Molecular Biochemistry II

Fatty Acid Oxidation

b 3

O C
1

fatty acid with a cis-9 double bond

A 16-C fatty acid with numbering conventions is shown. Most naturally occurring fatty acids have an even number of carbon atoms. The pathway for catabolism of fatty acids is referred to as the b-oxidation pathway, because oxidation occurs at the b-carbon (C-3).

O H2 C HC H2 C OH OH HO OH O C R H2 C O H2 C HC O O C O C O C R R R

glycerol

fatty acid

triacylglycerol

Triacylglycerols (triglycerides) are the most abundant dietary lipids. They are the form in which we store reduced C for energy. Each triacylglycerol has a glycerol backbone to which are esterified 3 fatty acids Most triacylglycerols are mixed. The 3 fatty acids differ in chain length & number of double bonds.

O H2 C HC H2 C OH OH HO OH O C R H2 C O H2 C HC O O C O C O C R R R

glycerol

fatty acid

triacylglycerol

Lipid digestion, absorption, transport will be covered separately.

Lipases hydrolyze triacylglycerols, releasing 1 fatty acid at a time, yielding diacylglycerols, & eventually glycerol.

CH2 OH HO CH CH2 OH

ATP 1

ADP
HO

CH2 OH CH CH2 O

NAD
PO3

H + CH OH 2 NADH
C O PO3

CH2 O

glycerol

glycerol-3-P

dihydroxyacetone-P

Glycerol, arising from hydrolysis of triacylglycerols, is converted to the Glycolysis intermediate dihydroxyacetone phosphate, by reactions catalyzed by: 1 Glycerol Kinase 2 Glycerol Phosphate Dehydrogenase.

b 3

O C
1

fatty acid with a cis-9 double bond

Free fatty acids, which in solution have detergent properties, are transported in the blood bound to albumin, a serum protein produced by the liver.

Several proteins have been identified that facilitate transport of long chain fatty acids into cells, including the plasma membrane protein CD36.

Fatty acid activation:

Fatty acids must be esterified to Coenzyme A before they can undergo oxidative degradation, be utilized for synthesis of complex lipids, or be attached to proteins as lipid anchors. Acyl-CoA Synthases (Thiokinases) of ER & outer mitochondrial membranes catalyze activation of long chain fatty acids, esterifying them to coenzyme A. This process is ATP-dependent, & occurs in 2 steps. There are different Acyl-CoA Synthases for fatty acids of different chain lengths.

Acyl-CoA Synthases Exergonic PPi (P~P) hydrolysis, catalyzed by Pyrophosphatase, makes the coupled reaction spontaneous. 2 ~P bonds of ATP are cleaved. The acyl-CoA product includes one "~" thioester linkage.

Fatty acid activation

O N

NH2 N N

fatty acid
O

C O

O N O CH2 H H OH O H H OH N

O O

P O

P O

P O

ATP
NH2 N N

2 Pi O R C

PPi O O P O O CH2 H H OH O H

acyladenylate

CoA

SH AMP O R C S

H OH

CoA

acyl-CoA

Summary of fatty aid activation: fatty acid + ATP acyladenylate + PPi PPi 2 Pi acyladenylate + HS-CoA acyl-CoA + AMP

Overall: fatty acid + ATP + HS-CoA acyl-CoA + AMP + 2 Pi

Mitochondrion
b-Oxidation pathway in matrix

b-Oxidation pathway:

Fatty acids are degraded in the mitochondrial matrix via the b-Oxidation Pathway.

Fatty acyl-CoA formed in cytosol by enzymes of outer mitochondrial membrane & ER

For most steps of the pathway there are multiple enzymes specific for particular fatty acid chain lengths.

Many of the constituent Mitochondrion enzymes are soluble proteins located in the b-Oxidation mitochondrial matrix. pathway in
But enzymes specific for very long chain fatty acids are associated with the inner membrane, facing the matrix.
matrix

Fatty acyl-CoA formed in cytosol by enzymes of outer mitochondrial membrane & ER

Fatty acyl-CoA formed outside can pass through the outer mitochondrial membrane (which has large VDAC channels), but cannot penetrate the inner membrane.

CH3 H3C + N CH3 CH2

OH CH CH2 COO +

R C O

carnitine

SCoA

Carnitine Palmitoyl Transferase

Transfer of the fatty acid moiety across the mitochondrial inner membrane involves carnitine.

R C CH3 H3C + N CH3 CH2 O CH CH2 COO + HSCoA O

fatty acyl carnitine

Carnitine Palmitoyl Transferases catalyzes transfer of a fatty acid between the thiol of Coenzyme A and the hydroxyl on carnitine.

cytosol O R-C-SCoA HO-carnitine 1 HSCoA R-C-O-carnitine O 2

mitochondrial matrix O HO-carnitine R-C-SCoA 3 R-C-O-carnitine HSCoA O

Carnitine-mediated transfer of the fatty acyl moiety into the mitochondrial matrix is a 3-step process: 1. Carnitine Palmitoyl Transferase I, an enzyme on the cytosolic surface of the outer mitochondrial membrane, transfers a fatty acid from CoA to the OH on carnitine. 2. An antiporter in the inner mitochondrial membrane mediates exchange of carnitine for acylcarnitine.

cytosol O R-C-SCoA HO-carnitine 1 HSCoA R-C-O-carnitine O 2

mitochondrial matrix O HO-carnitine R-C-SCoA 3 R-C-O-carnitine HSCoA O

3. Carnitine Palmitoyl Transferase II, an enzyme within the matrix, transfers the fatty acid from carnitine to CoA. (Carnitine exits the matrix in step 2.) The fatty acid is now esterified to CoA in the matrix.

O H3C C SCoA

acetyl-CoA
O

OOC

CH2

SCoA

malonyl-CoA

Control of fatty acid oxidation is exerted mainly at the step of fatty acid entry into mitochondria. Malonyl-CoA (which is also a precursor for fatty acid synthesis) inhibits Carnitine Palmitoyl Transferase I. Malonyl-CoA is produced from acetyl-CoA by the enzyme Acetyl-CoA Carboxylase.

AMP-Activated Kinase, H3C C a sensor of cellular energy acetyl-CoA levels, is allosterically ATP + HCO 3 activated by AMP, which is high in concentration ADP + Pi when [ATP] is low. Acetyl-CoA Carboxylase is inhibited when phosphorylated by AMP

SCoA

O OOC CH2 C

Acetyl-CoA Carboxylase (inhibited by AMP-Activated Kinase)

SCoA

malonyl-CoA

Activated Kinase, leading to decreased malonyl-CoA. The decrease in malonyl-CoA concentration leads to increased activity of Carnitine Palmitoyl Transferase I. Increased fatty acid oxidation then generates acetylCoA, for entry into Krebs cycle with associated ATP production.

AMP-Activated Kinase functions under a variety of conditions that lead to depletion of cellular ATP (reflected as increased AMP), including:
glucose deprivation, exercise, hypoxia & ischaemia.

AMP-Activated Kinase regulates various metabolic pathways to:

promote catabolism leading to ATP synthesis (e.g., stimulation of fatty acid oxidation) inhibit energy-utilizing anabolic pathways (e.g., fatty acid synthesis).

AMP-Activated Kinase in the hypothalamus of the brain is involved also in regulation of food intake.

H2O 2 & 3. bond between carbon atoms There are different Acyl-CoA H Dehydrogenases for O short (4-6 C), medium (6-10 C), long and very H3C (CH2 )n C CH2 C SCoA long (12-18 C) chain fatty acids. Very Long Chain Acyl-CoA Dehydrogenase is bound OH to the inner mitochondrial membrane. The others + are soluble enzymes Hlocated + NADH in the mitochondrial matrix. O O NAD+

b-Oxidation Pathway: Step 1. Acyl-CoA Dehydrogenase catalyzes oxidation of the fatty acid moiety of acyl-CoA to produce a double

H H3C (CH2)n C H FAD FADH2 H3C (CH2)n C H

3 b

H C H
2

O C
1

SCoA

fatty acyl-CoA

Acyl-CoA Dehydrogenase
H C O C SCoA

trans-2-enoyl-CoA

H H3C (CH2)n C H FAD FADH2 H3C (CH2)n C H

3 b

H
2 C

O C
1

SCoA

glutamate
H H3N+ C CH2 CH2 COO

fatty acyl-CoA

Acyl-CoA Dehydrogenase
H C O C SCoA
2

trans- -enoyl-CoA

C O

H2O FAD is the prosthetic group that functions as e acceptor for Dehydrogenase. Proposed H Acyl-CoA O mechanism: H C SCoA (CH 2)n C CH2carboxyl A3C Glu side-chain extracts a proton from the -carbon of the substrate, facilitating transfer of 2 e OH with H+ (a hydride) from the b position to FAD. The reduced FAD accepts a 2nd H+, yielding FADH2. H+ + NADH NAD+ O O

H H3C (CH2)n C H FAD FADH2 H3C (CH2)n C H H2O

3 b

H C H
2

O C
1

SCoA

fatty acyl-CoA

Acyl-CoA Dehydrogenase
H C O C SCoA

trans-2-enoyl-CoA

The carbonyl O of substrate is H the thioester O hydrogen bonded to the 2'-OH of the ribityl H3C C CH2this C SCoA (CH2)n moiety of FAD, giving part of FAD a role in positioning the substrate and increasing acidity OH of the substrate -proton.
H+ + NADH

dimethylisoalloxazine
H C H3C H3C C C C H C C N CH2 HC OH OH N C C

O C NH C N O

2e +2H

H C H3C H3C C C C H C C

H N C C N CH2 HC OH OH

C NH C N H O

FAD

HC HC H2C

OH O O P OO

O P OO

Adenine Ribose

FADH2

HC HC H2C

OH O O P OO

O P OO

Adenine Ribose

The carbonyl O of the thioester substrate is hydrogen bonded to the 2'-OH of the ribitol moiety of FAD, giving the sugar alcohol a role in positioning the substrate and increasing acidity of the substrate -proton.

H H3C (CH2)n C H FAD FADH2 H3C (CH2)n C H H2O

3 b

H C H
2

O C
1

SCoA

fatty acyl-CoA

Acyl-CoA Dehydrogenase
H C O C SCoA

trans-2-enoyl-CoA

O are on opposite The reactive Glu H and FAD sides of the substrate at the active site. H3C (CH2)n C CH2 C SCoA Thus the reaction is stereospecific, yielding a OH trans double bond in enoyl-CoA. H+ + NADH

Matrix
H+ + NADH NAD+ + 2H+ 2H+ + O2 H2O

2 e Q

III IV

++
4H
+

4H

cyt c

2H+

Intermembrane Space

FADH2 is reoxidized by transfer of 2 electrons to an electron transfer flavoprotein (ETF), which in turn passes the electrons to coenzyme Q of the respiratory chain.

Step 2. Enoyl-CoA Hydratase catalyzes stereospecific hydration of the trans double bond produced in the 1st step, yielding LhydroxyacylCoenzyme A.

H H3C (CH2)n C H FAD FADH2 H3C (CH2)n C H H2O H

3 b

H C H
2

O C
1

SCoA

fatty acyl-CoA

Acyl-CoA Dehydrogenase
H C O C SCoA

trans-2-enoyl-CoA

Enoyl-CoA Hydratase
O SCoA

H3C (CH2)n C CH2 C OH H+ + NADH

3-L-hydroxyacyl-CoA

H H2O H O SCoA

H3C (CH2)n C CH2 C

Step 3. Hydroxyacyl-CoA Dehydrogenase catalyzes oxidation of the hydroxyl in the b position (C3) to a ketone. NAD+ is the electron acceptor.

NAD+ H+ + NADH

OH

3-L-hydroxyacyl-CoA

Hydroxyacyl-CoA Dehydrogenase
O O SCoA

H3C (CH2)n C CH2 C HSCoA O H3C (CH2)n C

b -Ketothiolase

b-ketoacyl-CoA
O

SCoA + CH3 C

SCoA

fatty acyl-CoA (2 C shorter)

acetyl-CoA

H H3N+ C CH2 SH COO

O SCoA

H3C (CH2)n C CH2 C HSCoA O H3C (CH2)n C

b-ketoacyl-CoA
O

cysteine

SCoA + CH3 C

SCoA

Step 4. b-Ketothiolase catalyzes thiolytic cleavage.

fatty acyl-CoA (2 C shorter)

acetyl-CoA

b-Ketothiolase

A cysteine S attacks the b-keto C. Acetyl-CoA is released, leaving the fatty acyl moiety in thioester linkage to the cysteine thiol. The thiol of HSCoA displaces the cysteine thiol, yielding fatty acyl-CoA (2 C less).

A membrane-bound trifunctional protein complex with two subunit types expresses the enzyme activities for steps 2-4 of the boxidation pathway for long chain fatty acids. Equivalent enzymes for shorter chain fatty acids are soluble proteins of the mitochondrial matrix.

Summary of one round of the b-oxidation pathway:

fatty acyl-CoA + FAD + NAD+ + HS-CoA fatty acyl-CoA (2 C less) + FADH2 + NADH + H+ + acetyl-CoA The b-oxidation pathway is cyclic. The product, 2 carbons shorter, is the input to another round of the pathway. If, as is usually the case, the fatty acid contains an even number of C atoms, in the final reaction cycle butyryl-CoA is converted to 2 copies of acetyl-CoA.

Matrix
H+ + NADH NAD+ + 2H+

ADP + Pi ATP 2H+ + O2 H2O

F1 Fo

2 e Q

III IV

++
4H
+

4H

cyt c

2H

3H+

Intermembrane Space

NADH produced during fatty acid oxidation is reoxidized by transfer of 2e to respiratory chain complex I. Transfer of 2e from complex I to oxygen causes sufficient proton ejection to yield approximately 2.5 ATP. Recall that 4H+ enter the matrix per ATP synthesized, taking into account transmembrane flux of ADP, ATP & Pi.

Matrix
H+ + NADH NAD+ + 2H+

ADP + Pi ATP 2H+ + O2 H2O

F1 Fo

2 e Q

III IV

++
4H+ 4H+

cyt c

2H+

3H+

Intermembrane Space

FADH2 of Acyl-CoA Dehydrogenase is reoxidized by transfer of 2e via ETF to CoQ of the respiratory chain. H+ ejection from the matrix that accompanies transfer of 2e from coenzyme Q to oxygen, leads to production of approximately 1.5 ATP.

 Acetyl-CoA can enter Krebs cycle, yielding additional NADH, FADH2, and ATP.

Fatty acid oxidation is a major source of cell ATP.

Matrix
H+ + NADH NAD+ + 2H+

ADP + Pi ATP 2H+ + O2 H2O

F1 Fo

Problem (See web handout, tutorial)

2 e Q

III IV

++
4H+ 4H+

cyt c

2H+

3H+

Intermembrane Space

Catabolism of two 6-C glucose through Glycolysis, Krebs, & ox phos yields about 60 ~P bonds of ATP (30/glucose). Compare energy yield oxidizing a 12-C fatty acid. Assume:
1.5 ATP produced per FADH2 reoxidized in the respiratory chain (via coenzyme Q). 2.5 ATP produced per NADH reoxidized in the respiratory chain.

How many "high energy" (~) bonds are utilized in activating the fatty acid, by esterifying it to coenzyme 2 A? ()________

How many times is the b-oxidation pathway repeated during oxidation of a 12-C fatty acid? _________ How many each of NADH______, FADH2______, and Acetyl CoA______ are 6 5 b-oxidation pathway? produced, per 12-carbon 5 fatty acid, in the Oxidation of each acetyl CoA in Krebs cycle yields 3 NADH and one FADH2 (from succinate), resulting in additional production of _______NADH and _______FADH2. Thus 6 the yield is a total of _______NADH and _______FADH2.

18

In the respiratory chain, approx. 2.5 ~ bonds of ATP are produced per 23 11 NADH and 1.5 ~ bonds of ATP per FADH2 (electrons entering the respiratory chain via coenzyme Q). Thus from reoxidation of NADH and FADH2 a total of _______ ~ bonds of ATP are produced per 12-C fatty acid. 74 Add to this the ~P bonds of GTP produced in Krebs Cycle (one GTP per acetyl-CoA) for a total of _______ ~P bonds produced.

Summing input and80 output yields a total of _______ ~P bonds per 12-C fatty acid oxidized. Does fat yield more energy than carbohydrate? 78 _______

YES

Human genetic diseases have been identified that involve mutations in:
the plasma membrane fatty acid transporter CD36 Carnitine Palmitoyltransferases I & II (required for transfer of fatty acids into mitochondria)

Acyl-CoA Dehydrogenases for various chain lengths of fatty acids

Hydroxyacyl-CoA Dehydrogenases for medium & short chain length fatty acids

Medium Chain b-Ketothiolase

the trifunctional protein complex Electron Transfer Flavoprotein (ETF).

Human genetic diseases: Symptoms vary depending on the specific genetic defect but may include:
hypoglycemia and failure to increase ketone body production during fasting fatty degeneration of the liver heart and/or skeletal muscle defects maternal complications of pregnancy sudden infant death (SIDS).

Hereditary deficiency of Medium Chain AcylCoA Dehydrogenase (MCAD), the most common genetic disease relating to fatty acid catabolism, has been linked to SIDS.

The reactions presented accomplish catabolism of a fatty acid with an even number of C atoms & no double bonds. Additional enzymes deal with catabolism of fatty acids with an odd number of C atoms or with double bonds.
The final round of b-oxidation of a fatty acid with an odd number of C atoms yields acetyl-CoA & propionyl-CoA. Propionyl-CoA is converted to the Krebs cycle intermediate succinyl-CoA, by a pathway involving vitamin B12 (to be presented later).

 Most double bonds of naturally occurring fatty acids have the cis configuration.

As C atoms are removed two at a time, a double bond may end up in the wrong position or wrong configuration to be the correct substrate for EnoylCoA Hydratase. The reactions that allow unsaturated fatty acids to be fully catabolized by the b-oxidation pathway are summarized in the textbook.

Peroxisome
Single membrane Crystalline inclusion often present

Enzymes, some of which produce H2O2 , & always including Catalase, that degrades H2O2.

b-Oxidation of very long-chain fatty acids also occurs within peroxisomes. FAD is e acceptor for peroxisomal Acyl-CoA Oxidase, which catalyzes the 1st oxidative step of the pathway.

Within the peroxisome, FADH2 generated by fatty acid oxidation is reoxidized producing hydrogen peroxide: FADH2 + O2 FAD + H2O2 The peroxisomal enzyme Catalase degrades H2O2: 2 H2 O 2 2 H2 O + O 2 These reactions produce no ATP. Once fatty acids are reduced in length within the peroxisomes they may shift to the mitochondria to be catabolized all the way to CO2.

Carnitine is involved in transfer of fatty acids into and out of peroxisomes.

Serious genetic diseases are associated with defects in or deficiency of enzymes of the peroxisomal b-oxidation system.
Peroxisomes also contain enzymes for an essential -oxidation pathway that degrades fatty acids having methyl branches, such as phytanic acid, a breakdown product of chlorophyll.

Glucose-6-phosphatase glucose-6-P glucose Gluconeogenesis Glycolysis pyruvate fatty acids

During fasting acetyl CoA ketone bodies or carbohydrate cholesterol starvation, oxaloacetate citrate oxaloacetate is depleted in Krebs Cycle liver due to gluconeogenesis.
This impedes entry of acetyl-CoA into Krebs cycle.

Acetyl-CoA in liver mitochondria is converted then to ketone bodies, acetoacetate & b-hydroxybutyrate.

Ketone body synthesis: b-Ketothiolase. The final step of the boxidation pathway runs backward. HMG-CoA Synthase catalyzes condensation with a 3rd acetate moiety (from acetyl-CoA). HMG-CoA Lyase cleaves HMG-CoA to yield acetoacetate & acetyl-CoA.

O H3 C C SCoA + H3C

O C SCoA

acetyl-CoA

HSCoA

Thiolase
O O H2 C C

acetyl-CoA

O H3C C

H3C SCoA

SCoA

acetoacetyl-CoA

acetyl-CoA HSCoA
O

HMG-CoA Synthase
OH O H2 C C SCoA

H2 C

C CH3

HMG-CoA
O

HMG-CoA Lyase
O

O H2 C C CH3 + H3C

SCoA

acetoacetate

acetyl-CoA

b-Hydroxybutyrate CH3 + H Dehydrogenase C O NADH catalyzes reversible interconversion of CH2 the ketone bodies COO acetoacetate & acetoacetate b-hydroxybutyrate.

b-Hydroxybutyrate Dehydrogenase
CH3

NAD HO

CH CH2 COO

D-b -hydroxybutyrate

Ketone bodies are transported in the blood to other cells, where they are converted back to acetyl-CoA for catabolism in Krebs cycle, to generate ATP. While ketone bodies thus function as an alternative fuel, amino acids must be degraded to supply input to gluconeogenesis when hypoglycemia occurs, since acetate cannot be converted to glucose.