Metabolism Lecture 2023 PDF

Metabolism Dr Dima Moualla Lecture content Glycolysis Pentose Phosphate Pathway (PPP) Gluconeogenesis Glycogenesis Glycogenolysis Fatty acid oxidation (Beta oxidation) Introduction to metabolism The fate of all dietary components is METABOLISM. Metabolic pathways: - Anabol...

Metabolism Dr Dima Moualla Lecture content Glycolysis Pentose Phosphate Pathway (PPP) Gluconeogenesis Glycogenesis Glycogenolysis Fatty acid oxidation (Beta oxidation) Introduction to metabolism The fate of all dietary components is METABOLISM. Metabolic pathways: - Anabolic pathways: includes synthesis of compounds (requires energy) - Catabolic pathways: breakdown of bog molecules (produces energy) - Amphibolic pathways: connect both anabolic and catabolic reaction. Glucose participate in many biological processes: Formation of glycogen. Source for ribose (necessary for DNA synthesis) and NADPH. Formation of pyruvate (amino acids synthesis) and acetyl Co-A (precursor for fatty acids and cholesterol). Formation of triose phosphate necessary for glycerol formation triacyl glycerol formation. Glycolysis Aerobic glycolysis: - In the presence of oxygen - Leads to the formation of acetyl Co-A TCA cycle Anaerobic glycolysis: - In the absence of oxygen. - Leads to the formation of lactic acid (final product) GLYCOLYSIS Glycolysis Definition: It is defined as a sequence of reactions transforming glucose to lactate & pyruvate with the production of ATP. - 1 molecule of glucose will form 2 molecules of pyruvate Site: Cytosolic fraction of cell End result is twofold: Firstly, to produce energy Secondly, to produce intermediates for other biosynthetic pathways 7 Glycolysis Once transported into cell, glucose is phosphorylated by hexokinase The first five steps can be regarded as the ‘preparatory’ phase—consume 2 x ATP in order to convert 1 molecule into 2 molecules of 3-carbon sugar phosphates The second five steps are regarded as the ‘payoff’ phase—yields 4 x ATP (2 net) and 2 x NADH Step 1 Hexokinase catalyses the phosphorylation of glucose (from ATP) to form glucose 6-phosphate (first irreversible step) Step 2 Glucose 6-phosphate is converted to fructose 6- phosphate by phosphoglucose isomerase This reaction involves the conversion of an aldose to a ketose Step 3 Fructose 6-phosphate is phosphorylated by phosphofructokinase (PFK) to form fructose 1,6- bisphosphate and ADP (second irreversible step) Step 4 Fructose 1,6-bisphosphate (a six-carbon molecule) is split by aldolase into two three-carbon molecules: glyceraldehyde 3-phosphate and dihydroxyacetone phosphate Step 5 Of the two products from step 4, only glyceraldehyde 3- phosphate (G3P) is used for the remainder of glycolysis But dihydroxyacetone can be converted back to G3P Step 6 G3P is converted to 1,3- biphosphoglycerate. Enzyme involved is glyceraldehyde 3-phosphate dehydrogenase and uses Pi and NAD+ Step 7 The high-energy phosphate bond in 1,3- bisphosphoglycerate is then used to generate ATP 1,3-bisphosphoglycerate is converted by phosphoglycerate kinase to 3-phosphoglycerate Step 8 3-phosphoglycerate is converted by phosphoglycerate mutase to 2-phosphoglycerate This step moves the phosphate group to a different carbon on the same molecule Step 9 2-phosphoglycerate is converted to phosphoenolpyruvate (PEP) by enolase This dehydration reaction changes the low-energy phosphate ester bond of the former to the high-energy phosphate bond of PEP Step 10 The final reaction involves the conversion of phosphoenolpyruvate to pyruvate via pyruvate kinase (third irreversible step)  Fates of pyruvate Under anaerobic conditions, pyruvate can be converted to lactate by lactate dehydrogenase (LDH) The NAD+ from lactate production allows glycolysis to continue When O2 becomes available the lactate can be converted back to pyruvate Under aerobic conditions, pyruvate dehydrogenase (PDH) can convert pyruvate to acetyl coenzyme A (acetyl CoA) Acetyl CoA can then enter the TCA cycle in the mitochondria Fates of pyruvate cont. Converted to acetyl Co-A Converted to lactate Lactate can be turned back into glucose (Cori cycle) Fates of pyruvate cont. Converted to acetyl Co-A Converted to lactate Lactate can be turned back into glucose (Cori cycle) When ATP is in excess, acetyl CoA accumulates and can instead be used to make fatty acids and ketone bodies (see later lectures) Converted to ethanol (in yeast and some microorganisms under anaerobic conditions)(Pyruvate decarboxylase) Converted to oxaloacetate (pyruvate carboxylase).  Regulation of glycolysis There are various control points along the glycolytic pathway Primary control step is catalysed by PFK, but also hexokinase and pyruvate kinase PFK is allosterically inhibited by ATP Hexokinase is inhibited by glucose 6-phosphate (builds up after PFK inhibition) PK is also allosterically inhibited by ATP and activated by ADP/AMP Pentose Phosphate Pathway- PPP Pentose phosphate pathway (PPP) The PPP takes place in the cytoplasm of cells Starts here Pentose phosphate pathway (PPP) It consists of 2 oxidative, irreversible reactions, followed by a series of reversible reactions. The speed and the direction of these irreversible reactions are determined by availability of intermediates and how much they are needed. As well as generating NADPH, PPP provides the body with pentoses (Ribose-5-phosphate) required for the biosynthesis of nucleotides. PPP cont. Particularly important in cells that synthesise lipids and steroids Reducing power in cells is available as both NADH and NADPH Might sound similar, but have very distinct roles As you know, NADH is used in oxidative phosphorylation to produce ATP… But NADPH is used for powering biosynthesis They even have similar structures, but they can not be interconverted PPP 1. Irreversible oxidative reaction: - There are 2 reactions that leads to the formation of ribose-5-phosphate, CO2, 2NADPH (for each oxidized glucose). - These reactions are important especially in:  liver cells, mammary cells (cells that synthesizes fatty acids) Adrenal cortex: where steroid synthesis dependant on NADPH Red blood cells needs NADPH to keep glutathione in reduced state. 1. Irreversible oxidative reactions 2. Reversible non-oxidative reactions These reactions of the PPP occur in all cells that synthesize nucleotides and nucleic acids. Ribulose 5-phosphate undergoes isomerisation to ribose 5- phosphate nucleotides biosynthesis intermediates for glycolysis G-6-PD deficiency It is a genetic disorder lack of the enzyme glucose-6-phosphate dehydrogenase (G6PD) G6PD helps red blood cells to produce NADPH lack of NADPH, red blood cells will be susceptible to reactive oxygen species and oxidative stress. Therefore, proteins in membranes get oxidized RBC will be lysed. Gluconeogenesis, Glycogenesis and Glycogenolysis Gluconeogenesis The metabolic process by which organisms produce glucose from non-carbohydrate precursors. Gluconeogenesis is very important as glucose is the main energy source for the brain. Where do we get our glucose from? Sources of glucose 1. Diet: not continuous source. Depends on the meal. 2. glycogenolysis: glycogen is storage form for glucose. Used to get glucose quickly. 3. gluconeogenesis: supply body with glucose. But it is slow to respond to low blood sugar. Substrates used in gluconeogenesis 1. glycerol: which is formed from the hydrolysis of triacyl glycerol in adipose tissues. 2. Lactate: produced in muscles during exercise. 3. Amino acids: that are produced from hydrolysis of tissue proteins. Most reactions in glycolysis pathway are reversible and used in glucose synthesis from lactate or pyruvate but there are 3 reactions that are not reversible (steps 1, 3 and 10)  Gluconeogenesis and glycolysis are different processes. Each has its enzymes. There are 3 steps that cannot be reversed from glycolysis, which requires different enzymes. By pass step 10 1. conversion of pyruvate to phosphoenolpyruvate (PEP) - First pyruvate is converted to oxaloacetate via pyruvate carboxylase. - Then, oxaloacetate is converted to PEP via PEP carboxykinase. By pass step 1 and 3 Step 3: remove phosphate from fructose 1,6- biphosphate. - This is mediated by: fructose 1,6- biphosphatase Step 1: converting glucose -6- phosphate to glucose. - This is via glucose-6- phosphatase. Glycogenesis and glycogenolysis Glycogen in the body Most cells can store glycogen. Liver cells (5-8%) and muscle cells (1-3%). Glycogen is a big polymer of glucose. Glycogen enables the cells to store a lot of carbohydrates without affecting osmotic balance in cells. When glycogen storing cells are close to saturation, extra glucose is converted to lipids and stored in the liver and adipose tissues. I. Glycogenesis Occurs in cytosol. It requires: D-glucose and energy provided by ATP. 1. Glucos-6-P formation 2. Glucose -1-P formation 3. G-1-P interacts with UTP to form UDP-glucose 4. UDP-glucose deliver glucose to growing glycogen. II. Glycogenolysis This process occurs in liver and skeletal muscles (when blood sugar level decreases) for energy Glycogenolysis is not hydrolysis, but phosphorolysis- mediated by enzyme called phosphorylase. Phosphorylase breaks down α-1-4 glycosidic bonds. Glycogenolysis In resting state, phosphorylase is not active, and glycogen is stored. phosphorylase is activated by: - Epinephrine (adrenal medulla) - Glucagon (pancreas) The preliminary effect of these hormones is to enhance the formation of cAMP  cAMP initiates a series of reaction that lead to the activation of phosphorylase. cAMP is needed to transform the enzyme from inactive form to active form. It only takes a small amount of the hormone to activate phosphorylase.  TCA cycle and oxidative phosphorylation Citric acid cycle Also called tricarboxylic acid cycle or Krebs cycle. It is the final pathway where the oxidative metabolism of carbohydrates, amino acids, and fatty acids converge. The TCA cycle is an aerobic pathway. Citric acid cycle The cycle occurs totally in mitochondria. In close proximity to electron transport chain (ETC). Reduced co-enzymes produced by glycolysis, lipid metabolism and TCA cycle are oxidized in ETC. Most of the body’s catabolic pathways converge on the TCA cycle - Catabolism of some amino acids generate intermediates of the cycle. - TCA cycle supplies intermediates for a number of important synthetic reactions:  It contributes to glucose formation from the carbon skeleton of some amino acids.  It provides building blocks for the synthesis of some amino acids. So, The cycle should not viewed as a closed circle, but instead as a traffic circle with compounds entering and leaving as required. TCA cycle TCA cycle enzymes are located in a free or attached state to the inner mitochondrial membrane close to respiratory chain enzymes. Generation of acetyl-CoA Following glycolysis, pyruvate moves into the mitochondria. Pyruvate dehydrogenase catalyses the following reaction Pyruvate + CoA acetyl CoA + CO2 NAD NADH Pyruvate dehydrogenase- PDH Pyruvate needs to be moved to the mitochondria. Oxidative decarboxylation reaction. This is a complex enzyme that comprises 3 enzymes and requires various co-enzymes: Thiamine, FAD, NAD, Co-A and lipoic acid Citric Acid Cycle TCA reactions 1. Formation of citric acid: In this reaction oxaloacetate (C4) joins acetyl Co-A (C2) to form citrate (C6). It is mediated by citrate synthase. No energy is needed to be put into this system as acetyl-CoA is a high energy intermediate with enough free energy to drive the reaction 2. Formation of isocitrate Citrate then undergoes an isomerisation reaction to form isocitrate (enzyme: Aconitase) Water is removed, then added back, moving the hydroxyl group from one C atom to its neighbour Like citrate, isocitrate has a 6C skeleton 3. Isocitrate oxidative decarboxylation α-ketoglutarate (C5) is generated. This reaction involved: - Removing hydrogen to form NADH. - Removing carboxyl in the form of CO2. - Enzyme: Isocitrate Dehydrogenase - This reaction is a rate limiting step in TCA. - Enzyme is activated with ADP and first NADH generated inhibited by NADH and ATP. 4. α-ketoglutarate oxidative decarboxylation Formation of succinyl Co-A (C4). Enzyme: α-ketoglutarate Dehydrogenase This enzyme requires various cofactors: TPP, FAD, NAD, Co-A. This reaction results in: - Formation of NADH - Release of CO2 Second NADH generated 5. Succinate formation Enzyme: Succinate thiokinase energy stored in the activated succinyl Co-A molecule is released and used to drive the formation 6. Oxidation of succinate Succinate is oxidized to fumarate (C4). FAD is reduced to FADH2. Enzyme: succinate dehydrogenase. This is not a decarboxylation reaction. 7. Hydration of Fumarate Fumarate is now acted upon by enzyme fumarase to form malate. No energy or NADH formation in this step. 8. Oxidation of malate Formation of oxaloacetate. Enzyme: Malate dehydrogenase. In this reaction: release of NADH. So, oxaloacetate started the first reaction in TCA and is formed back again, so it is not consumed by the cycle. Third NADH generated Energy released by TCA cycle 2 carbon atoms enter TCA cycle and leaves as two CO2 Oxaloacetate is not consumed by the cycle. Oxidation of acetyl-CoA in TCA leads to: - 3 NADH (each NADH when oxidized results in 3 ATP) - 1 FADH2 (each FADH2 when oxidized results in 2 ATP) - 1 ATP directly from the cycle. So, 12 ATP will be formed for each acetyl Co-A that enter the cycle Energy released by TCA cycle Oxidation of acetyl-CoA in TCA leads to: - 3 NADH (each NADH when oxidized results in 3 ATP)  9 ATP - 1 FADH2 (each FADH2 when oxidized results in 2 ATP)  2ATP - 1 ATP directly from the cycle. So, 12 ATP will be formed for each acetyl Co-A that enter the cycle TCA in Metabolism All intermediate molecules in TCA are glucogenic, cause it leads to the formation of oxaloacetate which transforms to PEP via PEP carboxykinase. Pyruvate carboxylation to form oxaloacetate is important reaction to maintain oxaloacetate levels to keep TCA going. TCA in Metabolism Transamination of amino acids leads to the formation of TCA cycle intermediates. As these reactions are reversible, so TCA cycle is also considered a source for the carbon skeleton required to make amino acids. Oxidative Phosphorylation Electron transport chain Both NADH and FADH2 are generated in glycolysis and TCA cycle. These reduced coenzymes can enter the electron transport chain where each coenzyme donate a pair of electrons to a specialized set of electron carriers. Each electron carrier is associated with the inner mitochondrial membrane.  Complex I Complex II Co-Q (quinone) Complex III Cytochrome-c Complex IV (cytochrome oxidase) Complex V (ATP Synthase)  As electrons are passed down the electron transport chain, they lose much of their energy. Part of the energy is captured and stored by the production of ATP. This process is called oxidative phosphorylation  The electron carriers within the complexes are cytochromes and Fe containing proteins. The sequence of electron carriers roughly reflects their relative redox potentials. The more negative the standard redox of a molecule, the stronger a reducing agent it is. Electron transport carriers (complexes I-IV) Electron from NADH (FADH2) pass through complexes. This results in proton extrusion across the membrane, generating a proton motive force. This force is used to generate ATP via ATP synthase complex.  As the proton gradient is dissipated and protons move via the ATP Synthase complex from the intermembrane space into the matrix, the ATP Synthase complex undergoes a conformational change that facilitates the production of ATP.  Total Number of ATPs from the Oxidation Glucose Glucose Pyruvate 4 x substrate level phosphorylations 4 ATP 2 NADH 6 ATP 2 Pyruvate 2 Acetyl-CoA 2 NADH 6 ATP TCA Cycle (for every 2 X Acetyl CoA) -> OXPHOS 6 NADH 18 ATP 2 FADH 4 ATP 2 GTP 2 ATP TOTAL = 40 ATP -2ATP Lipid Metabolism Lipids The importance of lipids Triacylglycerol: The main energy storage in living organisms. Cholesterol: important constituent of cell membranes and a precursor to the synthesis of steroid hormones and bile salts. Glycolipids and phospholipids: important for the  Triacylglycerol are found in blood bound to lipoproteins, which keeps the insoluble lipids in a aqueous environment. Triacylglycerols are hydrolysed into glycerol and free fatty acids in the capillaries of adipose tissues and skeletal muscles. Free fatty acids are taken by these tissues. Glycerol is transferred to the liver and the kidney to participate in gluconeogenesis (synthesis of glucose). Fatty acids Fatty acids consist of long hydrocarbon chains with a terminal carboxylic group. Properties of the fatty acid depends on chain length and level of saturation. How the body uses fatty acids for energy? The process is oxidation (β-oxidation) Fatty acids are oxidised into acetyl-CoA. Fatty acids are oxidised in the mitochondria (while it is synthesized in the cytoplasm) Requirements: activated acyl-CoA molecules, NAD+, FAD. It produces ATP It is aerobic process as it requires oxygen.  Fatty acid oxidation leads to the formation of NADH, FADH2 ATP will be synthesized via oxidative phosphorylation. β-oxidation First step: Fatty acid activation. This step requires ATP. Fatty Acid + CoA + ATP Fatty Acyl-CoA + AMP + PPi Mediated by: Acyl-CoA synthetase (Thiokinase) Fatty acids are converted to an active intermediates (Fatty acyl-CoA). Activation step occurs in cytosol while oxidation occurs in mitochondria. β-oxidation Second step: translocation of activated fatty acids Activated acyl group cannot pass mitochondrial membrane directly (impermeable to CoA) A specialized carrier transport long-chain acyl group from cytosol into the mitochondrial matrix. This carrier is carnitine. Acyl group translocation Acyl group is first transferred from CoA to carnitine by carnitine palmitoyltransferase I (CPT-I) (an enzyme of outer mitochondrial membrane) This reaction forms acylcarnitine. Acylcarnitine is then transported into the mitochondrial matrix in exchange for free carnitine, by carnitine-acylcarnitine translocase. Carnitine palmitoyltransferase II (an enzyme of inner mitochondrial membrane) catalyses the transfer of the acyl group from carnitine to CoA. Acyl group translocation β-oxidation β-oxidation It consists of a sequence of 4 reactions involving the β carbon. It results in shortening the fatty acid chain by two carbons. Fatty acid chain is cleaved between the α and β carbon. Cleavage of the chain in β-oxidation starts at the β-oxidation 1. Oxidation that produces FADH2 (acyl-CoA dehydrogenase) 2. Hydration step (enoyl-CoA hydrolase) OH 3. Second oxidation that produces NADH (dehydrogenase) 4. A thiolytic cleavage that releases a molecule of acetyl-CoA (β-ketothiolase) β-oxidation Acetyl-CoA is oxidised in the presence of oxygen into CO 2 and H2O via TCA cycle complete oxidation of fatty acids. Each cycle of β-oxidation produces: 1 NADH, 1 FADH2 and a molecule of acetyl CoA which when oxidised produces more NADH and FADH2. more ATP Oxidation of unsaturated fatty acids Oxidation of monosaturated fatty acids (Oleic acid) requires one additional enzyme (isomerase). Isomerase enzyme converts the 3-trans derivative to the 2- trans derivative required by the enzyme hydrolase. Ketone bodies Acetyl-CoA when produced in excess for TCA cycle: - Acetoacetate - 3-hydroxybuterate ketone bodies - Acetone Normally, it exists in lower than 0.2mM/L Ketone bodies are important source of energy for the peripheral tissues (skeletal and cardiac muscle, kidney and even the brain). Synthesis of ketone bodies When acetyl-CoA is produced in excess such as diabetes. In uncontrolled diabetes, this causes ketoacidosis (acetone breath). Regulation of ketone bodies formation 1. The liver: take up to 30% of free fatty acids in blood. 2. In the liver: fatty acids are either oxidised or used to form ketone bodies or esterified to triacylglycerol (CPT-I activity if low in the fed state) 3. Acetyl-CoA produced is either oxidised in TCA cycle or make ketone bodies. Regulation of ketone bodies formation 1. The liver: take up to 30% of free fatty acids in blood. 2. In the liver: fatty acids are either oxidised or used to form ketone bodies or esterified to triacylglycerol (CPT-I activity if low in the fed state) 3. Acetyl-CoA produced is either oxidised in TCA cycle or make ketone bodies. Any questions