III METABOLIC BIOCHEMISTRY 3 3 Lipid Catabolism 3

III. METABOLIC BIOCHEMISTRY § 3. 3 Lipid Catabolism § 3. 3 a Fatty Acid Release § 3. 3 b Fatty Acid Transport § 3. 3 c Fatty Acid Oxidation § 3. 3 d Ketone Bodies

§ 3. 3 a Fatty Acid Release (Lipolysis)

Synopsis 3. 3 a - In the context of generating free energy, lipid catabolism can be subdivided into three stages: (1) Fatty Acid Release (luminal/cytosolic)—the breakdown of fats or triglycerides in the diet (in the lumen of small intestine after a meal) or adipose tissue (in the cytosol of adipocytes during starvation) into fatty acids by a group of enzymes collectively referred to as “lipases”—this process is technically called “lipolysis” (1) Fatty Acid Transport (cytosolic)—In the cytosol, the released fatty acids are first activated via covalent linkage to coenzyme A (Co. A) so as to generate acyl-Co. A harboring a “high-energy” thioester bond and then subsequently transported into the mitochondrial matrix via a carnitine shuttle (2) Fatty Acid Oxidation (mitochondrial)—In the mitochondrial matrix, fatty acids undergo multiple rounds of so-called “β-oxidation”, with each round producing reduced cofactors FADH 2 and NADH (destined for electron transport chain) as well as acetyl-Co. A (destined for Krebs cycle)

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

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

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 beginning @ C 9 within the fatty acid tail harboring cis-configuration - What does trans- 2 suggest? !

Triglyceride Breakdown -O Lipase -O + -O Fatty Acids - Triglycerides (or triacylglycerols) are fatty acid esters (usually with different fatty acid R groups) of glycerol—see § 1. 3! - The fats in the diet are largely triglycerides—they are also 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” - Dietary triglycerides are broken down in the lumen of small intestine (through the action of pancreatic lipase)—the resulting fatty acids and glycerol are absorbed into blood through the intestinal microvilli and transported to hepatocytes in the liver - In adipose tissue, the breakdown of triglycerides occurs within the cytosol of adipocytes (through the action of hormone-sensitive lipase expressed by adipocytes)—the resulting fatty acids and glycerol are secreted into the blood for transport to hepatocytes in the liver

Exercise 3. 3 a - Describe the chemical structure of Co. A - What is the functional group in Co. A 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?

§ 3. 3 b Fatty Acid Transport

Synopsis 3. 3 b - In the context of generating free energy, lipid catabolism can be subdivided into three stages: (1) Fatty Acid Release (luminal/cytosolic)—the breakdown of fats or triglycerides in the diet (in the lumen of small intestine after a meal) or adipose tissue (in the cytosol of adipocytes during starvation) into fatty acids by a group of enzymes collectively referred to as “lipases”—this process is technically called “lipolysis” (1) Fatty Acid Transport (cytosolic)—In the cytosol, the released fatty acids are first activated via covalent linkage to coenzyme A (Co. A) so as to generate acyl-Co. A harboring a “high-energy” thioester bond and then subsequently transported into the mitochondrial matrix via a carnitine shuttle (2) Fatty Acid Oxidation (mitochondrial)—In the mitochondrial matrix, fatty acids undergo multiple rounds of so-called “β-oxidation”, with each round producing reduced cofactors FADH 2 and NADH (destined for electron transport chain) as well as acetyl-Co. A (destined for Krebs cycle)

(A) FA Transport: 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 (Co. A) so as to generate the acyl -Co. A derivative within the cytosol—eg lauroyl. Co. A, myristoyl-Co. A, palmitoyl-Co. A, and stearoyl. Co. A (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 Co. A moiety (vide infra), acyl-Co. A 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-Co. A 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 Fatty Acid 1 Acyl-Co. A synthetase Acyl-Co. A (cytosolic) 2 Carnitine acyltransferase I Acyl-carnitine 3 Carnitine-acylcarnitine translocase (Mitochondrial Transit) Acyl-carnitine 4 Carnitine acyltransferase II Acyl-Co. A (mitochondrial matrix)

FA Transport: (1) Acyl-Co. A Synthetase - In order to be oxidized to provide free energy, fatty acids are first “primed” with Co. A in an ATP-dependent reaction to generate the acyl-Co. A derivative within the cytosol - The reaction is catalyzed by a family of enzymes called “acyl-Co. A 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 Co. A 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-Co. A within the cytosol—but how does acyl-Co. A get into the mitochondrial matrix (the site of Krebs cycle)?

FA Transport: (2) Carnitine Acyltransferase I Carnitine Acyl-Co. A Carnitine acyltransferase I Acyl-carnitine Co. A - Given the rather charged character of Co. A moiety, acyl-Co. A 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-Co. A 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-Co. A is conserved in the ester (or O-acyl) bond in acyl-carnitine

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

FA Transport: (4) Carnitine Acyltransferase II Acyl-carnitine Co. A Carnitine acyltransferase II Carnitine Acyl-Co. A - Inside the mitochondrial matrix, carnitine acyltransferase II catalyzes the reverse transfer of acyl group of acyl-carnitine back to Co. A to generate acyl. Co. A and free carnitine - Acyl-Co. A is then not only “chemically” but also “spatially” primed to be converted to acetyl-Co. A for subsequent entry into the Krebs cycle

FA Transport: Outline RCOOH 1 SCo. A 5 2 Carnitine acyltransferase I Carnitineacylcarnitine translocase Carnitine acyltransferase II 4 3 Acyl-Co. A 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 Co. A in the cytosol (2) Acyl group of cytosolic acyl-Co. A is transferred to carnitine acyl-carnitine (3) Acyl-carnitine is shuttled across the IMM into the mitochondrial matrix by carnitineacylcarnitine translocase (4) Acyl group of matrix acyl-carnitine is transferred to mitochondrial matrix Co. A acyl. Co. A, thereby freeing up free carnitine pool (5) Free carnitine within the matrix is shuttled back to the cytosol to repeat the cycle

Exercise 3. 3 b - 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?

§ 3. 3 c Fatty Acid Oxidation

Synopsis 3. 3 c - In the context of generating free energy, lipid catabolism can be subdivided into three stages: (1) Fatty Acid Release (luminal/cytosolic)—the breakdown of fats or triglycerides in the diet (in the lumen of small intestine after a meal) or adipose tissue (in the cytosol of adipocytes during starvation) into fatty acids by a group of enzymes collectively referred to as “lipases”—this process is technically called “lipolysis” (1) Fatty Acid Transport (cytosolic)—In the cytosol, the released fatty acids are first activated via covalent linkage to coenzyme A (Co. A) so as to generate acyl-Co. A harboring a “high-energy” thioester bond and then subsequently transported into the mitochondrial matrix via a carnitine shuttle (2) Fatty Acid Oxidation (mitochondrial)—In the mitochondrial matrix, fatty acids undergo multiple rounds of so-called “β-oxidation”, with each round producing reduced cofactors FADH 2 and NADH (destined for electron transport chain) as well as acetyl-Co. A (destined for Krebs cycle)

(B) FA Oxidation: Overview Acyl-Co. A - Within the mitochondrial matrix, oxidation of acyl -Co. A into acetyl-Co. A (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-Co. A 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-Co. A during each round - A common mechanism to cleave the C—C bond involves the following four steps: (1) Dehydrogenate: H 2 C—CH 2 HC=CH (2) Hydroxylate: HC=CH HC(OH)—CH 2 (3) Oxidize: HC(OH)—CH 2 C(O)—CH 2 (4) Cleave via nucleophilic attack: C(O)—CH 2 - Let us see that in action! 1 Acyl-Co. A dehydrogenase trans- 2 -Enoyl-Co. A 2 Enoyl-Co. A hydratase L- -Hydroxyacyl-Co. A 3 -Hydroxyacyl-Co. A dehydrogenase -Ketoacyl-Co. A 4 -Ketoacyl-Co. A thiolase Acetyl-Co. A

FA Oxidation: (1) Acyl-Co. A Dehydrogenase Dehydrogenation - Dehydrogenation of saturated C -C single bond within acyl-Co. A results in the formation of enoyl. Co. A 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-Co. A 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 FADH 2 - FADH 2 will be subsequently reoxidized back to FAD via the mitochondrial electron transport chain (ETC)

FA Oxidation: (2) Enoyl-Co. A Hydratase Hydration - Hydration of unsaturated C =C double bond within trans- 2 -enoyl. Co. A (prochiral) results in the formation of L- -hydroxyacyl-Co. A - Reaction catalyzed by enoyl-Co. A hydratase in a stereospecific manner producing exclusively the L-isomer - The addition of an –OH group at the C position “primes” L- -hydroxyacyl. Co. A for subsequent oxidation to a keto group—the C atom of which then serves as an electrophilic center for the release of first acetyl-Co. A L- -Hydroxyacyl-Co. A

FA Oxidation: (3) -Hydroxyacyl-Co. A Dehydrogenase Oxidation - Oxidation of –OH to a keto group at the C position within L- -hydroxyacyl-Co. A results in the formation of corresponding ketoacyl-Co. A - Reaction catalyzed by -hydroxyacyl-Co. A 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-Co. A -hydroxyacyl-Co. A dehydrogenase

FA Oxidation: (4) -Ketoacyl-Co. A Thiolase Thiolysis - Thiolysis (or breaking bonds with –SH group—cf hydrolysis and phosphorolysis) initiated by nucleophilic attack of the thiol group (-SH) of Co. A on the keto group within -ketoacyl-Co. A results in the cleavage of C -C bond, thereby releasing the first acetyl-Co. A (to enter the Krebs cycle) and an outgoing acyl-Co. A - Reaction catalyzed by -ketoacyl-Co. A thiolase - The outgoing acyl-Co. A is two C atoms shorter than the parent acyl-Co. A that entered the first round of -oxidation—this acyl-Co. A will undergo subsequent rounds of -oxidation (Steps 1 -4) to generate additional acetyl-Co. A molecules—how many? ! - Complete -oxidation of a 2 n: 0 fatty acid requires n-1 rounds—ie it will generate n acetyl-Co. A, n-1 NADH, and n-1 FADH 2! That would be bucketloads of energy—but exactly how much? !

FA Oxidation: Bucketloads of ATP Palmitic Acid (16: 0) Palmitoyl-Co. A 6 - 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? - Oxidation of each NADH and FADH 2 via the ETC respectively produces 2. 5 and 1. 5 molecules of ATP—see § 3. 6 ETC 10. 5 ATP 7 NADH ETC 17. 5 ATP -Oxidation 8 Acetyl-Co. A - Complete degradation of palmitic acid would require 7 rounds of -oxidation producing 7 FADH 2, 7 NADH and 8 acetyl-Co. A—the final round produces 2 acetyl-Co. A! - Further oxidation of each acetyl-Co. A via the Krebs cycle produces 3 NADH, 1 FADH 2 and 1 GTP (enzymatically converted to ATP) per molecule (and there are 8 acetyl-Co. A!)—see § 3. 5 7 FADH 2 Krebs cycle Fat Is hypercaloric! 24 NADH ETC 60 ATP 8 FADH 2 ETC 12 ATP 8 GTP 8 ATP Total Energy = 108 ATP

Exercise 3. 3 c - Summarize the chemical reactions that occur in each round of βoxidation. 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. Co. A, NADH, and FADH 2?

§ 3. 3 d Ketone Bodies

Synopsis 3. 3 d - While acetyl-Co. A 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”—the conversion of ketone bodies back into acetyl-Co. A is called “ketolysis” - Ketone bodies—essentially acetyl-Co. A-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 p. H 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 Typical Capillary Brain Capillary 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 - Being small and water-soluble, ketone bodies represent a neat trick to transport acetyl-Co. A 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-Co. A 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

Ketone Bodies: Ketogenesis (1) How is acetyl-Co. A converted to ketone bodies in the liver? SCo. A Acetyl-Co. A Ketone bodies include: - Acetoacetate - -hydroxybutyrate - Acetone (2) How are ketone bodies converted back to acetyl-Co. A in target tissues so as to be utilized as a source of fuel via the Krebs cycle? Acetoacetate ( -Ketobutyrate) Conversion of acetone back to acetyl-Co. A -hydroxybutyrate is easily converted occurs via lactate and pyruvate in the liver back to acetyl-Co. A via acetoacetate Acetoacetate decarboxylase (or spontaneously) CO 2 However, acetone is usually excreted via urine and/or exhaled NADH NAD+ -hydroxybutyrate dehydrogenase H 3 Acetone -Hydroxybutyrate

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

Ketone Bodies: (2) Acetoacetate Acetyl-Co. A [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-Co. A via the following enzymatic steps (ketolysis): 1 (1) -hydroxybutyrate dehydrogenase mediates the oxidation of hydroxybutyrate into acetoacetate (2) Ketoacyl-Co. A transferase condenses acetoacetate with Co. A (donated by succinyl-Co. A) to generate acetoacetyl. Co. A 2 (3) Thiolase breaks down acetoacetyl. Co. A into two acetyl-Co. A molecules using free Co. A as a nucleophile The newly generated acetyl-Co. A can now serve either as a Krebs cycle substrate for energy production (or as a precursor fatty acid biosynthesis!) 3

Exercise 3. 3 d - What are ketone bodies? - Which organs utilize ketone bodies as an alternative source of fuel? - How are ketone bodies synthesized and degraded?