Biochemistry - I Mondays and Wednesdays 9:30-10:45 AM (MR-1307) SPRING 2017 Lectures 21-22 Based on Profs. Kevin Gardner & Reza Khayat 1
Outline Vertebrate processing of dietary lipids Mobilization of triacylglycerols for catabolism Fatty acid oxidation (beta) Monounsaturated fatty acid oxidation Polyunsaturated fatty acid oxidation Odd numbered fatty acid oxidation Regulation of fatty acid synthesis/breakdown Mitochondria, organelles of substrate shuttling Omega oxidation Alpha Oxidation Ketone bodies Why learn about Fatty Acid Catabolism? Fatty acids store the majority of our energy, and improper oxidation of these molecules lead to a number of diseases. 2
Lipids Amphiphilic :hydrophilic (aqueous phase) and lipophilic (aliphatic phase) 3
Triacylglycerols (Triglycerides) Storage lipid (fats) 3 fatty acids (same or different) attached to each glycerol (three condensation reactions between alcohol and carboxylic acid - esterification) Nonpolar and water insoluble Lower specific gravity than water (oil floats on water) Stored in adipocytes (fat cells) Lipases (esterases) hydrolyze esters to release fatty acids and glycerol 1 3 2 4
Vertebrate Processing of Dietary Lipids Cholic acid One of several primary bile acids synthesized in liver from cholesterol Surround triacylglycerols to form micelle-like structures with embedded lipases (enzymes that hydrolyze lipid heads from tails) Role is to solubilize and hydrolyze fatty acids 5
Vertebrate Processing of Dietary Lipids epithelial cells 6
Chylomicrons Range in size from 100-500nm The surface is a layer of phospholipids Cholesterol gives rigidity to surface Triacylglycerols make up 80% of mass Embedded into structure are apolipoproteins, proteins responsible for transport of FA between organs Apolipoproteins act as signals for cells to uptake and metabolize chylomicron contents 7
Mobilization of Triacylglycerols from Adipocyte for Catabolism (0) Release of glucagon as result of low [glucose] (1) Glucagon binds specific receptor on adiopocyte (2) Stimulates adenylyl cyclase, via a G protein, to produce camp camp activates PKA (3) PKA phosphorylates hormone-sensitive lipase (4) PKA phosphorylates perilipin protein on surface of lipid droplet (5) lipase moves to lipid droplet and hydrolyzes triacylglycerols into component parts (6) Fatty acids leave adiopocyte, bind serum albumin (= a lipoprotein) in blood (7) Fatty acids enter myocyte via transporter (8) Fatty acids are oxidized to CO2 to generate ATP 8
Fate of Liberated Glycerol in the Adipocyte Reminder: D-glyceraldehyde 3-phosphate is the product of glycolysis R5! 9
Fatty Acid Oxidation The liberated fatty acid (from the triacylgycerol stored in adipocyte) can be transported into myocyte for oxidation The fatty acid needs to be modified by CoA for any chemistry to occur Fatty acyl-coa synthetase carries out this reaction in two steps Uses ATP to activate the fatty acid by forming a phosphodiester bond between fatty acid and AMP. Released are 2 inorganic phosphates Transfers CoA to fatty acid and kicks off AMP Both reactions are exergonic 10
mbrane (Fig. 17 6). CH 3 Fatty Acid oxidation CH 3 N CH 2 CH CH 2 COO CH 3 OH Carnitine Fatty acid-coa can either be used in cytosol to synthesize membrane lipids or transferred to the mitochondrion for oxidation Carnitine acyltransferase I attaches carnitine to FA-CoA for transfer Not known if modification occurs on membrane surface or in the inter membrane space Enzyme is inhibited by malonyl-coa, first intermediate in FA synthesis Carnitine acyltransferase II transfers mitochondrial CoA to FA, removing carnitine Carnitine is transported back out to be used again FA catabolism occurs in mitochondria FA anabolism occurs in cytoplasm w/acetyl-coa as starting reactant 11
Fatty Acid Oxidation ω Multiple types of fatty acid oxidation exist: α β ω Beta oxidation: Removes two carbons at a time to generate acetyl-coa. This is an oxidation mechanism, meaning that FADH2 and NADH are produced Oxidation occurs between α and β carbons Acetyl-CoA enters TCA for oxidation and NADH +FADH2 production, or to generate intermediates for anabolic reactions All the reducing agents (NADH and FADH2) can be used for a variety of biochemical steps, including ATP generation via the electrontransfer chain β α 12
β-fatty Acid Oxidation Used for saturated fatty acids, e.g. palmitoyl-coa (16 carbon fatty acid) Concentrate on the β-carbon and its vicinity. Reactions break the bond between the α- and the β-carbon (1) dehydrogenate between α- and β-carbon to make trans bond (2) hydrate the same bond (3) oxidize the OH on β-carbon to a ketone (= beta oxidation) (4) CoA-SH displaces acetyl-coa by attacking carbonyl at β-carbon Two carbons are removed in each cycle Total num. of cycles = (CN - 2) / 2 Each cycle produces 1 FADH2 and 1 NADH+H + FADH2 produces 1.5 ATP in electron transport chain passes electrons to ETC complex (pg. 713) NADH produces 2.5 ATP in electron transport chain NADH + H + + 1/2 O2 NAD + + H2O Acetyl-CoA also can go through TCA to generate more power 1 2 3 4 Palmitoyl-CoA 23O 2 108P i 108ADP CoA 108ATP 16CO 2 23H 2 O 13
Oxidation of Monounsaturated Fatty Acids Proceed as usual until a cis-double bond is reached Convert and reposition the cis-double bond to a trans-double bond If cis-δ 3 bond, then cis-δ 3, Δ 2 -Enoyl CoA isomerase enzyme carries next step If cis-δ 4 double bond, then 2,4 Dienoyl CoA reductase enzyme carries next step If cis-δ 2 double bond an enzyme converts to trans and beta-oxidation proceeds 4 3 2 1 Proceed as usual until another cis-double bond is reached (next slide) 14
Oxidation of Polyunsaturated Fatty Acids Proceed as usual until two consecutive cisdouble bonds are reached Convert and reposition the cis-double bond to a trans-double bond cis-δ 3 bond, then cis-δ 3, Δ 2 -Enoyl CoA isomerase moves double bond between C2 and C3 and isomerize it to trans. Steps 2-4 of beta oxidation can proceed normally. if cis-δ 4 double bond, then two enzymes are needed after step 1 of beta-oxidation generates a new trans Δ 2 double bond (generating a Δ 2, Δ 4 diene). One new enzyme 2,4 Dienoyl CoA reductase, converts the two double bonds into a single trans Δ 3 bond; next, a Δ 3, Δ 2 -Enoyl CoA isomerase isomerizes this into a trans Δ 2 bond that can normally be handled by steps 2-4 of beta oxidation. 15
Oxidation of Odd-numbered Fatty Acids Proceed as usual until 3 carbons left Use bicarbonate to add a CO2 to C2, generating 4C species Change enantiomer (D to L): Methyl malonyl-coa mutase uses coenzyme B12 to swap the C1 hydrogen and C3 methyl- CoA to generate succinyl-coa (product of reaction 4 in TCA). Succinyl-CoA can enter TCA 16
Regulation of Fatty Acid Synthesis/Breakdown glucagon High [glucose] in blood = stop FA and glycogen catabolism, and start anabolism Insulin activates insulin-dependent protein phosphatase Phosphatase dephosphorylates ACC-P (acetyl-coa carboxylase) ACC catalyzes formation of malonyl-coa malonyl-coa inhibits carnitine acyltransferase I, preventing FA entry into mitochondria = malonyl-coa promotes FA-anabolism Low [glucose] in blood = start fatty acid and glycogen catabolism Glucagon turns on PKA which phosphorylates and inactivates ACC 17
Regulation of Fatty Acid Synthesis/Breakdown 18
and gram-negative bacteria (see Fig. 1 6). In mitochondria, the four!-oxidation enzymes that act on short-chain fatty acyl CoAs are separate, soluble proteins (as noted earlier), similar in structure to the analogous enzymes of gram-positive bacteria (Fig. 17 15a). The gram-negative bacteria have four activities in three soluble subunits (Fig. 17 15b), and the eukaryotic enzyme system that acts on long-chain fatty acids the trifunctional protein, TFP has three enzyme activities in two subunits that are membraneassociated (Fig. 17 15c). The!-oxidation enzymes Enz2 Enz4 The enzymes of β-oxidation (b) Gram-negative bacteria (a) Gram-positive bacteria and mitochondrial short-chain-specific system Substrate (c) Mitochondrial very-long- Substrate Product Enz4 Enz1 Matrix Enz2 Enz4 Enz1 Product Enz1 Enz2 system of plants Substrate Intermediate Product Enz3 (d) Peroxisomal and glyoxysomal chain-specific system Substrate Enz1 Intermediate Intermediate Enz3 Product Enz3 Enz2 Enz4 MFP Enz2 Enz4 Enz6 Enz3 Intermediate Intermediate Enz3 Inner membrane Enz5 (d) Peroxisomal and glyoxysomal system of plants FIGURE 17 15 The enzymes of! oxidation. Shown here are the different subunit struc- tures of the enzymes of! oxidation in gram-positive and gram-negative bacteria, mitoenz1substrate, acyl-coa dehydrogenase chondria, and plant peroxisomes and glyoxysomes. Enz is acyl-coa dehydrogenase; Enz, enoyl-coa hydratase 2 Enz Enz, enoyl-coa hydratase; Enz, -!-hydroxyacyl-coa dehydrogenase; Enz, thiolase; Product Enz, -3-hydroxyacyl-CoA epimerase, and Enz, "," -enoyl-coa isomerase. (a) The Enz3, L-beta-hydroxyacyl-CoA dehydrogenase four enzymes of! oxidation in gram-positive bacteria are separate, soluble entities, as are MFP those of the short-chain-specific system of mitochondria. (b) In gram-negative bacteria, Enz Enz 4, thiolase Enz the four enzyme activities reside in three polypeptides; Enz and Enz are parts of a single Enz5, D-3-hydroxyacyl-CoA epimerase polypeptide chain. (c) The very-long-chain-specific system of mitochondria is also comenz posed of three polypeptides, one of which includes the activities of Enz and Enz ; in this Enz 3 2 Enz6, Δ, Δ -enoyl-coa isomerase case, the system is bound to the inner mitochondrial membrane. (d) In the peroxisomal and glyoxysomal!-oxidation systems of plants, and Enz are separate polypeptides, systems believed to have diverged from aenzcommon ancestor early in evolution Enz but Enz and Enz, as well as two auxiliary enzymes (Enz and Enz ), are part of a single polypeptide chain: the multifunctional protein, MFP. of life 1 1 2 5 4 3 D L 4 3 6 2 2 2 3 6 2 3 1 5 2 3 3 4 5 6 19
ω Oxidation of Fatty acids in the Smooth ER Minor pathway for healthy individual, but relied upon by diabetic, chronically alcoholic and starving individuals to eliminate toxic levels of free fatty acids Enzymes located in endoplasmic reticulum (ER) of liver and kidney of vertebrates Fatty acids with 10-12 carbons are preferred Either end can be attached to CoA, then enters mitochondria n-cycles of β-oxidation yields succinic acid (succinate) and adipic acid 12C Succinic acid enters TCA, adipic acid subsequently converted into succinate + acetyl CoA + 4C 6C 20
Alpha Oxidation of Fatty Acids in the Peroxisome Presence of methyl groups on α or β requires different chemistry Occurs in peroxisomes If CH3 on β, carry out α-oxidation to put on α If CH3 on α, carry out β-oxidation to produce propionyl-coa First step is to remove the first carbon via alpha oxidation Next step is to carry out a β-oxidation The methyl group on C-beta results in production of propinoyl-coa rather than Acetyl-CoA α-oxidation and β-oxidation switch back and forth to catabolize the branched fatty acid Refsum disease occurs from a genetic defect in phytanoyl-coa hydroxylase. This leads to high levels of phytanic acid severe neurological problems, including deafness and blindness δ β α β α CO2 from α- ketoglutarate alpha oxidation beta oxidation 21
Peroxisomes: Specialized Organelles for FA Oxidation Present in all eukaryotes Catabolism of long FA (> 22 carbon) Some plant cells have specialized peroxisomes ( glyoxysomes ), site of FA oxidation of the large supplies of FA found in seeds Not capable of oxidative phosphorylation Produced FADH2 generates H2O2 Catalase oxidizes reduces O2 to H2O with H2O2 Need to shuttle the NADH to the cytosol then mitochondrion for ATP generation Mitochondrion oxidizes FADH2 and NADH using a series of enzymes embedded in its cristae (membrane leaflets). The mitochondrion is very much a factory 22
Ketone Bodies Occurs during fasting, carbohydrate restrictive diets, prolonged exercise, type 1 diabetes, and starvation FA oxidation in liver produces acetyl-coa that enters TCA When the TCA cycle slow (e.g. TCA intermediates are being used for anabolism) in liver, [acetyl-coa] increases Acetyl-CoA is converted to ketone bodies in mitochondria (acetoacetate, acetone and D-β-Hydroxybutarate) HMG-CoA is precursor to sterol synthesis Liver cannot catabolize acetoacetate (does NOT have enzyme thiolase, top reaction) Acetoacetate and D-β-hydroxybutarate exported via blood to other organs and converted to acetyl-coa for TCA Acetone is toxic acetone exhaled, ketosis, sometimes useful in diagnosing diabetes Untreated diabetes mellitus (low insulin low glucose uptake by cells FA oxidation not inhibited [acetyl- CoA]inc TCA depleted for gluconeogenesis) and starvation (gluconeogenesis depletes TCA intermediates) produces high amount of ketone bodies 23
Ketone Bodies 24
Ketone Bodies OH O CH 3 C CH 2 C D- -Hydroxybutyrate H O D- -hydroxybutyrate dehydrogenase NAD NADH H CH 3 CH 3 -ketoacyl-coa transferase CH 3 C O C thiolase O O C S-CoA CH 2 CH 2 C O O Succinyl-CoA Succinate C CH 3 2 Acetyl-CoA O CoA-SH S-CoA C O S-CoA Acetoacetate Acetoacetyl-CoA Type 1 diabetes: a. Insulin-dependent b. Starts in childhood c. Autoimmune disease, antibodies attack pancreas such that it can t make insulin Type 2 diabetes: a. Onset diabetes due to living habits b. More common form c. Non-insulin dependent. Pancreas makes insulin but unable to signal, usually because the target cells do not have the receptors expressed. 25