Fatty acid oxidation doc. Ing. Zenóbia Chavková, CSc.
Physiological functions of fatty acids 1. Structural components of cell membranes (phospholipids and sphingolipids) 2. Energy storage (triacylglycerols) 3. Signaling molecules (long-chain fatty acids) Structure of 5 FAs found in dietary lipids: Palmitic acid leic acid Linoleic acid Arachidonic acid Eicosapentaenoic acid
4 3 2 C 1 fatty acid with a cis- 9 double bond A 16-C fatty acid with numbering conventions Most naturally occurring fatty acids have an even number of carbon atoms Free fatty acids, are transported in the blood bound to albumin, a serum protein produced by the liver Long chain fatty acids are transported into cells, bound to several proteins, including the plasma membrane protein CD36
The pathway for catabolism of fatty acids is referred to as the -oxidation pathway, because oxidation occurs at the -carbon (C-3)
Fatty acid activation: In the 1 step, FAs must be activated (converted into CoA derivatives) in the cytoplasm before beeing oxidized in the mitochondria Acyl-CoA Synthases (Thiokinases, Fatty acyl CoA ligases), enzymes of ER & located on the outer mitochondrial membrane catalyze activation of long chain FAs, esterifying them to coenzyme A
There are different Acyl-CoA Synthases for FAs of different chain lengths The conversion of free FA into fatty acyl-coa is ATP-dependent process, requiring the cleavage of ATP & occurs in 2 steps Summary of fatty acid activation: 1. fatty acid + ATP acyladenylate + PP i +PP i 2 P i 2. acyladenylate + HS-CoA acyl-coa + AMP verall: fatty acid + ATP + HS-CoA acyl-coa + AMP + 2 P i
Acyl-CoA Synthases Fatty acid activation fatty acid R C N N NH 2 N N Exergonic PP i (P~P) hydrolysis is catalyzed by Phyrophosphatase P P P CH 2 H H H H H H ATP NH 2 2~P bonds of ATP are cleaved 2 P i PP i N N N N The acyl-coa product includes one "~" thioester linkage R C CoA SH AMP P CH 2 H H H H H H acyladenylate R C S CoA acyl-coa
The Carnitine Shuttle Fatty acyl-coa formed outside the mitochondria can pass through the outer mitochondrial membrane but cannot penetrate the inner membrane (Inner mitochondrial membrane is impermeable to fatty acyl-coa) Transport of FA moiety across the inner mitochondrial membrane requires the Carnitine Shuttle
Fatty acid - oxidation is considered to occur in the mitochondrial matrix Mitochondrion -xidation pathway in matrix Fatty acid must enter the matrix to be oxidized Fatty acyl-coa formed in cytosol by enzymes of outer mitochondrial membrane & ER However enzymes of the pathway specific for -oxidation very long chain fatty acid are associated with the inner membrane (facing the matrix)
Carnitine-mediated transfer of the fatty acyl moiety into the mitochondrial matrix is a 3-step process: 1. Carnitine Palmitoyl Transferase I, an enzyme on the cytosolic surface of the outer mitochondrial membrane, transfers a fatty acid from acyl-scoa to the -H on carnitine 2. An antiporter in the inner mitochondrial membrane mediates exchange of carnitine for acylcarnitine 3. Carnitine Palmitoyl Transferase II, an enzyme within the mitochondrial matrix, transfers the fatty acid from acylcarnitine to CoA The fatty acid is now esterified to CoA in the matrix
ATP + CoA AMP + PPi palmitate ACS [1] palmitoyl-coa palmitoyl-coa Intermembranecarnitine Space CPT-I [2] Activation of palmitate to palmitoyl CoA and mitochondrial uptake Cytoplasm UTER MITCHNDRIAL MEMBRANE ACS - Acyl CoA synthase CoA CPT - Carnitine Palmitoyl Transferase I,II CAT- Carnitine Acyl Translocase palmitoyl-carnitine CAT [3] INNER MITCHNDRIAL MEMBRANE MATRIX carnitine CPT-II [4] palmitoyl-carnitine palmitoyl-coa CoA
β-oxidation: Step 1 Acyl-CoA Dehydrogenase catalyzes oxidation of the fatty acyl-coa H to produce H a = bond between C 2 & 3 H 2 H There are different Acyl-CoA Dehydrogenases: For short (4-6 C), Medium (6-10 C), H Long and very long (12-18 C) chain fatty acids Very Long Chain Acyl-CoA Dehydrogenase is bound to the inner mitochondrial membrane The others are soluble enzymes located in the mitochondrial matrix FAD is the electron acceptor H H 3 C (CH 2 ) n C C C SCoA FAD FAD H 2 H 3 H 3 C (CH 2 ) n C C C SCoA H 3 C (CH 2 ) n C CH 2 C SCoA H + + NADH NAD + H 3 C (CH 2 ) n C CH 2 C SCoA HSCoA H H 2 1 fatty acyl-coa Acyl-CoA Dehydrogenase trans- 2 -enoyl-coa H 3 C (CH 2 ) n C SCoA + CH 3 C SCoA
H 3 C C SCoA acetyl-coa ATP + HC 3 ADP + P i C malonyl-coa Acetyl-CoA Carboxylase (inhibited by AMP-Activated Kinase) CH 2 C SCoA Control of FA oxidation is exerted mainly at the step of FA entry into mitochondria Malonyl-CoA inhibits Carnitine Palmitoyl Transferase I. (Malonyl-CoA is also a precursor for FA synthesis) AMP-Activated Kinase, a sensor of cellular energy levels, catalyzes phosphorylation of Acetyl-CoA Carboxylase under conditions of high AMP (when ATP is low) Phosphorylation inhibits Acetyl-CoA Carboxylase, thereby decreasing malonyl-coa production The decrease in malonyl-coa releases Carnitine Palmitoyl Transferase I from inhibition
Step 2. Enoyl-CoA Hydratase catalyzes hydration of the trans double bond yielding L-hydroxyacyl- Coenzyme A H H 3 H 3 C (CH 2 ) n C C C SCoA FAD H H FAD H 2 H H 3 C (CH 2 ) n C C C SCoA H H 2 H H 3 C (CH 2 ) n C CH 2 C SCoA H 2 1 fatty acyl-coa Acyl-CoA Dehydrogenase trans- 2 -enoyl-coa Enoyl-CoA Hydratase 3-L-hydroxyacyl-CoA H + + NADH
Step 3. H 2 H H Hydroxyacyl-CoA Dehydrogenase catalyzes oxidation of the -H in the β-position (C 3) to a ketone NAD+ is the electron acceptor H 3 C (CH 2 ) n C CH 2 C SCoA NAD + H H + + NADH H 3 C (CH 2 ) n C CH 2 C SCoA HSCoA 3-L-hydroxyacyl-CoA Hydroxyacyl-CoA Dehydrogenase H 3 C (CH 2 ) n C SCoA + CH 3 C SCoA -ketoacyl-coa -Ketothiolase fatty acyl-coa acetyl-coa (2 C shorter)
Step 4. b-ketothiolase catalyzes thiolytic cleavage H 3 C (CH 2 ) n C CH 2 C SCoA HSCoA -ketoacyl-coa H 3 C (CH 2 ) n C SCoA + CH 3 C SCoA fatty acyl-coa acetyl-coa (2 C shorter) -Ketothiolase Acetyl-CoA is released, leaving the fatty acyl moiety in thioester linkage to the cysteine -SH The thiol of HS-CoA displaces the cysteine thiol, yielding fatty acyl-coa (2 C less)
β-xidation-summary FAD-dependent dehydrogenation Hydration NAD-dependent dehydrogenation Cleavage
Palmitoylcarnitine Inner mitochondrial membrane Carnitine translocase Respiratory chain Matrix side Palmitoylcarnitine Palmitoyl-CoA FAD oxidation FADH 2 hydration H 2 2 ATP 3 ATP recycle 6 times oxidation cleavage NAD + NADH CoA CH 3 -(CH) 12 -C-S-CoA + Acetyl CoA l Citric acid cycle 2 C 2
Summary of one round of the -oxidation pathway: fatty acyl-coa + FAD + NAD + + HS-CoA fatty acyl-coa (2 C less) + FADH 2 + NADH + H + + acetyl-coa The -oxidation pathway is cyclic The product, 2 carbons shorter, is the input to another round of the pathway
ATP YIELD DURING THE -XIDATIN F FAs The energy yield from FA oxidation is high From palmitoyl CoA to 8 acetyl CoA ATP (yield) 7 NADH, which each provide 3 ATP when oxidized by the electron transport chain 21 7 FADH 2, which each provide 2 ATP when oxidized by the electron transport chain 14 From the 8 acetyl-coa, which each provide 12 ATP when converted to C 2 and H 2 by the TCA cycle 96 Total energy yield from 1 molecule of palmitoyl CoA 131
Fatty acid oxidation is a major source of cell ATP The reactions presented accomplish catabolism of a fatty acid with an even number of C atoms & no double bonds Additional enzymes deal with catabolism of fatty acids with an odd number of C atoms or with double bonds
XIDATIN F FATTY ACIDS WITH AN DD NUMBER F CARBNS A small proportion of natural lipids contain odd numbers of C atoms The -oxidation proceeds by the same reaction steps than that of FA with even number of C atoms, until the final round of -oxidation is reached yielding acetyl-coa and propionyl-coa Propionyl-CoA is converted to the Krebs cycle intermediate succinyl-coa, by ATP-dependent 2 step pathway involving vitamin B 12
xidation of odd chain FAs Propionyl CoA is carboxylated, forming methylmalonyl-coa Propionyl CoA carboxylase (biotin-dependent) catalyzes the reaction: propionyl CoA + ATP + C 2 methylmalonyl CoA + AMP + PPi The carbons of methylmalonyl CoA are rearranged, forming succinyl CoA, (can enter the TCA for further oxidation) Methylmalonyl CoA mutase (adenosyl cobalamin-dependent) catalyzes the reaction: methylmalonyl CoA succinyl CoA
Most double bonds of naturally occurring fatty acids have the cis configuration As C atoms are removed two at a time, a double bond may end up: In the wrong position Wrong configuration for the enoyl-coa to be a substrate for Enoyl-CoA Hydratase
XIDATIN F UNSATURATED FATTY ACIDS The oxidation of unsaturated FAs is essentially the same process as for saturated fats, except when a double bond is encountered In such case, the bond is isomerised by a specific enoyl-coa isomerase and oxidation continues Wrong position Wrong configuration
The oxidation of unsaturated fatty acids: Provides less energy than that of the saturated because they are less highly reduced Therefore fewer reducing equivalents can be produced from these structures
- AND - FATTY ACID XIDATIN IN PERXISMES -oxidation which occurs in peroxisomes involves NADPH, molecular 2, cytochromes, free fatty acid (not the CoA derivative), -hydroxylase and -oxidase The precise mechanism of -oxidation has not been yet established The -oxidation step generates a substrate for the -oxidation
Phytanic acid: Fatty acid present in the tissues of ruminants and in dairy products Therefore, an important dietary component of fatty acid intake A rare inborn error of lipid metabolism, Refsum s disease, results from a defect in the -hydroxylase step of -oxidation
There are differences in the peroxisomal and mitochondrial processes of -oxidation: Peroxisomes can initiate the oxidation of fatty acids longer than 18 C atoms, but mitochondria cannot Carnitine is required for the translocation of FAs into mitochondria but not into peroxisomes (by simple diffusion) The first oxidation step in the peroxisomal pathway, differs from that of the mitochondrial: Uses 2 as the oxidant (FADH 2 ), Products include the trans, -unsaturated fatty acyl-coa and H 2 2, degraded by catalase to 2 and H 2
Within the peroxisome, FADH 2 generated by FA oxidation is reoxidized producing H 2 2 : FADH 2 + 2 FAD + H 2 2 The peroxisomal enzyme Catalase degrades H 2 2 : 2 H 2 2 2 H 2 + 2 These reactions produce no ATP! Both mitochondrial and peroxisomal -oxidation require acyl-coa synthetases that use ATP and generate AMP, PP i and acyl-coa The unsaturated fatty acyl-coa in peroxisomes is metabolized by the familiar steps of -oxidation
- oxidation by the endoplasmic reticulum It is a third, but a minor pathway for FAs oxidation This process involves: NADPH, 2 and cytochrome P-450 The substrate include: Medium- and long-chain free FAs The -methyl group is first hydroxylated and then oxidized to a carboxylate, which is decarboxylated as in -oxidation After attachment to CoA, -oxidation can continue from both ends of the molecule The product of -oxidation is a dicarboxylate! -oxidation is one mechanism for removing branches containing multiple carbons!
Hereditary deficiency of Medium Chain Acyl-CoA Dehydrogenase (MCAD), the most common genetic disease relating to FA catabolism, can lead to sudden death in infants (SIDS) In the first years of life this deficiency will become apparent following a prolonged fasting period Symptoms include vomiting, lethargy, frequently coma Excessive urinary excretion of medium-chain dicarboxylic acids as well as their glycine and carnitine esters is diagnostic of this condition
Medium-chain length Acyl-CoA dehydrogenase deficiency is found in approximately 1 in 10,000 births and is therefore more prevalent than phenylketonuria It causes a decrease in FA oxidation severe hypoglycemia and is the cause of up to 10% of cases of sudden infant death syndrome (SIDS) In the case of this enzyme deficiency, taking care to avoid prolonged fasting is sufficient to prevent clinical problems
KETNE BDIES - AN ALTERNATE FUEL FR CELLS When carbohydrate utilization is low or deficient, the level of oxaloacetate will also be low, resulting in a reduced flux through the TCA cycle This in turn leads to release of ketone bodies from the liver for use as fuel by other tissues Ketone body is a nonsystematic term for acetoacetate, -hydroxybutyrate, and acetone
During fasting, carbohydrate starvation, or diabetic ketoacidosis, oxaloacetate is depleted in liver due to gluconeogenesis Gluconeogenesis oxaloacetate Acetyl-CoA in liver mitochondria is converted then to ketone bodies Glucose-6-phosphatase glucose-6-p glucose pyruvate Glycolysis fatty acids acetyl CoA ketone bodies cholesterol citrate Krebs Cycle
Ketone body synthesis: β-ketothiolase Acetoacetyl-CoA is formed by condensation of 2 moles of acetyl-coa through a reversal of the thiolase catalyzed reaction of fat oxidation HMG-CoA Synthase, an enzyme found in large amounts only in the liver, catalyzes condensation with a 3 rd acetate moiety (from acetyl-coa) HMG-CoA Lyase H 3 C C SCoA + H 3 C C SCoA acetyl-coa acetyl-coa HSCoA Thiolase H 3 C C acetyl-coa C H 2 C acetoacetate H 3 C SCoA HSCoA C C H 2 C C H C CH 3 H 2 C C SCoA acetoacetyl-coa H 2 C HMG-CoA Synthase C SCoA HMG-CoA HMG-CoA Lyase CH 3 + H 3 C C SCoA acetyl-coa cleaves HMG-CoA to yield acetoacetate & acetyl-coa
Ketone bodies: Carried by the circulation to extrahepatic cells Converted back to acetyl-coa for catabolism in Krebs cycle Acetoacetate and 3-hydroxybutyrate are oxidized to produce energy Acetone is a metabolic dead end that is: Expired from the lungs r excreted in the urine It cannot be oxidized to produce energy!
Some of the HMG-CoA leaves the mitochondria, where it is converted to mevalonate (the precursor for cholesterolsynthesis) by HMG-CoA reductase -Hydroxybutyrate dehydrogenase catalyzes reversible inter-conversion acetoacetate and -hydroxybutyrate Acetoacetate is reduced to -hydroxybutyrate via -hydroxybutyrate dehydrogenase that uses NADH with the eventual production of acetyl CoA -Hydroxybutyrate Dehydrogenase CH 3 C CH 2 H + NADH NAD + H CH 3 CH CH 2 C acetoacetate C D- -hydroxybutyrate
Ketone bodies are utilized by extrahepatic tissues through the conversion of 1. -hydroxybutyrate to acetoacetate 2. of acetoacetate to acetoacetyl-coa 1. Step involves the reversal of the -hydroxybutyrate dehydrogenase reaction 2. Step involves the action of acetoacetate:succinyl-coa transferase, also called ketoacyl-coa-transferase acetoacetate + succinyl-coa <------> acetoacetyl-coa + succinate
-Hydroxybutyrate NAD + Succinyl CoA synthetase = NADH loss of GTP Acetoacetate -Hydroxybutyrate Succinyl CoA dehydrogenase CoA transferase CoA Succinate 2 Acetyl CoA Acetoacetyl CoA Thiolase xidation of ketone bodies in brain, muscle, kidney, and intestine Citric Acid Cycle Acetoacetate:succinyl-CoA transferase is enzyme present in all tissues except the liver Its absence allows the liver to produce ketone bodies but not to utilize them This ensures that extrahepatic tissues have access to ketone bodies as a fuel source during prolonged starvation
Ketone bodies synthesis occurs exclusively in liver mitochondria Ketone bodies are important sources of energy because: They are soluble in aqueous solution (do not need to be incorporated in lipoproteins or carried by albumin as do the other lipids) They are produced in the liver when the amount acetyl-coa present exceeds the oxidation capacity of the liver They are used in extrahepatic tissues (the skeletal and cardiac muscles, renal cortex, even the brain)
Adipose Tissue X Free fatty acids Liver Insulin Ketone Bodies Pancreas Mechanism for prevention of ketoacidosis from excess ketone body production Ketoacidosis is prevented in starvation by ketones promoting secretion of insulin that in turn slows down lipolysis, the source of the carbons for ketone production
Ketone bodies are relatively strong acids (pka around 3.5), and their increase lowers the ph of the blood. This acidification of the blood is dangerous chiefly because it impairs the ability of hemoglobin to bind oxygen
TRIACYLGLYCERL (TRIGLYCERIDE) DEGRADATIN
MBILIZATIN F STRED FATS FAs stored primarily within adipocytes of adipose tissue in the form of neutral triacylglycerols serve as the body s major fuel storage reserve Triacylglycerols are the form in which we store reduced C for energy In response to energy demands by peripheral tissues, FAs must be released from storage as triacylglycerols
H 2 C HC H H H C R H 2 C HC C C R R H 2 C H H 2 C C R glycerol fatty acid triacylglycerol Lipases hydrolyze triacylglycerols, releasing fatty acids at a time, yieldingdiacylglycerols, monoacylglyceros, eventually glycerol
The release of FAs from triacylglycerols is hormonally sensitive event, initiated by hormone-sensitive lipase that removes a FA from either C-1 or C-3 of the TAG Additional lipase, specific for monoacylglycerol or diacylglycerol removes the remaining FAs
The release of metabolic energy, in the form of FAs, is controlled by a complex series of interrelated cascades that result in the activation of hormone-sensitive lipase The stimulus to activate this cascade in adipocytes can be glucagon, epinephrine or -corticotropin These hormones bind cell-surface receptors that are coupled to the activation of adenylate cyclase upon ligand binding
The net result of the action of these enzymes is: 3 moles of free FA 1 mole of glycerol The free fatty acids: Diffuse from adipose cells Combine with albumin in the blood Transported to other tissues, where they passively diffuse into cells In the presence of high plasma levels of insulin and glucose, hormone-sensitive lipase is dephosphorylated and becomes inactive
Fate of glycerol Glycerol cannot be metabolized by adipocytes because they lack glycerol kinase Glycerol is transported through the blood to the liver which can phosphorylate it The resulting glycerol phosphate: Can be use to form TAG in the liver r can be converted to DHAP by reversal of the glycerol dehydrogenase reaction DHAP can participate in glycolysis or gluconeogenesis
CH 2 H ATP ADP CH 2 H NAD + H + + NADH CH 2 H H CH CH 2 H H CH 1 2 CH 2 P 3 C CH 2 P 3 glycerol glycerol-3-p dihydroxyacetone-p Glycerol, from hydrolysis of TAGs, is converted to the glycolysis intermediate dihydroxyacetone phosphate, by reactions catalyzed by: 1 Glycerol kinase 2 Glycerol phosphate dehydrogenase