Biomol5

Fatty Acid Degradation and Biosynthesis

Department of Biochemistry Jeerus Sucharitakul

Fatty acid breakdown Oxidation of hydrocarbon chain in fatty acid •Free energy is stored in forms of FADH and NADH •The final product from oxidation process is acetyl CoA. •Oxidation occurs in matrix of mitochondria Processes in fatty acid oxidation I Activation of fatty acid (cytosol) II Transport of activated fatty acid from cytosol into intermembrane space and matrix III Oxidation of hydrocarbon chains (matrix of mitochondria)

Activation of fatty acids •Fatty acid is activated by forming a thioester link with CoA •The reaction is catalyzed by acyl CoA synthase or fatty acid thiokinase which is presented on the outer membrane of mitochondria. •Free energy for driving the reaction comes from hydrolysis of pyophosphate (PPi) of ATP. O R

O

C O- + ATP + CoA

R

C

S CoA + AMP + Pi2

∆Go = -15 kJ/mol Pi2 + H2O

Pi + Pi

∆Go = -19 kJ/mol

Activation of fatty acids

Transport of fatty acid into mitochondria •Small and medium chains (≤10 C) acyl CoA; free diffusion •Longer chains; enzymatic reaction, carnitine shuttle CH3 H3C

N+ CH2

H C

CH3

OH

H2 C COO-

Carnitine

•Facilitated diffusion by integral membrane protein (carnitine transporter) •Carnitine carries acyl CoA by covalent linkage to acyl group.

Transport of fatty acid into mitochondria

Carnitine transferase I; outer membrane of mitochondria Carnitine transferase II; inner membrane of mitochondria Carnitine-mediated entry process is the rate-limiting step.

Oxidation of fatty acids •Cleavage bond of hydrocarbon chain at β-C (β-oxidation) by two carbon producing of acetyl CoA. •Hydrocarbon is an inert compound therefore, it is activated by reduction forming of double bonds.

β-oxidation Acetyl CoA

FADH2

NADH

Oxidative phosphorylation

Oxidation of fatty acids

I Oxidation

II Hydration

III Oxidation

IV Thiolysis

Oxidation of fatty acids β -oxidation of saturated fatty acids

β -oxidation of unsaturated fatty acids -Most lipid in animals and plants Are unsaturated fatty acids -Double bonds are in cis conformation.

β -oxidation of unsaturated fatty acids C H

C H

H2 C

H2 C

Monosaturated fatty acids

O

H2 C

C

SCoA

β-oxidation

C H

O

H2 C

C H

C

SCoA

cis-∆3-enoyl CoA Isomerase H2 C

O

H C

•Double bond at C-3 prevents reduction the bond between C-2 and C-3

C

C

SCoA

H

trans-∆2-enoyl CoA

•Enzyme enoyl-CoA hydratase is only specific for substratetrans conformation. •Enzyme isomerase translocate double bond from cis to trans conformation without reduction.

β -oxidation of monounsaturated fatty acids

β -oxidation of polyunsaturated fatty acids

β -oxidation of polyunsaturated fatty acids

β -oxidation of fatty acids Even-number fatty acids

Odd-number fatty acids

β-oxidation C3 Acetyl CoA

Acetyl CoA + Propionyl CoA Carboxylation TCA cycle

Succinyl CoA

Conversion of propionyl-CoA into succinyl-CoA

B12 is a cofactor of methylmalonyl-CoA mutase.

Vitamin B12 deficiency •Serious disease; pernicious anemia •Vit B12 can not be synthesized by animals or plants but only few microorganism can do it. •Vit B12 is synthesized by intestinal bacteria •Absorption of Vit B12 in intestinal lumen requires a glycoprotein, intrinsic factor. Pernicious anemia -Genetic deficiency disease, for intrinsic factor -Low production of hemogolbin -Low production of erythrocytes -Severe impairment of central nervous system

Animals can not use acetyl CoA for gluconeogenesis. •Animals can not convert acetyl CoA into pyruvate or oxaloacetate. •No net conversion of carbons from acetyl CoA produced from fatty acid oxidation into oxaloacetate. •Plants have the pathway for conversion of acetyl CoA into oxaloacetate for glucose synthesis and for other precursors, glyoxylate cycle.

Ketone bodies •Excess production of Acetyl-CoA from β-oxidation can be converted into ketone bodies in the liver. •Ketone bodies can be used as a fuel by other tissues (extrahepatic tissues) such as brain, heart, renal cortex and muscle under starvation.

Formation of ketone bodies from acetyl Co-A

Degradation of β -hydroxybutyrate as a fuel by extrahepatic tissues Liver acts as a producer of β-hydroxybutyrate but can not be a consumer because it lacks of enzyme thiolase.

Conditions for ketone bodies production I Starvation; deplete of glucose, glycogen and intermediates in TCA cycle for gluconeogenesis -At this condition all tissues divert to use fat as a energy source. -Liver produces ketone bodies for extrahepatic tissues II Untreated diabetes; lack of insulin -Extrahepatic tissues can not take up glucose efficiently from Blood. -lack of intermediate in TCA cycle due to high rate of gluconeogensis. -Accumulation of acetyl-CoA -Characteristic odor to the breath from volatile acetone

Acidosis -Lowered blood pH from β-hydroxybutyrate -Coma and death -High level of ketone bodies in blood and urine, ketosis -Collectively called ketoacidosis -People during very low-calorie diets, body uses fat from Adipose tissue as a major source of energy are risky to ketosis.

Lipid biosynthesis Lipids have many various functional roles in cells. •Composition of cell membranes •Pigments; retinal, carotene •Cofactor; vitamin K •Bile salts •Hormones •Intracellular mediators

Lipid biosynthesis •Fatty acid synthesis occurs in different pathways and different compartment from fatty acid breakdown. •In most higher eukarytoes, fatty acid biosynthesis occurs in cytosol. •The first intermediate for lipid biosynthesis is three-carbon compound, malonyl-CoA. •Formation of malonyl-CoA is a rate-limiting step in this process. •Malonyl-CoA is synthesized by acetyl-CoA carboxylase.

O -

O

C

H2 C

O C

Malonyl-CoA

S

CoA

Lipid biosynthesis

Acetyl-CoA carboxylase •Multifunctional enzyme •Coenzyme; biotin (CO2 carrier)

Lipid biosynthesis Enzyme catalyzing fatty acid synthesis is fatty acid synthase. Substrates for fatty acid synthase -Malonyl-CoA -NADPH -Acetyl-CoA -ATP Palmitate (saturated C16) is the principle product of the fatty acid synthase system in animal cells.

Fatty acid synthase •Multifunctional enzyme •The product from chain elongation is held by ACP domain (acyl carrier protein) •Enzyme is located on cytosolic surface of SER.

Fatty acid synthase

Channeling of substrates for fatty acid synthesis Pentose phosphate pathways (cytosol)

Carbohydrates, amino acids, β-oxidation (mitochondria) Acetyl-CoA

NADPH

Fatty acid synthesis

Reoxidation of malate to pyruvate by malate enzyme Malonyl CoA

Source of NADPH in cytosol Cytosolic NADPH is generated by (hepatocyte and adipocyte) I Pentose phosphate pathway; animals II Malic enzyme; plants 2NADPH + H+

2NADP+ Glucose 6-phosphate

COO-

NADP+

Ribulose 5-phosphate

NADPH + H+

HCOH CH2 COO-

Malate

COOC

Malic enzyme

O

+ CO2

COO-

Pyruvate

Acetyl CoA shuttle •Acetyl CoA used in fatty acid synthesis is formed in matrix of mitochondria. •Inner membrane of mitochondria is impermeable to acetyl CoA. •Acetyl CoA is passed into cytosol in form of citrate. •Citrate is hydrolyzed by enzyme citrate lyase releasing acetyl-CoA in cytosol Citrate synthase Oxaloacetate + Acetyl-CoA

Citrate lyase Citrate + CoA-SH + ATP

Citrate + CoA-SH

Oxaloacetate + Acetyl-CoA + ADP + Pi

Acetyl CoA shuttle

Regulation of fatty acid synthesis Citrate Citrate lylase

Insulin

Acetyl-CoA Acetyl-CoA carboxylase

Glucagon Epinephrine

Malonyl-CoA

Palmitoyl-CoA

Long chain fatty acid synthesis Palmitate (C16) is the principal product of the fatty acid synthase system in animal cells. •Long chain fatty acids are synthesized by other system, fatty acid elongation system which is in SER and mitochondria. •The mechanism for elongation is similar to that of fatty acid synthase.

Synthesis of unsaturated fatty acid (desaturation) •Palmitate (C16) and stearate (C18) are precursors for unsaturated fatty acids. •Most common monounsaturated fatty acids of animal tissues are palmitoleate, 16:1∆9 and oleate, 18:1 ∆9. •The double bond is introduced by oxidation, which is catalyzed by enzyme fatty acyl-CoA desaturase in ER. •NADP+ is an final electron acceptor.

Synthesis of unsaturated fatty acid (desaturation) •Only plants can synthesize polyunsaturated fatty acids, linoleate and linolenate. •Both of those fatty acids are essential fatty acids for mammals as precursors for other polyunsaturated fatty acids. •Arachidonic acid (ecosanoid) is an essential of regulatory lipids.

Ecosanoids (C20) are biological signaling in cells. Arachidonic acid Cycloxygenase

I Prostaglandins; -pain, inflammation, fever, wake-sleep cycle II Thromboxanes; -vasoconstriction -blood clotting -platelet aggregration

Lipoxygenase

Leukotrienes; -Contraction of smooth muscle in lung -Allergic reaction or hypersensitivity

Ecosanoids (C20) are biological signaling in cells. Cyclic pathway of cycloxygenase

Ecosanoids (C20) are biological signaling in cells. Linear pathway of lipooxygenase

Biosynthesis of triacylglycerols •Synthesis of storage lipids •Synthesis of membrane phospholipids

•Glycerol 3-phosphate is a precursors for biosynthesis of triacylglycerols. •Reactions is catalyzed by two pathways using enzyme glycerol 3-phosphate dehydrogenase for reduction and glycerol kinase.

Biosynthesis of triacylglycerols

Fatty acyl-CoA

Phosphatidic acid is an intermediate for triacylglycerol, and for glycerophopholipids (components of cell membranes).

Biosynthesis of triacylglycerols

Phosphatidic acid is an intermediate for triacylglycerol, and for glycerophopholipids (components of cell membranes).

Hormonal regulation triacylglycerol synthesis

Triacylglycerol cycle

Biosynthesis of membrane phospholipids Constructions of membrane phospholipids I Synthesis of backbone molecule (glycerol or sphingosine) II Attachment of fatty acids through ester or amide III Addition of head group through phosphodiester or ester IV Alteration of head group to the desired product.

CDP (cytidine diphosphate) is used for action either diacylglycerol or head group.

Regulations of fatty acid degradation and biosynthesis

Biosynthesis of cholesterol and steroids •27-carbon compound •Component of cellular membranes •Precursor for steroid hormones •Precursor for bile acid •One of risk factors for cardiovascular diseases -Mammalian cells can synthesize cholesterol from simple molecules, acetyl-CoA. -The isoprene unit is an intermediate for cholesterol synthesis. CH3 H2C

C

CH

CH2

Isoprene unit

Biosynthesis of cholesterol and steroids

Committed step for cholesterol synthesis

Mevalonate is oxidized into C5 isoprene unit by decarboxylation.

HMG CoA reductase •Committed step and regulation step for cholesterol Synthesis •Feedback inhibition by high level cholesterol •Target for therapy of hypercholesterolemia, competitive inhibition to decrease rate for cholesterol synthesis -Lovastatin; fungal products •Much of cholesterol synthesis in vertebrates takes place in the liver.

Steroid hormones

Bile salts •Bile acids are polar derivatives of cholesterol with amino acid. •Synthesis from liver in form of glycolate and taurocholate. •Bile salts are stored in and concentrated in the gall bladder, and released into intestine for lipid digestion. OH

HO

O N H

OH

-

O O

OH

Glycolate

Amide linkage with glycine

HO

O N H

SO3-

OH

Taurocholate

Amide linkage with taurine

Vitamin D •Vitamin D (cholecalciferol) •Synthesis by skin from 7-dehydrocholesterol in a photochemical reaction •Regulation of gene expression 7-dehydrocholesterol UV light

Cholecalciferol (Vitamin D3) Liver and Kidney

Dihydroxycholecalciferol (reactive compound)