reductase -Ketoacyl (Pant) Thioesterase -C Enzyme (Cys) Transacylase (Ser) Reductase

Order of domains in primary structure of mammalian Fatty Acid Synthase

The primary structure of the mammalian Fatty Acid Synthase protein is summarized above. Fatty Acid Synthase in mammals is a homodimer. X-Ray crystallographic analysis at 3.2 resolution shows the dimeric Fatty Acid Synthase to have an X-shape. The 2 copies of the protein are displayed at right in different colors.
Fatty Acid Synthase PDB 2VZ8

Enoyl ACP N- Condensing Malonyl/acetyl-CoA Dehydratase Reductase -Ketoacyl (Pant) Thioesterase -C Enzyme (Cys) Transacylase (Ser) Reductase

Order of domains in primary structure of mammalian Fatty Acid Synthase

The domain arrangement is shown below. Each copy of the dimeric protein has an S shape, with the N-terminal KS (Condensing Enzyme / Ketoacyl Synthase) domain folded back to form part of the central interaction domain.
KR = -Ketoacyl Reductase; ER = Enoyl Reductase; DH = Dehydratase; KS = -Ketoacyl Synthase (Condensing Enzyme); MAT = Malonyl/Acetyl-CoA Transacylase.
KR

ER
DH KS

ER
DH KS

KR

arm

MAT

MAT Arrangement of domains in Fatty Acid Synthase

leg

Enoyl ACP N- Condensing Malonyl/acetyl-CoA Dehydratase Reductase -Ketoacyl (Pant) Thioesterase -C Enzyme (Cys) Transacylase (Ser) Reductase

Order of domains in primary structure of mammalian Fatty Acid Synthase

KR = -Ketoacyl Reductase; ER = Enoyl Reductase; DH = Dehydratase; KS = -Ketoacyl Synthase (Condensing Enzyme); MAT = Malonyl/Acetyl-CoA Transacylase.

The X-ray analysis does not resolve the C-terminal ACP (acyl carrier protein) & Thioesterase domains, predicted from the primary structure to be near the KR domains. These domains may be too flexible to be resolved by crystallography. KR KR ER ER
DH KS MAT DH KS MAT Arrangement of domains in Fatty Acid Synthase

arm

leg

Fatty Acid Synthase complex is somewhat asymmetric.

There is evidence for conformational changes relating to catalysis. Protein flexibility may facilitate transfer of ACPattached reaction intermediates among the several active sites in each half of the complex. For images see: website (ETH Zurich) website (Asturias lab,
Scripps)

article (Maier, Leibundgut

& Ban; requires subscription to Science).
Fatty Acid Synthase PDB 2VZ8

Explore with Chime the structure of the Mammalian Fatty Acid Synthase III.

Palmitate, a 16-C saturated fatty acid, is the final product of the Fatty Acid Synthase reactions. 1 1. a. How many acetyl-CoA used for initial priming of enzyme? 1 _____ b. How many acetyl-CoA used for synthesis of each 7 malonate? ____ c. How many 8 malonate used (how many reaction cycles) per synthesis of one 16-C palminate? ________ d. Total acetyl-CoA used for priming & for syntheisis of 1 malonate, a + b(c): ________ 7 2. a. How many ~P bonds of ATP used for synthesis of each 2 malonate? ________ b. Total ~P bonds of ATP used for synthesis of one 16-C 14 palmitate,

Summary (ignoring H+ & water):

Write a balanced equation for synthesis of palmitate from acetyl-CoA, listing net inputs and outputs: 8 acetyl-CoA + 14 NADPH + 7 ATP palmitate + 14 NADP+ + 8 CoA + 7 ADP + 7 Pi Summary based on malonate as an input: acetyl-CoA + 7 malonyl-CoA + 14 NADPH palmitate + 7 CO2 + 14 NADP+ + 8 CoA Fatty acid synthesis occurs in the cytosol. AcetylCoA generated in mitochondria is transported to the cytosol via a shuttle mechanism involving citrate.

-Oxidation & Fatty Acid Synthesis Compared

Oxidation Pathway Fatty Acid Synthesis pathway location acyl carriers (thiols) e acceptors/donor -OH intermediate 2-C product/donor mitochondrial matrix Coenzyme-A FAD & NAD+ L acetyl-CoA cytosol phosphopantetheine (ACP) & cysteine NADPH D malonyl-CoA (& acetyl-CoA)

Fatty Acid Synthase is transcriptionally regulated. In liver: Insulin, a hormone produced when blood glucose is high, stimulates Fatty Acid Synthase expression. Thus excess glucose is stored as fat. Transcription factors that that mediate the stimulatory effect of insulin include USFs (upstream stimulatory factors) and SREBP-1. SREBPs (sterol response element binding proteins) were first identified for their regulation of cholesterol synthesis. Polyunsaturated fatty acids diminish transcription of the Fatty Acid Synthase gene in liver cells, by suppressing production of SREBPs.

In fat cells:
Expression of SREBP-1 and of Fatty Acid Synthase is inhibited by leptin, a hormone that has a role in regulating food intake and fat metabolism. Leptin is produced by fat cells in response to excess fat storage.

Leptin regulates body weight by decreasing food intake, increasing energy expenditure, and inhibiting fatty acid synthesis.

Elongation beyond the 16-C length of the palmitate product of Fatty Acid Synthase is mainly catalyzed by enzymes associated with the endoplasmic reticulum (ER). ER enzymes lengthen fatty acids produced by Fatty Acyl Synthase as well as dietary polyunsaturated fatty acids. Fatty acids esterified to coenzyme A serve as substrates. Malonyl-CoA is the donor of 2-carbon units in a reaction sequence similar to that of Fatty Acid Synthase except that individual steps are catalyzed by separate proteins.

10 9

O C OH

oleate 18:1 cis D9

Desaturases introduce double bonds at specific positions in a fatty acid chain. Mammalian cells are unable to produce double bonds at certain locations, e.g., D12. Thus some polyunsaturated fatty acids are dietary essentials, e.g., linoleic acid, 18:2 cis D9,12 (18 C atoms long, with cis double bonds at carbons 9-10 & 12-13).

10 9

O C OH

oleate 18:1 cis D9

Formation of a double bond in a fatty acid involves the following endoplasmic reticulum membrane proteins in mammalian cells: NADH-cyt b5 Reductase, a flavoprotein with FAD as prosthetic group. Cytochrome b5, which may be a separate protein or a domain at one end of the desaturase. Desaturase, with an active site that contains two iron atoms complexed by histidine

The desaturase catalyzes a mixed function oxidation reaction.

There is a 4-electron reduction of O2 2 H2O as a fatty acid is oxidized to form a double bond. 2e pass from NADH to the desaturase via the FAD-containing reductase & cytochrome b5, the order of electron transfer being: NADH FAD cyt b5 desaturase 2e are extracted from the fatty acid as the double bond is formed. E.g., the overall reaction for desaturation of stearate (18:0) to form oleate (18:1 cis D9) is: stearate + NADH + H+ + O2 oleate + NAD+ + 2H2O

Fatty Acid Synthesis Pathway

Acetyl CoA Carboxylase

first reaction of fatty acid synthesis
AcCoA + ATP + CO2 malonyl-CoA + ADP + Pi

malonyl-CoA serves as activated donor of acetyl groups in FA synthesis

Fatty Acid Synthesis

figure 20-3
Glycolysis
cytoplasmic

PDH
mitochondrial

FA synthesis
cytoplasmic Citrate Shuttle
moves AcCoA to cytoplasm produces 50% NADPH via malic enzyme

Pyruvate malate cycle

Fatty Acid Synthesis Pathway

FA Synthase Complex
figure 20-4

Priming reactions
transacetylases

(1) condensation rxn (2) reduction rxn (3) dehydration rxn (4) reduction rxn

Regulation of FA synthesis:

Acetyl CoA Carboxylase

Allosteric regulation
stimulated by citrate
feed forward activation

inhibited by palmitoyl CoA

hi B-oxidation (fasted state) or esterification to TG limiting

Inducible enzyme
Induced by insulin Repressed by glucagon

Regulation of FA synthesis:

Acetyl CoA Carboxylase

Covalent Regulation
figure 20-5
Activation (fed state)
insulin induces protein phosphatase activates ACC

Inactivation (starved state)

glucagon increases cAMP activates protein kinase A inactivates ACC

Fatty acids are synthesized and degraded by different mechanisms: Synthesis

Cytosol Intermediates are covalently linked to ACP Fatty acid synthase contain multienzyme activities Utilizes NADP+ as coenzyme Requires both ATP

Oxidation
Mitochondrial matrix Bonded to CoA Degradative enzymes are not associated Utilizes NAD+ and FAD as coenzymes Generates ATP Aerobic process

KETOGENESIS
It occurs when there is a high rate of fatty acid oxidation in the liver

These three substances are collectively known as the ketone bodies (also called acetone bodies or acetone). Enzymes responsible for ketone bodies formation are associated with mitochondria.

KETOGENESIS IS REGULATED AT THREE CRUCIAL STEPS:

1. Adipose tissue: Factors regulating mobilization of free fatty acids from adipose tissue are important in controlling ketogenesis 2. Liver: After acylation, fatty acids undergo oxidation or esterified to triacylglycerol or ketone bodies. a. CPT-1 regulates entry of long-chain acyl groups into mitochondria prior to -oxidation. Its activity is low in the fed state, and high in starvation.

Fed state: Malonyl-CoA formed in the fed state is a potent inhibitor of CPT-1. Under these conditions, free fatty acids enter the liver cell in low concentrations and are nearly all esterified to acylglycerols and transported out as VLDL. Starvation: Free fatty acid concentration increases with starvation, acetyl-CoA carboxylase is inhibited and malonyl-CoA decreases releasing the inhibition of CPT-I and allowing more -oxidation. These events are reinforced in starvation by decrease in insulin/glucagon ratio. This causes inhibition of acetyl-CoA carboxylase

In short, -oxidation from free fatty acids is controlled by the CPT-I gateway into the mitochondria, and the balance of free fatty acid uptake not oxidized is esterified. 3. Acetyl-CoA formed from -oxidation of fatty acids is either oxidized in TCA cycle or it forms ketone bodies.

Discovered enzymes

Synthesis way Fatty acid biosynthesis Fatty acid desaturation Synthesis of membrane + storage lipids -Oxidation Other lipids Others

found (members) 7 (11) 4 (14) 13 (21) 10 (24) 6 (7) 6 (7)

EST-evidence 5 4 10 8 5 50

Fatty acid biosynthesis

HCO3
-

Acetyl -CoA CO2Malonyl -CoA

Activating enzyme:

Acetyl-CoA-carboxylase

HCO3- is activated via acetyl-CoA to malonyl-CoA

Constructing enzyme: 7 subunits synthesizes a C-chain

Fatty acid synthase (FAS)

Break off enzyme: Stopps the circle of FAS

Acyl-CoA-thioesterase

Products of a C-chain-length from 14 or 16 C atoms

Fatty acid synthase (FAS)

Group of organisms bacteria, plants

7 subunits coded in

seven proteins

Saccharomyces cerevisae

two proteins

vertebrates + Laccaria bicolor one protein

Fatty acid synthase complex

Fungi Laccaria bicolor Coprinopsis cinerea Separated in subunits no no Length (AS) 3935 3942 Members 5 (1) 2 (1)

Phaenerochaete chrysosporium
Ustilago maydis Cryptococcus neoformans Neurospora crassa Saccharomyces cerevisae

no
no yes ( + ) yes ( + ) yes ( + )

3894
3901 2461/1381 2075/1901 2051/1887

4 (1)
2 (1) 1/1 1/1 1/1

Fatty acid synthase (FAS)

+ Laccaria bicolor

all seven activities coded in one hugh gene (> 12 kbp)

FAS coded in two subunits ( + )

Fatty acid synthase (FAS)

+ Laccaria bicolor

all seven activities coded in one hugh gene (> 12 kbp)

FAS coded in two subunits ( + )

The group of basidiomycetes is splitted by the structure of FAS.

Analysis of fatty acid patterns via HPLC 200

18:2 (9Z,12Z) 18:1 (9Z) 16:0 16:1 (7Z) 16:1 (9Z) 16:1 (11Z) 17:0=IS 17:1 (9Z) 18:3 (9Z, 12Z, 15Z)

160 relative abundance

120

80

20:1 (11Z) 20:2 (11Z, 14Z) 21:0

22:2 (13Z, 16Z) 22:0 22:1 (13Z)

40
15:0
14:0

0 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 retention time [min]

the highest amount of fatty acids shows palmitic acid (16:0), stearic acid (18:1D9Z) and linoleic acid (18:2D9Z, 12Z) as in Neurospora crassa the longest fatty acids shows a chain length of 24 C atoms 16:1D11Z was also found, a fatty acid, which may be involved in mycorrhizal processes

18:0

23:0 23:1 (14Z) 24:0 24:1 (15Z)

20:0

Analysis of fatty acid patterns

14:0 ELO

15:0

Fatty acid 2- hydrolase Delta9-fatty acid desaturase Delta12-fatty acid desaturase

1 5 5 1 1

16:0
ELO 18:0 ELO

16:1 (9Z)

Delta15-fatty acid desaturase Fatty acid-elongase

18:1 (9Z) ELO 20:1 (11Z)

12

18:2 (9Z, 12Z) ELO 20:2 (11Z, 14Z) ELO

22:2 (13Z, 16Z)

15

18:3 (9Z, 12Z, 15Z)

ELO

ELO

22:0
ELO 24:1 (15Z)

Two possibilities of 16:1 (11Z)- fatty acid synthesis

A)

Acyl-CoA-thioesterase stops FAS at a chain length of 14:0 Needed enzymes: Fatty acid-elongase Delta9- fatty acid desaturase

14:0

14:1 (9Z) ELO 16:1 (11Z)

16:0

11

16:1 (11Z)

B) Acyl-CoA-thioesterase stops FAS at a chain length of 16:0 Needed enzymes:

D11-

fatty acid desaturase

LIPOGENESIS

principally in adipose tissue and liver

lipogenesis cytoplasm; requires acetyl CoA adipose: FA stored as triacylglycerols via esterification liver: produces TAG packaged into VLDL and exported compounds metabolized to acetyl CoA can serve as a fat precursor glucose = primary source of carbons for fat synthesis.

Glucose CYTOPLASM Fatty Acids PPP Glycolysis CO2 NADPH FAS Malate Pyruvate ME NADP+

MITOCHONDRIAL MATRIX NAD, CoA PDH Pyruvate ATP, CO2 PC ADP, Pi Oxaloacetate CS Acetyl CoA NADH, CO2

NAD+ Malonyl CoA MDH ADP, Pi ACC NADH CO2, ATP Oxaloacetate Acetyl CoA ADP+Pi CL ATP, CoA Citrate

Citrate

Figure 6. Export of acetyl CoA as citrate for fatty acid biosynthesis, generation of NADPH and pathway of lipogenesis. (similar to diagram discussed for cholesterol synthesis exxept this involves PDH reaction)

KEY MITOCHONDRIAL REACTIONS

PYRUVATE CARBOXYLASE pyruvate + CO2 + ATP oxaloacetate + ADP + Pi

PYRUVATE DEHYDROGENASE pyruvate + NAD+ + coenzyme A (CoA) acetyl CoA + CO2 + NADH

KEY CYTOPLASMIC REACTIONS INDIRECTLY NEEDED FOR LIPOGENESIS

Citrate Lyase citrate + CoA + ATP acetyl CoA + oxaloacetate + ADP + Pi

Malate dehydrogenase oxaloacetate + NADH malate + NAD+ Malic Enzyme malate + NADP+ pyruvate + NADPH

KEY CYTOPLASMIC REACTIONS DIRECTLY NEEDED FOR LIPOGENESIS AND FATTY ACID ACTIVATION

Acetyl CoA Carboxylase: acetyl CoA + HCO3- + ATP malonyl CoA + ADP + Pi
Fatty Acid Synthase: acetyl CoA + 7 malonyl CoA + 14 NADPH + 14 H+ palmitate + 7 CO2 + 8 CoA + 14 NADP+ Acyl CoA Synthetase: (also used for fatty acids other than palmitate) palmitate + ATP + CoA palmitoyl CoA + AMP + PPi

CE acp

A C P

condensation

CE acp

A C P

reduction dehydration reduction

CE acp

A C P

CO2 CO2 C=O C=O C=O CO2 CH3 CH2 C=O C=O CH3C=O - CO2 COO CH CH2 2 CH3 acetyl malonyl C=O C=O CoA CoA CH3 CH3 C=O CH2 C=O CH3

2 NADPH C=O
CH2

2 NADP+ C=O CH2 CH2 CH3

C=O
CH3 4-C unit

Figure 7. General mechanism for the fatty acid synthase reaction. CE is condensing enzyme. ACP is acyl carrier protein. This row represents the initial steps for priming the reaction with acetyl CoA and the addition of two carbons from malonyl CoA.

CE acp
4-C unit

A C P

condensation

CE acp

A C P

reduction dehydration reduction

CE acp 6-C unit

A C P

CO2
malonyl CoA

2 NADPH 6-C unit

2 NADP+

Figure 7. General mechanism for the fatty acid synthase reaction. CE is condensing enzyme. ACP is acyl carrier protein. This row depicts a typical cycle of adding two more carbons to the fatty acid chain.

CE acp 6-C unit

A C P

5 more cycles adding 10 more carbons 5malonyl CoA 5CO2 10NADPH 10NADP+

CE acp

A C P thioesterase cleavage

malonyl CoA

16-C unit palmitate CE acp A C P

palmitate

Figure 7. General mechanism for the fatty acid synthase reaction. CE is condensing enzyme. ACP is acyl carrier protein. This row shows the release of the finished product, palmitate, through cleavage by thioesterase.

Sources of NADPH for the Biosynthesis of Fatty Acids.

malic enzyme: Malate + NADP+ Pyruvate + CO2 + NADPH pentose phosphate pathway: Glucose-6-P + 2 NADP+ Ribulose-5-P + 2 NADPH + CO2

Glycerol ATP ADP glycerol kinase Glycerol-3-P fatty acyl CoA

Dihydroxyacetone phosphate fatty acyl CoA CoA

Acyldihydroxyacetone phosphate NADPH

NADP+

CoA Lysophosphatidic acid fatty acyl CoA

CoA Phosphatidic Diacylglycerol phosphatase acid fatty acyl CoA CoA

Pi

Triacylglycerol
Figure 8. Formation of phosphatidic acid from glycerol-3-P or DHAP, and its conversion to triacylglycerol

Ketone Bodies
Starvation causes accumulation of acetyl CoA
not enough carbohydrates to keep Krebs Cycle going acetyl CoA forms acetoacetate, bhydroxybutyrate, and acetone.

http://www.biocarta.com/pathfiles/ketoneb odiesPathway.asp

Diabetes and Ketone Bodies

When there is not enough insulin in the blood and it must break down fat for its energy. Ketones build up in the blood and then spill over into the urine so that the body can get rid of them. Acetone can be exhaled through the lungs. This gives the breath a fruity odor. Ketones that build up in the body for a long time lead to serious illness and coma. (Diabetic ketoacidosis)

Ketone Bodies

A special source of fuel and energy for certain tissues Some of the acetyl-CoA produced by fatty acid oxidation in liver mitochondria is converted to acetone, acetoacetate and -hydroxybutyrate These are called "ketone bodies" Source of fuel for brain, heart and muscle Major energy source for brain during starvation Synthesis in Figure 24.28 They are transportable forms of fatty acids!

Ketone Bodies - II
Interesting Aspects of Their Synthesis Occurs only in the mitochondrial matrix First step - Figure 24.28 - is reverse thiolase Second reaction makes HMG-CoA These reactions are mitochondrial analogues of the (cytosolic) first two steps of cholesterol synthesis Third step - HMG-CoA lyase - is similar to the reverse of citrate synthase

Ketone Bodies and Diabetes

"Starvation of cells in the midst of plenty" Glucose is abundant in blood, but uptake by cells in muscle, liver, and adipose cells is low Cells, metabolically starved, turn to gluconeogenesis and fat/protein catabolism In type I diabetics, OAA is low, due to excess gluconeogenesis, so Ac-CoA from fat/protein catabolism does not go to TCA, but rather to ketone body production Acetone can be detected on breath of type I diabetics

Lipogenesis aka Fatty Acid Synthesis

Function: Fuel for heart Sequence: Glucose Pyruvate Acetyl CoA Fatty Acids Major Location: Liver

Note:
Fatty Acid Synthesis (cytosol) Fatty Acid Oxidation (mitochondrial matrix) Opposite of each other but enzymes are completely different!

Lipogenesis the shuttle

Remember the shuttles from my last tutorial? Here we go again... Getting Acetyl CoA out of the mitochondria and into the cytosol: Mitochondria Pyruvate Acetyl CoA Citrate Diffuses Cytosol

Fatty Acids
Acetyl CoA Citrate

Lipogenesis
Step One Acetyl CoA (2C) Malonyl CoA (3C)

Regulatory step Enzyme: Acetyl CoA Carboxylase Cofactor: Biotin (CO2 carrier, gets CO2 from HCO3-)

Activators: citrate + insulin

Inhibitors: palmitoyl CoA (v effective ve feedback) + glucagon + adrenaline

Lipogenesis
Step Two Activation by Acyl Carrier Protein (ACP)

Acetyl CoA

Acetyl-ACP

ACP contains phosphopantetheine prosthetic group (the swinging arm)

Malonyl CoA + Acetyl-ACP loadsa reactions Step Three (palmitic acid C16) This is the Fatty Acid Synthase Complex

Lipogenesis
Step Three Formation of palmitic acid C16 Enzyme: FA Synthase (catalyses the elongation process of two successive carbon atoms in each cycle) The reaction repeats 7 times, until palmitic-ACP (C16) Sequence: Condensation, Reduction, Dehydration, Reduction Palmitic acid (C16) is released from ACP and FA Synthase complex by palmitoyl deacylase (a thioesterase) 8 Acetyl CoA + 7 ATP + 14 NADPH + 14 H+ + H2O

Palmitate + 7 ADP + 7 Pi + 8 CoASH + 14 NADP+

Lipogenesis
Note: An odd-numbered fatty acid can be produced if the starting molecule is propionyl-CoA (3C) and not acetyl CoA (2C) You may see the beginning part of the reaction written as Acetyl CoA + 7 Malonyl CoA this is the same as 8 Acetyl CoA! Further Metabolism Desaturation in ER Function of unsaturated fatty acids: cell membrane constituent

Further elongation (eg stearate (C18))

Lipogenesis
Regulation There is feedback inhibition of palmitoyl CoA to: 1) Acetyl CoA carboxylase 2) FA Synthase Molecule X

3) Pentose phosphate pathway (source for NADPH)

Acetyl CoA carboxylase is regulated by hormones Molecule Y

This kind of feedback is seen all over the joint

LIPID BIOSYNTHESIS
Fatty acid biosynthesis-basic fundamentals Fatty acid biosynthesis-elongation and desaturation Triacylglycerols Phospholipids Cholesterol Cholesterol metabolism

Fatty Acid Biosynthesis

Synthesis Cytosol Requires NADPH Acyl carrier protein D-isomer CO2 activation Keto saturated Beta Oxidation Mitochondria NADH, FADH2 CoA L-isomer No CO2 Saturated keto

Rule:
Fatty acid biosynthesis is a stepwise assembly of acetyl-CoA units (mostly as malonyl-CoA) ending with palmitate (C16 saturated)

3 Phases
Activation

Elongation
Termination

ACTIVATION
O

Cofactor
Biotin
NH C 2C 2C 2C 2C H H H H O S N H C 2C 2C 2 E ZY E HC 2 H H H N M HN

CH3C~SCoA O

ATP
ADP + Pi
-OOC-CH

HCO3-

Biocytin
2C~SCoA

LY S

O
O

O C N

O NH

CO2

active carbon
S

C 2C 2C 2C 2C H H H H O

Acetyl-CoA carboxylase

Carboxybiocytin

Acetyl-CoA Carboxylase
The rate-controlling enzyme of FA
In Bacteria -3 proteins (1) Carrier protein with Biotin (2) Biotin carboxylase (3) Transcarboxylase In Eukaryotes - 1 protein (1) Single protein, 2 identical polypeptide chains (2) Each chain Mwt = 230,000 (230 kDa) (3) Dimer inactive (4) Activated by citrate which forms filamentous form of protein that can be seen in the electron microscope

synthesis

Yeast Fatty Acid Synthase Complex 2,500 kDa Multienzyme Complex

6 molecules of 2 peptide chains called A and B

(66)

A: (185,000) Acyl Carrier protein -ketoacyl-ACP synthase (condensing enzyme) -ketoacyl-ACP reductase
B: (175,000) -hydroxy-ACP dehydrase enoyl-ACP reductase palmitoyl thioesterase Fatty Acid Synthase Complex

Acyl Carrier Protein

Phosphopantetheine O HS-CH2-CH2-N-C-CH2-CH2-N-C-C-C-CH2-O-P-O-CH2-SerO OH H O Cysteamine
H

H HO CH3

ACP

Acyl carrier protein 10 kDa

O O HS-CH2-CH2-N-C-CH2-CH2-N-C-C-C-CH2-O-P-O-P-O-CH2 O O OH H O O O O-P-O OH
H

H HO CH3

Adenine
H

Coenzyme A

OH

Initiation

Overall Reaction
Malonyl-CoA + ACP
-OOC-CH 2C~S-

CH3C~SCoA O

ACP

+ HS-CoA

CO2

HS-CoA
ACP

Acyl Carrier Protein

CH3C-CH2C~SO O

NOTE: Malonyl-CoA carbons become new COOH end Nascent chain remains tethered to ACP CO2, HS-CoA are released at each condensation

-Carbon
CH3C-CH2C~SACP

Elongation
O
O Reduction
-Ketoacyl-ACP reductase

NADPH
D isomer

H CH3C-CH2C~SHO O

ACP Dehydration -Hydroxyacyl-ACP dehydrase

-H2O NADPH

H CH3C- C- C~S- ACP = H O

Reduction

Enoyl-ACP reductase

CH3CH2CH2C~S- ACP O

TERMINATION
-KS

Ketoacyl ACP Synthase

Transfer to Malonyl-CoA

Transfer to KS -S-ACP

-CH2CH2CH2C~S- ACP O

Split out CO2

CO2

Free to bind Malonyl-CoA

When C16 stage is reached, instead of transferring to KS, the transfer is to H2O and the fatty acid is released

Fatty Acid Synthase

-Ketoacyl -ACP synthase
KS

O S-C-CH2-CH2-CH3
KS

Acetyl-CoA HS CoA-SH

Acetyl-CoAACP transacylase

O CH3-CH2-CH2-C-S Enoyl-ACP reductase NADPH H+ O CH3-CH=CH-C-S -Hydroxyacyl-ACP H2O

dehydrase

NADP+

O S -C-CH3
KS -SH
KS

Initiation or priming

ACP

O S-C-CH3

SH Malonyl-CoA
Malonyl-CoACoA-SH ACP transacylase

OH

CH3-CH -CH2-C-S NADP+ NADPH H+

S
C=O CH2 C=O CH3 CO2

O S -C-CH2-COO-Keto-ACP synthase (condensing enzyme)

KS -SH

-Ketoacyl -ACP reductase

Elongation

Substrate Entry AT MT

Reduction

Thioesterase palmitate release

CE
CH2 HS Translocation HS

DH KR ER ACP

TE
SH CH2 CE

SH Translocation

TE

ACP ER KR DH

MT

AT

Thioesterase palmitate release

Reduction

Substrate Entry

Overall Reactions
Acetyl-CoA + 7 malonyl-CoA + 14NADPH + 14H+ 7H + Palmitate + 7CO2 + 14NADP+ + 8 HSCoA + 6H2O

7 Acetyl-CoA + 7CO2 + 7ATP 7 malonyl-CoA +7ADP + 7Pi + 7H+

8 Acetyl-CoA + 14NADPH + 7H+ + 7ATP Palmitate + 14NADP+ + 8 HSCoA + 6H2O + 7ADP + 7Pi

PROBLEM: Fatty acid biosynthesis takes place in the cytosol. Acetyl-CoA is mainly in the Mitochondria
acetyl-CoA

How is acetyl-CoA made available to the cytosolic fatty acyl synthase?

SOLUTION: Acetyl-CoA is delivered to cytosol from the mitochondria as CITRATE

CH2COO HO-C-COO

HS-CoA

Acetyl-CoA

mitochondria
CH2COO HO-C-COO CH2COO Acetyl-CoA

CH2COO

Citrate lyase
COO C=O OAA Malate CH2 dehydrogenase COO
NADH

L-malate CO2

OAA
CO2 Pyr

COO HO-C-H L-malate CH2 COO Malic enzyme

NADP+

NADPH + H+ COO C=O Pyruvate CH3

Cytosol

Post-Synthesis Modifications
C16 satd fatty acid (Palmitate) is the product

Elongation Unsaturation Incorporation into triacylglycerols Incorporation into acylglycerol phosphates

Elongation of Chain (two systems)

R-CH2CH2CH2C~SCoA Malonyl-CoA* O (cytosol) HS-CoA OOC-CH2C~SCoA CH3C~SCoA O O CO2 Acetyl-CoA (mitochondria) R-CH2CH2CH2CCH2C~SCoA O O 1 NADPH Elongation systems are NADH found in smooth ER and 2 - H2O
3 NADPH

mitochondria

R-CH2CH2CH2CH2CH2C~SCoA O

Desaturation
Rules:
The fatty acid desaturation system is in the smooth membranes of the endoplasmic reticulum There are 4 fatty acyl desaturase enzymes in mammals designated D9 , D6, D5, and D4 fatty acyl-CoA desaturase Mammals cannot incorporate a double bond beyond D9; plants can. Mammals can synthesize long chain unsaturated fatty acids using desaturation and elongation

Rule:

The Desaturase System requires O2 and resembles an electron transport system Cyt b5 reductase
(FAD)

NADH
2

Cyt b5
3

O2

Saturated FA-CoA
1 2

NOTE: 1. System is in ER membrane

2. Both NADH and the fatty acid contribute electrons 3. Fatty acyl desaturase is considered a mixed function oxidase

Fatty acid desaturation system

C18-stearoly-CoA C18 D9-oleyl-CoA + O2 + 2H+ + 2H2O Desaturase

Desaturase
2 cyt b5 Fe2+ 2 cyt b5 Fe2+ Cyt b5 2H+ + cyt b5 reductase FAD cyt b5 reductase FADH2

Cyt b5 reductase

NADH + H+

NAD+

Desaturase
Palmitoleate 16:1(D9)

Palmitate 16:0

Elongase
Stearate 18:0

Desaturase Desaturase

Essential fatty acid

Oleate 18:1(D9)
Linoleate 18:2(D9,12)

Permitted transitions in mammals

Desaturase
-Linolenate 18:3(D9,12,15) Other lipids

Desaturase

-Linolenate 18:3(D6,9,12)

Elongase Desaturase
Eicosatrienoate 20:3(D8,11,14)
Arachidonate 20:4(D5,8,11,14)

ketogenesis
~In liver ~When there is high rate of fatty acid oxidation ~Used as respiratory substrate by extrahepatic tissue ~Normal level 1mg/dl. ~In starvation ketone bodies is utilised by brain and heart;but liver utilises amino acid. ~Heart always FA.
Mitochondrial enz.

[NAD+]/[NADH]

Can also arise from terminal 4 C OF FA.

INTERMEDIATE AND PRECURSUR FOR ACETOACETATE

PREDOMINANT KETONE BODY IN BLOOD AND URINE

~Acetocetate once formed cannot be reactivated except in cytosol (cholesterol syn.) ~Increased blood level ,increased oxidation. ~Saturation of oxidative machinary 12mmol/l ~Ketonemia is due to increased production rather than decreased utilisation. ~Acetone is volatile. ~Severity of ketosis,mesurment of ketonemia not ketonuria. LIVER ~Rotheras test.

Regulation of ketogenesis ~Adipose tissue lipolysis. ~CPT-1activity low in well fed state as there is increase in insulin/glucagon ratio. ~Liver oxidises FFA when increased within constraints of oxidative phosphorylation by ketone body production

Carnitine deficiency; hypoglycemia,lipid accumulation,muscular weakness.oral supplementation required. CPT-1 deficiency: reduced fatty skeletalacid oxidation, ketogenesis, hypoglycemia. CPT-II deficiency affects primarily skeletal muscle Inherited defects of enzymes of -oxidation & ketogenesis leads to non ketotic hypoglysemia, fatty liver.

Dicarboxylic aciduria deficiency of medium chain acylcoA DH.

FATTY ACID BIOSYNTHESIS occurs primarily in the cytoplasm of : liver adipose (fat) central nervous system lactating mammary gland Intermediates covalently linked to acyl carrier protein Activation of each acetyl CoA. acetyl CoA + CO2 Malonyl CoA Four-step repeating cycle, extension by 2-carbons /cycle Condensation Reduction Dehydration reduction The enzymes of fatty acid synthesis are packaged together in a complex called as fatty acid synthase (FAS). The product of FAS action is palmitic acid. (16:0). Modifications of this primary FA leads to other longer (and shorter) FA and unsaturated FA. The fatty acid molecule is synthesized 2 carbons at a time FA synthesis begins from the methyl end and proceeds toward the carboxylic acid end.

PRODUCTION OF MALONYL COA:REGULATORY,IRREVERSIBLE

Mn++

acetylCoA carboxylase

.BIOTIN .BIOTIN CARBOXYLASE .BC CARRIER PROTEIN .TRANS CARBOXYLASE .REGULATORY ALLOSTERIC SITE

For fatty acid biosynthesis, acetylCoA has to be transported from the mitochondria to the cytoplasm. This is done via a shuttle system called the Citrate Shuttle. Malonyl CoA is synthesized by the action of acetylCoA carboxylase. Biotin is a required cofactor. AcetylCoA carboxylase is under allosteric regulation. Citrate is a positive effector and palmitoyl CoA is a negative effector

homodimeric enzyme, seven catalytic activities, and eight sites two carriers ACP1 acts as a holding station for acetyl- or fatty acylgroups. ACP2, binds the growing fatty acyl chain during the condensation and reduction

Net reaction: Acetyl CoA + 7 malonyl CoA + 14 NADPH + 14 H+ Palmitate + 7 CO2 + 8 CoA + 14 NADP+ + 6H2O

Acetyl-CoA:ACP transacylase, transfers an acetyl group to cysteinyl-S on ACP1. malonyl-group transferred to the pantetheinylS of ACP2 by Malonyl-CoA:ACP transacylase. carbon dioxide leaves the malonyl group, with the electrons from its bond attacking the acyl group on ACP1 (Ketoacyl-ACP synthase) -ketoacyl group ready to go through the reverse of the reactions of -oxidation. Thus the keto-group is reduced to an alcohol using NADPH (-ketoacyl-ACP reductase), followed by the elimination of the alcohol (Enoyl-ACP hydrase) to give the cis-2,3-enoyl group. The enoyl is then reduced with NADPH substituting for FADH2 (Enoyl-ACP reductase) to give the saturated acyl group. Finally the acyl group is transferred from the pantotheinyl-S of ACP2 to the cysteinyl-S on ACP1 (ACP-acyltransferase) leaving ACP2 available to pick up the next malonyl moiety.

After seven turns of the cycle palmitate is released.