Oxidation and biosynthesis of fatty acids 1

Stages of fatty acid oxidation (1) Activation of fatty acids takes place on the outer mitochondrial membrane (2) Transport into the mitochondria (3) Degradation to two-carbon fragments (as acetyl CoA) in the mitochondrial matrix (b-oxidation pathway) 2

(1) Activation of Fatty Acids Fatty acids are converted to CoA thioesters by acyl-CoA synthetase (ATP dependent) The PPi released is hydrolyzed by a pyrophosphatase to 2 Pi Two phosphoanhydride bonds (two ATP equivalents) are consumed to activate one fatty acid to a thioester 3

(2) Transport of Fatty Acyl CoA into Mitochondria The carnitine shuttle system. Fatty acyl CoA is first converted to acylcarnitine (enzyme carnitine acyltransferase I (bound to the outer mitochondrial membrane). Acylcarnitine enters the mitochondria by a translocase. The acyl group is transferred back to CoA (enzyme - carnitine acyltransferase II). 4

Carnitine shuttle system Path of acyl group in red 5

(3) The Reactions of b oxidation The b-oxidation pathway (b-carbon atom (C3) is oxidized) degrades fatty acids two carbons at a time   6

1. Oxidation of acyl CoA by an acyl CoA dehydrogenase to give an enoyl CoA Coenzyme - FAD 7

2. Hydration of the double bond between C-2 and C-3 by enoyl CoA hydratase with the 3-hydroxyacyl CoA (b-hydroxyacyl CoA) formation 8

3. Oxidation of 3-hydroxyacyl CoA to 3-ketoacyl CoA by 3-hydroxyacyl CoA dehydrogenase Coenzyme – NAD+ 9

Enzyme - b-ketothiolase. 4. Cleavage of 3-ketoacyl CoA by the thiol group of a second molecule of CoA with the formation of acetyl CoA and an acyl CoA shortened by two carbon atoms. Enzyme - b-ketothiolase. 10

The shortened acyl CoA then undergoes another cycle of oxidation The number of cycles: n/2-1, where n – the number of carbon atoms 11

b-Oxidation of saturated fatty acids Fatty acyl CoA 12

One round of b oxidation: 4 enzyme steps produce acetyl CoA from fatty acyl CoA Each round generates one molecule each of: FADH2 NADH Acetyl CoA Fatty acyl CoA (2 carbons shorter each round) Fates of the products of b-oxidation: - NADH and FADH2 - are used in ETC - acetyl CoA - enters the citric acid cycle - acyl CoA – undergoes the next cycle of oxidation 13

ATP Generation from Fatty Acid Oxidation Net yield of ATP per one oxidized palmitate Palmitate (C15H31COOH) - 7 cycles – n/2-1 The balanced equation for oxidizing one palmitoyl CoA by seven cycles of b oxidation Palmitoyl CoA + 7 HS-CoA + 7 FAD+ + 7 NAD+ + 7 H2O 8 Acetyl CoA + 7FADH2 + 7 NADH + 7 H+ ATP generated 8 acetyl CoA 10x8=80 7 FADH2 7x1.5=10.5 7 NADH 7x2.5=17.5 108 ATP ATP expended to activate palmitate -2 Net yield: 106 ATP 14

LIPID METABOLISM: FATTY ACID OXIDATION 15

b-OXIDATION OF ODD-CHAIN FATTY ACIDS Odd-chain fatty acids occur in bacteria and microorganisms Final cleavage product is propionyl CoA rather than acetyl CoA Three enzymes convert propionyl CoA to succinyl CoA (citric acid cycle intermediate) 16

Propionyl CoA Is Converted into Succinyl CoA 1. Propionyl CoA is carboxylated to yield the D isomer of methylmalonyl CoA. The hydrolysis of an ATP is required. Enzyme: propionyl CoA carboxylase Coenzyme: biotin 17

2. The D isomer of methylmalonyl CoA is racemized to the L isomer Enzyme: methylmalonyl-CoA racemase 18

Enzyme: methylmalonyl CoA mutase Coenzyme: vitamin B12 (cobalamin) 3. L isomer of methylmalonyl CoA is converted into succinyl CoA by an intramolecular rearrangement Enzyme: methylmalonyl CoA mutase Coenzyme: vitamin B12 (cobalamin) 19

OXIDATION OF FATTY ACIDS IN PEROXISOMES Peroxisomes - organelles containing enzyme catalase, which catalyzes the dismutation of hydrogen peroxide into water and molecular oxygen Acyl CoA dehydrogenase transfers electrons to O2 to yield H2O2 instead of capturing the high-energy electrons by ETC, as occurs in mitochondrial b-oxidation. 20

METABOLISM OF LIPIDS: SYNTHESIS OF FATTY ACIDS 21

Fatty Acid Synthesis Occurs mainly in liver and adipocytes, in mammary glands during lactation Occurs in cytoplasm FA synthesis and degradation occur by two completely separate pathways When glucose is plentiful, large amounts of acetyl CoA are produced by glycolysis and can be used for fatty acid synthesis 22

Three stages of fatty acid synthesis: A. Transport of acetyl CoA into cytosol B. Carboxylation of acetyl CoA C. Assembly of fatty acid chain 23

A. Transport of Acetyl CoA to the Cytosol Acetyl CoA from catabolism of carbohydrates and amino acids is exported from mitochondria via the citrate transport system Cytosolic NADH also converted to NADPH Two molecules of ATP are expended for each round of this cyclic pathway 24

Citrate transport system 25

Sources of NADPH for Fatty Acid Synthesis 1. One molecule of NADPH is generated for each molecule of acetyl CoA that is transferred from mitochondria to the cytosol (malic enzyme). 2. NADPH molecules come from the pentose phosphate pathway. 26

B. Carboxylation of Acetyl CoA Enzyme: acetyl CoA carboxylase Prosthetic group - biotin A carboxybiotin intermediate is formed. ATP is hydrolyzed. The CO2 group in carboxybiotin is transferred to acetyl CoA to form malonyl CoA. Acetyl CoA carboxylase is the regulatory enzyme. 27

C. The Reactions of Fatty Acid Synthesis Five separate stages: (1) Loading of precursors via thioester derivatives (2) Condensation of the precursors (3) Reduction (4) Dehydration (5) Reduction 28

During the fatty acid synthesis all intermediates are linked to the protein called acyl carrier protein (ACP-SH), which is the component of fatty acyl synthase complex. The pantothenic acid is a component of ACP. Intermediates in the biosynthetic pathway are attached to the sulfhydryl terminus of phosphopantotheine group. 29

Acetyl transacylase and malonyl transacylase catalyze these reactions. The elongation phase of fatty acid synthesis starts with the formation of acetyl ACP and malonyl ACP. Acetyl transacylase and malonyl transacylase catalyze these reactions. Acetyl CoA + ACP  acetyl ACP + CoA Malonyl CoA + ACP  malonyl ACP + CoA 30

Condensation reaction. Acetyl ACP and malonyl ACP react to form acetoacetyl ACP. Enzyme - acyl-malonyl ACP condensing enzyme. 31

Acetoacetyl ACP is reduced to D-3-hydroxybutyryl ACP. Reduction. Acetoacetyl ACP is reduced to D-3-hydroxybutyryl ACP. NADPH is the reducing agent Enzyme: -ketoacyl ACP reductase 32

D-3-hydroxybutyryl ACP is dehydrated to form crotonyl ACP Dehydration. D-3-hydroxybutyryl ACP is dehydrated to form crotonyl ACP (trans-2-enoyl ACP). Enzyme: 3-hydroxyacyl ACP dehydratase 33

The final step in the cycle reduces crotonyl ACP to butyryl ACP. Reduction. The final step in the cycle reduces crotonyl ACP to butyryl ACP. NADPH is reductant. Enzyme - enoyl ACP reductase. This is the end of first elongation cycle (first round). 34

In the second round butyryl ACP condenses with malonyl ACP to form a C6--ketoacyl ACP. Reduction, dehydration, and a second reduction convert the C6--ketoacyl ACP into a C6-acyl ACP, which is ready for a third round of elongation. 35

Final reaction of FA synthesis Rounds of synthesis continue until a C16 palmitoyl group is formed Palmitoyl-ACP is hydrolyzed by a thioesterase Overall reaction of palmitate synthesis from acetyl CoA and malonyl CoA Acetyl CoA + 7 Malonyl CoA + 14 NADPH + 14 H+ Palmitate + 7 CO2 + 14 NADP+ + 8 HS-CoA + 6 H2O 36

Organization of Multifunctional Enzyme Complex in Eukaryotes The synthase is dimer with antiparallel subunits. Each subunit has three domains. ACP is located in domain 2. Domain 1 contains transacylases, ketoacyl-ACP synthase (condensing enzyme) Domain 2 contains acyl carrier protein, -ketoacyl reductase, dehydratase, and enoyl reductase. Domain 3 contains thioesterase activity. 37

38

Fatty Acid Elongation and Desaturation The common product of fatty acid synthesis is palmitate (16:0). Cells contain longer fatty acids and unsaturated fatty acids they are synthesized in the endoplasmic reticulum. The reactions of elongation are similar to the ones seen with fatty acid synthase (new carbons are added in the form of malonyl CoA). For the formation of unsaturated fatty acids there are various desaturases catalizing the formation of double bonds. 39

THE CONTROL OF FATTY ACID METABOLISM Acetyl CoA carboxylase plays an essential role in regulating fatty acid synthesis and degradation. The carboxylase is controlled by hormones: glucagon, epinephrine, and insulin. Another regulatory factors: citrate, palmitoyl CoA, and AMP 40

Global Regulation is carried out by means of reversible phosphorylation Acetyl CoA carboxylase is switched off by phosphorylation and activated by dephosphorylation Insulin stimulates fatty acid synthesis causing dephosphorylation of carboxylase. Glucagon and epinephrine have the reverse effect (keep the carboxylase in the inactive phosphorylated state). Protein kinase is activated by AMP and inhibited by ATP. Carboxylase is inactivated when the energy charge is low. 41

Local Regulation Acetyl CoA carboxylase is allosterically stimulated by citrate. The level of citrate is high when both acetyl CoA and ATP are abundant (isocitrate dehydrogenase is inhibited by ATP). Palmitoyl CoA inhibits carboxylase. 42

Response to Diet Fed state: Starvation: Insulin level is increased Inhibits hydrolysis of stored TGs Stimulates formation of malonyl CoA, which inhibits carnitine acyltransferase I FA remain in cytosol (FA oxidation enzymes are in the mitochondria) Starvation: Epinephrine and glucagon are produced and stimulate adipose cell lipase and the level of free fatty acids rises Inactivate carboxylase, so decrease formation of malonyl CoA (lead to increased transport of FA into mitochondria and activate the b-oxidation pathway) 43