1. III. METABOLIC BIOCHEMISTRY
§3.3 Lipid Catabolism
§3.3a Fatty Acid Release
§3.3b Fatty Acid Transport
§3.3c Fatty Acid Oxidation
§3.3d Ketone Bodies

2. §3.3a Fatty Acid Release (Lipolysis)

3. Synopsis 3.3a
In the context of generating free energy, lipid catabolism can be subdivided into three stages:
Fatty Acid Release—the cytosolic breakdown of triglycerides (or fats) in the diet or adipose tissue (during starvation) into fatty acids by a group of enzymes collectively referred to as “lipases”—this process is technically called “lipolysis”
Fatty Acid Transport—In the cytosol, the released fatty acids are first activated via covalent linkage to coenzyme A (CoA) so as to generate acyl-CoA harboring a “high-energy” thioester bond and then subsequently transported into the mitochondrial matrix via a carnitine shuttle
Fatty Acid Oxidation—In the mitochondrial matrix, fatty acids undergo multiple rounds of so-called “β-oxidation”, with each round producing reduced cofactors FADH2 and NADH (destined for electron transport chain) as well as acetyl-CoA (destined for Krebs cycle)

4. 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

5. Fatty Acids: A Few Common Members
[HA]
Base/Salt
[A-]
Acyl Group
[R—C(O)—]
Structure
x:m O - 10
12:0
Lauric Acid
Laurate
Lauroyl O - 12
14:0
Myristic Acid
Myristate
Myristoyl O - 14
16:0
Palmitic Acid
Palmitate
Palmitoyl O - 16
18:0
Stearic Acid
Stearate
Stearoyl

6. Fatty Acids: Nomenclature   
cis-9-dodecanoate
While the x:m symbolism provides insights into the length and the degree of unsaturation of a fatty acid (see §1.3), 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 bond
z => 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 C9 within the fatty acid tail harboring cis-configuration
What does trans-2 suggest?!

7. Triglyceride Breakdown
Lipase
Fatty Acids -O +
Triglycerides (or triacylglycerols) are fatty acid esters (usually with different fatty acid R groups) of glycerol—see §1.3!
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”
After their release from their parent triglycerides within the cytosol, fatty acids are next transported into the mitochondrial matrix for their subsequent oxidation to release free energy (vide infra)

8. Exercise 3.3a Describe the chemical structure of CoA
What is the functional group in CoA that participates in covalent linkage with fatty acids
In the context of fatty acid nomenclature, what does the trans-7 notation imply?
How are fatty acids released from their parent fats?

9. §3.3b Fatty Acid Transport

10. Synopsis 3.3b
In the context of generating free energy, lipid catabolism can be subdivided into three stages:
Fatty Acid Release—the cytosolic breakdown of triglycerides (or fats) in the diet or adipose tissue (during starvation) into fatty acids by a group of enzymes collectively referred to as “lipases”—this process is technically called “lipolysis”
Fatty Acid Transport—In the cytosol, the released fatty acids are first activated via covalent linkage to coenzyme A (CoA) so as to generate acyl-CoA harboring a “high-energy” thioester bond and then subsequently transported into the mitochondrial matrix via a carnitine shuttle
Fatty Acid Oxidation—In the mitochondrial matrix, fatty acids undergo multiple rounds of so-called “β-oxidation”, with each round producing reduced cofactors FADH2 and NADH (destined for electron transport chain) as well as acetyl-CoA (destined for Krebs cycle)

11. (A) FA Transport: Overview
Acyl-CoA synthetase
Fatty Acid 1 2 3 4
Acyl-CoA (cytosolic)
Acyl-CoA (mitochondrial matrix)
Acyl-carnitine
Carnitine acyltransferase I
Carnitine-acylcarnitine translocase
(Mitochondrial Transit)
Carnitine acyltransferase II
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—eg lauroyl-CoA, myristoyl-CoA, palmitoyl-CoA, and stearoyl-CoA (12:0, 14:0, 16:0, and 18:0)
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

12. FA Transport: (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)?

13. FA Transport: (2) Carnitine Acyltransferase I
Acyl-CoA
Carnitine
acyltransferase I
Acyl-carnitine
CoA
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

14. FA Transport: (3) Carnitine-Acylcarnitine Translocase
Cytosol
(intermembrane
space)
Mitochondrial
Matrix
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

15. FA Transport: (4) Carnitine Acyltransferase II
Acyl-carnitine
CoA
Carnitine
acyltransferase II
Carnitine
Acyl-CoA
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

16. FA Transport: Outline 1 5 2 4 3 Carnitine Carnitine Carnitine-
RCOOH
Carnitine-
acylcarnitine
translocase 1
SCoA 5
Carnitine
acyltransferase I
Carnitine
acyltransferase II 2 4 3
Acyl-CoA is transported from the cytosol (or the intermembraneous space) to the mitochondrial matrix by the carnitine shuttle system as follows:
Fatty acid is “primed” with CoA in the cytosol
Acyl group of cytosolic acyl-CoA is transferred to carnitine  acyl-carnitine
Acyl-carnitine is shuttled across the IMM into the mitochondrial matrix by carnitine- acylcarnitine translocase
Acyl group of matrix acyl-carnitine is transferred to mitochondrial matrix CoA  acyl-CoA, thereby freeing up free carnitine pool
Free carnitine within the matrix is shuttled back to the cytosol to repeat the cycle

17. Exercise 3.3b
Describe the activation of fatty acids. What is the energy cost for the process?
How does carnitine shuttle transport fatty acids into the mitochondrial matrix?
Distinguish between the roles and subcellular localization of carnitine acyltransferases I and II?

18. §3.3c Fatty Acid Oxidation

19. Synopsis 3.3c
In the context of generating free energy, lipid catabolism can be subdivided into three stages:
Fatty Acid Release—the cytosolic breakdown of triglycerides (or fats) in the diet or adipose tissue (during starvation) into fatty acids by a group of enzymes collectively referred to as “lipases”—this process is technically called “lipolysis”
Fatty Acid Transport—In the cytosol, the released fatty acids are first activated via covalent linkage to coenzyme A (CoA) so as to generate acyl-CoA harboring a “high-energy” thioester bond and then subsequently transported into the mitochondrial matrix via a carnitine shuttle
Fatty Acid Oxidation—In the mitochondrial matrix, fatty acids undergo multiple rounds of so-called “β-oxidation”, with each round producing reduced cofactors FADH2 and NADH (destined for electron transport chain) as well as acetyl-CoA (destined for Krebs cycle)

20. (B) FA Oxidation: Overview
Acyl-CoA dehydrogenase 1 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
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:
Dehydrogenate: H2C—CH2  HC=CH
Hydroxylate: HC=CH  HC(OH)—CH2
Oxidize: HC(OH)—CH2  C(O)—CH2
Cleave via nucleophilic attack: C(O)—CH2
- Let us see that in action!

21. 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)

22. FA Oxidation: (2) Enoyl-CoA Hydratase
L--Hydroxyacyl-CoA
Hydration
Hydration of unsaturated C=C double 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

23. FA Oxidation: (3) -Hydroxyacyl-CoA Dehydrogenase
L--Hydroxyacyl-CoA
-hydroxyacyl-CoA dehydrogenase
Oxidation
Oxidation of –OH to a keto group at the C position 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)

24. FA Oxidation: (4) -Ketoacyl-CoA Thiolase
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?!

25. FA Oxidation: Bucketloads of ATP6
Palmitic Acid (16:0)
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
-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
Fat Is hypercaloric!

26. Exercise 3.3c
Summarize the chemical reactions that occur in each round of β-oxidation. Explain why this process is called β-oxidation?
How is ATP recovered from the products of β-oxidation?
How many rounds of β-oxidation are needed to completely oxidize an 18:0 fatty acid? How many of the following are produced: acetyl-CoA, NADH, and FADH2?

27. §3.3d Ketone Bodies

28. Synopsis 3.3d
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?!!

29. Ketone Bodies: Physiological Significance
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
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:
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
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

30. Ketone Bodies: Ketogenesis
SCoA
Acetyl-CoA
How is acetyl-CoA converted to ketone bodies in the liver?
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?
Ketone bodies include:
Acetoacetate
-hydroxybutyrate
Acetone
Acetoacetate
Conversion of acetone back to acetyl-CoA occurs via lactate and pyruvate in the liver
-hydroxybutyrate is easily converted back to acetyl-CoA via acetoacetate
CO2
Acetoacetate
decarboxylase
(or spontaneously) 3
Acetone
NADH
NAD+
-hydroxybutyrate
dehydrogenase H
-Hydroxybutyrate
However, acetone is usually excreted via urine and/or exhaled

31. Ketone Bodies: (1) Acetyl-CoA  Acetoacetate [Liver]
Glutaric Acid (5C) 1
The conversion of acetyl-CoA to ketone bodies such as acetoacetate in the liver occurs via three major enzymatic steps:
Thiolase condenses two molecules of acetyl-CoA into acetoacetyl-CoA
Hydroxymethylglutaryl-CoA synthase adds another molecule of acetyl-CoA to acetoacetyl-CoA to generate -hydroxy--methylglutaryl-CoA
Hydroxymethylglutaryl-CoA lyase breaks down -hydroxy--methylglutaryl-CoA into acetyl-CoA and acetoacetate—one of the three ketone bodies 2 3

32. Ketone Bodies: (2) Acetoacetate  Acetyl-CoA [Heart|Brain]
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:
-hydroxybutyrate dehydrogenase mediates the oxidation of -hydroxybutyrate into acetoacetate
Ketoacyl-CoA transferase condenses acetoacetate with CoA (donated by succinyl-CoA) to generate acetoacetyl-CoA
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!) 1 2 3

33. Exercise 3.3d What are ketone bodies?
Which organs utilize ketone bodies as an alternative source of fuel?
How are ketone bodies synthesized and degraded?