METABOLIC REGULATIONS OF CARBOHYDRATE, FATS AND PROTEIN : METABOLIC REGULATIONS OF CARBOHYDRATE, FATS AND PROTEIN DR. HARENDRA VYAS OCT-2011 How do we use food components in catabolic and anabolic pathways?: How do we use food components in catabolic and anabolic pathways? Involves specific chemical reactions: - Each reaction is catalyzed by a specific enzyme. - Other compounds, besides those being directly metabolized, are required as intermediates or catalysts in metabolic reactions - adenosine triphosphate (ATP) - nicotinamide adenine dinucleotide (NAD + ) - flavin adenine dinucleotide (FAD + ) - Coenzyme A ATP: ATP ATP is the energy currency of the cell The structure of ATP is similar to that of nucleic acids The energy in ATP is “carried” in the phosphate groups - to convert ADP into ATP requires energy - the energy is stored as potential energy in the phosphate group bond - removal of the third phosphate releases that energy NADH, FADH2: NADH, FADH 2 NAD + can accept a hydrogen ion and become reduced to NADH: NAD + + 2[H + ] + 2e- NADH + H + The added hydrogen ion (and electrons) can be carried to and used in other reactions in the body. FAD+ is similarly reduced to FADH 2 . NADH and FADH carry hydrogen ions and electrons to the enzymes in the electron transport chain of the mitochondria, allowing ATP production there. Coenzyme A: Coenzyme A The enzyme coenzyme A converts acetyl groups (2-carbon structures) into acetyl CoA, which can then be used in metabolic reactions During the course of acetyl CoA production, energy is released and is used to convert NAD + to NADH Cellular Respiration: Cellular Respiration Generating ATP from food requires glycolysis, the Krebs Cycle, and the electron transport chain. Overall reaction: C 6 H 12 O 6 + 6 O 2 ----> 6 CO 2 + 6 H 2 O + 38 ATP + heat The Main point: the break down of glucose releases LOTS of energy: - about 40% in usable form (ATP) - about 60% as heat What happens to pyruvic acid?: What happens to pyruvic acid? In aerobic respiration (oxygen present): - pyruvic acid moves from cytoplasm to mitochondria - pyruvic acid (3 carbons) is converted to acetyl group (2 carbons), producing CO 2 in the process - acetyl group is converted to acetyl CoA by coenzyme A - acetyl CoA is used in the Krebs cycle. Glycolysis: General Functions: Glycolysis: General Functions Provide ATP energy Generate intermediates for other pathways Hexose monophosphate pathway Glycogen synthesis Pyruvate dehydrogenase Fatty acid synthesis Krebs’ Cycle Glycerol-phosphate (TG synthesis) Glycolysis: Specific tissue functions: Glycolysis: Specific tissue functions RBC’s Rely exclusively for energy Skeletal muscle Source of energy during exercise, particularly high intensity exercise Adipose tissue Source of glycerol-P for TG synthesis Source of acetyl-CoA for FA synthesis Liver Source of acetyl-CoA for FA synthesis Source of glycerol-P for TG synthesis Function of glycolysis: Function of glycolysis In most tissues it is part of ATP production system it must be sensitive to the energy status of the cell (ii) In liver and adipose tissue- it is often part of the process that converts excess carbs to fat In these tissues there will be different controls Glycolysis: Glycolysis Dual role : 1. degrades glucose to generate ATP 2. provides building blocks for synthetic reactions. Glycolysis is regulated to meet these two major cellular needs. Reactions catalyzed by Hexokinase Phosphofructokinase Pyruvate kinase are control sites. In metabolic pathways, enzymes catalyzing essentially irreversible steps are potential sites of control. Main site of control Glycolysis: Embden-Meyerhof Pathway : Glycolysis : Embden -Meyerhof Pathway Oxidation of glucose Products: 2 Pyruvate 2 ATP 2 NADH Cytosolic What are the potential regulatory steps?: What are the potential regulatory steps? Approach: Approach (1) What is the function of glycolysis -Regulation must relate to function (2) What are the regulatory steps and how are they regulated? Glycolysis: Glycolysis We have identified the irreversible steps: HK, PFK, PK Control of these key enzymes can make the glycolysis proceed faster – How? Slide 16: Glucose 6-phosphate is also a precursor for the synthesis of glycogen in liver and it is used in the pentose phosphate pathway to produce NADPH, reducing power. The main site of regulation is PFK because it is the first committed step in glycolysis (step unique to a pathway). If HK were the main site of regulation, synthesis of glycogen and formation of reducing power (NADPH) would also be shut down. In the liver, glucokinase is present instead of HK. Glucokinase is specific for glucose with a K m value of 5 mM. HK uses also other hexoses as substrates and has a K m value for glucose of 0.1 mM. Glucokinase provides G-6-P for the synthesis of glycogen , the storage form of glucose, when glucose is abundant. When supply of glucose is limited (low [glu]) very little glucose is metabolized by GK because of the high K m value and glucose is metabolized by HK in the brain and muscle . Regulation of metabolism provides energy where it is first required . Glucokinase Substitutes Hexokinase in Liver Properties of Glucokinase and Hexokinase : Properties of Glucokinase and Hexokinase Slide 18: Regulation of Glysolysis – Hexokinase Hexokinase (HK), the first enzyme in the glycolytic pathway, is inhibited by high concentrations of G-6-P . This type of inhibition is called product inhibition . When PFK in inhibited, the [G-6-P] increases, resulting in inhibition of HK. Why is not HK the primary regulation site for glycolysis? Regulation of Glycolysis - PFK: Regulation of Glycolysis - PFK Hexokinase, phosphofructokinase, and pyruvate kinase are regulated by reversible binding of allosteric effectors (in milliseconds) and covalent modification by phosphorylation (in seconds). Their amounts are varied by transcriptional control (in hours). Phosphofructokinase (PFK) is the most important control element in mammals. In the liver, PFK (340 kDa tetramer ) is: Activated by AMP, F-2,6-BP . Inhibited by ATP, citrate, H + . Allosteric enzyme. A monomeric enzyme cannot be allosterically regulated Regulation of PFK by ATP: Regulation of PFK by ATP High levels of ATP lower the affinity for F-6-P. This effect is elicited by binding of ATP to a regulatoty site that is different from the catalytic site. The hyperbolic kinetics is converted to a sigmoidal kinetics. The inhibitory effect of ATP is reversed by AMP, so the activity of the enzyme increases when the ATP/AMP ratio is lowered (when cells need to synthesize ATP). Glycolysis is stimulated when energy charge falls . High levels of H + also inhibits PFK to prevent excessive formation of lactate by lactic fermentation and consequent drop in blood pH (acidosis). Slide 21: Regulation of PFK by Citrate Citrate, an early intermediate in the citric acid cycle, inhibits PFK . High levels of citrate mean that biosynthetic precursors are abundant, therefore additional glucose should not be degraded for this purpose. Citrate enhances the inhibitory effect of ATP. Regulation of PFK-1 in Muscle: Regulation of PFK-1 in Muscle Relatively constitutive Allosterically stimulated by AMP High glycolysis during exercise Allosterically inhibited by ATP High energy, resting or low exercise Citrate Build up from Krebs’ cycle May be from high FA beta-oxidation -> hi acetyl-CoA Energy needs low and met by fat oxidation Regulation of PFK-1 in Liver: Regulation of PFK-1 in Liver Inducible enzyme Induced in feeding by insulin Repressed in starvation by glucagon Allosteric regulation Like muscle w/ AMP, ATP, Citrate Activated by Fructose-2,6-bisphosphate Role of F2,6P2 in Regulation of PFK-1: Role of F2,6P 2 in Regulation of PFK-1 PFK-2 catalyzes F6P + ATP -> F2,6P 2 + ADP PFK-2 allosterically activated by F6P F6P high only during feeding (hi glu, hi GK activity) PFK-2 activated by dephophorylation Insulin induced protein phosphatase Glucagon/cAMP activates protein kinase to inactivate Therefore, during feeding Hi glu + hi GK -> hi F6P Insulin induces prot. P’tase and activates PFK-2 Activates PFK-2 –> hi F2,6P 2 Activates PFK-1 -> hi glycolysis for fat synthesis Coordinated Regulation of PFK-1 and FBPase-1: Coordinated Regulation of PFK-1 and FBPase-1 Both are inducible, by opposite hormones Both are affected by F2,6P 2 , in opposite directions Slide 26: Regulation of PFK by Fructose 2,6-Bisphosphate (F-2,6-BP) F-2,6-BP, not to be mistaken with the product of the reaction (F-1,6-BP) is a potent allosteric activator of PFK in liver, discovered in 1980. F-2,6-BP increases the affinity of PFK for F-6-P and decreases the inhibitory effect of ATP. F-2,6-BP is an allosteric activator that shifts the conformational equilibrium of PFK from the T to the R state . Slide 27: Phosphofructokinase 2 (PFK2) and Fructose Bisphosphatase 2 (FBPase2) F-2,6-BP is formed by phosphorylation of F-6-P by phosphofructokinase 2 ( PFK2 ). F-2,6-BP is hydrolyzed to F-6-P by fructose bisphosphatase 2 ( FBPase2 ). PFK2 and FBPase2 are present in a single 55 kDa polypeptide chain, which also contains a regulatory domain at the N-terminus. Bifunctional enzyme . PFK2 resembles PFK and FBPase2 resembles phosphoglycerate mutase (probably originated by gene fusion). F-6-P accelerates the synthesis of F-2,6-BP: example of feedforward stimulation . Regulated also by phosphorylation of a serine residue . Scarce glucose increase in blood levels of hormone glucagone cAMP cascade phosphorylation of bifunctional enzyme activation of FBPase2 and inhibition of PFK2 lower level of F-2,6-BP lower activity of PFK glycolysis is slowed down . Slide 28: Regulation of Glycolysis - Pyruvate Kinase (PK) Controls the outflow from glycolysis. Pyruvate can be oxidized further or used for syntheses. Isoenzymes (57 kDa): L-type (liver) activated by dephosphorylation. M-type (muscle, brain) insensitive to Phosphorylation. L-type (liver) is regulated by phosphorylation: Scarse glucose glucagon triggers cAMP cascade phosphorylation of PK decrease in activity. Hormone-triggered phosphorylations of PK & PFK2/FBPase2 prevent liver to consume glucose when it is more urgently needed by brain and muscle. Regulation of glycolysis by Adenine Nucleotides: Regulation of glycolysis by Adenine Nucleotides In tissues where the primary function of glycolysis is ATP production (muscle, Brain etc), it need to be sensitive to [ATP], directly or indirectly. ATP is too important to be allowed to undergo large changes in concentration. [AMP] can reflect [ATP], inversely as follows: Slide 30: This reaction is close to equlibrium in vivo: K eq = 0.44 A small change in [ATP] translates into large % increase in [AMP] How?: How? Eg. Total [Adenine nucleotide] = 5 mM ATP = 4.8; ADP = 0.185; AMP = 0.015 Due to the action of Adenylate kinase a 4% reduction in [ATP] will lead to a 300% increase in [AMP]. ATP = 4.6; ADP = 0.34; AMP = 0.06 Slide 32: Thus [AMP] serves as a very sensitive indicator of small changes in [ATP] . Are the rate limiting glycolytic steps sensitive to AMP? i) Phosphofructokinase is allosterically inhibited by ATP and activated by AMP ii) Hexokinase is allosterically inhibited by glucose-6-P Calculate the free energy changes across the 10 reactions of glycolysis: Calculate the free energy changes across the 10 reactions of glycolysis e.g. Phosphohexoisomerase K eq for G6P-F6P = 0.5 Concentrations [G6P] = 8.3 x 10 -5 M [F6P] = 1.4 x 10 -5 M Δ G o = -2.303 RT log K eq = -(2.303 x 8.31 x 310 x (-0.3)) = 1776 J/mole Δ G = Δ G o + 2.303 RT log [product]/[reactant] = -2.810 J/mole = -2.81 kJ/mole Slide 35: Free energy profile - almost all of the free-energy released during glycolysis occur in just 3 steps HK, PFK, PK These three reactions are displaced far from the thermodynamic equilibrium The other reactions of glycolysis are close to equilibrium If the close-to-equilibrium reaction is activated it can not make a difference to the overall rate of the pathway However, if we can activate the far-from-equilibrium (irreversible) reactions , we can make the pathway go faster. Slide 36: Hexokinase Inhibitors Glucose 6-phosphate Pyruvate kinase Phosphofructokinase ATP CITRIC ACID CYCLE (ATP, citrate) ATP, citrate, PEP Activators ADP, AMP, Fructose 2,6P Regulation of Glycolysis Slide 37: Glycogen Glycolysis Gluconeogenesis Lactate (anaerobic) Pentose Phosphate Pathway The Fates of Glucose Citric acid cycle (aerobic) Slide 38: Gluconeogenesis: Synthesis of glucose from non-hexose precursors (lactate, fatty acids and amino acids) Slide 39: Pentose Phosphate Pathway (a) Produces NADPH, a reducing agent needed for anabolic reactions. (b) Pentose type sugars. Required for the production nucleic acids Pentose Phosphate Pathway Glycolysis Slide 40: Muscle glycogen reserves are rebuilt during times of less intense activity Gluconeogenesis in liver converts lactate to glucose Heavy activity in muscle consumes muscle glycogen The liver is the central control point for regulating blood glucose levels Slide 41: Liver Brain Muscle tissue glucose lactate Dietary and intrinsic carbohydrates, proteins, and lipids The liver is the central control point for regulating blood glucose levels glycogen lactate glycogen glucose rest active It’s critical that blood glucose levels are maintained within a defined concentration range. Slide 42: Low Blood Glucose: (Hungry. Several hours after eating) Increased gluconeogenesis Increase glycogen break down Slide 44: Epinephrine (aka Adrenaline) mediates the fight or flight response Effects similar to glucagon. It mobilizes glucose from glycogen. In contrast, it also promotes glucose catabolism in muscle tissue (for production of ATP for activity). Low Blood Glucose: (Panic reaction) Slide 45: Glucagon/Epinephrine control of glycogen synthesis/degradation Slide 46: Optional reading Page 449-454 Signal cascade initiated by epinephrine Slide 47: Signal cascade initiated by epinephrine Slide 48: Glucagon control of glycolysis/gluconeogenesis (the fructose 2,6-bisphosphatate connection) Glucogon controls the production of fructose 2,6-bisphosphatate Slide 49: Insulin lowers blood glucose levels High Blood Glucose: Slow flux through Glycolysis: Slow flux through Glycolysis Rapid Flux through Glycolysis: Rapid Flux through Glycolysis Increased demand for ATP Low [ATP] and High [AMP] Regulation of Cellular Glucose Uptake: Regulation of Cellular Glucose Uptake Brain & RBC: GLUT-1 has high affinity (low Km)for glucose and are always saturated. Insures that brain and RBC always have glucose. Liver: GLUT-2 has low affinity (hi Km) and high capacity. Uses glucose when fed at rate proportional to glucose concentration Muscle & Adipose: GLUT-4 is sensitive to insulin Glucose Utilization: Glucose Utilization Phosphorylation of glucose Commits glucose for use by that cell Energy consuming Hexokinase: muscle and other tissues Glucokinase: liver Regulation of Cellular Glucose Utilization in the Liver: Regulation of Cellular Glucose Utilization in the Liver Feeding Blood glucose concentration high GLUT-2 taking up glucose Glucokinase induced by insulin High cell glucose allows GK to phosphorylate glucose for use by liver Post-absorptive state Blood & cell glucose low GLUT-2 not taking up glucose Glucokinase not phophorylating glucose Liver not utilizing glucose during post-absorptive state Regulation of Cellular Glucose Utilization in the Liver: Regulation of Cellular Glucose Utilization in the Liver Starvation Blood & cell glucose concentration low GLUT-2 not taking up glucose GK synthesis repressed Glucose not used by liver during starvation Regulation of Cellular Glucose Utilization in the Muscle: Regulation of Cellular Glucose Utilization in the Muscle Feeding and at rest High blood glucose, high insulin GLUT-4 taking up glucose HK phosphorylating glucose If glycogen stores are filled, high G6P inhibits HK, decreasing glucose utilization Starving and at rest Low blood glucose, low insulin GLUT-4 activity low HK constitutive If glycogen stores are filled, high G6P inhibits HK, decreasing glucose utilization Regulation of Cellular Glucose Utilization in the Muscle: Regulation of Cellular Glucose Utilization in the Muscle Exercising Muscle (fed or starved) Low G6P (being used in glycolysis) No inhibition of HK High glycolysis from glycogen or blood glucose Regulation of Glycolysis: Regulation of Glycolysis Regulation of 3 irreversible steps PFK-1 is rate limiting enzyme and primary site of regulation. Pyruvate Dehydrogenase: The enzyme that links glycolysis with other pathways: Pyruvate Dehydrogenase: The enzyme that links glycolysis with other pathways Pyruvate + CoA + NAD -> AcetylCoA + CO2 + NADH The PDH Complex: The PDH Complex Multi-enzyme complex Three enzymes 5 co-enzymes Allows for efficient direct transfer of product from one enzyme to the next The PDH Reaction: The PDH Reaction E1: pyruvate dehydrogenase Oxidative decarboxylation of pyruvate E2: dihydrolipoyl transacetylase Transfers acetyl group from TPP to lipoic acid E3: dihydrolipoyl dehydrogenase Transfers acetly group to CoA, transfers electrons from reduced lipoic acid to produce NADH Regulation of PDH Muscle: Regulation of PDH Muscle Resting (don’t need) Hi energy state Hi NADH & AcCoA Inactivates PDH Hi ATP & NADH & AcCoA Inhibits PDH Exercising (need) Low NADH, ATP, AcCoA Regulation of PDH Liver: Regulation of PDH Liver Fed (need to make FA) Hi energy Insulin activates PDH Starved (don’t need) Hi energy No insulin PDH inactive Krebs Cycle (KC): Krebs Cycle (KC) Also known as TCA cycle, or citric acid cycle Reactions of KC occur in mitochondrial matrix Common final degradative pathway for breakdown of monomers of CHO, fat and protein to CO 2 and H 2 0 Electrons removed from acetyl groups and attached to NAD + and FAD Small amount of ATP produced from substrate level phosphorylation KC also provides intermediates for anabolic functions (eg gluconeogenesis) Pyruvate Acetyl CoA: Pyruvate Acetyl CoA Pyruvate produced in cytosol and transported into mitochondria Cannot directly enter KC First converted to acetyl CoA by pyruvate dehydrogenase complex From: Summerlin LR (1981) Chemistry for the Life Sciences. New York: Random House p 548. Pyruvate Oxidation: Pyruvate Oxidation Pyruvate + CoA + NAD+ acetylCoA + CO 2 + NADH + H + Krebs Cycle: Krebs Cycle Acetyl CoA combines with oxaloacetic acid, forming citric acid A series of reactions then occurs resulting in: - one ATP produced - three NADH and one FADH 2 produced (go to electron transport chain) - two CO 2 molecules produced Citric Acid Cycle: Citric Acid Cycle Hans Krebs proposed the “citric acid cycle” for the complete oxidation of pyruvate in animal tissues in 1937 (1953 Nobel Prize laureate). The tricaboxylic acid (TCA) Cycle , Krebs Cycle Slide 69: Historical perspective: 1930: Elucidation of Glycolysis Study of oxidation of glucose in muscle, addition of Malonate inhibited the respiration (i.e. O 2 uptake). Malonate is an inhibitor of Succinate oxidation to Fumerate 1935: Szent-Gyorgyi: demonstrated that little amounts (catalytic amounts) of succinate, fumerate, malate or oxaloacetate acelerated the rate of respiration. He also showed the sequence of inter-conversion: Succinate --- Fumerate --- malate ---oxaloacetate. 1936: Martius & Knoop: Found the following sequence of reaction: Citrate to cis-aconitase to Isocitrate to a Ketogluterate to succinate 1937: Krebs: Enzymatic conversion of Pyruvate + Oxaloacetate to citrate and CO 2 Discovered the cycle of these reactions and found it to be a major pathway for pyruvate oxidation in muscle. The three stages of respiration: The three stages of respiration Stage I All the fuel molecules are oxidized to acetyl-CoA. Stage II The acetyl-CoA is completely oxidized into CO 2 , electrons were collected by NAD and FAD via the citric acid cycle. Stage III Passage of electrons through the electron transport system to yield ATP from oxidative phosphorylation . Krebs Cycle: Preparatory Step: Krebs Cycle: Preparatory Step Pyruvic acid is converted to acetyl CoA in three main steps: Decarboxylation Carbon is removed from pyruvic acid Carbon dioxide is released Oxidation Hydrogen atoms are removed from pyruvic acid NAD + is reduced to NADH + H + Formation of acetyl CoA – the resultant acetic acid is combined with coenzyme A, a sulfur-containing coenzyme, to form acetyl CoA Krebs Cycle: Krebs Cycle An eight-step cycle in which acetic acid is decarboxylated and oxidized, generating: Three molecules of NADH + H + One molecule of FADH 2 Two molecules of CO 2 One molecule of ATP For each molecule of glucose entering glycolysis, two molecules of acetyl CoA enter the Krebs cycle Citric Acid Cycle: Citric Acid Cycle The common pathway leading to complete oxidation of carbohydrates, fatty acids, and amino acids to CO 2 . Some ATP is produced, More NADH is made ,NADH goes on to make more ATP in electron transport and oxidative phosphorylation A pathway providing many precursors for biosynthesis Krebs Cycle: Krebs Cycle Figure 25.7 Krebs Cycle: Krebs Cycle citric acid cycle overview : citric acid cycle overview TCA Cycle Summary: TCA Cycle Summary 1 acetate through the cycle produces 2 CO 2 , 1 GTP, 3NADH, 1FADH 2 Formation of citrate: Formation of citrate Oxaloacetate condenses with acetyl CoA to form Citrate Non-equilibrium reaction catalysed by citrate synthase Inhibited by: ATP NADH Citrate - competitive inhibitor of oxaloacetate From: Summerlin LR (1981) Chemistry for the Life Sciences. New York: Random House p 550. Citrate isocitrate: Citrate isocitrate Citrate isomerised to isocitrate in two reactions (dehydration and hydration) Equilibrium reactions catalysed by aconitase Results in interchange of H and OH Changes structure and energy distribution within molecule Makes easier for next enzyme to remove hydrogen From: Summerlin LR (1981) Chemistry for the Life Sciences. New York: Random House p 550. isocitrate -ketoglutarate: isocitrate -ketoglutarate Isocitrate dehydrogenated and decarboxylated to give -ketoglutarate Non-equilibrium reactions catalysed by isocitrate dehydrogenase Results in formation of: NADH + H + CO 2 Stimulated (cooperative) by isocitrate, NAD + , Mg 2+ , ADP, Ca 2+ (links with contraction) Inhibited by NADH and ATP From: Summerlin LR (1981) Chemistry for the Life Sciences. New York: Random House p 550. -ketoglutarate succinyl CoA: -ketoglutarate succinyl CoA Series of reactions result in decarboxylation, dehydrogenation and incorporation of CoASH Non-equilibrium reactions catalysed by -ketoglutarate dehydrogenase complex Results in formation of: CO 2 NADH + H + High energy bond Stimulated by Ca 2+ Inhibited by NADH, ATP, Succinyl CoA (prevents CoA being tied up in matrix) From: Summerlin LR (1981) Chemistry for the Life Sciences. New York: Random House p 550. Succinyl CoA succinate: Succinyl CoA succinate Equilibrium reaction catalysed by succinate thiokinase Results in formation of: GTP CoA-SH From: Summerlin LR (1981) Chemistry for the Life Sciences. New York: Random House p 550. Succinate fumarate: Succinate fumarate Succinate dehydrogenated to form fumarate Equilibrium reaction catalysed by succinate dehydrogenase Only Krebs enzyme contained within inner mitochondrial membrane Results in formation of FADH 2 From: Summerlin LR (1981) Chemistry for the Life Sciences. New York: Random House p 550. Fumarate malate: Fumarate malate Fumarate hydrated to form malate Equilibrium reaction catalysed by fumarase Results in redistribution of energy within molecule so next step can remove hydrogen From: Summerlin LR (1981) Chemistry for the Life Sciences. New York: Random House p 550. Malate oxaloacetate: Malate oxaloacetate Malate dehydrogenated to form oxaloacetate Equilibrium reaction catalysed by malate dehydrogenase Results in formation of NADH + H + From: Summerlin LR (1981) Chemistry for the Life Sciences. New York: Random House p 550. ATP generated by the cycle: ATP generated by the cycle 3 NADH 3 NAD + ETS 3*2.5=7.5 ATP FADH 2 FAD 1.5 ATP Substrate level phosphorylation 1 GTP 10 ATP Equivalents Total ETS Energetics: Energetics Energy is conserved in the reduced coenzymes NADH, FADH 2 and one GTP NADH, FADH 2 can be oxidized to produce ATP by oxidative phosphorylation ETS 1.5 1.5 1.5 7.5 7.5 7.5 Summary of ATP Production: Summary of ATP Production Figure 25.10 Regulation of the TCA Cycle: Regulation of the TCA Cycle Again, 3 irreversible reactions are the key sites Citrate synthase - regulated by availability of substrates - acetyl-CoA and oxaloacetate, citrate is a competitive inhibitor; Allosteric: - NADH , ATP,succinyl-CoA Isocitrate dehydrogenase – NADH,ATP inhibit, ADP and NAD + Ca ++ activate -Ketoglutarate dehydrogenase - NADH and succinyl-CoA inhibit, AMP Ca ++ activate Slide 90: Glucose glycolysis 2ATP( Substrate-level phosphorylation ) 2 2NADH ( oxphos) 3 -5 2Pyruvate oxidative decarboxylation 2 NADH ( oxphos) 5 2 Acetyl CoA TCA cycle 20 6 NADH 2 FADH 2 2 GTP total 30-32ATP 25ATP 5-7ATP CO2 ATP generated by complete oxidation of glucose Regulation of Krebs Cycle: Regulation of Krebs Cycle Cycle always proceeds in same direction due to presence of 3 non-equilibrium reactions catalysed by Citrate synthase Isocitrate dehydrogenase -ketoglutarate dehydrogenase From: Summerlin LR (1981) Chemistry for the Life Sciences. New York: Random House p 550. Regulation of Krebs Cycle: Regulation of Krebs Cycle Flux through KC increases during exercise 3 non-equilibrium enzymes inhibited by NADH KC tightly coupled to ETC If NADH decreases due to increased oxidation in ETC flux through KC increases Isocitrate dehydrogenase and -ketoglutarate dehydrogenase also stimulated by Ca2 + Flux increases as contractile activity increases From: Summerlin LR (1981) Chemistry for the Life Sciences. New York: Random House p 550. Aerobic Nature of the Cycle: Aerobic Nature of the Cycle NADH and FADH 2 must be reoxidized by the electron transport chain. Succinate Dehydrogenase is part of electron transport chain in the inner membrane of mitochondria. Slide 94: Major regulatory sites are irreversible reactions Anaplerotic reactions : Anaplerotic reactions Anaplerotic (filling up) reactions replenish citric acid cycle intermediates Amphibolic Nature of TCA Cycle means it both Anabolic and Catabolic. TCA cycle provides several of Intermediates for Biosynthesis Anaplerotic reactions: Anaplerotic reactions PEP carboxylase - converts PEP to oxaloacetate , Anaplerotic reaction in plants and bacteria Pyruvate carboxylase - converts pyruvate to oxaloacetate , a major anaplerotic reaction in mammalian tissues Malic enzyme converts pyruvate into malate Slide 99: In Mitochondria In Cytosol Slide 100: Reaction of pyruvate dehydrogenase complex (PDC) Reactions of TCA cycle: 8 reactions: Citrate synthase Aconitase Iso-citrate dehydrogenase a ketoglutarate dehydrogenase Succinyl-Coenzyme A synthetase Succinate dehydrogenase Fumerase Malate dehydrogenase Slide 102: Pyruvate dehydrogenase Complex (PDC) It is a multi-enzyme complex containing three enzymes associated together non-covalently: E-1 : Pyruvate dehydrogenase, uses Thiamine pyrophosphate as cofactor bound to E1 E-2 : Dihydrolipoyl transacetylase, Lipoic acid bound, CoA as substrate E-3 : Dihydrolipoyl Dehydrogenase FAD bound, NAD + as substrate Advantages of multienzyme complex: Higher rate of reaction: Because product of one enzyme acts as a substrate of other, and is available for the active site of next enzyme without much diffusion. Minimum side reaction. Coordinated control. Slide 104: Conservation of energy of oxidation in the CAC: The two carbon acetyl group generated in PDC reaction enter the CAC, and two molecules of CO2 are released in on cycle. Thus there is complete oxidation of two carbons during one cycle. Although the two carbons which enter the cycle become the part of oxaloacetate, and are released as CO2 only in the third round of the cycle. The energy released due to this oxidation is conserved in the reduction of 3 NAD+, 1 FAD molecule and synthesis of one GTP molecule which is converted to ATP. Slide 105: Efficiency of Biochemical engine in Living Systems: Oxidation of one glucose yields 2840 kJ/mole energy Energy obtained by biological engine: 32ATP X 30.5 kJ/Mol = 976 kJ/mol Thus 34% efficiency is obtained if calculations are done using standard conditions. But if concentrations in the cellular condition are taken in account, the efficiency is close to 65%. Slide 106: Anaerobic bacteria has incomplete citric acid cycle for production of biosynthetic precursors. They do not contain a-ketoglutarate dehydrogenase. Slide 107: The amphibolic nature of Citric acid cycle: This pathway is utilized for the both catabolic reactions to generate energy as well as for anabolic reactions to generate metabolic intermediates for biosynthesis. If the CAC intermediate are used for synthetic reactions, they are replenished by anaplerotic reactions in the cells (indicated by red colours). Slide 108: Fig. 16.16 Glyoxalate cycle Slide 111: Regulation of CAC: Rate controlling enzymes: Citrate synthatase Isocitrate dehydrogenase a -keoglutaratedehydrogenase Regulation of activity by: Substrate availability Product inhibition Allosteric inhibition or activation by other intermediates Interconversion Pathways of Nutrients: Interconversion Pathways of Nutrients Carbohydrates are easily and frequently converted into fats Their pools are linked by key intermediates They differ from the amino acid pool in that: Fats and carbohydrates are oxidized directly to produce energy Excess carbohydrate and fat can be stored Interconversion of Nutrients: Interconversion of Nutrients Lipogenesis : once glycogen stores are filled, glucose and amino acids are converted to lipids Rate limiting enzyme: acetyl CoA carboxylase amino acids acetyl fatty CoA acids glucose glucose 6-phosphate glyceraldehyde 3-phosphate glycerol triglycerides acetyl CoA carboxylase Interconversion of Nutrients (cont.): Interconversion of Nutrients (cont.) Gluconeogenesis : amino acids and glycerol can be used to produce glucose (liver) More glucose is produced via gluconeogenesis than glycogenolysis Rate-limiting enzyme: phosphoenolpyruvate carboxykinase Glycerol glyceraldehyde glucose 3- phosphate 6-phosphate Amino pyruvic acid glucose acids oxaloacetate phosphoenol pyruvate PEPCK Interconversion Pathways of Nutrients: Interconversion Pathways of Nutrients Figure 25.16 Slide 116: Hormonal Regulation of Nutrients Right after a meal (resting): - blood glucose elevated - glucagon, cortisol, GH, epinephrine low - insulin increases (due to increased glucose) - Cells uptake glucose, amino acids. - Glucose converted to glycogen, amino acids into protein, lipids stored as triacylglycerol. - Blood glucose maintained at moderate levels. Slide 117: A few hours after a meal (active): - blood glucose levels decrease - insulin secretion decreases - increased secretion of glucagon, cortisol, GH, epinephrine - glucose is released from glycogen stores (glycogenolysis) - increased lipolysis (beta oxidation) - glucose production from amino acids increases ( oxidative deamination; gluconeogenesis ) - decreased uptake of glucose by tissues - blood glucose levels maintained Hormonal Regulation of Nutrients LIPID METABOLISM: LIPID METABOLISM Lipid Metabolism: Lipid Metabolism Over 95% of stored energy in the body is in the form of triacylglycerol During lipid catabolism ( lipolysis ), triacylglycerol is broken down into free fatty acids and glycerol Free fatty acids are metabolized by beta-oxidation: 1) fatty acid (18 C) + coenzyme A 2) fatty acid (18 C)-coA 3) fatty acid (16 C) and acetyl-coA Acetyl-CoA used in citric acid cycle This reaction also yields NADH => electron transport chain Excess acetyl-CoA forms ketone bodies Lipid Metabolism: Lipid Metabolism Most products of fat metabolism are transported in lymph as chylomicrons Lipids in chylomicrons are hydrolyzed by plasma enzymes and absorbed by cells Only neutral fats are routinely oxidized for energy Catabolism of fats involves two separate pathways Glycerol pathway Fatty acids pathway Introduction: 121 Fatty acids play several important roles: Building blocks for phospholipids and glycolipids Target proteins to membranes High energy source of fuel Fatty acid derivatives are used as hormones and intracellular messengers Introduction Slide 122: Lipids are non-polar (hydrophobic) compounds, soluble in organic solvents . Classification of Lipids Simple Lipids A. Neutral fats - Triglycerides B. Waxes 2. Conjugated Lipids (polar lipids) A. Phospholipids - contain a phosphoric acid molecule and a fat molecule. B. Glycolipid- contain a carbohydrate and a fat molecule. cerebrosides globosides gangliosides C. Sulfolipids - contain a sulfate radical. D. lipoprotein 3. Derived Lipids A. Fatty acids B. Glycerol C. Cholesterol and other steroid (Vit. D) D. Vitamins A, E, K Slide 123: Fatty acids consist of a hydrocarbon chain with a carboxylic acid at one end. Chain length from C4 to C24 . Alternatively,C atoms are numbered from COOH C is no. 1 , The Length of the Carbon Chain long-chain(16-above), medium-chain(8-14), short-chain(2-6) The Degree of Unsaturation ~saturated ~unsaturated -- monounsaturated, polyunsaturated The Location of Double Bonds omega-3 fatty acid, omega-6 fatty acid Branched ,hydroxy, cyclic Slide 124: Saturated fatty acids Name end in “Anoic”. Acetic 2 Propinoic 3 (OCFA)iso-BCFA Butyric 4 Valeric 5 (OCFA)iso-BCFA Caproic 6 Caprilic 8 Capric 10 Lauric 12 Myristic 14 Palmitic 16 (25%) Stearic 18 (5%) Arachidic 20 Lignoceric 24 Unsaturated fatty acids Name end in ”Enoic” Monounsaturated Palmitoleic 16 D9( ω 7) Oleic 18 D9( ω 9) Erucic 22 D13( ω 9) Nervonic 24 D15( ω 9) Polyunsaturated Linoleic 18 D9,12( ω 6) α - linolenic 18 D9,12,15( ω 3) ( γ - D 9,12,6( ω 6)) Arachidonic 20 D5,10,11,14( ω 6) Timnodonic 20 D5,8,11,14,17( ω 3)EPA Clupanodonic 22 D7,10,13,16,19( ω 3)DPA Cervonic 22 D4,7,10,13,16,19( ω 3)DHA Slide 125: Omega-3: Eicosopentaenoic acid (EPA) Docosahexaenoic acid (DHA) Alpha-linolenic acid (ALA) flaxseed--most, canola (rapeseed), soybean, walnut, wheat germ body can make some EPA and DHA from ALA Omega-6 corn, safflower, cottonseed, sesame, sunflower Linoleic acid Introduction of first double bond is always at or near D9 by desaturase in presence of O2,NADH,cyt b5. Slide 126: Omega-3 Fatty Acids ~ Associated with : anti-inflammatory, antithrombotic, antiarrhythmic, hypolipidemic, vasodilatory properties ~Inflammatory conditions ~Ulcerative colitis, Crohn’s ~Cardiovascular disease ~Type 2 diabetes * Mental function ~Renal disease * Growth and development Essential Fatty Acid Deficiency ~Classical symptoms include: growth retardation, reproductive failure, skin lesions, kidney and liver disorders, subtle neurological and visual problems ~People with chronic intestinal diseases ~Depression-inadequate intake alters brain activity or depression alters fatty acid metabolism ~Attention Deficit Hyperactivity Disorder ~lower levels of omega-3--more behavioral problems Slide 127: Geometric isomerism Cis -configuration—naturally occuring Trans -form—metabolic intermediate. By product of saturation of FA ’hardening’ Slide 128: Triacylglycerol ~Esters of trihydric alcohol,glycerol with various fatty acid. ~fatty acids are stored primarily in adipocytes as triacylglycerol. ~Triacylglycerol must be hydrolyzed to release the fatty acids. ~Adipocytes are found mostly in the abdominal cavity and subcutaneous tissue. ~Adipocytes are metabolically very active; their stored triacylglycerol is constantly hydrolyzed and resynthesized ~role of HORMONE SENSITIVE LIPASE Introduction: 129 Overview of fatty acid synthesis Introduction 1. Triglycerides: 130 Triglycerides are a highly concentrated store of energy 9 kcal/g vs 4 kcal/g for glycogen Glycogen is also highly hydrated, 2 g H 2 O/g glycogen 1. Triglycerides 2. Utilization of Fatty Acids as Fuel: 131 Three stages of processing Triglycerols are degraded to fatty acids and glycerol in the adipose tissue and transported to other tissues. Fatty acids are activated and transported into the mitochondria. Fatty acids are broken down into two-carbon acetyl– CoA units and fed into the citric acid cycle. 2. Utilization of Fatty Acids as Fuel Lipid Metabolism: Lipid Metabolism Glycerol is converted to glyceraldehyde phosphate Glyceraldehyde is ultimately converted into acetyl CoA Acetyl CoA enters the Krebs cycle Fatty acids undergo beta oxidation which produces: Two-carbon acetic acid fragments, which enter the Krebs cycle Reduced coenzymes, which enter the electron transport chain Lipid Metabolism: Lipid Metabolism Figure 25.12 Slide 134: CHOLESTEROL PHOSPHOLIPID TRIACYLGLYCEROL LIPID METABOLISM FATS ARE WELL-SUITED TO SERVE AS STORAGE FUELS, BUT THEIR MOBILIZATION IS CHALLENGING Slide 135: PROCESSING OF DIETARY LIPIDS IN VERTEBRATES Muscle: oxidation Adipocyte: storage Slide 136: Adipose tissue (fat cells) Epinephrine and glucagon bind fat cell receptor to trigger cAMP- mediated activation of PKA. PKA activates lipase by phosphorylation Lipase interacts with stored lipids in intracellular droplet, to generate fatty acids and glycerol Perilipin phosphorylation also induces mobilization of stored lipids. Mobilized fatty acids in the blood are bound to serum albumin for transport to muscle cells Lipogenesis and Lipolysis: Lipogenesis and Lipolysis Excess dietary glycerol and fatty acids undergo lipogenesis to form triglycerides Glucose is easily converted into fat since acetyl CoA is: An intermediate in glucose catabolism The starting molecule for the synthesis of fatty acids Lipolysis, the breakdown of stored fat, is essentially lipogenesis in reverse Oxaloacetic acid is necessary for the complete oxidation of fat Without it, acetyl CoA is converted into ketones (ketogenesis) Lipogenesis and Lipolysis: Lipogenesis and Lipolysis Figure 25.13 Lipid Metabolism: Synthesis of Structural Materials: Phospholipids are important components of myelin and cell membranes The liver: Synthesizes lipoproteins for transport of cholesterol and fats Makes tissue factor, a clotting factor Synthesizes cholesterol for acetyl CoA Uses cholesterol for forming bile salts Certain endocrine organs use cholesterol for synthesizing steroid hormones Lipid Metabolism: Synthesis of Structural Materials State of the Body: State of the Body Figure 25.15 Lipid Metabolism: Lipid Metabolism Lipid Metabolism: Lipid Metabolism Where & when are fatty acids synthesized?: Where & when are fatty acids synthesized? Synthesis of Fatty Acids (FA) occurs primarily in the liver and lactating mammary gland, less so in adipose tissue FA are synthesized from acetyl CoA derived from excess protein and carbohydrate FA synthesis uses ATP and NADPH as energy sources FA synthesis requires lots of acetyl CoA: FA synthesis requires lots of acetyl CoA Transfer of acetyl CoA from mitochondria to cytosol involves the citrate shuttle Occurs when citrate concentration in mitochondria is high due to inhibition of isocitrate dehydrogenase by high levels of ATP. (Note: High ATP levels are also required for FA synthesis.) First step in FA synthesis is synthesis of malonyl CoA: First step in FA synthesis is synthesis of malonyl CoA Energy to form C-C bonds is supplied indirectly by synthesizing malonyl CoA from acetyl CoA using ATP and CO 2 The reaction is catalyzed by Acetyl CoA carboxylase FA synthesis: FA synthesis After 7 cycles, palmitoyl-S-ACP is produced and palmitate is released by palmitoyl thioesterase Overall reaction is: 8 acetyl CoA + 14 NADPH + 14H + + 7ATP palmitate + 8CoA + 14 NADP + + 7ADP + 7 P i + 7H 2 O FA synthesis: FA synthesis Sources of NADPH for FA synthesis are the hexose monophosphate pathway and the malic enzyme reaction that converts malate to pyruvate + NADPH in the cytosol Fatty Acid Oxidation: Fatty Acid Oxidation 2.2 Activation of Fatty Acids: 149 Acyl CoA synthetase reaction occurs in the on the mitochondrial membrane. 2.2 Activation of Fatty Acids 2.3 Transport into Mitochondrial Matrix: 150 Carnitine carries long-chain activated fatty acids into the mitochondrial matrix 2.3 Transport into Mitochondrial Matrix Beta-oxidation of fatty acids: Beta-oxidation of fatty acids β-oxidation of FA produces acetyl CoA and NADH and FADH 2 , which are sources of energy (ATP) First, FA are converted to acyl CoA in the cytoplasm: Carnitine shuttle: Carnitine shuttle For transport into mitochondria, CoA is replaced with carnitine by acylcarnitine transferase I Inside mitochondria a corresponding enzyme (II) forms acyl CoA Malonyl CoA inhibits acylcarnitine transferase I So, when FA synthesis is active, FA are not transported into mitochondria Defects in FA transport (including carnitine deficiency) are known Reactions of beta-oxidation: Reactions of beta-oxidation The cycle of reactions is repeated until the fatty acid is converted to acetyl CoA Beta Oxidation: Beta Oxidation Energy yield from beta-oxidation of fatty acids: Energy yield from beta-oxidation of fatty acids For palmitate (16:0) the overall reaction is: Palmitate + 8CoA + 7NAD + + 7FAD + 7H 2 O 8 Acetyl CoA + 7NADH + 7FADH 2 + 7 H + Energy yield as ATP for palmitate: 7 FADH 2 = 1.5 x 7 = 10.5 ATP 7 NADH = 2.5 x 7 = 17.5 ATP 8 Acetyl CoA = 10 x 8 = 80 ATP Total: 108 ATP But, two high energy bonds used in acyl CoA formation, so overall yield is 106 ATP . Why do we subtract two ATPs? Energy yield from beta-oxidation of fatty acids: Energy yield from beta-oxidation of fatty acids Energy yield as ATP for palmitic acid: 7 FADH 2 = 1.5 x 7 = 10.5 ATP 7 NADH = 2.5 x 7 = 17.5 ATP 8 Acetyl CoA = 10 x 8 = 80 ATP Total: 108 ATP Two high energy bonds used in acyl CoA formation, so overall yield is 106 ATP Beta-oxidation of unsaturated fatty acids: Beta-oxidation of unsaturated fatty acids Unsaturated FA yield a bit less energy than saturated FA because they are already partially oxidized Less FADH 2 is produced Why do the Lippincott and Garrett & Grisham texts give different ATP yields for complete oxidation of palmitate?: Why do the Lippincott and Garrett & Grisham texts give different ATP yields for complete oxidation of palmitate? Beta oxidation occurs in mitochondria, so NADH and FADH 2 can be used directly in electron transport, and acetyl CoA can also be used directly for production of energy via TCA cycle. Theoretical yield of ATP from NADH or FADH 2 : 2 ATP per FADH 2 3 ATP per NADH Energy yield as ATP for palmitic acid: 7 FADH 2 = 2 x 7 = 14 ATP 7 NADH = 3 x 7 = 21 ATP 8 Acetyl CoA = 12 x 8 = 96 ATP Total: 131 ATP Two high energy bonds used in fatty acyl CoA (palmitoyl CoA) formation, so overall yield is 129 ATP (according to the Lippincott book) Actual yield of ATP from NADH or FADH2 is thought to be lower than the theoretical yield because:: Actual yield of ATP from NADH or FADH 2 is thought to be lower than the theoretical yield because: – Membranes leak some H + without forming ATP – Some of the proton gradient drives other mitochondrial processes So, actual yield is thought to be closer to: 1.5 ATP per FADH 2 2.5 ATP per NADH Actual energy yield as ATP for palmitic acid is therefore: 7 FADH 2 = 1.5 x 7 = 10.5 ATP 7 NADH = 2.5 x 7 = 17.5 ATP 8 Acetyl CoA = 10 x 8 = 80 ATP Total: = 108 ATP Minus the two high energy bonds used in fatty acyl CoA formation = 106 ATP Beta-oxidation of odd-chain fatty acids: Beta-oxidation of odd-chain fatty acids Odd-chain FA degradation yields acetyl CoAs and one propionyl CoA Propionyl CoA is metabolized by carboxylation to methylmalonyl CoA (carboxylase is a biotin enzyme) Methyl carbon is moved within the molecule by methylmalonyl CoA mutase (one of only two Vitamin B 12 cofactor enzymes) to form succinyl CoA Are fatty acids glucogenic?: Are fatty acids glucogenic? Fatty acids are not glucogenic in animals Why can’t we make glucose from fatty acids? Why are the statements above only ~99% true? Regulation of Beta Oxidation: Regulation of Beta Oxidation Largely by concentration of free fatty acids available Malonyl CoAinhibits carnitine transferase which will inhibit entry of acyl CoA into mitochondria Slide 163: In tissues that use ketone bodies, acetoacetate is condensed with CoA by transfer from succinyl CoA acetoacetyl CoA can then be converted to two acetyl CoAs 2.1 Breakdown of Triacylglycerols: 164 In the adipose tissue, lipases are activated by hormone signaled phosphorylation 2.1 Breakdown of Triacylglycerols Breakdown of Triacylglycerols: 165 The glycerol is absorbed by the liver and converted to glycolytic intermediates. Breakdown of Triacylglycerols Breakdown of Triacylglycerols: 166 The lipases break the triacylglycerols down to fatty acids and glycerol The fatty acids are transported in the blood by serum albumin Breakdown of Triacylglycerols Fatty acid oxidation: 167 Each round in fatty acid degradation involves four reactions 2. Hydration to L–3–Hydroxylacyl CoA Fatty acid oxidation 2.4 Fatty acid oxidation: 168 Each round in fatty acid degradation involves four reactions 3. Oxidation to 3–Ketoacyl CoA 2.4 Fatty acid oxidation 2.4 Fatty acid oxidation: 169 Each round in fatty acid degradation involves four reactions 4. Thiolysis to produce Acetyl–CoA 2.4 Fatty acid oxidation 2.4 Fatty acid oxidation: 170 Each round in fatty acid degradation involves four reactions The process repeats itself 2.4 Fatty acid oxidation 2.4 Fatty acid oxidation: 171 Each round in fatty acid degradation involves four reactions 2.4 Fatty acid oxidation 2.5 ATP Yield: 172 The complete oxidation of the sixteen carbon palmitoyl–CoA produces 106 ATP's 2.5 ATP Yield 3.1 Special Cases: 173 Unsaturated fatty acids (monounsaturated) 3.1 Special Cases 3.1 Special Cases: 174 Unsaturated fatty acids (polyunsaturated) 3.1 Special Cases 3.2 Odd-Chain: 175 3.2 Odd-Chain 3.5 Ketone Bodies: 176 3.5 Ketone Bodies Use of fatty acids in the citric acid cycle requires carbohydrates for the the production of oxaloacetate. During starvation or diabetes, OAA is used to make glucose Fatty acids are then used to make ketone bodies (acetoacetate and D–3–hydroxybutarate) Ketone bodies: Ketone bodies Acetoacetyl CoA is formed by incomplete FA degradation or by condensation of two acetyl CoAs by thiolase Acetoacetyl CoA condenses with a third acetyl CoA to form hydroxymethylglutaryl CoA ( HMG-CoA ) HMG-CoA is cleaved to produce acetoacetate + acetyl CoA Reduction of acetoacetate to β-hydroxybutyrate, or spontaneous decarboxylation to acetone, produces the other two ketone bodies Ketone bodies: Ketone bodies Excessive ketone bodies can be produced in diabetes mellitus or starvation (a lot of acetyl CoA in liver) When rate of production exceeds utilization, ketonemia, ketonuria, and acidemia can result 3.6 Ketone Bodies as a Fuel Source: 179 The liver is the major source of ketone bodies. It is transported in the blood to other tissues Acetoacetate in the tissues Acetoacetate is first activated to acetoacetate by transferring the CoASH from succinyl–CoA. It is then split into two Acetyl–CoA by a thiolase reaction 3.6 Ketone Bodies as a Fuel Source 3.7 Fatty Acids Cannot be Used to Synthesize Glucose: 180 Even though the citric acid cycle intermediate oxaloacetate can be used to synthesize glucose, Acetyl–CoA cannot be used to synthesize oxaloacetate. The two carbons that enter the citric acid cycle as Acetyl–CoA leave as CO 2 . 3.7 Fatty Acids Cannot be Used to Synthesize Glucose 4. Fatty Acid Synthesis.: 181 Fatty acid are synthesized and degraded by different pathways. Synthesis takes place in the cytosol . Intermediates are attached to the acyl carrier protein (ACP). In higher organisms, the active sites for the synthesis reactions are all on the same polypeptide. The activated donor in the synthesis is malonyl –ACP. Fatty acid reduction uses NADPH + H + . Elongation stops at C 16 ( palmitic acid) 4. Fatty Acid Synthesis. 4.1 Formation of Malonyl Coenzyme A: 182 Formation of malonyl–CoA is the committed step in fatty acid synthesis. 4.1 Formation of Malonyl Coenzyme A 4.2 Acyl Carrier Protein: 183 The intermediates in fatty acid synthesis are covalently linked to the acyl carrier protein (ACP) 4.2 Acyl Carrier Protein 4.3 Elongation: 184 In bacteria the enzymes that are involved in elongation are separate proteins; in higher organisms the activities all reside on the same polypeptide. To start an elongation cycle, Acetyl–CoA and Malonyl–CoA are each transferred to an acyl carrier protein 4.3 Elongation 4.3 Elongation: 185 Acyl-malonyl ACP condensing enzyme forms Acetoacetyl-ACP. 4.3 Elongation 4.3 Elongation: 186 The next three reactions are similar to the reverse of fatty acid degradation, except The NADPH is used instead of NADH and FADH 2 The D–enantiomer of Hydroxybutarate is formed instead of the L–enantiomer 4.3 Elongation 4.3 Elongation: 187 The elongation cycle is repeated six more times, using malonyl–CoA each time, to produce palmityl–ACP. A thioesterase then cleaves the palmityl–CoA from the ACP. 4.3 Elongation 4.4 Multifunctional Fatty Acid Synthase: 188 Domain 1 Substrate entry (AT & MT) and condensation unit (CE) Domain 2 Reduction unit (DH, ER & KR) Domain 3 Palmitate release unit (TE) 4.4 Multifunctional Fatty Acid Synthase 4.4 Multifunctional Fatty Acid Synthase: 189 4.4 Multifunctional Fatty Acid Synthase 4.5 Fatty Acid Synthase Mechanism: 190 4.5 Fatty Acid Synthase Mechanism 4.6 Stoichiometry of FA synthesis: 191 The stoichiometry of palmitate synthesis: Synythesis of palmitate from Malonyl–CoA Synthesis of Malonyl–CoA from Acetyl–CoA Overall synthesis 4.6 Stoichiometry of FA synthesis 4.7 Citrate Shuttle: 192 Acetyl–CoA is synthesized in the mitochondrial matrix, whereas fatty acids are synthesized in the cytosol Acetyl–CoA units are shuttled out of the mitochondrial matrix as citrate: 4.7 Citrate Shuttle 4.8 Sources of NADPH: 193 The malate dehydrogenase and NADP + –linked malate enzyme reactions of the citrate shuttle exchange NADH for NADPH 4.8 Sources of NADPH 6. Elongation and Unsaturation: 194 Endoplasmic reticulum systems introduce double bonds into long chain acyl–CoA's Reaction combines both NADH and the acyl–CoA's to reduce O 2 to H 2 O. 6. Elongation and Unsaturation 6.1 Elongation and Unsaturation: 195 Elongation and unsaturation convert palmitoyl–CoA to other fatty acids. Reactions occur on the cytosolic face of the endoplasmic reticulum. Malonyl–CoA is the donor in elongation reactions 6.1 Elongation and Unsaturation 5. Regulation of Fatty Acid Synthesis: 196 Regulation of Acetyl carboxylase Global + insulin - glucagon - epinephrine Local + Citrate - Palmitoyl–CoA - AMP 5. Regulation of Fatty Acid Synthesis 5.1 Regulation of Fatty Acid Synthesis: 197 5.1 Regulation of Fatty Acid Synthesis Slide 198: Both acetate and malonate groups are linked to the enzyme by thioesters The reduction sequence amounts to adding 4 electrons per step, and decarboxylating the 3C malonate to 2C, also at every step. The chain grows from the carboxyl terminal end Palmitate (16:0) is released Slide 199: 4-step sequence Condensation of activated acyl groups, with decarboxylation, gives a b -keto product Reduction to alcohol Elimination of water to create double bond Reduction of double bond to saturation First step – the CO 2 released is the same carbon added from HCO 3 - by acetyl-CoA carboxylase Condensation coupled to decarboxylation becomes thermo. favorable The extra energy needed to make fatty acid synthesis favorable is provided by ATP (acetyl-CoA malonyl-CoA) Slide 200: 7 protein chains are needed in the overall synthetic scheme ACP – acyl carrier protein, thioester linkage KR, HD, ER do steps 2, 3, 4 of the synthetic pathway within each individual cycle In the overall scheme, the first step is the activity of the transacetylase to transfer a 2C unit from acetyl-CoA Uses acetyl-CoA pool in the cytoplasm Slide 201: First step of fatty acid synthesis – AT activity transfers acetate from acetyl-CoA onto the KS subunit Second step – MT activity transfers malonyl group from malonyl-CoA to ACP Slide 202: PRIMED COMPLEX WITH BOTH 2C ACETATE AND 3C MALONATE ESTERIFIED Slide 203: After priming, the KS subunit performs the first step of the 4-step synthesis by condensing the acetyl and malonyl groups Loss of CO 2 4-carbon unit remains attached to the ACP Slide 204: The next three steps (steps 2, 3, 4 in the synthesis) are performed in turn by the KR, HD, and ER subunits of the large complex Substrate channeling End-product is butyryl- group attached on ACP (butyryl-ACP) Last step is the AT activity transfers the 4C butyryl group from ACP to KS to prepare for the next round (recall AT also transfers acetate from free Ac-CoA onto KS) Slide 205: Beginning of the second round – another malonate is transferred onto ACP by MT activity KS does condensation of malonate with butyryl group to form 6-carbon fatty acid. Note growth is at the carboxyl end And so on… A hydrolytic activity releases palmitate (16C stage) OVERALL REACTION 8Ac-CoA + 7 ATP + 14 NADPH + 14 H + palmitate + 8 CoA + 7 ADP + 7 P i + 14 NADP + + 6 H 2 O Note that CO 2 is taken up in production of malonyl-CoA but is released in the condensation step – no net use Malonyl-CoA is synthesized and then used immediately Only 6 H 2 O produced because one is needed in final hydrolysis Slide 206: Control mechanism at the level of fatty acid transport – malonyl-CoA inhibits carnitine acyl-transferase I Stops fatty acid oxidation and synthesis from occurring simultaneously Slide 207: Fatty acid elongation system – used to make stearate (18:0) or longer fatty acids Found in both mitochondria and in smooth endoplasmic reticulum Elongation steps are interspersed with desaturation to introduce double bonds Slide 208: Introduction of the double bond into a fatty acid chain is oxidative Electrons are transferred through redox cofactors from NADPH, which is also oxidized Oxygen is reduced to water This enzyme is a “mixed-function” oxidase Oxidase enzyme family – catalyze oxidations where molecular oxygen is the electron acceptor, but no oxygens appear in the oxidized product (occurs in b -oxidation in plant peroxisomes Mono/dioxygenases – oxygens are directly incorporate into substrate DESATURATION OF FATTY ACIDS Reduction of O 2 is accomplished with electrons derived from both saturated fatty-acyl CoA and from NADPH Slide 209: Phospholipids are synthesized by esterification of an alcohol to the phosphate of phosphatidic acid (1,2-diacylglycerol 3-phosphate). Most phospholipids have a saturated fatty acid on C-1 and an unsaturated fatty acid on C-2 of the glycerol backbone Simplest phospholipid PHOSPHOLIPIDS CLASSIFICATION NITROGEN CONTAINING GLYCERO PHOSPHATIDS ~lecithin ~Cephalin ~Phosphotidyl serine NON-NITROGEN CONTAINING GLYCERO PHOSPHATIDS ~PHOSPHATIDYL INOSITOL ~PHOSPHATIDYL GLYCEROL ~CARDIOLIPIN LONG CHAIN ALCOHOL CONTAINING plasmalogen ALCOHOL SPHINGOSINE sphingomyelin Slide 210: Acyl-CoA synthetases are found in the endoplasmic reticulum, peroxisomes, and inside and on the outer membrane of mitochondria. β -hydroxy- -trimethylammonium butyrate or carnitine(SYN. LYSINE & METHIONINE) (thiokinase) impairment in fatty acid oxidation leads to hypoglycemia. Glyburide,tolbutamide OXIDATION OF FATTY ACID Slide 211: -OXIDATION OF FATTY ACIDS INVOLVES SUCCESSIVE CLEAVAGE WITH RELEASE OF ACETYL-COA fatty acid oxidase Generation of FADH2 & NADH Oxidation of Fatty Acids Produces a Large Quantity of ATP ATP PRODUCTION 1FADH2—2ATP 1NADH--- 3ATP net 5(7x5=35) ACETYL COA—TCA—12ATP.(12x8=96) UTILISATION—2ATP. Eg palmitic acid(16 c)7 cycles,8 acetylcoA(35+96-2=129 ATP ) hypoglycin Slide 212: FATTY ACID BIOSYNTHESIS occurs primarily in the cytoplasm of : liver adipose (fat) central nervous system lactating mammary gland • Intermediates covalently linked to acyl carrier protein • Activation of each acetyl CoA. • acetyl CoA + CO2 Malonyl CoA • Four-step repeating cycle, extension by 2-carbons /cycle – Condensation – Reduction – Dehydration – reduction The enzymes of fatty acid synthesis are packaged together in a complex called as fatty acid synthase (FAS). • The product of FAS action is palmitic acid. (16:0). • Modifications of this primary FA leads to other longer (and shorter) FA and unsaturated FA. • The fatty acid molecule is synthesized 2 carbons at a time• FA synthesis begins from the methyl end and proceeds toward the carboxylic acid end. Slide 213: Mn ++ For fatty acid biosynthesis, acetylCoA has to be transported from the mitochondria to the cytoplasm. This is done via a shuttle system called the Citrate Shuttle . Malonyl CoA is synthesized by the action of acetylCoA carboxylase. Biotin is a required cofactor. AcetylCoA carboxylase is under allosteric regulation. Citrate is a positive effector and palmitoyl CoA is a negative effector PRODUCTION OF MALONYL COA:REGULATORY,IRREVERSIBLE acetylCoA carboxylase .BIOTIN .BIOTIN CARBOXYLASE .BC CARRIER PROTEIN .TRANS CARBOXYLASE .REGULATORY ALLOSTERIC SITE Slide 214: homodimeric enzyme, seven catalytic activities, and eight sites two carriers ACP1 acts as a holding station for acetyl- or fatty acyl- groups. ACP2, binds the growing fatty acyl chain during the condensation and reduction Net reaction: Acetyl CoA + 7 malonyl CoA + 14 NADPH + 14 H+ Palmitate + 7 CO2 + 8 CoA + 14 NADP+ + 6H2O Slide 215: Acetyl-CoA:ACP transacylase , transfers an acetyl group to cysteinyl-S on ACP1. malonyl-group transferred to the pantetheinyl-S of ACP2 by Malonyl-CoA:ACP transacylase . carbon dioxide leaves the malonyl group, with the electrons from its bond attacking the acyl group on ACP1 (Ketoacyl-ACP synthase) b -ketoacyl group ready to go through the reverse of the reactions of b -oxidation. Thus the keto-group is reduced to an alcohol using NADPH ( b -ketoacyl-ACP reductase) , followed by the elimination of the alcohol (Enoyl-ACP hydrase) to give the cis -2,3-enoyl group. The enoyl is then reduced with NADPH substituting for FADH2 (Enoyl-ACP reductase) to give the saturated acyl group. Finally the acyl group is transferred from the pantotheinyl-S of ACP2 to the cysteinyl-S on ACP1 (ACP-acyltransferase) leaving ACP2 available to pick up the next malonyl moiety. After seven turns of the cycle palmitate is released . Biotin and CO2 (again…): 06.4.26 216 Biotin and CO 2 (again…) A trifunctional protein: acetyl-CoA carboxylase One subunit carries the biotin, attached via the e -amino group of a lysine residue One subunit activates CO 2 by transferring it to the biotin Which serves as a long flexible arm to carry the CO 2 to the third subunit Fig. 21-1 Acetyl-CoA Activation: Making Malonyl: 06.4.26 217 Acetyl-CoA Activation: Making Malonyl This third subunit, a transcarboxylase, does exactly that: transfers the CO 2 to acetyl-CoA, converting it into malonyl-CoA, to be used in the next step of the reaction Fig. 21-1 After Activation, Biosynthesis!: 06.4.26 218 After Activation, Biosynthesis! To make a fatty acid, first a 2-carbon unit is activated, becoming malonyl-CoA Conceptually mirroring b -oxidation, a four-step process then lengthens the nascent fatty acid chain by 2 carbons Employing a remarkable enzyme complex containing 7 different activities And a long flexible prosthetic tether derived from pantothenate (where else is this used?) At “Start”, Who’s Holding Whom? And How?: 06.4.26 219 At “Start”, Who’s Holding Whom? And How? The acetyl- and malonyl-CoA thio-esters can “load” onto the thiol groups of a cysteine residue in KS ( b -ketoacyl-ACP synthase) and ACP-4’PPT respectively This primes the system for the subsequent reactions Fig. 21-4 Fig. 21-3 FAS Step 1: A Condensation & Elimination Reaction: 06.4.26 220 Step 1: A Condensation & Elimination Reaction Fig. 21-2 Step 2: A Reduction Reaction: 06.4.26 221 Step 2: A Reduction Reaction Fig. 21-2 Note: FAS FAS Step 3: A Dehydration Reaction: 06.4.26 222 Step 3: A Dehydration Reaction Fig. 21-2 FAS FAS Step 4: A Reduction Reaction (Again): 06.4.26 223 Fig. 21-2 Step 4: A Reduction Reaction (Again) Observe that all of the previous four reactions have been carried out tethered to the 4’PPT of ACP And that the original acetyl group attached to KS is at the terminal end of the chain FAS FAS Note: Now Go Back to Start…: 06.4.26 224 Now Go Back to Start… After the first complete cycle, the fully reduced butyryl group is now transferred back to the Cys residue of KS, Thus freeing up the 4’PPT tether of ACP to accept another moiety of malonyl-CoA …and the cycle can continue (see Fig. 21-5) Fig. 21-6 Recall: Characteristics of Fatty Acid Biosynthesis: 06.4.26 225 Recall: Characteristics of Fatty Acid Biosynthesis As is typical for biosynthetic pathways, the reaction sequences are Endergonic Reductive And they employ ATP as the metabolic energy source The electron carrier NADPH as reductant Large and sophisticated enzyme complexes Fatty Acid Synthase: From Subunits to Domains: 06.4.26 226 Fatty Acid Synthase: From Subunits to Domains Remarkably, although each of the activities arose separately at the bacterial level, by the time vertebrates finished evolving, a single very large protein was enough to encompass all of the activities of the fatty acid synthase. Fig. 21-7 Cofactors as Biological Tethers - A general principle -: 06.4.26 227 Cofactors as Biological Tethers - A general principle - Lipoate – “swinging arm” of pyruvate dehydrogenase Biotin – carries CO 2 in an important anaplerotic reaction Pantothenate (Vit B 5 ) – tethers the growing chain in fatty acid synthetase Fig. 16-17 Origins of Biosynthetic Reducing Power: 06.4.26 228 Origins of Biosynthetic Reducing Power Reducing power is generated during oxidative catabolism, and is needed during reductive anabolism So why is NADPH rather than NADH used for this anabolic process? Fig. 21-9 Where Does Cytosolic Acetyl-CoA Come From?: 06.4.26 229 Where Does Cytosolic Acetyl-CoA Come From? Acetyl-CoA comes from citrate, which can come out of the TCA cycle (under what conditions?) But there’s a “location” problem, And a problem of reducing power… Fig. 21-10 cytosol One Transporter is Not Enough!: 06.4.26 230 One Transporter is Not Enough! And malic enzyme can also be part of the solution Under what conditions would the cell want to have abundant acetyl-CoA in the cytosol? Fig. 21-10 Think about the regulation…: 06.4.26 231 Think about the regulation… Why should feedback be as shown, and why should citrate, especially, play such a central role? Both citrate and malonyl-CoA regulate the choice of oxidizing metabolic fuel vs. its storage as fatty acids, and involves allosteric signals Acetyl-CoA carboxylase is also regulated by phosphorylation, which causes depolymerization of its filaments and thus inactivation Fig. 21-11 How Are Choices About Fatty Acid Metabolism Made?: 06.4.26 232 How Are Choices About Fatty Acid Metabolism Made? Fatty acids are a valuable fuel, and are burned only when their energy is needed In the cytosol of liver cells, fatty acyl-CoA’s are Either taken into mitochondria for b -oxidation Or converted into TAGs and phospholipids by cytosolic enzymes This metabolic fork is governed by the rate of uptake of fatty acyl-CoA’s into mitochondria Which can be inhibited by malonyl-CoA… Recall: Where does malonyl-CoA come from?: 06.4.26 233 Recall: Where does malonyl-CoA come from? Abundant cytoplasmic acetyl-CoA is converted to malonyl-CoA in a biotin-dependent carboxylation reaction This is the first step in fatty acid biosynthesis Under what conditions would there be lots of acetyl-CoA in the cytoplasm? Fig. 21-1 cytoplasm Recall the Acyl-Carnitine/ Carnitine Transporter: 06.4.26 234 Recall the Acyl-Carnitine/ Carnitine Transporter Responsible for the magic trick of supplying fatty acyl-CoA’s to the mitochondrial matrix, where b -oxidation takes place Transport is the rate-limiting step in fatty acid oxidation This is the point of regulation by malonyl-CoA, which inhibits acyl-carnitine transferase I Why malonyl-CoA? The Crosstalk Between Two Pathways: 06.4.26 235 The Crosstalk Between Two Pathways With plentiful energy from carbohydrates, when not all the glucose can be oxidized or stored as glycogen, The excess is channeled into biosynthesis of fatty acids (for storage as TAGs) As often, this is not simply the reverse of b -oxidation, but entails as its first step Carboxylation of acetyl-CoA to produce malonyl-CoA (see Ch. 21) Rather than reversing thiolase , which has other consequences (discussed later) Overall Control of Fatty Acid Oxidation: 06.4.26 236 Overall Control of Fatty Acid Oxidation Fatty acyl-CoA TAG’s and PL’s Malonyl-CoA (from activation of fatty acid bio- synthesis from excess glucose) II I transporter Inner mt membrane b -oxidation High NADH burn store Note need For NAD+ Fig. 17-8 Summary of Control Points: 06.4.26 237 Summary of Control Points Mobilization from TAGs in the adipocyte by TAG-lipase is under hormonal control – epinephrine and glucagon. The carnitine shuttle (which controls entry of fatty acids to the mitochondrial matrix) is inhibited by malonyl-CoA (the first intermediate in fatty acid biosynthesis). Malonyl-CoA is high when carbo-hydrate is plentiful. High NADH inhibits b -hydroxy acyl-CoA dehydrogenase High acetyl-CoA inhibits thiolase Can You Now Answer These Questions?: 06.4.26 238 Can You Now Answer These Questions? What is the rate-limiting step in b -oxidation of fatty acids? At what 3 places is fatty acid oxidation regulated, and how? How does fatty acid metabolism interface with glucose metabolism? What are ketone bodies? Where and why do they arise? Are ketone bodies good or bad? Explain. Regulation of Pyruvate Acetyl CoA: Regulation of Pyruvate Acetyl CoA PDH reaction regulated to spare pyruvate from being irreversibly lost Glucose important for brain and once converted to Acetyl CoA cannot be used for glucose synthesis PDH regulated by phosphorylation and allosteric control Dephosphorylation activates PDH Phosphatase enzyme activated by high Ca2 + Phosphorylation inactivates PDH Kinase activated by acetyl CoA and NADH PDH allosterically inhibited by: ATP Acetyl CoA NADH From: Summerlin LR (1981) Chemistry for the Life Sciences. New York: Random House p 548.