Part III => METABOLISM and ENERGY

§3.4 Lipid Catabolism

§3.4a Fatty Acid Degradation

§3.4b Ketone Bodies

Section 3.4a:

Fatty Acid Degradation

Synopsis 3.4a

- Triglycerides (or fats) in the diet or adipose tissue are broken down into fatty acids by a group of enzymes referred to as “lipases”

- Degradation of such fatty acids releases free energy—how?

- In the cytosol, fatty acids to be degraded are linked to coenzyme A (CoA) and then transported into the mitochondrion via a carnitine shuttle for oxidation

- In the mitochondrion, each round of so-called “β-oxidation” of fatty acids produces FADH2, NADH, and acetyl-CoA

- Acetyl-CoA is subsequently oxidized via the Krebs cycle and the energy released is stored in the form of GTP, FADH2 and NADH—see §3.5

- FADH2 and NADH ultimately donate their electrons to produce ATP via the electron transport chain (ETC)—see §3.6

Coenzyme A—a common metabolic cofactor

Coenzyme A (CoA) is involved in numerous metabolic pathways, including:(1) Biosynthesis of fatty acids(2) Oxidation of fatty acids(3) Oxidation of pyruvate

cis-9-dodecanoate

- While the x:m symbolism provides insights into the length and the degree of unsaturation of a fatty acid (see §1.4), an alternative nomenclature is needed to indicate both the position and the stereochemistry of the double bond(s)

- In this nomenclature, the position and stereochemical configuration of C=C double bond is indicated by the z-n notation:

=> unsaturation within the C=C bondz => cis/trans stereochemistry about the C=C bond n => numeric position of first C atom within C=C bond from carboxyl end

- For example, the cis-9 notation is indicative of a C=C double bond beginning @ C9 within the fatty acid tail harboring cis-configuration

- What does trans-2 suggest?!

Fatty Acid Nomenclature

Lipase

Fatty Acids

-O

-O

-O

+

Triglyceride Breakdown

- Triglycerides (or triacylglycerols) are fatty acid esters (usually with different fatty acid R groups) of glycerol—see §1.4!

- Triglycerides are largely stored in the adipose tissue where they function as “high-energy” reservoirs

- In order to release such energy to be used as “free energy”, triglycerides are first de-esterified or hydrolyzed into free fatty acids by lipases via a process known as “lipolysis”

- Once released from their parent triglycerides within the cytosol, fatty acid (FA) degradation to generate acetyl-CoA (for subsequent oxidation via the Krebs cycle) requires TWO umbrella stages (additional stages are needed for the oxidation of unsaturated fatty acids—a subject that is beyond the scope of this lecture):

(A) FA Import(B) FA Oxidation

(A) FA Import: Overview

- Prior to their oxidation within the mitochondria, the fatty acids are first imported from the cytosol

- Such import requires the “priming” of fatty acids with coenzyme A (CoA) so as to generate the acyl-CoA derivative within the cytosol

- Recall that acyl is a functional group with the general formula R-C=O, where R is an alkyl sidechain (or in this case, the non-polar tail of fatty acids)

- Given the rather charged character of CoA moiety (vide infra), acyl-CoA produced in the cytosol cannot cross (or diffuse through) the inner mitochondrial membrane (IMM) to reach the mitochondrial matrix (the site of Krebs cycle)

- Accordingly, acyl-CoA is subjected to reversible conversion to acyl-carnitine in order to exploit the carnitine shuttle system located within the IMM to translocate it to the mitochondrial matrix

Acyl-CoA synthetase

Fatty Acid

1

2

3

4

Acyl-CoA (cytosolic)

Acyl-CoA (mitochondrial matrix)

Acyl-carnitine

Acyl-carnitine

Carnitine acyltransferase I

Carnitine-acylcarnitine translocase

(Mitochondrial Transit)

Carnitine acyltransferase II

FA Import: (1) Acyl-CoA Synthetase

- In order to be oxidized to provide free energy, fatty acids are first “primed” with CoA in an ATP-dependent reaction to generate the acyl-CoA derivative within the cytosol

- The reaction is catalyzed by a family of enzymes called “acyl-CoA synthetases” or “thiokinases”

- First step mediated via nucleophilic attack of O atom of fatty acid carboxylate anion on the -phosphate of ATP to generate the acyladenylate mixed anhydride intermediate and PPi—which undergoes exergonic hydrolysis to Pi to drive the reaction to completion

- Second-step involves nucleophilic attack by the thiol (-SH) group of CoA on the carbonyl C atom of acyladenylate mixed anhydride intermediate to generate acyl-COA and AMP

- The overall result is that the free energy of fatty acid is conserved via the generation of a “high-energy” thioester bond of acyl-CoA within the cytosol—but how does acyl-CoA get into the mitochondrial matrix (the site of Krebs cycle)?

FA Import: (2) Carnitine Acyltransferase I

- Given the rather charged character of CoA moiety, acyl-CoA produced in the cytosol cannot cross (or diffuse through) the inner mitochondrial membrane (IMM) to reach the mitochondrial matrix (the site of Krebs cycle)

- Accordingly, acyl-CoA is first converted to acyl-carnitine by carnitine acyltransferase I—an enzyme located at the outer (intermembraneous space) surface of IMM—in order to exploit the carnitine shuttle system for its delivery into the mitochondrial matrix

- Carnitine, a quaternary amine, has no known physiological function other than its role in the shuttling of fatty acids from the intermembraneous space to mitochondrial matrix

- Note that the free energy of thioester bond in acyl-CoA is conserved in the ester (or O-acyl) bond in acyl-carnitine

Carnitine

Acyl-carnitine

Acyl-CoA

CoA

Carnitine

acyltransferase I

FA Import: (3) Carnitine-Acylcarnitine Translocase

Acyl-carnitine is shuttled across the inner mitochondrial membrane (IMM)—from the cytosol (or the intermembraneous space) to the mitochondrial matrix—by the carnitine-acylcarnitine translocase

Carnitine-

acylcarnitine

translocase

Acyl-carnitine

Cytosol(intermembrane

space)

Mitochondrial

Matrix

Acyl-carnitine

FA Import: (4) Carnitine Acyltransferase II

- Inside the mitochondrial matrix, carnitine acyltransferase II catalyzes the reverse transfer of acyl group of acyl-carnitine back to CoA to generate acyl-CoA and free carnitine

- Acyl-CoA is then not only “chemically” but also “spatially” primed to be converted to acetyl-CoA for subsequent entry into the Krebs cycle

Carnitine

Acyl-carnitine

Acyl-CoA

CoA

Carnitine

acyltransferase II

FA Import: Outline

Acyl-CoA is transported from the cytosol (or the intermembraneous space) to the mitochondrial matrix by the carnitine shuttle system as follows:

(1) Fatty acid is “primed” with CoA in the cytosol

(2) Acyl group of cytosolic acyl-CoA is transferred to carnitine acyl-carnitine

(3) Acyl-carnitine is shuttled across the IMM into the mitochondrial matrix by carnitine-acylcarnitine translocase

(4) Acyl group of matrix acyl-carnitine is transferred to mitochondrial matrix CoA acyl-CoA, thereby freeing up free carnitine pool

(5) Free carnitine within the matrix is shuttled back to the cytosol to repeat the cycle

Carnitine-

acylcarnitine

translocase2

3

5

4Carnitine

acyltransferase I

Carnitine

acyltransferase II

RCOOH

SCoA1

(B) FA Oxidation: Overview

- Within the mitochondrial matrix, oxidation of acyl-CoA into acetyl-CoA (a Krebs cycle substrate) occurs via four distinct steps—each requiring the involvement of a specific mitochondrial enzyme

- This process is referred to as “-oxidation”—due to the fact that the acyl group of acyl-CoA is oxidized at its -carbon atom in a repetitive fashion so as to degrade fatty acids with the removal of a two-carbon unit in the form of acetyl-CoA during each round

- A common mechanism to cleave the C—C bond involves the following four steps:

(1) Dehydrogenate: H2C—CH2 HC=CH(2) Hydroxylate: HC=CH HC(OH)—CH2

(3) Oxidize: HC(OH)—CH2 C(O)—CH2

(4) Cleave via nucleophilic attack: C(O)—CH2

- Let us see that in action!

Acyl-CoA dehydrogenase1

2

3

4

trans-2-Enoyl-CoA

Acetyl-CoA

-Ketoacyl-CoA

L--Hydroxyacyl-CoA

Enoyl-CoA hydratase

-Ketoacyl-CoA thiolase

Acyl-CoA

-Hydroxyacyl-CoA

dehydrogenase

FA Oxidation: (1) Acyl-CoA Dehydrogenase

Dehydrogenation

- Dehydrogenation of saturated C-C single bond within acyl-CoA results in the formation of enoyl-CoA harboring a C=C double bond

- Since such dehydrogenation begins at C atom numbered 2, the product is prefixed with trans-2 to indicate the stereochemical configuration and position of the C=C double bond

- Reaction catalyzed by acyl-CoA dehydrogenase using FAD as an oxidizing agent (more powerful than NAD+) or electron acceptor—thus the energy released due to the oxidation of acyl group is conserved in the form of FADH2

- FADH2 will be subsequently reoxidized back to FAD via the mitochondrial electron transport chain (ETC)

FA Oxidation: (2) Enoyl-CoA Hydratase

L--Hydroxyacyl-CoA

Hydration

- Hydration of unsaturated C=Cdouble bond within trans-2-enoyl-CoA (prochiral) results in the formation of L--hydroxyacyl-CoA

- Reaction catalyzed by enoyl-CoA hydratase in a stereospecific manner producing exclusively the L-isomer

- The addition of an –OH group at the C position “primes” L--hydroxyacyl-CoA for subsequent oxidation to a keto group—the C atom of which then serves as an electrophilic center for the release of first acetyl-CoA

FA Oxidation: (3) -Hydroxyacyl-CoA Dehydrogenase

Oxidation

- Oxidation of –OH to a keto group at the Cposition within L--hydroxyacyl-CoA results in the formation of corresponding -ketoacyl-CoA

- Reaction catalyzed by -hydroxyacyl-CoA dehydrogenase using NAD+ as an oxidizing agent or electron acceptor—the energy of electron transfer is conserved in NADH

- NADH will be subsequently reoxidized back to NAD+ via the mitochondrial electron transport chain (ETC)

L--Hydroxyacyl-CoA

-hydroxyacyl-CoA

dehydrogenase

Thiolysis

- Thiolysis (or breaking bonds with –SH group—cf hydrolysis and phosphorolysis) initiated by nucleophilic attack of the thiol group (-SH) of CoA on the keto group within -ketoacyl-CoA results in the cleavage of C-C bond, thereby releasing the first acetyl-CoA (to enter the Krebs cycle) and an outgoing acyl-CoA

- Reaction catalyzed by -ketoacyl-CoA thiolase

- The outgoing acyl-CoA is two C atoms shorter than the parent acyl-CoA that entered the first round of -oxidation—this acyl-CoA will undergo subsequent rounds of -oxidation (Steps 1-4) to generate additional acetyl-CoA molecules—how many?!

- Complete -oxidation of a 2n:0 fatty acid requires n-1 steps—ie it will generate n acetyl-CoA, n-1 NADH, and n-1 FADH2! That would be bucketloads of energy—but exactly how much?!

FA Oxidation: (4) -Ketoacyl-CoA Thiolase

Palmitoyl-CoA

8 Acetyl-CoA

7 NADH

7 FADH2

8 FADH2

24 NADH

8 GTP

10.5 ATP

17.5 ATP

60 ATP

12 ATP

8 ATP

Total Energy = 108 ATP

Krebs

cycle

ETC

ETC

ETC

ETC

-Oxidation- Palmitic acid is a saturated fatty acid harboring

16 carbon atoms (16:0)

- It is the most commonly occurring fatty acid in living organisms

- So how much energy does -oxidation of a single chain of palmitic acid (16 C atoms) generate?

- Complete degradation of palmitic acid would require 7 rounds of -oxidation producing 7 FADH2, 7 NADH and 8 acetyl-CoA—the final round produces 2 acetyl-CoA!

- Further oxidation of each acetyl-CoA via the Krebs cycle produces 3 NADH, 1 FADH2 and 1 GTP (enzymatically converted to ATP) per molecule (and there are 8 acetyl-CoA!)—see §3.5

- Oxidation of each NADH and FADH2 via the ETC respectively produces 2.5 and 1.5 molecules of ATP—see §3.6

6

Palmitic Acid (16:0)

FA Oxidation: Bucketloads of ATP

Fat Is hypercaloric!

Exercise 3.4a

- Describe the activation of fatty acids. What is the energy cost for the process?

- How do cytosolic acyl groups enter the mitochondrion for degradation?

- Summarize the chemical reactions that occur in each round of β-oxidation. Explain why the process is called β-oxidation?

- How is ATP recovered from the products of β-oxidation?

Section 3.4b:

Ketone Bodies

Synopsis 3.4b

- While acetyl-CoA produced via fatty acid oxidation is by and large funneled into the Krebs cycle in most tissues, it can also be converted to the so-called ketone bodies in a process referred to as “ketogenesis”

- Ketone bodies—essentially acetyl-CoA-in-disguise—include small water-soluble molecules such as acetoacetate, -hydroxybutyrate , and acetone

- Ketogenesis primarily occurs within the mitochondrial matrix of liver cells under conditions of starvation during glucose shortage—the metabolic state under which the body derives some of its energy from the use of ketone bodies as metabolic fuels is called “ketosis”—eg the body being in a state of ketosis vs state of glycolysis

- Conditions such as alcohol consumption, ketogenic (fat-rich) diet, prolonged starvation, and diabetes mellitus can result in the production of ketone bodies in a rather high concentration in the blood—such metabolic state is referred to as “ketoacidosis”

- Ketoacidosis results in a decrease in blood pH and is fraught with serious pathological consequences—fruit-like smell of breath due to acetone may be a sign of ketoacidosis!

- Why is there a need to produce ketone bodies?!!

Ketone Bodies: Physiological Significance

- Being small and water-soluble, ketone bodies represent a neat trick to transport acetyl-CoA from liver to peripheral tissues (to be used as a metabolic fuel) such as the:

(1) Heart (virtually no glycogen reserves)—since heart primarily relies on fatty acids for energy production, ketone bodies serve as an alternative source of fuel that can be readily “burned” via the Krebs cycle to generate energy

(2) Brain (low glycogen reserves that likely mediate neuronal activity rather than glucose metabolism)—since fatty acids and acetyl-CoA cannot enter the brain due to the presence of the so-called blood-brain-barrier (BBB), the ability of ketone bodies to diffuse (via monocarboxylate transporters) through the BBB renders them perfect candidates as an alternative source of fuel (when glucose is in short supply) and as precursors for fatty acid biosynthesis

BBB is an highly selective filter/barrier that separates the circulating blood in the brain from the extracellular fluid—only water, gases, and lipophilic molecules such as steroid hormones can usually cross the BBB by passive diffusion

Typical Capillary Brain Capillary

SCoA

Acetyl-CoA

Acetoacetate

Ketone Bodies: Ketogenesis

NADH

NAD+

-hydroxybutyrate

dehydrogenase

H

-Hydroxybutyrate

CO2

Acetoacetate

decarboxylase

(or spontaneously)

3

Acetone

-hydroxybutyrate is easily converted back to acetyl-CoA via acetoacetate

Conversion of acetone back to acetyl-CoA occurs via lactate and pyruvate in the liver

Ketone bodies include:- Acetoacetate- -hydroxybutyrate- Acetone

(1) How is acetyl-CoA converted to ketone bodies in the liver?

(2) How are ketone bodies converted back to acetyl-CoA in target tissues so as to be utilized as a source of fuel via the Krebs cycle?

However, acetone is usually excreted via

urine and/or exhaled

The conversion of acetyl-CoA to ketone bodies such as acetoacetate in the liver occurs via three major enzymatic steps:

(1) Thiolase condenses two molecules of acetyl-CoA into acetoacetyl-CoA

(2) Hydroxymethylglutaryl-CoA synthase adds another molecule of acetyl-CoA to acetoacetyl-CoA to generate -hydroxy--methylglutaryl-CoA

(3) Hydroxymethylglutaryl-CoA lyase breaks down -hydroxy--methylglutaryl-CoA into acetyl-CoA and acetoacetate—one of the three ketone bodies

Ketone Bodies: (1) Acetyl-CoA Acetoacetate [Liver]

Glutaric Acid (5C)1

2

3

1

2

3

Ketone bodies such as acetoacetate and -hydroxybutyrate (produced by the liver) travel in the bloodstream to reach tissues such as the heart and brain, where they are converted back to acetyl-CoA via the following enzymatic steps:

(1) -hydroxybutyrate dehydrogenasemediates the oxidation of -hydroxybutyrate into acetoacetate

(2) Ketoacyl-CoA transferase condensesacetoacetate with CoA (donated by succinyl-CoA) to generate acetoacetyl-CoA

(3) Thiolase breaks down acetoacetyl-CoA into two acetyl-CoA molecules using free CoA as a nucleophile

The newly generated acetyl-CoA can now serve either as a Krebs cycle substrate for energy production (or as a precursor for fatty acid biosynthesis!)

Ketone Bodies: (2) Acetoacetate Acetyl-CoA [Heart|Brain]

Exercise 3.4b

- What are ketone bodies?

- Which organs utilize ketone bodies as an alternative source of fuel?

- How are ketone bodies synthesized and degraded?