Class Note for BIOC 460 at UA
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Date Created: 02/06/15
Biochemistry 460 Dr Tischler LIPOLYSIS BETAOXIDATION KETONES LIPOGENESIS Related Reading Chapter 22 619644 in Stryer 639h edition OBJECTIVES 1 For the lipolytic pathway lipolysis describe the pathway identify where it occurs name the principal enzyme involved and explain the role of albumin and fatty acid binding protein in the transport and metabolism of free fatty acids liberated by lipolysis 2 For the degradation of fatty acyl CoAs describe the roles of acyl CoA synthetase carnitine palmitoyl transferases CPTI and CPTII and carnitine acylcarnitine translocase CAT and discuss the relationship of the products of the Boxidation pathway to energy production 3 For ketone body metabolism identify where and when ketone body formation ketogenesis occurs state the role of ketogenesis identify where ketone oxidation occurs and explain why normally individuals do not develop ketoacidosis even when producing ketone bodies 4 Describe the reactions catalyzed by malic enzyme and acetyl CoA carboxylase 5 For the fatty acid synthase reaction list the substrates and key products identify the sources of NADPH for the reaction and describe its general mechanism 6 Describe how fatty acids are stored as a source of fuel during starvation or stress PHYSIOLOGICAL PREMISE Would you believe that diabetics having a ketotic crisis have actually been arrested for DUIs even though they have consumed no alcohol Indeed a blood analysis would show no alcohol Why would this occur During a ketotic crisis a byproduct of the excess ketone production is acetone Having nowhere else to go it is expired through the lungs It is the acetone that arresting officers have smelled on the breath of these individuals and despite their protestations have innocently believed them to be consuming alcohol LIPOLY SIS Lipolysis is a simple process whereby the fatty acids attached to glycerol in triacylglycerols are hydrolytically removed yielding free fatty acids plus glycerol Lipolysis largely occurs in adipose tissue for the mobilization of fatty acids to serve as a fuel in the body as well as a precursor for the synthesis of ketone bodies Additionally lipolysis may also occur in muscle or liver where smaller amounts of fatty acids are stored to produce energy for the use of the cell in which they are stored Hormone sensitive cyclic AMP regulated lipase initiates lipolysis by cleaving off the rst fatty acid Then this lipase and other lipases remove the remaining two fatty acids from the glycerol backbone The fatty acids and glycerol are then released from the adipose tissue into the blood Glycerol is watersoluble and therefore can freely travel through the blood However fatty acids are very hydrophobic because of their long hydrocarbon tails Consequently they must bind to albumin a protein released from liver to be carried through the blood Lipolysis Beta Oxidation Ketones amp Lipogenesisl DEGRADATION 0F FATTY ACIDS Overview ofdegmdalian of fatty acids lipoproteins chylomicrons or VLDL I 2 MITOCHONDRION CAPILLARY acetyl00A 7 i oxidatio A 3 r61 gtFA 39 acyl00A 1 1 CYTOPLASM carnitine transporter from fat cells I FA fatty aCId LPL lipoprotein lipase cell membrane FABP fatty acid binding protein Figure 1 Overview of fatty acid degradation Fatty acids are delivered bound to albumin or released from lipoproteins Fatty acid binding protein carries the fatty acids within the cytoplasm Fatty acids are transported as the carnitine derivative into the mitochondrion for subsequent oxidation Albumin delivers free fatty acids FA from fat cells following lipolysis Fig 1 1 Lipoproteins also deliver fatty acids via chylomicrons or very low density lipoproteins VLDL by the action of lipoprotein lipase that is located in the capillary cell wall Fig 1 2 Fatty acids are solubilized within the cell by binding to fatty acid binding protein FABP Fig 1 3 Fats in the liver may also be synthesized lipogenesis or released from triacylglycerols or phospholipids Fatty acids are then activated to their acyl CoA form in a reaction catalyzed by acyl CoA synthetase ACS fatty acid CoA ATP fatty acyl CoA AMP 2 P Fig 1 4 Fig 2 1 This reaction also activates fatty acids derived from lipogenesis Recall that CoA is also used for converting acetate to the chemically reactive acetyl CoA via pyruvate dehydrogenase for use in the citric acid cycle Following activation of the fatty acids they are transported into the mitochondria via the carnitine transport system Fig 1 5 see Fig 2 for more detail In the mitochondria the fatty acids are oxidized via the betaoxidation pathway Fig 1 6 see Fig 3 for more detail Acetyl CoA produced by betaoxidation feeds into the citric acid cycle TCA cycle for energy production Fig 1 7 or may be used by the liver in the synthesis of ketone bodies see Fig 5 for more detail Lipolysis Beta Oxidation Ketones amp LipogenesisZ Uptake of fatty acids into mitochondria Palmitoyl CoA like all acyl CoA molecules cannot directly pass through the inner mitochondrial membrane Instead fatty acids are transported across the membrane attached to camitine Palmitate is rst activated to palmitoyl CoA on the outside of the outer mitochondrial membrane Fig 2 1 Palmitoyl CoA diffuses through the outer membrane Then palmitoylcamitine is formed by the reaction of palmitoyl CoA with camitine Via carnitine palmitoyl transferase I CPTI Fig 2 2 CPTI is located in the outer mitochondrial membrane ATPC0A AMPPPi palmitoylCoA Cytoplasm Outer Mitochondrial Membrane CPTl 2 CoA palmitoylCoA lntermem brane camitine palmitoylcarnitine S ace 9 4 In ner Mitochondrial 3 Mem brane Matrix I 4 I x camitine almitoylcarnitine palmitoylCoA CoA LEGEND ACS acyl CoA synthetase CPT camitinepalmitoyl transferase CA carnitineacylcarnitine translocase Figure 2 Activation of palmitate to palmitoyl CoA and its mitochondrial uptake Via the camitinecylcarnitine translocase steps 4 and Sin Fig l Lipolysis Beta Oxidation Ketones amp Lipogenesis3 The carnitine transporter carnitine acylcarnitine translocase Fig l 5 Fig 2 3 is an integral protein of the mitochondrial inner membrane that exchanges palmitoylcarnitine from the intermembrane space for carnitine in the mitochondria matrix Palmitoylcarnitine is the principal molecule transported into the matrix on this translocase though other fatty acylcamitines use it as well The palmitoylcarnitine is converted back to palmitoyl CoA in the mitochondria in a reaction catalyzed by carnitine palmitoyl transferase II CPTII that is attached to the matrix side of the inner mitochondrial membrane Fig 2 4 Thus palmitoyl CoA is regenerated in the mitochondrial matrix and carnitine is liberated to be transferred back to the intermembrane space Other speci c carnitinefatty acyl transferases participate in the conversion of less common fatty acyl molecules to their carnitine derivative A speci c transferase is needed for palmitoyl CoA because this fatty acid is the most prevalent stored in triacylglycerols Camitine palmitoyl transferase defects lead to considerable muscle weakness since fatty acids are a major fuel during muscle utilization Furthermore because fatty acid oxidation is obligatory for gluconeogenesis to occur in the liver such defects can contribute to hypoglycemia in fasting Betaoxidation 0f evenchain fatty acids Saturated no HCCH bonds fatty acyl CoA molecules of any chain length that enter the mitochondrial matrix can be substrates for the Boxidation pathway Fig 3 Palmitoyl CoA is given as a basic example It is rst transported into the mitochondrial matrix as the carnitine derivative and then reactivated to palmitoyl CoA The rst reaction in the pathway is an oxidation reaction acyl CoA dehydrogenase that uses FAD as a coenzyme There are several different dehydrogenases that catalyze this reaction depending on the length of the fatty acid hydrocarbon tail short medium or longchain The remaining reactions include a hydratase another dehydrogenase step with NAD as the coenzyme and nally a thiolase that removes acetyl CoA from the end of the chain The products of each of these steps are acetyl CoA and a fatty acyl CoA molecule that is two carbons shorter than the one that initiated the 4 step sequence Thus in this example palmitoyl CoA is shortened after one cycle to a fatty acyl CoA with 14 carbons The Boxidation reactions recycle to consecutively remove 2carbon units as acetyl CoA On the nal cycle which begins with the 4carbon fatty acyl CoA intermediate two acetyl CoA molecules are formed as the product when this intermediate is cleaved Thus the l6carbon palmitoyl CoA molecule needs to cycle just 7 times to produce 8 molecules of acetyl CoA During each cycle one molecule each of FADHZ and NADH are produced The overall reaction of Boxidation of palmitoyl CoA is palmitoyl CoA 7 FAD 7 NAD 7 H20 7 CoA 8 acetyl CoA 7 FADH2 7 NADH Energy is produced indirectly from oxidation of fatty acids in several ways The FADHZ produced is oxidized subsequently by the respiratory chain to produce 2 ATP via oxidative phosphorylation per each FADHZ Similarly each NADH is oxidized via the respiratory chain to produce 3 ATP via oxidative phosphorylation Thus the 7 FADHZ and the 7 NADH will ultimately yield a total of 35 molecules of ATP Additional ATP also can be produced when the acetyl CoA is oxidized via the citric acid cycle which as you should recall produces 3 NADH l FADHZ and l GTP for each acetyl CoA oxidized In liver this acetyl CoA maybe used instead for the synthesis of ketone bodies ketogenesis as described below Lipolysis Beta Oxidation Ketones amp Lipogenesis4 Palm itoylcarnitine c arnitine inner membrane 1 translocase respiratory chain Palmitoylcarnitine 1 Palm itoylCoA m atrix Figure 3 Processing and Boxidation of palm itoyl CoA an evenchain fatty acid FAD oxidation FADHZ hydration H20 1 i recycle NAD 6 tlmes oxidation N ADH thiolase COA CH3CH212CSCOA Acetyl CoA l l O Very longchain fatty acids 20carbon or longer are processed via a modified Boxidation pathway in peroxisomes with acetyl CoA and peroxide as products The process ends with an 8carbon fatty acid that is then converted to its carnitine form and further oxidized in the mitochondria The acetyl CoA products are converted to acetyl carnitine and oxidized via the citric acid cycle after transport and conversion to acetyl CoA KET ONE METABOLISM K etogen esis Ketogenesis occurs only in liver mitochondria and only when the production of acetyl CoA from fatty acids exceeds the capacity of the citric acid cycle to oxidize it Fig 4 The excess acetyl CoA then is used to produce ketones Hydroxym ethylglutaryl CoA HIVIG CoA an intermediate in ketogenesis is formed via mitochondrial HIVIG CoA synthase Hydroxymethylglutaryl CoA is also formed in the cytoplasm as a precursor of cholesterol biosynthesis In ketogenesis HMG CoA is cleaved by HNIG CoA lyase to form acetoacetate with acetyl CoA as the other product BHydroxybutyrate the primary ketone body in the blood is formed from acetoacetate via Bhydroxybutyrate dehydrogenase which requires NADH as a coenzyme Lipolysis Beta Oxidation Ketones amp Lipogenesis5 MITOCHONDRION oxidation to C02 Fatty acid 2 A C A b BOXIdatIon cety o Citric acid cycle excess amounts Thio39ase of acetyl CoA CoA Acetoacetyl CoA acetyl CoA HMGCoA synthase Figure 4 Ketone body Co A formation ketogenesis in liver mitochondria from H d th I I t I C A excess acetyl CoA derived y roxyme y g u any 0 from the Boxidation of HMGCoAlyase fatty Ids acetyl CoA nonenzyma c Acetoacetate N ADH Acetone BHyd roxybutyrate dehydrogenase NAD BHydroxybutyrate Ketane body oxidation Only under conditions of high rates of lipolysis eg longterm starvation or in uncontrolled diabetes are there sufficient amounts of ketones in the blood to be effective as a fuel If glucose ketones and fatty acids are all available in the blood ketones are the preferred fuel that is they will be used preferentially in many tissues over glucose andor fatty acids The primary tissues using ketones when they are available are brain muscle kidney and intestine but not the liver Bhydroxybutyrate is oxidized to acetoacetate by Bhydroxybutyrate dehydrogenase in the mitochondria This reaction is the reverse of the one catalyzed by this enzyme in ketogenesis Hence the reaction produces NADH Acetoacetate is converted to acetoacetylCoA which is cleaved into two acetylCoA molecules that can be oxidized for energy Lipolysis Beta Oxidation Ketones amp Lipogenesis6 K etasis Both Bhydroxybutyrate and acetoacetate are acids Acetoacetate is spontaneously nonenzymatic cleaved to acetone Fig 5 which is nonacidic When abnormal amounts of ketones are produced in the body ie ketosis they may appear in the urine or be expired as acetone see physiological premise When excessive buildup of ketone bodies leads to a fall in the pH of the blood due to the acidic ketone bodies ketoacidosis results In normal individuals ketosis is prevented as follows 1 excess ketone bodies promote the pancreatic release of insulin 2 insulin subsequently inhibits lipolysis to decrease the release of fatty acids 3 the supply of fatty acids reaching the liver is decreased so that there are not sufficient amounts of acetyl CoA for the synthesis of ketones Fig 6 A diabetic loses this important control because of their inability to secrete insulin type I juvenile diabetes or because their adipose cells have lost their ability to respond normally to insulin type II adultonset diabetes Adipose F f tty LIVER ree a tlssue acids Figure 5 Mechanism for prevention of ketosis due to excess ketone body production that can lead to 7 ketoacidosis Ketone Insulln Bodies 4 BIOSYNTHESIS AND STORAGE OF FATTY ACIDS Lipogenesis Synthesis of fatty acids lipogenesis principally occurs in adipose tissue and liver In adipose tissue the fatty acids are stored immediately as triacylglycerols formed via esteri cation as discussed later in this lecture Liver also produces triacylglycerol that is packaged into VLDL and exported into the blood as dsicussed in the preceding lecture Any compound metabolized to acetyl CoA can serve as a precursor for fat synthesis However glucose is the primary source of carbons for fat biosynthesis Glucose is converted to pyruvate via glycolysis and pyruvate is then transported into the mitochondrial matrix However lipogenesis occurs in the cytoplasm and requires acetyl CoA Because acetyl CoA cannot be directly transported across the mitochondrial membrane its carbons must be carried to the cytoplasm via a different mechanism This is accomplished by incorporating the two carbons from acetyl CoA into citrate Citrate is then transported from the matrix to the cytoplasm as a carrier of the carbons destined for fatty acids Fig6 The production of citrate requires equal amounts of oxaloacetate and acetyl CoA for the citrate synthase reaction Therefore pyruvate must be converted to both oxaloacetate via pyruvate carboxylase and acetyl CoA via pyruvate dehydrogenase Recall that pyruvate carboxylase is also important in gluconeogenesis and requires biotin as a cofactor and ATP as an energy source The pyruvate dehydrogenase mechanism was described in an earlier lecture Lipolysis Beta Oxidation Ketones amp Lipogenesis7 pyruvate CO2 ATP oxaloacetate ADP P pyruvate carboxylase pyruvate NAD coenzyme A CoA acetyl CoA CO2 NADH pyruvate dehydrogenase Glucose CYTOPLASM MITOCHONDRIAL MATRIX h PPP Glycolysis NAD CoA NADH CO2 Faltty x 2 2 I A pDH I co P ruvate Pyruvate Acetyl CoA ATP CO2 PC NADP ADP Pi Malonyl CoA NAD ADP Pi MDH 0xaoacetate ACC NADH ATP CO2 0xaoacetate Acetyl CoA ADPP CL ATP CoA Citrate Citrate LEGEND O trahslocase ACC acetyl CoA carboxylase CL citrate lyase ME malic enzyme CS citrate synthase PC pyruvate carboxylase FAS fatty acid synthase PDH pyruvate dehydrogenase MDH malate dehydrogenase PPP pehtose phosphate pathway Figure 6 Export of acetyl CoA incorporated into citrate for fatty acid biosynthesis generation of NADPH and pathway of lipogenesis When there is excessive intake of dietary glucose then a lot of citrate is produced and is available to participate in lipogenesis In the cytoplasm citrate is cleaved by citrate lyase CL to regenerate acetyl CoA and oxaloacetate in an energy requiring reaction Coenzyme A is required as a cosubstrate for this lyase reaction Oxaloacetate is then reduced to malate via malate dehydrogenase MDH that uses NADH as the cosubstrate Malate is oxidized to pyruvate by NADP via malic enzyme ME The NADPH produced by malic enzyme is obligatory for fatty acid synthesis citrate CoA ATP acetyl CoA oxaloacetate ADP P citrate lyase oxaloacetate NADH malate NAD malate dehydrogenase malate NADP pyruvate NADPH malic enzyme Lipolysis Beta Oxidation Ketones amp LipogenesisS Acetyl CoA carboxylase ACC is the committed step for fat biosynthesis with malonyl CoA as its product Fig 6 The mechanism for acetyl CoA carboxylase is identical to that of pyruvate carboxylase in that biotin a prosthetic group is obligatory for the reaction as well as C02 bicarbonate acetyl CoA CO2 ATP malonyl CoA ADP P acetyl CoA carboxylase Fatty acid synthase is a dimeric enzyme consisting of 7 enzyme activities Two of these activities are responsible for attaching acetyl CoA and malonyl CoA to the compleX to initiate the reaction and for attaching malonyl CoA in subsequent steps to build the fatty acid molecule A third activity condensing enzyme CE contains an acyl carrier protein ACP to which the growing fatty acid carbon chain is attached initially this is acetyl CoA Fig 7 A second acyl carrier protein provides the site for attachment of malonyl CoA the source of 2carbon units for the growing chain The condensing enzyme initially joins two carbons from the malonyl group with the two carbons of the acetyl group This condensation results in release of C02 the third malonyl carbon The condensation product is a 4carbon intermediate Fig 7 row 1 Finally in a series of three reactions that use two molecules of NADPH reductase steps a 4carbon fatty acid is formed Fig 7 row 1 The new 4carbon unit now moves to the condensing enzyme site thus allowing a second molecule of malonyl CoA to be attached for the neXt cycle Fig 7 row 2 The reaction then continues through ve more cycles that include attachment of ve more molecules of malonyl CoA and condensation of the growing acyl chain with these new malonyl groups The cycling ends when palmitate l6 carbons is formed Fig 7 row 3 The palmitate is cleaved from the condensing enzyme in a reaction catalyzed by a thioesterase Fig 7 row 3 A total of seven cycles are required to form palmitate and thus oxidation of 14 molecules of NADPH 2 per cycle occurs Note that the product of lipogenesis is the free fatty acid NOT the fatty acyl CoA form The fatty acid must be activated by acyl CoA synthetase a reaction described before Overall reactions acetyl CoA 7 malonyl CoA 14 NADPH palmitate 7 CO2 8 CoA 14 NADP fatty acid synthdse palmitate ATP CoA palmitoyl CoA AMP 2 P acyl CoA synthetase Lipolysis Beta Oxidation Ketones amp Lipogenesis9 A reduction 3 condensation dehydration CE c CE P CE P reduction acp P acp acp C02 ZNADPH ZNADP 4 b car on acetyl malonyl 4carbon unit CoA CoA unit A condensation A reduaior39l CE 3 c CE c dehydration P CE P P reduction acp acp acp C02 ZNADPH ZNADP 6 b car on 4carbon malonyl 6carbon unit unit CoA unit A A 5 more cycles A thioesterase c c adding 10 carbons c cleavage P P P i palmitate 6carbon malonyl 16carbon unit unit coA palmitate Figure 7 General mechanism for the fatty acid synthase reaction CE is condensing enzyme ACP is acyl earlier protein The rst row is the initial steps for priming the reaction with acetyl CoA and the addition of two carbons from malonyl CoA The second row depicts a typical cycle of adding two more carbons to the fatty acid chain The nal row shows the release of the nished product palmitate through cleavage by thioesterase Sources 0fNADPHf0r Lipagenesis Malic enzyme generates NADPH during lipogenesis when it processes the carbons from oxaloacetate a product of the citrate lyase reaction Fig 1 However malic enzyme provides only about half of the NADPH required for lipogenesis The remaining NADPH needed for the fatty acid synthase reaction comes from the oxidative branch of the pentose phosphate pathway Esteri catian of Fatty Acids for Storage The backbone of triacylglycerols and phospholipids hosphoglycerides is glycerol Both glycerol 3 phosphate and dihydroxyacetone phosphate are precursors for these pathways The pathway in Fig 8 depicts just the triacylglycerol pathway Phosphatidic acid is an intermediate in the biosynthesis of both molecules The primary fatty acid esteri ed to triacylglycerols is palmitoyl CoA These triacylglycerols are then stored as a fuel for mobilization during starvation or stress Unsaturated fatty acids are not stored as triacylglycerols because their oxidation for energy is more complicated In times of starvation or stress the rapid mobilization of fats for fuels is essential for survival Since palmitoyl CoA is the major product of lipogenesis it is the primary storage form of lipid fuel Lipolysis Beta Oxidation Ketones amp LipogenesislO In contrast phospholipids contain unsaturated containing CC double bonds fatty acids In the membrane phospholipids play roles other than for structural purposes Phospholipases catalyze the speci c cleavage of side groups from phospholipids We learned earlier that phospholipase C acts on membrane phosphatidylinositol to generate the second messengers diacylglycerol and 1P3 Additionally phospholipase A2 action releases arachidonic acid see below Snake venoms contain phospholipases and destroy phospholipids in membrane of tissues they in ltrate possibly causing tissue necrosis at the site of the bite When venoms attack inner mitochondrial membrane the large amounts of fatty acids released act as natural uncouplers causing severe loss of the ability of the mitochondrion to produce energy Glycerol Dihydroxyacetone phosphate ATP fatty acyl CoA glycerol kinase ADP Glycerol3P C 0 A fatty acyl CoA Acyldihydroxyacetone phosphate NADPH CoA Lysophosphat1d1c acid N ADP fatty acyl CoA CoA Phosphatidic acid Diacylglycerol phosphatase fatty acyl CoA CoA Triacylglycerol Figure 8 Formation of phosphatidic acid from glycerol3phosphate or dihydroxyacetone phosphate and its conversion to triacylglycerol Lipolysis Beta Oxidation Ketones amp Lipogenesisll
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