BIOL214 Lecture Notes PDF

The Biochemistry or Energy and Metabolism Introduction to Biochemistry of Energy and Metabolism Metabolism Highly coordinated cellular activity in which multi-enzyme systems (metabolic pathways) cooperate to: - Obtain energy by degrading energy-rich nutrients or by capturing sola...

The Biochemistry or Energy and Metabolism Introduction to Biochemistry of Energy and Metabolism Metabolism Highly coordinated cellular activity in which multi-enzyme systems (metabolic pathways) cooperate to: - Obtain energy by degrading energy-rich nutrients or by capturing solar energy (photosynthesis) - Convert nutrient molecules to new molecules within cells - Polymerise monomers to polymers (polysaccharides, lipids, proteins, nucleic acids) - Synthesise and degrade biomolecules - Maintain distinctive composition of different cell compartments Living Organisms Require Energy - Energy comes from the environment - Energy comes in the form of chemical fuels (food or stored molecules) or light - Energy is required for: - Mechanical work in muscle contraction and cellular movements - Active transport of molecules and ions across cell membranes - Synthesis of macromolecules and other molecules from simple precursors Three Forms of Energy in Biology Energy carriers - Contain ≥1 energy-rich covalent bond Macromolecules - Highly reduced (electron-rich) molecules - Large branched polysaccharides (glycogen in animals and starch in plants) - Fatty acids (from which most animal cells derive their energy between meals) - Polysaccharides and fatty acids are both degraded to acetyl-CoA in the mitochondria Electrochemical gradients - Electrical force (membrane potential) - Chemical force (ion concentration) Different Forms of Energy Carriers - ATP (adenosine triphosphate) - Energy release following transfer of groups - NADH (nicotinamide adenine dinucleotide) - NADPH (nicotinamide adenine dinucleotide phosphate) - FADH2 (flavin adenine dinucleotide) - All three release high energy electrons and H+ - Coenzyme A (CoA) - Carrier of acyl (R−C=O) and acetyl (CH3−C=O) groups - Energy release from high energy bond of the acyl or acetyl group - Others (guanosine triphosphate, carboxylated biotin, uridine diphosphate glucose, Sadenosylmethionine) ATP is the Most Abundant Energy Carrier - Formed by oxidation of carbon fuels - Supplies free energy to enzymatic pathways by transfer of groups (Pi , PPi or AMP) - Links energy-releasing pathways with energy-requiring pathways NADH and NADPH are Carriers of High Energy Electrons - Act to shuttle electrons and H+ between anabolic and catabolic processes - NADH and NADPH both derived from vitamin B3 (niacin) - The extra phosphate group on NADPH - No effect on the electron-transfer properties of NADPH compared to NADH - Allows NADPH to bind different substrates compared to NADH - Helpful tip: NADH is used mainly in ATP generation - Helpful tip: NADPH is used mainly in synthesis of organic molecules Coenzyme A is an Activated Carrier Important in the Oxidation of Pyruvate and Fatty Acids Majority of molecule facilitates recognition by specific enzymes - red is the important information Coenzyme A Is An Activated Carrier of Acyl or Acetyl Groups Majority of molecule facilitates recognition by specific enzymes Helpful to Remember Central Role of Carbon in Biochemistry - Most molecules within cells are comprised of carbon (organic molecules) - Carbon is outstanding among all elements in its ability to form large molecules - It is small, has 4 electrons and 4 vacancies in its outermost shell to form 4 covalent bonds (sharing of electrons between atoms) - Can form highly stable C-C covalent bonds to form linear and branched chains, and rings - C skeletons can bind functional groups such as - Methyl (CH3), hydroxyl (OH), carboxyl (COOH), carbonyl (CO), phosphate (PO3 2- ), sulphydral (SH), amino (NH2) Small Organic Molecules Are Used to Form Macromolecules: Revision Catabolic and Anabolic Metabolism - Catabolic and anabolic pathways constitute the metabolism of a cell - Enzymes catalyse these two pathways - These two pathways are almost always distinct from each other (compartmentalisation) - Compartmentation or compartmentalisation allows cells or organisms to perform specific processes more efficiently Catabolic Pathways Converge and Anabolic Pathways Diverge - (Don’t need to know) Catabolic and Anabolic Metabolism (to help with understanding) Electron Flow is Important in Metabolism - Flow of electrons is ultimately responsible for all work done in living organisms - In non-photosynthetic organisms the source of electrons is food and stored molecules (reduced or electron-rich compounds) - Electrons move from a range of metabolic intermediates to specialised electron carriers (NADH, NADPH and FADH2) Helpful Tip to Follow Oxidation and Reduction in Biochemistry - Oxidation of organic molecules results in the loss of electrons - Reduction of organic molecules results in the gain of electrons - Helpful tip: When a cellular molecule loses an electron, it often loses a proton (H+) - AH → A + e- + H+ (and vice versa) - In organic molecules: - oxidation occurs if the number of C-H bonds decreases - reduction occurs if the number of C-H bonds increases Helpful to Remember Water is a Reactant in Biochemistry - Water can participate directly in chemical reactions - Condensation reaction: formation of a bond between -OH and -OH expelling water - Hydrolysis reaction: cleavage of this bond accompanied by addition of water elements Enzymes Promote Metabolic Reactions - Cellular chemical reactions require much higher temperatures than that found in cells - This is overcome by the use of specialised proteins called enzymes - Enzymes: - Function as catalysts to speed up a specific reaction (1012 times faster) - Can catalyse the reaction of 1000s of substrate molecules every second - Often conjugated to a cofactor (inorganic ion) or coenzyme (complex molecule) to function - Usually involved in a series of connected reactions Enzyme-Substrate Complex - Active site provides a specific environment in which a given reaction can occur more rapidly - Substrate is the molecule that is bound to the active site and acted upon by the enzyme Enzymes are Catalysts E + S ⇌ ES ⇌ EP ⇌ E + P (E, enzyme; S, substrate; P, product; ES and EP transient states) - Enzymes enhance reaction rates by lowering activation energies - Enzymes do not affect the equilibrium of a reaction (and thus can be bidirectional) Factors Affecting the Activity of Enzymes (and Metabolic Processes) - Amounts of enzymes: primarily by rate of transcription of enzyme genes - Catalytic activities of enzymes: allosteric control, feedback inhibition, covalent modification or proteolytic cleavage - Accessibility of substrates: into cells or subcellular compartments (compartmentation or compartmentalisation) - Allosteric Control of Enzymes - Allosteric control is the regulation of an enzyme by a regulatory molecule that interacts at a site (allosteric site) other than the active site (at which catalytic activity occurs) - Allosteric modulators can be either stimulatory (positive) (as shown to the left) or inhibitory (negative) Feedback Inhibition of Enzymes - Inhibition of the first irreversible reaction (or committed step) in linear pathways - Feedback inhibition and activation of branched pathways - Inhibition of common initial step by its own product and activated by the product of another pathway - Enzyme multiplicity in branched pathways - Inhibition of the committed step catalysed by isoenzymes - Cumulative feed inhibition of branched pathways - Partial inhibition of a common step by each of the final products Covalent Modification of Enzymes (and Proteins) Phosphorylation Regulates the Activity of Many Enzymes (and Proteins) Some enzymes are deactivated by phosphorylation and activated by dephosphorylation Some Enzymes (Zymogens) are Regulated by Proteolytic Cleavage Proprotein or proenzyme: - Precursors that are cleaved to form other proteins Zymogens: - Inactive precursor that is cleaved to form an active protease - Often involved in pathways mediating digestive, coagulation, immune and cell death pathways - Focus of Chymotrypsin Practical - Discussed further in Protein Metabolism lectures Summary - Metabolism involves the coordination of multi-enzyme systems to generate energy, and to degrade and synthesise molecules - Catabolism refers to the degradation of molecules, anabolism refers to the synthesis of molecules - Biological energy can be in the form of energy carriers, macromolecules or electrochemical gradients - Electron flow, energy carriers, water and enzymes play key roles in metabolism Cell Respiration part 1 The process by which organic fuels, such as carbohydrates (e.g. sugars), are broke down for the transformation and storage of cellular energy (ATP) - This happens in the mitochondria, in eukaryotes. - This capacity to release energy from energy-rich molecules is in all organisms Cell biology: revision Only plant cells have chloroplasts Glucose: where do the plants get it from? Glucose: where do animals get it from? Plants: Starch - A polysaccharide made of glucose Mitochondrion (plural: mitochondria) The Ecosystem Revise: redox reactions Chemical reactions that transfer electrons between reactants are called oxidation-reduction reactions, or redox reactions - OIL RIG (oxidation is loss, reduction is gain) for electrons Respiration Cell respiration: the process by which organic fuels, such as carbohydrates, are broken down for the production of cellular energy (ATP). Respiration Part 1 - Summary Plants can make their own glucose from sunlight: happens in chloroplasts - Stores glucose as starch - Animals eat this starch to obtain glucose and convert to glycogen Fuel can then be used to make ATP via cellular respiration in plants or animals: - Occurs in the mitochondria - CO2 and H2O also produced - 1x glucose + 6O2 → 6x H2O + 32 ATP - Redox: oxidation-reduction - OIL RIG - Oxidation Is Loss, Reduction Is Gain (of electrons) - O2 is very, very hungry for electrons Cell Respiration part 2 Cellular respiration: 3 main stages 1. In the cytoplasm: Glycolysis - Breaks down glucose into two molecules of pyruvate 2. In the mitochondria: Citric acid cycle - Completes the breakdown of pyruvate 3. In the mitochondria: Oxidative phosphorylation - Accounts for most ATP synthesis NADH and FADH2 - Storing your electron boody for later Stage 1: Glycolysis in the cytoplasm cuts glucose into pyruvate Pyruvate oxidation makes acetyl-CoA in the mitochondria Acetyl CoA feeds into the citric acid cycle Stage 2: In the Mitochondria The Citric Acid Cycle: highly simplified Stage 1 & 2 Summary 1. Glycolysis - Glucose broken down into pyruvate - Pyruvate is transported from the cytoplasm into the mitochondria (transition step) - Pyruvate oxidation produces Acetyl CoA, NADH2, and CO2 2. Citric acid cycle - Uses Acetyl CoA from the pyruvate oxidation - Occurs in mitochondria - Every turn of the cycle: - Acetyl (2 carbons) is broken down - CO2 released - NAD+ is converted to NADH - FAD is converted into FADH2 - ADP is converted into ATP Note: only 2 ATP are converted for every glucose molecule The citric acid cycle occurs in the mitochondrial matrix: NADH and FADH2 feed into oxidative phosphorylation ATP Balance Sheet How much ATP? For one single glucose, we get: This is not much. Most energy so far extracted is in the form of: NADH and FADH2 Oxidative phosphorylation I: Electron Transport Chain Oxidative phosphorylation II: Chemiosmosis - The electron transport chain has created a proton (H+) gradient across the inner mitochondrial membrane  - Energy coupling: The H+ ions want to move into the matrix (an exergonic reaction). - This releases energy to drive ATP synthesis (an endergonic reaction) - The ATP synthesis in the inner mitochondrial membrane uses the proton gradient to do work: phosphorylate ADP into ATP 3. Oxidative Phosphorylation summary - During glycolysis and the citric acid acid cycle, electrons are temporarily stored in NADH and FADH2 - Energy conserved in these molecules is converted into ATP via oxidative phosphorylation - Two stage process: electron transport and chemiosmosis Electron Transport needs Oxygen Organisms that respire aerobically require oxygen because oxygen is the final electron acceptor in the electron transport chain (oxidative phosphorylation) For aerobic organisms: ATP produced via glycolysis/citric acid cycle is not enough No oxygen = not enough ATP Stage 3 Summary - Oxidative phosphorylation - Electron transport chain (ETC) - Embedded in the inner membrane of mitochondria (eukaryotes) or plasma membrane (prokaryotes)  - ETC is made up of a series of membrane protein complex and diffusible electron carriers (shuttles) - NADH and FADH2 transfer electrons to the ETC - As electrons are passed from one protein complex to another, protons (H+) are pumped out across the inner mitochondrial membrane - Results in a proton gradient: a high concentration of H+ in the intermembrane space - Oxygen is the final electron acceptor in electron transport, eventually forming water But what if there’s no oxygen? Anaerobic respiration Anaerobic Respiration Other Energy Sources Cellular Respiration Summary (don’t need to remember the numbers just the processes) Glycolysis (in the cytoplasm) - Breaks down glucose and forms pyruvate + ATP Pyruvate oxidation (in the mitochondria) - Converts pyruvate into acetyl CoA - If O2 available, progresses to … Citric Acid Cycle (in the mitochondria) - Uses O2 and acetyl CoA to produce NADH, FADH2, CO2 and ATP Oxidative phosphorylation (in the mitochondria) - Utilises the electron transport chain in the inner mitochondrial membrane - Uses O2 and electrons from NADH and FADH2 to produce a proton gradient - Chemiosmosis: between the mitochondrial matrix and the intermembrane space - Produces a proton gradient that drives production of many ATP Glycolysis - Metabolism doesn’t work in isolation Carbohydrate Metabolism Cellular respiration Process in which cells breakdown glucose to form ATP - Adenosine tri-phosphate (ATP) is the energy currency of organisms Occurs in three major stages: - acetyl CoA production (glycolysis) - acetyl CoA oxidation (TCA cycle) - electron transfer/oxidative phosphorylation Other precursors can be amino acids and glycerol, but relax on that for now How much ATP do you make from 1 snake lolly? (theoretically) Don’t need to know how to do this calculation: - Glucose amount = 5.45 grams - Molecular mass of glucose = 180.16 g/mol - Moles of glucose = 5.45 g/180.16g/mol = 0.0302508 - Molecules of glucose = 0.0302508 moles X 6.022x1023 (Avogadro’s #) = 1.822x1022 molecules - If 1 glucose molecule makes 38 ATP… - 38 ATP X 1.822x1022 molecules = 6.92x1023 ATP or… - 692,000,000,000,000,000,000,000 ATP molecules! - Even at 50% efficiency, that’s a lot of ATP. Glycolysis - Glycolysis; Greek, Glykis = “sweet”, Lysis = “splitting” - Occurs in the cytosol of every cell - Energy is released from glucose and captured as ATP/NADH - Serves as a model to understand many metabolic pathways The importance of glycolysis 1. Main method of creating Acetyl-CoA 2. Glucose is the only metabolic energy source for: - Tissues: brain, kidney, & rapidly contracting skeletal muscles - Cells: erythrocytes & sperm cells 3. Tightly regulated – as ATP hydrolyses easily, we can use glycolysis to regulate ATP production Glycolysis overview 1. Phosphorylation of glucose This is an important regulatory enzyme; Know this reaction; know names and structures Chemical logic: Happens at C6 as C1 is a carbonyl (C=O) and can’t be phosphorylated 2. Conversion of G6P to Fructose-6-phosphate (F6P) Isomerization: a reaction that changes the shape of a single molecule but doesn't permanently add or remove any atoms. Chemical logic: isomerisation moves carbonyl to C2, prep for next step 3. Phosphorylation of F6P to Fructose-1,6-bisphosphate This is an important regulatory enzyme; Know this reaction; know names and structures - PFK-1 = “gatekeeper” of glycolysis (committed step) - Mutations associated with cancer Chemical logic: phosphorylates so both ends have a phosphate and interconvertible once cleaved 4. Cleavage of fructose-1,6-bisphosphate (F-1,6-BP) Chemical logic: bunch of steps to chop this bad boy up 5. Interconversion of the triose phosphates Chemical logic: rearranges dihydroxyacetone phosphate to G3P to funnel both products into a single pathway 6. Oxidation of G3P to 1,3-bisphophoglycerate (1,3-BPG) - inorganic phosphate introduced (not from ATP) - NAD+ is a cofactor here - NADH produced is to be used in oxidative phosphorylation (or to make lactate) Chemical logic: pinches some e- and Hand replace with free-floating phosphate so we can make ATP in step 7. Sidebar: ATP synthesis Question: If we have inorganic phosphate floating around, why don’t we just make ATP from AMP/ADP directly? Answer: Complex pathways allow for tight regulation - ATP hydrolyses quickly, so cellular respiration allows us to make ATP when needed Sidebar: NAD+ NADH 7. Phosphoryl transfer from 1,3-BPG to ADP Chemical logic: Finally, time to make some ATP 8. Conversion of (3-PG) to 2-phosphoglycerate Chemical logic: Sets up final steps by moving the phosphoryl group 9. Dehydration of 2-PG to phosphoenolpyruvate (PEP) Chemical logic: Dehydration activates the phosphoryl for transfer to ADP in next step 10. Transfer of phosphoryl group from PEP to ADP This is an important regulatory enzyme; Know this reaction; know names and structures(~ish) Chemical logic: ATP production Glycolysis is elevated in tumour cells (don’t need to memorise the image) - Tumour cells can grow faster than a blood supply can be made leads to anaerobic metabolism - Only 2 ATP made in anaerobic conditions, so cancer cells must ramp up glycolysis - E.g. glucose transporters, hexokinase - Compounds that inhibit key steps in glycolysis can kill cancer cells by limiting energy production Oxidation of multiple carbohydrates involves glycolysis (don’t need to know) Glycolysis summary - Glucose is broken down from a 6-carbon molecule to two 3-carbon molecules called pyruvate - There is an investment phase of 2 ATP and a gain of 4 ATP, netting 2 ATP Glycolysis summary - NADH is also produced Key steps include - Step 1. G G6P (via hexokinase) - Irreversible, costs ATP - Step 3. F6P F1,6-BP (via PFK1) - Irreversible, commitment step, highly regulated - Step 10. PEP Pyruvate (via PK) - Irreversible, makes ATP, last step Fates of pyruvate Oxygen is required to unlock the full energy potential of glucose via cellular respiration Fates of pyruvate (anaerobic) Ethanol production Yeast are capable of ethanol fermentation Two-step reduction of pyruvate to ethanol - Humans do not have pyruvate decarboxylase - Humans do have alcohol dehydrogenase Lactate production - If no O2 around, ETC can’t accept e- from NADH – process halts - So, the cell makes lactate by using all this accumulated NADH - feeds NAD+ back into glycolysis until O2 is available - Lactate formed by active skeletal muscle can be recycled - The Cori cycle – covered in week 4! Summary Glycolysis - occurs in the cytosol - from 1 glucose to 2 pyruvate molecules - 2NADH +2H+ + 2ATP generated. - does NOT require O2 - regulated to maintain constant cellular [ATP] - is the only source of ATP for some cell/tissue types - Tumours have very high rates of glycolysis Pyruvate - can be converted to Lactate (anaerobic conditions) - can be converted to ethanol by yeast - can be converted to acetyl-CoA to feed into TCA cycle (next lecture) The TCA cycle Glycolysis doesn’t make much energy Cellular respiration Acetyl-CoA Coenzyme A The function of CoA is to accept and carry acetyl groups Coenzymes Coenzymes are not a permanent part of the enzymes’ structure - They associate, fulfill a function, and dissociate Conversion of pyruvate to acetyl-CoA Pyruvate now needs conversion to be useful! Catalyzed by the Pyruvate Dehydrogenase Complex (PDC) - Requires 5 coenzymes - Thiamine pyrophosphate (TPP), lipoyllysine, and FAD are prosthetic groups (covalently bound to PDC) - NAD+ and CoA-SH are co-substrates - Oxidative decarboxylation of pyruvate Pyruvate Dehydrogenase Complex - Large! (up to 10 MDa) - Located in the mitochondrial matrix of eukaryotes - Subunits organized into 3 functional proteins Overall reaction of PDC Enzyme 1: 1. Decarboxylation of pyruvate to a hydroxyethyl intermediate 2. Oxidation of hydroxyethyl to ketone; reduction of lipoyllysine Enzyme 2: 3. Formation of acetyl-CoA Enzyme 3: 4. Reduction of FAD; Oxidation of the lipoyllysine 5. Oxidation of FADH2; reduction of NAD The PDC uses substrate channelling so: 1. the intermediates never leave the surface 2. the local concentration of substrates is kept high 3. it prevents the “theft” of the activated acetyl group by other enzymes that use this group as a substrate B Vitamins PDC: - Thiamine pyrophosphate (TPP) is a B1 derivative - NAD+ contains niacin which is B3 - Pantothenic acid is B5 – part of CoA - Riboflavin (B2) is involved in the ETC - Biotin (B7) is important in gluconeogenesis Thiamine-deficient animals are unable to oxidize pyruvate normally - The brain is especially susceptible - Beri-beri (“I cannot, I cannot”) disease is characterised by loss of neural function - Elevated blood levels of pyruvate often indicate defects in pyruvate oxidation Revision: The mitochondrion The TCA Cycle The Tricarboxylic acid (TCA) cycle - Or Krebs, or citric acid cycle Funnels Acetyl-CoA through oxidation steps to produce - 2 x CO2 - 3 x NADH - 1 x FADH2 - 1 x GTP 2 turns per starting glucose molecule Occurs in mitochondrial matrix - Except for succinate dehydrogenase, which is located on the inner mitochondrial membrane TCA cycle steps (focus on this diagram more than the two above) 1. C -C bond formation to make citrate 2. Isomerization via cis -aconitate 3. & 4. Oxidative decarboxylations to give 2 NADH 5. Substrate -level phosphorylation to give GTP 6. Dehydrogenation to give reduced FADH 2 7. Hydration 8. Dehydrogenation to give NADH 2. Isomerization via cis-aconitate - Aconitase (2) catalyses the conversion of citrate (a tricarboxylic acid) to isocitrate - 2 step reaction with cis-aconitate intermediate Prosthetic group and enzyme binding example - Aconitase (2) contains an iron-sulfur (Fe-S) centre as its coenzyme/prosthetic group - The Fe-S centre binds the substrate and catalyses the addition or removal of H2O - The citrate molecule is shown in blue at the active site of aconitase α-Ketoglutarate Dehydrogenase (4) Complex like pyruvate dehydrogenase - Same coenzymes, identical mechanisms Active sites different to accommodate different-sized substrates But Jay, why do I have to know the intermediates? 1. Academics are cruel 2. I’ve been told to teach you 3. TCA is the heart of cellular metabolism - Intersects many pathways - Many points of regulation - Anabolic pathways - Medical implications TCA cycle intermediates are key biosynthetic precursors (don’t need to memories diagrams) - Compounds in the blue boxes are synthesised from intermediates of the TCA cycle. - The red arrows show reactions that replenish the TCA cycle (called anaplerotic reactions) TCA cycle tips to know for the exam Key substrates and enzymes Key products - NADH at steps 3, 4 and 8 - GTP at step 5 - FADH2 at step 6. Example question: Name the enzyme that oxidises malate to produce oxaloacetate and NADH Answer: malate dehydrogenase 8. Malate Shuttle NADH made in glycolysis can’t get into mitochondria Instead, the cytosolic AND mitochondrial forms of malate dehydrogenase form part of a shuttle: - Cytoplasmic MDH uses up NADH to convert oxaloacetate to malate - Malate is transported into the mitochondrion - Mitochondrial MDH oxidises malate to oxaloacetate to recreate NADH for further use in cellular respiration The Malate/Aspartate shuttle Case Study: Fluoroacetate (1080) For use in controlling feral dingo populations Symptoms of 1080 poisoning: - Excessive howling - Manic running - Vomiting - Seizures - Loss of coordination - Difficulty breathing - Eventual death Binds tightly to aconitase, slowing the TCA cycle, causing death by: 1. Elevating citrate levels, - causes hypocalcaemia & heart failure 2. Decreasing ATP Summary - Cellular respiration occurs in 3 stages; the second stage is the TCA cycle - Pyruvate enters the TCA cycle by being converted to Acetyl-CoA via the pyruvate dehydrogenase complex. - Efficiency of PDC is maintained by substrate channelling - The net reaction in the TCA cycle is: - Acetyl-CoA + 3NAD+ + FAD + GDP + Pi + 2H2O CoA-SH + 3NADH + 3H+ + FADH2 + GTP + 2CO2 - There are two oxidative decarboxylation steps. - Intermediates of the TCA cycle are important in intermediary metabolism as biosynthetic precursors. Oxidative Phosphorylation Cell respiration (recap) Process in which cells breakdown glucose to form ATP - Adenosine tri-phosphate (ATP) is the energy currency of organisms Occurs in 3 major stages: 1. acetyl CoA production (glycolysis) 2. acetyl CoA oxidation (TCA cycle) 3. electron transfer/oxidative phosphorylation ← We’re finally here! How’s the bank account? So far, 1 glucose molecule has made: Glycolysis - 2 ATP (net) - 2 NADH (1 per G3P) Pyruvate decarboxylation - 2 NADH (1 per pyruvate) TCA cycle (for 2 turns) - 6 NADH - 2 FADH2 - 2 GTP (becomes ATP) Oxidative Phosphorylation (OxPhos) Reduced e– carriers from TCA cycle, acetyl CoA production, glycolysis… - ADP is like a partially charged car battery - ATP is like a fully charged car battery - The ETC is like a charger - It provides energy to convert ADP into ATP Where is this happening? The Electron Transport Chain/OxPhosph – 2 Step Process 5 protein complexes in oxidative phosphorylation Coenzyme Q (ubiquinone) A mobile electron carrier - Can freely diffuse within the lipid bilayer of the inner mitochondrial membrane (lipid soluble) - Can shuttle reducing equivalents between other less mobile components - Carries two e- from each complex I & II to complex III Cytochrome c - The second mobile electron carrier in oxidative phosphorylation - A soluble heme-containing protein in the intermembrane space - Heme iron can be either ferrous (Fe3+, oxidized) or ferric (Fe2+, reduced) - Cytochrome c carries a single electron from the complex III to complex IV Complex I: NADH dehydrogenase Complex II: Succinate Dehydrogenase Complex III: Ubiquinone:cytochrome c oxidoreductase Complex IV: Cytochrome oxidase The Electron Transport Chain/OxPhosph – 2 Step Process The order of electron flow (don’t need to memories table and diagram) - The components of the ETC are arranged in order of increasing standard electrode potentials. - e- flow spontaneously from molecules with lower (more -ve) potentials to those with higher (more +ve) potentials. - The order of electron flow - The order of e- transport has also been worked out using inhibitors of known standard reduction potential - Those before the block become reduced and those after the block are oxidised Electrons are passed along As they lose energy as they go The energy can be used to do work Oxygen is the last electron acceptor How does electron transport make ATP? Chemiosmotic coupling - Redox reactions pump protons (H+) into the intermembrane space - Changes pH and electrical charge across the membranes - This causes an electrochemical gradient - Known as chemiosmotic coupling - Gradient discharged by protons flowing through FO part of ATP synthase Complex V: ATP synthase - ATP synthase (or F-type ATPase) - “F” comes from “phosphorylation Factor” FO - membrane spanning domain - Transports H+ from IMS to matrix, dissipating the proton gradient - “O” in FO (not zero!) comes from Oligomycin sensitivity - Energy transferred to F1 to catalyze phosphorylation of ADP F1 - ATP synthesizing domain - soluble complex in the matrix Complex V: ATP synthase ATP synthase - A total of 3 H+ are transported per ATP generated - Another H+ is used to transport phosphate (H2PO4 - required for ATP synthesis) - Net yield: 4H+ per ATP Note: ATP yields are theoretical. Different ratios exist for different ATP synthases Binding-change model - The proton-motive force causes rotation of the central shaft (→) - Contacts each αβ subunit pair in succession - This produces a conformational change which - ejects ATP from the β-ATP site - the β-ADP site is converted to the β-ATP conformation, which promotes condensation of bound ADP + Pi to form ATP - the β-empty site becomes a β-ADP site, which loosely binds ADP + Pi entering from the solvent - ATP cannot be released from one site unless and until ADP and Pi are bound at the other ATP yield (don’t have to know how to do this math) - For each NADH oxidised in oxidative phosphorylation, ~2.5 ATP are made - For each FADH2 oxidised in oxidative phosphorylation, ~1.5 ATP are made - From previous steps - 10 NADH: 10 x 2.5 ATP = 25 ATP - 2 FADH2: 2 x 1.5 ATP = 3 ATP - = 28 ATP + 4 ATP (made in other steps) - = ~30-32 ATP produced by the full oxidation of 1 glucose molecule Note: ATP yields are theoretical – some protons leak across the inner mitochondrial membrane Uncoupling in Oxidative Phosphorylation - Uncoupling refers to the disconnection between the ETC and ATP synthesis - Uncouplers are molecules that can transport protons across the inner mitochondrial membrane without going through ATP synthase – dissipating the proton gradient - Reduces ATP production - Increases O2 consumption - Generates heat CN blocks Complex IV to O2 – not an uncoupler Venturicidin/oligomycin – blocks ATP synthase DNP uncouples – O2 consumed but no ATP made Dinitrophenol (DNP) - DNP was used extensively in diet pills from 1933 to 1938 - DNP acts as a protonophore, allowing protons to leak across the inner mitochondrial membrane, bypassing ATP synthase - Acute administration of 20–50 mg/kg in humans can be lethal - Concerns about dangerous side-effects and rapidly developing cataracts resulted in DNP being discontinued Uncoupling in Oxidative Phosphorylation An uncoupling protein (UCP1 = thermogenin) “Non-shivering thermogenesis” Brown fat distribution in human infant: major organs/blood vessels protected Summary - The ETC consists of four protein complexes in (or on) the inner m.m - Energy from electrons donated by NADH and FADH2 are used to pump protons into the intermembrane space - The final electron acceptor is O2, which is reduced to form water - Electron transport is “coupled” with the phosphorylation of ADP to form ATP - ~38 per glucose molecule in total - The energy released as H+ returns to the mitochondrial matrix drives ATP synthesis - Oxidative phosphorylation can be uncoupled by various uncoupling reagents/proteins Regulation of Cellular Respiration Principles of regulation: Homeostasis Keeping the concentrations of most metabolites in a steady state maintains homeostasis - Fuels enter cells and waste products leave - Gross composition of a cell does not change much over time Dynamic ‘steady state’: - The rate of synthesis of a metabolite equals the rate of breakdown of this metabolite Pathways are in ‘steady state’ unless perturbed - After interference, a new steady state will be established to satisfy changed requirements Flow of metabolites through pathways is regulated to maintain homeostasis - Sometimes, the levels of required metabolites must be altered very rapidly - Need to increase the capacity of glycolysis during action - Need to reduce the capacity of glycolysis after the action In many cases the ultimate products of metabolic pathways (directly or indirectly) inhibit their own biosynthetic pathways - E.g. ATP inhibits the commitment step of glycolysis ATP:ADP – they rule it allATP:ADP – they rule it all ATP/ADP conc set the rate of e- transfer through cellular respiration via a series of coordinated controls Control points of glycolysis (need to know right diagram for exam plus ATP in left) Regulation of glycolysis Short-term (~seconds): - metabolite fluctuations balance ATP production and consumption - allosteric: ATP, NADH, Citrate, ADP Long-term (~minutes to hours): - hormones (glucagon, epinephrine, insulin) & gene expression control glycolytic enzyme levels Hormonal regulation - Hormones regulate metabolism indirectly by influencing cellular processes - They don’t bind to enzymes directly - Two important hormones include the peptides glucagon and insulin Insulin lowers blood glucose Produced by β-cells of the pancreas, insulin combines with its receptor on the cell surface, stimulating uptake of glucose into cells by the GLUT4 transporter In hepatocytes, insulin stimulates glycogen formation A major problem in glucose metabolism is: - Type I diabetes → no insulin production - Type II diabetes → insulin resistance Glucagon raises blood glucose - Produced by α-cells of the pancreas - In liver, stimulates glycogen breakdown (next week) - In liver, lowers Fructose 2,6 bisphosphate → inhibiting glycolysis & stimulating gluconeogenesis - Inhibits pyruvate kinase - Prevents phosphoenolpyruvate from being converted to pyruvate Glycolysis control point – reaction 1 - Enzyme: Hexokinase - Reaction: phosphorylation of glucose to G6P - Significance: 1st irreversible step in glycolysis G6P, the product of the reaction, inhibits hexokinase But Jay, if G6P inhibits hexokinase, how does it not immediately halt? 1. Inhibition is not “all or nothing”– it takes time 2. G6P is being used up in subsequent steps, accumulation from a bottleneck will start to act on hexokinase 3. Isozymes with varying affinity Regulation of Hexokinase - Isozymes Different tissues have different isozymes with varying affinities for glucose: Hexokinase I, II, and III are found in most tissues - Inhibited allosterically by G6P - High affinity for glucose; half saturated at ~0.1 mM (Hexokinase II) - Blood glucose is ~4-5 mM → Enzyme is saturated/acting near its max rate When G6P increase above normal levels, these isozymes are (reversibly) inhibited - Brings glucose 6-phosphate formation into balance with the rate of utilisation Hexokinase IV: Glucokinase Hexokinase IV, also known as glucokinase, expressed in the liver High Km (enzyme doesn’t like substrate as much): - Requires a higher glucose conc to reach half-max velocity compared to other hexokinases  - Therefore, it’s less active at low glucose levels, allowing the brain to prioritise glucose uptake Not inhibited by glucose 6-phosphate - Can function at, and be responsive to higher glucose concentrations Regular toll booth = hexokinase Fast pass = glucokinase Toll collector = enzyme active site - Regular toll get congested when traffic (glucose) is high - Fast pass can process traffic faster, - but only opens when traffic is high Hexokinase isozymes: kinetics Hexokinase IV (Liver) Km = ~10 mM. - Main function: to regulate blood glucose levels Activity is regulated by glucose conc (i.e. a small change in conc can result in a large change in activity) Hexokinase I (muscle) Km = ~0.1 mM. - Main function: catabolise glucose & provide pyruvate for the TCA cycle Expression of glucokinase is controlled by insulin (hormonal rather than allosteric) Role of Glucose-6-phosphate - G6P is an important branch point in metabolism - Phosphorylation of glucose doesn’t commit it solely to glycolysis Glycolysis control point – reaction 3 Regulation of phosphofructokinase-1 (PFK-I) - Fructose-6-phosphate → Fructose-1,6-bisphosphate is the commitment step in glycolysis - While ATP is a substrate, ATP is also a negative effector - Glycolysis is down-regulated if there is plenty of ATP Regulation of phosphofructokinase-1 Allosteric inhibitors (X) - ATP (both a substrate and an eventual product of glycolysis) - Citrate (TCA cycle intermediate); an indicator the cell is meeting its energy needs Allosteric activators (▲) - ADP and AMP (↑ in conc when ATP utilisation outpaces production) - Fructose-2,6-bisphophate (↑ in conc when blood glucose conc increase) Allosteric regulation by ATP - ATP binds to an allosteric site, lowering the affinity of the active site for fructose-6-phosphate - The same is true for citrate binding - AMP/ADP act allosterically to relieve this inhibition - [ATP] = lower PFK-1 activity - [ADP/AMP] = higher PFK-1 activity Allosteric regulation by fructose 2,6-bisphosphate (F2,6BP) - NOT a glycolytic intermediate - Specifically produced to regulate glycolysis and gluconeogenesis (week 4) - F26BP is synthesized from F6P by PFK-2 in response to insulin - Broken down by fructose 2,6-bisphosphatase (FBPase-2) Glycolysis control point – reaction 10 Pyruvate kinase (step 10) 10 controls outflow from glycolysis Hormonal/allosteric regulation of pyruvate kinase - The L (liver) isozyme, but not the M (muscle) isozyme, is subject to covalent modification – phosphorylation, inhibiting conversion of PEP to pyruvate - Allosterically activated (▲) by Fructose-1,6- bisphosphate creates high flow through from glycolysis - Allosterically inhibited (X) by: - ATP - Acetyl-CoA and long-chain fatty acids - Alanine (enough amino acids) - Low blood glucose causes release of the hormone glucagon → - Activates cAMP-dependent protein kinase (PKA) → - Phosphorylates pyruvate kinase → - Glucose metabolism in the liver is slowed; glucose is conserved Regulation of the pyruvate dehydrogenase complex - PDH complex incorporates pyruvate & CoA-SH to give Acetyl-CoA - Regulated allosterically - ATP, acetyl-CoA, and NADH inhibitors - AMP, CoA, NAD+ activators - And via phosphorylation (next slide) Regulation of the pyruvate dehydrogenase complex Regulation by covalent modification (phosphorylation): - Reversible phosphorylation of a serine residue in one of the 2 subunits of E1 - PDH kinase (PDK) and PDH phosphatase (PDP) add and remove phosphate - PDH kinase is regulated by ATP - High [ATP] →→PDH kinase more active → phosphorylated PDH → less acetyl-CoA - Low [ATP] → PDH kinase less active/phosphatase removes phosphate → more acetyl-CoA Regulation of the TCA cycle - Rates of glycolysis and the TCA cycle are integrated so that wasteful consumption of glucose does not occur - Supply of pyruvate matches demand for acetyl-CoA - Indicators of plentiful energy supply (ATP, NADH, Succinyl-CoA) again inhibit key reactions - Indicators of energy depletion (AMP, CoA, NAD+) activate key reactions - Regulated at highly thermodynamically favourable and irreversible steps (1, 3, and 4) - Citrate synthase, isocitrate dehydrogenase, and α-ketoglutarate - Citrate synthase (step 1) is also inhibited by succinyl-CoA - Succinyl-CoA communicates flow at this point back to the start of the cycle - α-ketoglutarate is an important branch for amino acid metabolism, so it shouldn’t be halted completely Oxidative phosphorylation control - ETC/Ox Phosph is tightly controlled - E.g. An adult requires ~8,7000 KJ of metabolic energy per day - ATP → ADP + Pi -30.5 KJ/mol - This is ~285 moles (~144 kg) of ATP - but at any one-time, total ATP is < 0.1 mole (50 grams) Oxidative phosphorylation is not controlled allosterically, but by SUBSTRATE AVAILABILITY - The substrates are ADP, Pi , O2, NADH, FADH2 At rest - the rate of ATP hydrolysis is minimal, so ADP and Pi are low - Therefore, the substrates for phosphorylation are low and, correspondingly, electron transport is slow As activity is increased - ATP is hydrolysed to release energy - ADP increases, resulting in phosphorylation to form ATP Analogy: - when no one is recycling, the factory cannot manufacture many new materials - Once goods are used up and recycled, the factory now has the materia Summary - Cells turn over metabolites in a steady state - Glycolysis is regulated at 3 steps - Step 1: Hexokinase: Types I-III inhibited by product G6P, glucokinase in liver is not - Step 3: Phosphofructokinase-1: - Allosterically inhibited by citrate and ATP - Allosterically activated by AMP/ADP and F26BP - Hormonally regulated by insulin (F26BP increases) - Step 10: Pyruvate kinase - Allosterically inhibited by ATP, Acetyl-CoA, and alanine - Allosterically activated by Fructose 1,6-bisphosphate (created from the last regulatory checkpoint) - Hormonally, pyruvate kinase is phosphorylated in response to glucagon (in the liver), preventing conversion of PEP into pyruvate – slowing glycolysis and conserving glucose - Pyruvate dehydrogenase complex is activated/inhibited allosterically or via phosphorylation - The TCA cycle is regulated at 3 points allosterically or by substrate availability - Oxidative phosphorylation is regulated only by substrate availability Gluconeogenesis You are now experts in breaking down glucose What if we need glucose instead? Gluconeogenesis - Formation of new sugar - Gluco (sugar); neo (new); genesis (develop) - Process that converts non-carbohydrate precursors to glucose - Occurs mainly in the liver Gluconeogenesis is expensive - 2 Pyruvate + 4 ATP + 2 GTP + 2 NADH + 2 H+ + 4 H2O → - Glucose + 4 ADP + 2 GDP + 6 Pi + 2 NAD+ - but necessary Gluconeogenesis precursors Animals can produce glucose from - Sugars: pyruvate, lactate, or oxaloacetate - Protein: amino acids that can be converted to TCA cycle intermediates (or glucogenic amino acids) Animals cannot produce glucose from fatty acids - Fatty acids break down into acetyl-CoA, which cannot be converted into glucose - Plants, yeast, and bacteria can produce glucose from fats (through the glyoxylate cycle) Whilst it is true that fatty acids can’t turn to glucose, glycerol from triglycerides can! Why do we need gluconeogenesis? - Simple, to provide the body with glucose when needed - What happens when glucose supplies are limited? - Hungry between meals? Long fasts? During vigorous exercise? - Brain, nervous system, erythrocytes, renal medulla, embryonic tissues rely completely upon glucose for metabolic energy - The brain alone requires 120 g of glucose per day - 60% of all the glucose used in the body is stored as glycogen in the muscle/liver - Supply of glucose from glycogen stores is not always sufficient - Glycogen stores depleted during fasts/vigorous exercise - With no glycogen…we need to get glucose from somewhere! Case Study: The Poor Life Decisions of Dr Perry (for interest not on exam) I have about 1,800 calories available in my muscle (glucose/glycogen) and liver (glycogen) 50 km ultramarathon: - 1,800 cals – 4,216 calories burned - = 2,416 cals I needed to consume - Energy intake: - 3 wraps with banana, peanut butter, & dulce de leche = 1,589 cals - 5 energy gels = 500 cals - Cliff bar = 260 cals - Total: 2,349 - 67 cal deficit so I was okay (physically…) 21 km obstacle course: - 1,800 cals – 2,539 calories burned - = leaving 739 cals I needed to consume - Energy intake: - 2 * snake lollies = 84 cals - 655 cals deficit - Symptoms: fatigue, heart palpitations, nausea, pale skin (even by my standard) - Medical tent tested blood sugar level – exercise-induced hypoglycemia So how did I not die once all stored glycogen was consumed? Gluconeogenesis! Glycolysis vs gluconeogenesis Gluconeogenesis and glycolysis share common steps (7/10) - But gluconeogenesis is not a reversal of glycolysis Steps 1, 3, & 10 of glycolysis are “irreversible” - These are bypassed by different enzymes in gluconeogenesis - Bypass #1: → pyruvatePEP - pyruvate carboxylase and PEP carboxykinase via oxaloacetate intermediate - Bypass #2: - F1,6BP → F6P - FBPase-1 removes a phosphate - Bypass #3: - G6P → Glucose - G6Pase removes a phosphate Note: oxaloacetate is a TCA intermediate Note: oxaloacetate is a TCA intermediate 3 irreversible steps of glycolysis are bypassed by different enzymes in gluconeogenesis (don’t need to know) Three irreversible steps of glycolysis are bypassed by four different enzymes in gluconeogenesis Note: Getting from pyruvate to glucose requires consuming 4 ATP & 2 GTP, plus 2NADH Bypass # 1a: Pyruvate to oxaloacetate Pyruvate carboxylase converts pyruvate (3C) to oxaloacetate (4C) - If “carboxy” means CO2, pyruvate carboxylase is just adding a CO2 onto pyruvate - Pyruvate is transported into the mitochondria - Pyruvate then carboxylated using a biotin cofactor Biotin = Vitamin B7 Consuming large quantities of raw egg (containing avidin, that complexes biotin) can cause deficiency - Carboxylation using a biotin (vitamin B7) cofactor - Biotin = carrier of activated CO2 - (don’t need to know the chemical structure) Acetyl-CoA: a positive allosteric effector, stimulates pyruvate carboxylase activity - accumulates when fatty acids are to be used as fuel ADP: a negative allosteric effector, inhibits enzyme activity Bypass #1b: Oxaloacetate to phosphoenolpyruvate (PEP) Phosphoenolpyruvate carboxykinase converts oxaloacetate to PEP - If carboxy means CO2 & kinase means moving; carboxykinase = moving CO2 - Phosphorylation from GTP and decarboxylation - Occurs in mitochondria or cytosol Note: Previous step essentially ‘activates’ pyruvate to oxaloacetate Pyruvate → PEP But Jay, why carboxylate only to decarboxylate right after? 1. Direct conversion would be highly endergonic (requiring energy) – an oxaloacetate intermediate provides an energetically favourable route 2. Compartmentalisation (next slides) 3. Helps to replenish a key intermediate of TCA cycle Pyruvate to PEP – alternate pathways Involves two alternate pathways: Pyruvate to PEP involves an extra step: - conversion to malate and back to oxaloacetate for cytosolic PEP carboxykinase - Net effect is +1 NADH in the cytosol Lactate to PEP directly uses mitochondrial PEP carboxykinase - muscle & erythrocytes - Conversion of lactate in cytosol produces NADH NADH is essential for later gluconeogenic steps Bypass # 2: Fructose 1,6-bisphosphate to Fructose 6-phosphate aka – getting rid of that phosphate - By fructose bisphosphatase-1 (FBPase-1) - Co-ordinately/oppositely regulated with PFK-1 (glycolysis step 3) This reaction provides a critical control point in the reciprocal regulation of gluconeogenesis and glycolysis! More on this in a moment Bypass # 3: Glucose 6 -phosphate to Glucose - By glucose 6-phosphatase - Critical control point in glucose metabolism - Opposite direction to Hexokinase (glycolysis step 1) Bypass # 3: Glucose 6-phosphate to glucose cont… Glucose 6-phosphatase - Found on the luminal side of the endoplasmic reticulum in hepatocytes, renal cells and the epithelial cells of the small intestine, but not other tissues - Why? Gluconeogenesis - Stops here in most tissues - Glucose (but not glucose 6-phosphate) is transported from cells - Glucose 6-phosphate is often converted to glycogen (next lecture) Precursors of gluconeogenesis: Lactate Lactate is a result of glycolysis in - Erythrocytes - An aerobic muscle, especially during vigorous exercise NADH is produced by lactate dehydrogenase in the cytosol The Cori cycle Lactate is carried in the blood to the liver where it is converted back to glucose during recovery from strenuous exercise Precursors of gluconeogenesis: amino acids - Deamination: a.a. lose their amino group, forming keto acids - Keto acids are converted into various intermediates (e.g. pyruvate) which can be used in gluconeogenesis - α-Ketoglutarate, succinyl-CoA and fumarate are all TCA cycle intermediates and give rise to oxalocetate (don’t need to know structures or table) Jay, can you elaborate more on ketones and ketosis? 1. No. This is a carbohydrate module. 2. I don’t fully understand it. 3. Do you seriously want more enzymes to remember? Precursors of gluconeogenesis: Glycerol - β oxidation of fatty acids gives rise to acetyl-CoA (Fatty acid degradation) (Ron’s lectures) - In mammals, there are no pathways that can convert acetyl-CoA to pyruvate - Therefore, fatty acids resulting from hydrolysis of triglycerides cannot be used in gluconeogenesis… However, glycerol… Glycerol resulting from the hydrolysis of triglycerides can be used in gluconeogenesis (don’t need to memories structure) Regulation of glycolysis & gluconeogenesis is coordinated Glycolysis & gluconeogenesis are opposing pathways - Glycolysis has 3 steps with large negative ΔG’ - Essentially irreversible due to thermodynamics - Gluconeogenesis uses detours around these steps - These bypass reactions also have large negative ΔG’ to ensure unidirectionality - Regulation is necessary to determine the direction - Mostly influenced by F6P ←→ F1,6-BP Reciprocal regulation by allosteric modulators Reciprocal regulation by fructose 2,6-bisphosphate (F26BP) Allosteric regulation by fructose 2,6-bisphosphate (F26BP) - NOT a glycolytic intermediate - A regulator specifically produced to regulate glycolysis and gluconeogenesis - F26BP is synthesized from F6P by PFK-2 in response to insulin - Broken down by fructose 2,6-bisphosphatase (FBPase-2) F26BP allosterically regulates fructose 1,6-bisphosphatase - Activates phosphofructokinase (glycolysis) - Inhibits fructose 1,6-bisphosphatase (gluconeogenesis) When F26BP binds to its allosteric site on PFK-1: - Increases the enzyme’s affinity for F6P - Decreases the enzyme’s affinity for ATP When F26BP binds to its allosteric site on FBPase-1: - Decreases the enzyme’s affinity for F6P - Increases FBPase-1’s sensitivity to AMP Transcriptional regulation of gluconeogenesis (don’t need to memorise) - >150 human genes are regulated by insulin - Many glycolytic genes are affected by hormones, including PFK-2/FBPase-2 Regulation by fructose 2,6-bisphosphate (F26BP) The levels of F26BP are hormonally (insulin/glucagon) and enzymatically regulated Enzymatic regulation of fructose 2,6-bisphosphate (F26BP) - F26BP is synthesized from F6P by PFK-2 (results in increased glycolysis by increasing activity of PFK-1) - Broken down by fructose 2,6-bisphosphatase (FBPase-2) (results in increased gluconeogenesis by removing inhibition of FBPase-1) Hormonal regulation of fructose 2,6-bisphosphate (F26BP) - Production of insulin in response to high blood glucose promotes the active form of PFK-2 - results in increased F26BP → PFK-1 activity → glycolysis → normal blood glucose - Production of glucagon in response to low blood glucose promotes the active form of FBPase-2 - results in decreased F26BP higher FBPase-1 activity gluconeogenesis normal blood glucose Summary - Gluconeogenesis is the synthesis of glucose from non-carbohydrate precursors - Pyruvate, lactate, amino acids & glycerol (but not fatty acids in animals) - Gluconeogenesis is energetically expensive, but necessary for specific tissues - Gluconeogenesis and glycolysis share 7 common steps, but not those that are irreversible: hexokinase (step 1), PFK-1 (step 3), pyruvate kinase (step 10) - These reactions are bypassed by 4 different enzymes; pyruvate carboxylase [1a] + PEP carboxykinase [1b], glucose 6-phosphatase , fructose 1,6-bisphosphatase - Conversion of pyruvate to PEP has two alternate pathways, pyruvate or lactate - Regulation of gluconeogenesis is coordinated with glycolysis - Glycolysis and gluconeogenesis are reciprocally regulated by fructose 2,6-bisphosphate Glycogen Metabolism Carbohydrate Metabolism Glycogen is a source of fuel in animals Source of fuel in animals (starch in plants) Skeletal muscle - ≤2% wet weight - Major source of glucose for contraction Liver - ≤10% wet weight - Plays a role in maintaining blood glucose levels - Provides glucose to other tissues between meals or fasting (especially the brain) Glycogen is a source of fuel in animals - The total amount of energy stored as glycogen is less than that of triacyglycerols (fatty acids) - Glycogen storage* = ~500 g @ ~4 cals/g - Fat storage* = 16,000 g @ 9 cals/g - Muscle glycogen can be exhausted in 11 residues) chain to a more interior branch point (α1-6) of the same or another glycogen chain Glycogenin facilitates the formation of new glycogen chains Primer: short oligomer of sugars (or nucleotides) to which an enzyme adds additional monomeric subunits - Glycogen synthase cannot initiate a new cluster… - Requires a preformed α(1→4) polyglucose chain (≥8 residues) - Glycogenenin is both an enzyme and a primer - New glycogen chains begin with the autocatalytic transfer of UDP-glucose to glycogenin, followed by several additions of glucose residues to form a primer that can be acted upon by glycogen synthase Summary Breakdown - (1) Glycogen phosphorylase breaks α(1→4)- linked glucose units to produce glucose 1- phosphate - Glycogen debranching enzyme catalyses two successive reactions: - (2a) Transfer three glucose subunits to a nearby non-reducing end - (2b) Cleaves off the remaining α(1→6)-linked glucose - (3) Phosphoglucomutase converts glucose 1- phosphate to glucose 6-phosphate Synthesis - Glycogenin forms a primer of 8-12 α(1→4)- linked glucose units - (1) UDP-glucose pyrophosphorylase synthesizes UDP-glucose from UTP and glucose 1-phosphate - (2) Glycogen synthase catalyses the addition of UDP-glucose to a non-reducing end of glycogen to make a new α(1→4)-linkage - (3) Glycogen branching enzyme catalyses the transfer of a cluster of glucose molecules from a long chain to a more interior branch point (α1-6) How do cells know when to break down OR synthesise glycogen? Glycogen phosphorylase is regulated allosterically & hormonally - Glucagon/Epinephrine signalling pathway - Starts phosphorylation cascade via cAMP (next slide) - Activates glycogen phosphorylase (b= inactive; a = active) - Glycogen phosphorylase (a) cleaves glucose residues off glycogen → G1P → G6P → glycolysis Epinephrine (adrenaline) & glucagon induce glycogen degradation Glycogen phosphorylase in liver as a glucose sensor - Binding of glucose to an allosteric site on phosphorylase a induces a conformational change - Exposes phosphorylated serine side chains to the action of phosphorylase a phosphatase (PP1) - Phosphorylase a phosphatase (PP1) dephosphorylates enzyme - Phosphorylase a to phosphorylase b Insulin induces glycogen synthesis - ↑BG releases insulin stimulates glycogen synthesis by inactivation of glycogen synthase kinase 3 (GSK3) - Inactivation of GSK3 allows PP1 to dephosphorylate and activate glycogen synthase Epinephrine (adrenaline) & glucagon induce glycogen degradation …and inhibit glycogen synthesis - Cascade initiated by glucagon/epinephrine activates protein kinase A – this deactivates the enzyme involved in glycogen synthesis Carbohydrate metabolism differs between tissues Regulation of glycogen synthase Phosphoprotein phosphatase 1 is central to glycogen metabolism (diagrams is a summary and won’t directly be in the exam) These allosteric & hormonal signals coordinate carbohydrate metabolism globally Note: you don’t need to “memorise” this schematic, but you should already understand these independent pathways to some degree Summary - Glycogen is a polymer of α(1→4)-linked subunits of glucose, with α(1→6)-linked branches, constructed around a core primer based upon the enzyme glycogenin - G6P is converted to G1P via phosphoglucomutase (reversibly), before activation via the addition of a nucleotide through UDP-Glucose pyrophosphorylase, forming UDPglucose – the building block of glycogen - Glycogen branching and debranching enzymes are responsible for forming and degrading/remodelling the α(1→6)-linked branches - Epinephrine/glucagon induce glycogen breakdown (via a signalling cascade, activating phosphorylase), and impair glycogen synthesis (via inactivating glycogen synthase) - Insulin and the availability of glucose induces glycogen synthesis, with phosphoprotein phosphatase 1 (PP1) dephosphorylating/activating glycogen synthase, and dephosphorylating/inactivating phosphorylase