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Part III => METABOLISM and ENERGY §3.4 Noncarbohydrate Metabolism §3.4a Fatty Acid Degradation §3.4b Ketone Bodies §3.4c Amino Acid Breakdown 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 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 §1.4! - Triglycerides are largely stored in the adipose tissue where they function as “high-energy” reservoirs—due to being more reduced (carry more electrons, or more hydrogens!) than their carbohydrate and protein counterparts, they yield significantly more energy per unit mass upon oxidative catabolism (see §3.6) - In order to release such energy to be used as “free energy”, triglycerides must first be deesterified or hydrolyzed into free fatty acids via the action of lipases - 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 Derivatization (B) FA Oxidation (A) FA Derivatization: Overview Fatty Acid - In order to be oxidized to provide free energy, fatty acids are first “primed” with coenzyme A (CoA) 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 polar tail of fatty acids) - Given the rather charged character of CoA moiety (vide infra), acyl-CoA produced in the cytosol cannot cross 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 1 Acyl-CoA synthetase Acyl-CoA (cytosolic) 2 Carnitine acyltransferase I Acyl-carnitine 3 Carnitine-acylcarnitine translocase (Mitochondrial Transit) Acyl-carnitine 4 Carnitine acyltransferase II Acyl-CoA (mitochondrial matrix) FA Derivatization: (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 α-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 Derivatization: (2) Carnitine Acyltransferase I Carnitine Acyl-CoA Carnitine acyltransferase I Acyl-carnitine CoA - Given the rather charged character of CoA moiety, acyl-CoA produced in the cytosol cannot cross 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 FA Derivatization: (3) Carnitine-Acylcarnitine Translocase Mitochondrial Matrix Cytosol (intermembrane space) Carnitineacylcarnitine translocase Acyl-carnitine Acyl-carnitine 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 Derivatization: (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 acylCoA 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 FA Derivatization: Outline RCOOH 1 SCoA 5 2 Carnitine acyltransferase I Carnitineacylcarnitine translocase Carnitine acyltransferase II 4 3 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 carnitineacylcarnitine 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 (B) FA Oxidation: Overview - Within the mitochondrial matrix, oxidation of acylCoA 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 “β-oxidation”—due to the fact that the acyl group of acyl-CoA is oxidized at its β-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 FA Nomenclature (cf x:m symbolism introduced in §1.4) α ∆9 β γ δ - The position of C=C double bond is indicated by the notation ∆n, where n denotes the numeric position of the first C atom within C=C from the carbonyl end - Thus, ∆9 is indicative of a C=C double bond beginning @ C9 within the fatty acid tail - On the other hand, cis-∆9 is indicative of a C=C double bond at the same position but with cis-configuration - What does trans-∆2 suggest?! Acyl-CoA 1 Acyl-CoA dehydrogenase trans-∆2-Enoyl-CoA 2 Enoyl-CoA hydratase L-β-Hydroxyacyl-CoA 3 β-Hydroxyacyl-CoA dehydrogenase β-Ketoacyl-CoA 4 β-Ketoacyl-CoA thiolase Acetyl-CoA FA Oxidation: (1) Acyl-CoA Dehydrogenase Dehydrogenation - Dehydrogenation of saturated Cα-Cβ single bond within acyl-CoA results in the formation of enoyl-CoA harboring a Cα=Cβ 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 Cα=Cβ double bond - Reaction catalyzed by acyl-CoA dehydrogenase using FAD as an oxidizing agent 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 Hydration - Hydration of unsaturated Cα=Cβ double bond within trans-∆2-enoylCoA (prochiral) results in the formation of L-β-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 Cβ position “primes” L-β-hydroxyacylCoA for subsequent oxidation to a keto group—that can then serve as a nucleophilic center for the release of first acetyl-CoA L-β-Hydroxyacyl-CoA FA Oxidation: (3) β-Hydroxyacyl-CoA Dehydrogenase Oxidation - Oxidation of –OH to a keto group at the Cβ position within L-β-hydroxyacyl-CoA results in the formation of corresponding βketoacyl-CoA - Reaction catalyzed by β-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-β-Hydroxyacyl-CoA β-hydroxyacyl-CoA dehydrogenase FA Oxidation: (4) β-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 β-ketoacyl-CoA results in the cleavage of Cα-Cβ bond, thereby releasing the first acetyl-CoA (to enter the Krebs cycle) and an outgoing acyl-CoA - Reaction catalyzed by β-ketoacyl-CoA thiolase - The outgoing acyl-CoA is two C atoms shorter than the parent acyl-CoA that entered the first round of β-oxidation—this acyl-CoA will undergo subsequent rounds of β-oxidation (Steps 14) to generate additional acetyl-CoA molecules—how many?! - Complete β-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: Bucketloads of ATP Palmitic Acid (16:0) Palmitoyl-CoA 6 - Palmitic acid is a saturated fatty acid harboring 16 carbon atoms (16:0) 7 FADH2 ETC 10.5 ATP 7 NADH ETC 17.5 ATP β-Oxidation - It is the most commonly occurring fatty acid in living organisms - So how much energy does β-oxidation of a single chain of palmitic acid (16 C atoms) generate? 8 Acetyl-CoA - Complete degradation of palmitic acid would require 7 rounds of β-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 Krebs cycle Fat Is hypercaloric! 24 NADH ETC 60 ATP 8 FADH2 ETC 12 ATP 8 GTP 8 ATP Total Energy = 108 ATP 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 βoxidation. 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 include small water-soluble molecules such as acetoacetate, acetone and β-hydroxybutyrate - Ketogenesis primarily occurs within the mitochondrial matrix of liver cells under conditions of starvation or in the case of non-carbohydrate diet during glucose shortage—why produce ketone bodies?!! - Being small and water-soluble, ketone bodies serve as important metabolic fuels for tissues 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 passively diffuse 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 an highly selective filter/barrier separating the circulating blood in the brain from the extracellular fluid Ketone Bodies: Ketogenesis SCoA Acetyl-CoA Ketone bodies include: - Acetoacetate - β-hydroxybutyrate - Acetone (1) How is acetyl-CoA converted to ketone bodies such as acetoacetate (2) How is acetoacetate subsequently utilized as a source of fuel via the Krebs cycle? Acetoacetate Conversion of acetone back to acetyl-CoA β-hydroxybutyrate is easily converted occurs via lactate and pyruvate in the liver back to acetyl-CoA via acetoacetate Acetoacetate decarboxylase (or spontaneously) NADH CO2 NAD+ β-hydroxybutyrate dehydrogenase H 3 Acetone β-Hydroxybutyrate Ketone Bodies: (1) Acetyl-CoA Acetoacetate [Liver] 1 Glutaric Acid (5C) 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 (2) Hydroxymethylglutaryl-CoA synthase adds another molecule of acetyl-CoA to acetoacetyl-CoA to generate β-hydroxyβ-methylglutaryl-CoA 3 (3) Hydroxymethylglutaryl-CoA lyase breaks down β-hydroxy-β-methylglutaryl-CoA into acetyl-CoA and acetoacetate—the ketone body Ketone Bodies: (2) Acetoacetate Acetyl-CoA [Heart|Brain] Ketone bodies such as acetoacetate and β-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 (1) β-hydroxybutyrate dehydrogenase mediates the oxidation of βhydroxybutyrate into acetoacetate (2) Ketoacyl-CoA transferase condenses acetoacetate with CoA (donated by succinyl-CoA) to generate acetoacetyl-CoA 2 (3) Thiolase breaks down acetoacetylCoA 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!) 3 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? Section 3.4c: Amino Acid Breakdown Synopsis 3.4c - After their release from dietary/cellular proteins, free amino acids can be broken down into the following metabolites for energy production (or in biosynthetic pathways): - α-Ketoglutarate - Succinyl-CoA - Fumarate - Oxaloacetate - Pyruvate - Acetoacetate - Acetyl-CoA - Of these seven metabolites, four are Krebs cycle intermediates: - α-ketoglutarate - Succinyl-CoA - Fumarate - Oxaloacetate - Pyruvate and acetoacetate (a ketone body) can be easily converted to acetyl-CoA–the spark that starts the Krebs cycle “ignition” by virtue of its ability to donate a two-carbon unit in the form of an acetyl group - In a nutshell, the breakdown products of amino acids essentially serve as a “fuel” for the Krebs cycle—but be aware that acetyl-CoA can also be converted into fatty acids! Protein Digestion Chymotrypsin —Ala—Ser—Phe—Ser—Lys—Gly—Ala—Arg—Trp—Thr—Asp—Tyr—Gly—Lys—Cys— Trypsin - Dietary proteins are degraded into free amino acids by the collaborative action of three major digestive proteases: pepsin, trypsin, and chymotrypsin - Trypsin preferentially cleaves peptide bonds on the C-terminus of Lys and Arg (see §2.2) - Chymotrypsin preferentially cleaves peptide linkages on the C-terminus of large hydrophobic residues such as Trp, Phe, and Tyr (see §2.2) - With preference for hydrophobic and aromatic residues, pepsin displays a high degree of promiscuity (or broad specificity) in its ability to cleave peptide bonds - The released amino acids enter the bloodstream from the digestive tract to be absorbed by other tissues - Once inside the cells, excess dietary amino acids are broken down into metabolic intermediates, many of which enter the Krebs cycle for energy production - During starvation, the degradation of cellular proteins serves as an alternative source of such free amino acids that are ultimately broken down into metabolic intermediates for energy production Products of Amino Acid Breakdown In the context of their catabolic breakdown, amino acids can be divided (albeit with a substantial overlap!) into two major groups: (1) Glucogenic amino acids—these are amino acids that can be directly broken down into glucose precursors such as pyruvate, αketoglutarate, succinyl-CoA, fumarate, or oxaloacetate—used in the synthesis of glucose (gluconeogenesis) (2) Ketogenic amino acids—these are amino acids that can be directly broken down into ketogenic precursors such as acetyl-CoA or acetoacetate—used in the synthesis of ketone bodies (ketogenesis) Helpful Hints: (a) Of the 20 standard amino acids, only Leu and Lys are NOT glucogenic—ie they are exclusively ketogenic! (b) Of the other 18 amino acids, only five amino acids are both glucogenic and ketogenic—Trp, Ile, Phe, Thr and Tyr (use WIFTY as a mnemonic!)