Part III => METABOLISM and ENERGY. 3.4 Lipid Catabolism 3.4a Fatty Acid Degradation 3.4b Ketone Bodies

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1 Part III => METABOLISM and ENERGY 3.4 Lipid Catabolism 3.4a Fatty Acid Degradation 3.4b Ketone Bodies

2 Section 3.4a: Fatty Acid Degradation

3 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 FADH 2, NADH, and acetyl-coa - Acetyl-CoA is subsequently oxidized via the Krebs cycle and the energy released is stored in the form of GTP, FADH 2 and NADH see FADH 2 and NADH ultimately donate their electrons to produce ATP via the electron transport chain (ETC) see 3.6

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 Acid Nomenclature cis- 9 -dodecanoate - While the x:m symbolism provides insights into the length and the degree of unsaturation of a fatty acid (see 1.4), 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 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?!

6 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 - In order to release such energy to be used as free energy, triglycerides are first deesterified 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

7 (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 Fatty Acid 1 Acyl-CoA synthetase Acyl-CoA (cytosolic) - 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 2 Acyl-carnitine 3 Acyl-carnitine 4 Carnitine acyltransferase I Carnitine-acylcarnitine translocase (Mitochondrial Transit) Carnitine acyltransferase II Acyl-CoA (mitochondrial matrix)

8 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)?

9 FA Import: (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 (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

10 FA Import: (3) Carnitine-Acylcarnitine Translocase Cytosol (intermembrane space) Mitochondrial Matrix 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

11 FA Import: (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

12 RCOOH FA Import: Outline 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

13 (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 Acyl-CoA 1 Acyl-CoA dehydrogenase trans- 2 -Enoyl-CoA - 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: 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! 2 L- -Hydroxyacyl-CoA 3 -Ketoacyl-CoA 4 Acetyl-CoA Enoyl-CoA hydratase -Hydroxyacyl-CoA dehydrogenase -Ketoacyl-CoA thiolase

14 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 FADH 2 - FADH 2 will be subsequently reoxidized back to FAD via the mitochondrial electron transport chain (ETC)

15 FA Oxidation: (2) Enoyl-CoA Hydratase 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 L- -Hydroxyacyl-CoA

16 FA Oxidation: (3) -Hydroxyacyl-CoA Dehydrogenase Oxidation L- -Hydroxyacyl-CoA - Oxidation of OH to a keto group at the C position within L- -hydroxyacyl-coa results in the formation of corresponding - ketoacyl-coa -hydroxyacyl-coa dehydrogenase - 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)

17 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 FADH 2! That would be bucketloads of energy but exactly how much?!

18 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) - 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 FADH 2, 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 FADH 2 and 1 GTP (enzymatically converted to ATP) per molecule (and there are 8 acetyl-coa!) see Oxidation of each NADH and FADH2 via the ETC respectively produces 2.5 and 1.5 molecules of ATP see Acetyl-CoA Krebs cycle -Oxidation 7 FADH 2 7 NADH 24 NADH 8 FADH 2 8 GTP ETC ETC ETC ETC 10.5 ATP 17.5 ATP Fat Is hypercaloric! 60 ATP 12 ATP 8 ATP Total Energy = 108 ATP

19 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?

20 Section 3.4b: Ketone Bodies

21 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?!!

22 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 Ketone Bodies: Physiological Significance 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: (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

23 Ketone Bodies: Ketogenesis (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? SCoA Acetyl-CoA Ketone bodies include: - Acetoacetate - -hydroxybutyrate - Acetone Conversion of acetone back to acetyl-coa occurs via lactate and pyruvate in the liver Acetoacetate decarboxylase (or spontaneously) Acetone CO 2 However, acetone is usually excreted via urine and/or exhaled 3 Acetoacetate -hydroxybutyrate is easily converted back to acetyl-coa via acetoacetate NADH NAD + -hydroxybutyrate dehydrogenase H -Hydroxybutyrate

24 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: (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 2 3

25 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: (1) -hydroxybutyrate dehydrogenase mediates the oxidation of - hydroxybutyrate into acetoacetate 1 (2) Ketoacyl-CoA transferase condenses acetoacetate with CoA (donated by succinyl-coa) to generate acetoacetyl-coa 2 (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!) 3

26 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?