Metabolic Biochemistry Lectures 2-9 Notes PDF

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Metabolic Biochemistry lecture notes for lecture 2-9 only....

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Metabolic Biochemistry Lecture Notes Lecture 2 Part 1 – Introduction to Metabolic Biochemistry Metabolic Biochemistry - Bioenergetics, the role of energy in determining metabolic pathways and the major energy donating compounds used in metabolic reactions - The major metabolic pathways regulating synthesis and breakdown of carbohydrates, lipids, proteins, amino acids and nucleotides - The biochemical properties of enzymes and the regulation of enzyme activity - The mechanisms that compartmentalise metabolic activity and integrate fuel metabolism - Metabolic adaptations that result from different dietary regiments e.g. starvation, diabetes, endurance sport - Genetic defects that disrupt pathways to cause metabolic disease - The basic techniques and methods of experimentation used for enzyme analysis

Definitions - Catabolism – breaks molecules into smaller units, which are then either oxidised to release energy or used in other anabolic reactions e.g. glycolysis (glucose breakdown) - Anabolism – synthesis of complex compounds from smaller unit e.g. carbohydrates, lipids, proteins, amino acids (anabolic processes require energy) - Metabolites – substrates, intermediates and products of metabolic processes Free energy - Thermodynamic quantity equivalent to the capacity of a system to do work - Symbol for Gibbs Free energy = G (change in G = ∆G) Source of Energy - Carbohydrates o Animals obtain carbs by consuming foods such as rice, wheat, potatoes and other grains/plant based foods) o Yield glucose upon digestion o Glucose is further digested to produce energy in the form of ATP o Simple Carbs  Monosaccharides  Disaccharides  Preferred source (immediate) – chocolate, honey, bananas o Complex Carbs  Starch  Cellulose  Humans cannot directly digest cellulose as we lack the enzyme to break down the beta acetal linkages  Glycogen is the main storage form of glucose in animals, fungi and bacteria

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Proteins o Major building block of all tissues and cells o Yield amino acid and nitrogen upon digestion o Obtained mainly from eggs, milk, soybeans, legumes, meat & fish Lipids and fats o Concentrated sources of energy o Yield fatty acids and acetyl CoA upon digestion o Obtained from plant and animal products such as oils, meat, eggs, diary

Cell Membranes - Protect the cell and compartmentalise organelles o Plasma cell membrane o Lysosomal membranes o Nuclear membranes o Endoplasmic reticulum ATP -

Energy currency of the cell Transfers energy from chemical bonds to energy-requiring reactions inside the cell

NAD -

Carrier of energy in the form of hydrogens and electrons

Metabolic Pathways - Series of reactions that are normally catalysed by enzymes - Share intermediates and products so the study of one reaction in isolation is arbitrary - Catabolic reactions involve breaking down metabolites in a process that yields free energy (exergonic) - Energy released in this process is utilised to synthesise ATP - A few intermediates are shared in the catabolism of many substrates - Products of one reaction can act as the substrate for another reaction - Metabolic pathways are highly regulated and coordinated to meet cellular demand Metabolomics - Systemic study of the unique chemical fingerprints that specific cellular processes leave behind - Metabolome represents the collection of all metabolites in a biological cell, tissue, organ or organism which are the end products of cellular processes - Metabolic profiling can give an instantaneous snapshot of the physiology of that cell Lecture 2 Part 2 – Bioenergetics and Metabolism Thermodynamics - Principles that describe flow and exchange of heat, energy and matter - Systems are: o Isolated – no exchange of either energy or matter o Closed – no exchange of matter, may exchange energy

o Open – may exchange energy or matter (living systems) Two Fundamental Laws of Thermodynamics - First Law: Conservation of energy o For any chemical or physical change, total energy remains constant; energy can change for or be transported by cannot be created/destroyed - Second Law: Universe tends towards increasing disorder o In all natural processes, the entropy of the universe increases o Entropy = unusable energy  E.g. energy lost as heat Gibbs Free Energy (G) - Energy goes into making macromolecules and energy goes out when these are broken down - Positive ∆G = unfavorable reaction o Energy is required to drive the reaction - Negative ∆G = favorable reaction o Reaction is spontaneous, free energy is released Lecture 3 Part 1 – Reaction Coupling Reaction Coupling - Requires chemical and electrical energy Activation Energy - Always positive Metabolic Coupling Reactions - Oxidation-reduction reactions always occur together (coupled) - Key role in flow of energy in biological reactions - Electrons carry energy with them from one molecule to another - Energy production during cellular respiration and photosynthesis are entirely based on oxidation-reduction reactions Lecture 3 Part 2 – Enzyme Kinetics Enzymes - Catalyse reactions – increase the rate of a reaction without themselves being changed - Recyclable and highly specific - All enzymes are proteins but not all proteins are enzymes Equilibrium with Enzymes - With enzymes the point of equilibrium is reached faster - This does NOT mean that there is more products, it just means that we got the products quicker

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Therefore, enzyme speed up the rate of reaction, but they do NOT alter the equilibrium end-point Enzyme lower the activation energy but do NOT change the ∆G

Enzyme Specificity - Specificity due to o Size and shape o Electrostatic repulsion and attraction o Hydrophobicity and hydrophilicity Sterospecificity of Enzymes - Isomers – molecules with same chemical formula but different spatial arrangement - Enantiomers are mirror images - Enzymes are also stereospecific Thaliodomide - Drug used to ease morning sickness in late 50’s - Shortly after, 10,000 babies bron with phocomelia (only 50% survived) Rate Constants ≠ Equilibrium Constant - Rate constant K o Rate of reaction o Rate of conversion of reactants to products o Measured in molves per second - Equilibrium constant Keq o Ratio of concentration of products to concentration of reactants Reaction Rate – Velocity - V = k.[S] o V = velocity (rate of reaction) o K = rate constant o S = substrate concentration - Rate is amount of substrate converted per unit of time Steady State Enzyme Kinetics - State of reaction where concentration of enzyme-substrate complex is constant o Linear section of graph o Lag phase – enzyme and substrate are docking, no product made o Linear phase – steady state, same concentration of product made over time, o Plateau phase – steady state lost, no more substrate available to make product Michaelis-Menten Equation - Leonor Michaelies and Maud Menten developed an alternate graph to describe enzyme kinetics for a single substrate reaction Lecture 3 Part 3 – Enzyme Kinetics and Inhibition

Enzyme Inhibitors - A compound that binds to the enzyme and decreases its activity - Decrease in activity can be caused by o Inhibitor preventing the substrate from binding o Inhibitor preventing the enzyme from catalysing the reaction - Inhibitors binding can be reversible or irreversible - Most inhibitors fall into one of three categories o Competitive inhibitor o Non-competitive inhibitor o Uncompetitive inhibitor Reversible Inhibitor - Competitive inhibitors bind to enzyme active site  Substrate cannot bind  Product cannot be formed o Resembles substrate - Non-competitive inhibitors bind to another site on the enzyme o Can bind to the free enzyme or enzyme substrate complex o Product cannot be formed - Uncompetitive inhibitors bind to another site on the enzyme o Can only bind to the substrate complex o When the active site is occupied by the substrate o Product cannot be formed Lecture 4 Part 1 – Glycolysis Oxidation of Glucose - Complete oxidation of glucose yields energy via aerobic respiration - Glucose molecule primed by addition of phosphate groups from ATP and split into 2 pyruvate molecules Cellular Respiration - Conversion of energy in the chemical bonds of glucose into the phosphate bonds in ATP - Glucose is oxides to produce CO2 and H2O - This energy release from this oxidation is used to power the synthesis of ATP Classification of Enzymes - Kinases o Catalyses the transfer of a phosphate group - Oxidoreductases o Catalyses oxidation-reduction reactions - Isomerases o Catalyses intramolecular rearrangement Glycolysis Energy Output

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Preparatory (investment) phase o 2 ATP molecules in  Steps 1 and 3 Payoff phase o 4 ATP molecules out  Steps 7 & 10 o 2 NADH molecules out  Reduced electron carriers  Step 6

Lecture 4 Part 2 – Gluconeogenesis Gluconeogenesis - Generation of glucose from non-carbohydrate sources - Mainly occurs in liver, also small intestine and kidneys - Stored glycogen in the liver is not enough to feed the body and brain – need other sources - Synthesis of glucose from pyruvate uses 7/10 enzymes used in glycolysis - Why not 10? Three of the reactions in glycolysis have a large negative deltaG that they are essentially irreversible Hexokinase Regulation - Muscle hexokinase has an isoenzyme in the liver, glucokinase Regulation by Transcription - High blood glucose concentration o Hexokinase gene active o Increase hexokinase production o Activate glycolysis - Low blood glucose concentration o G6Pase gene active o Increase G6Pase production o Activate gluconeogenesis o Maintain blood glucose levels Pentose Phosphate pathway - Glucose 6-phosphate is used for other purposes apart from energy production - Glucose 6-phosphate is oxidised and donates electrons to NADPH resulting in a pentose sugar - Uses of NADPH and pentose sugars o Reduce reactive oxygen species, decreasing their toxicity (NADPH) o Synthesis of fatty acids (NADPH) o Cell proliferation and growth (both)

Lecture 5 – Pyruvate Dehydrogenase Complex and Citric Acid Cycle

Pathway - Glycolysis o Anaerobic phase o Occurs in cytoplasm - Pyruvate dehydrogenase and citric acid cycle: o Aerobic phase o Occurs in mitochondria o Pyruvate transported via cell membrane channels Citric acid cycle - The engine that feeds the oxidative phosphorylation process Content - Pyruvate dehydrogenase complex o Role of co-enzymes o Reactions involved - Citric acid cycle o Reactions of the cycle o Energy output o Regulation Pyruvate Dehydrogenase Complex - Made of 3 enzymes in 3 sub units o Complex of multiple copies of the same enzyme - Arranged to act as a channel o Easily control flow of substrates o Substrates are kept close to complex Pyruvate Catabolism - Dehydrogenation (hydrogen removal) and decarboxylation (CO2 removal) o Generates NADH (reduced electron carrier) - Under anaerobic conditions, pyruvate converted to lactic acid - Highly exergonic (essentially irreversible deltaG) Co-Enzymes of PDH Complex - Organic non-protein molecules that interacts with a protein to make an enzyme - Associated with 5 co-enzymes: o Thiamine pyrophosphate o Lipoamide o Co-enzyme A o FAD o NAD+ Thiamine Pyrophosphate (TPP) - Sourced from thiamine (vitamin B1) - Acidic carbon interacts with middle carbon pyruvate

Lipoamide (lipoyllysine) - Permanently bound to dihydrolipoyl transacetylase - Dithiol group gets oxidised or reduced Coenzyme A - Not bound to enzymes in PDH complex - Carries acetyl groups - Binds acetate to make acetyl-coA Flavin Adenine Dinucleotide (FAD) - Permanently bound to PDH complex - Can accept 1 or 2 hydride ions (H-) and thus 1 or 2 electrons - Sourced from riboflavin (vitamin B2) Nicotinamide Adenine Dinucleotide (NAD+) - Carries 1 hydride ion (H-) and thus 1 electron - Not bound to PDH complex, can transport electron from FADH2 - Sourced from niacin (vitamin B3) PDH Complex 1. Pyruvate is decarboxylated and product (acetyl group) binds to TPP 2. Acetyl group oxidised to acetate and electrons are transferred to thiol groups in lipoamide (reduced) 3. Acetate binds coenzyme A to make acetyl-coA (enters citric acid cycle) coenzymes need to be recycled 4. Two H- ions removed from reduced lipoamide (recycled) and transferred to FAD 5. Electrons transferred to NAD+ Citric Acid Cycle/Krebs Cycle/Tricarboxylic Acid Cycle - Common oxidation pathway or carbohydrates, proteins and fatty acids - 2 phases: o Acetyl-CoA catabolism o Regenerating oxaloacetate - Amphibolic o Catabolic o Anabolic Anaplerotic Reactions - Reactions that replenish substrates used for other pathways o Gluconeogenesis o Fatty acid synthesis o Amino acid synthesis o Nucleotides Phase 1 – Acetyl-CoA Catabolism - Acetyl-CoA broken down

o 2 carbon dioxides released o 2 NADH created Phase 2 – Regeneration of Oxaloacetate - Oxaloacetate regenerated o 1 GTP (ATP) created o 1 FADH2 created o 1 NADH created Total Energy Output per Cycle - 1 GTP molecule o Quickly converted to ATP - 3 NADH molecules o Steps 3, 4 and 8 - 1 FADH2 molecule o Step 6 - Continuous cycle o Oxygen needed for oxidative phosphorylation which provides substrates for CAC! PDH Complex Regulation - Conversion of pyruvate by the PDH complex is allosterically regulated o Negative regulators  An abundance of ATP, NADH or acetyl-CoA and fatty acids  Fatty acids also a source of acetyl-CoA o Positive regulatory  An abundance of AMP or Co-enzyme A Regulation within Cycle - Exergonic reactions that need reactants otherwise whole cycle is slowed down - Steps 1, 3 and 4 regulated - Rate limiting steps o Build-up of products - Rapid response to produce energy Lecture 6 Part 1 – Oxidative Phosphorylation and ETC Biological Oxidation: Redox Reactions - Glucose oxidation o Combustion with oxygen to produce most reduced form possible o Electrons in biological redox reactions are transferred as H- (hydride) ions (dehydrogenations) o The electron removed does not exist freely but are bound to electron carriers (NAD/NADH, FAD/FADH) - Oxidoreductases transfer electrons - Energy may be captured and used to make ATP or to form other chemical bonds

Oxidation and Reduction reactions - Redox potential: relative tendency to gain or lose electrons - Measured in volts or millivolts Reaction Coupling - Energy of an exergonic reaction is used to power an endergonic reaction - Oxidative phosphorylation: process coupling removal of hydrogen ions/electrons from one molecule and giving phosphate molecules to another molecule Mitochondria - Outer membrane o Permeable - Inner membrane o Impermeable, entry/exit only through membrane channels o Highly folded structure for increased surface area - Matrix o Enzymes for citric acid cycle and electron transport chain Oxidative phosphorylation - Energy of oxidation drives ATP synthesis - Involves flow of electrons through a chain of membrane bound carriers in mitochondria o Electrons provide energy for proton pump o Diffuse back into mitochondrial matrix o Travel through ATP synthesase energy is released ETC -

Transfer of electrons from NADH to FADH2 to membrane bound enzymes Electron transport from one protein to the next results in the production of a protein gradient across the inner mitochondrial membrane ETC is the final electron transport to the terminal electron acceptor, O2 which is reduced to H2O

ETC: FADH2 Bypass - Succinate dehydrogenase (from TCA cycle) and FAD are permanently bound (form complex 2) o Enzyme inserted into mitochondrial inner membrane o Allows electrons to pass from FADH2 into ETC ETC -

NAD must be recycled from NADH While these electrons are transferred to the ETC, this energy of transfer is used to pass H+ ions (protons) across inner membrane against conc. Gradient

Flow of Electrons - NAD+/H and FAD/H2 are hydrophilic and cannot sit in cell membrane - Electron carrying molecules within the mitochondrial inner membrane

Electron Carrier Ubiquinone (Q10) - Small and hydrophobic - Carries electrons and H+ - Ubiquinone naturally made in body Electron Carrier: Cytochrome C - Water soluble - Carries 1 electron Iron-Sulfur (Fe-S centres) - Participate in single electron transfers through protein - 2 states: oxidised and reduced Fe - Found in complex 1-3 - Complex IV has a copper-sulfur centre Ischemia - Lack of O2 to cells due to lack of blood flow - Low O2 leads to no ETC or oxidative phosphorylation o E- transfer to O2 stops o H+ pumping stops o Proton motive force collapses - Cell relies on anaerobic glycolysis - ATP synthase operated in reverse Lecture 6 Part 2 – Electron Transport Chain ETC Complexes - I – NADH dehydrogenase o Accepts electron from NADH - II – Succinate dehydrogenase o Accepts electrons from FADH2 - III – cytochrome bc1 complex o Accepts electrons from ubiquinone - IV – cytochrome oxidase complex o Accepts electrons from cytochrome C Complex I - NADH to ubiquinone - Contains flavin mononucleotide and at least 6 fe-s centres - L shaped - Ubiquinone becomes ubiquinol diffuses through membrane into complex II - Catalyses 2 simultaneous reactions o Exergonic transfer of H- from NADH to ubiquinone and H+ from matrix to ubiquinol o Endergonic transfer of 4H+ from matrix to inter-membrane space - Proton pump driven by energy of exergonic transfer

Complex II - Succinate to ubiquinone - 4 different polypeptide chains o SDHA & SDHB o SDHC & SDHD Complex III - Ubiquinol to cytochrome C - Swaps from a 2 electron carrier to a 1 electron carrier - 2 reactions coupled - Complex contains 3 environments o Top o Middle o Bottom - Q has TWO binding sites - P side - N side Complex IV - Cytochrome oxidase - Cytochrome C to oxygen - Contains Cu2+ ions and heme groups to facilitate electron transfer - 4 electrons from Cytochrome C and 4 H+ from matrix to convert O2 to water Uncoupling: Heat Production - Mitochondria also involved with heat production - Uncoupled mitochondria in brown adipose tissue produce heat - Brown adipose tissue in which fuel oxidation - Thermogenin Chemiosmotic Gradient: Proton Motive Force - Powers synthesis of ATP from ADP and inorganic phosphate - Transfer of electrons from NADH to O2 is highly exergonic Chemiosmotic Model - Electron passage through the ETC is accompanied by proton movement from the matrix to the inter membrane space - Electron transport and ATP synthesis are inherently coupled Complex V - ATP synthase - Readily reversible - Powered by chemiosmotic gradient - H+ travels through ATP synthase to get into matrix - ATP synthase is a molecular rotary motor

Lecture 7 – Glycogen Metabolism Glycogen - Principal storage form of gluxose in the body - Long branched polymer of glucose units - Found principally in liver and skeletal muscle Glycogen Storage - Stored in large cytosolic granules containing enzymes that synthesise and degrade glycogen - In the body stored mainly in skeletal and liver muscle - Muscle glycogen – provides a quick source of energy for skeletal muscle contraction - Liver glycogen – acts as a reservoir of glucose for other tissues when dietary glucose in unavailable Glycogen Metabolism - Synthesis and breakdown of glycogen occur by different pathways which utilise different enzymes - Breakdown of glycogen is termed Glycogenolysis - Synthesis of glycogen is termed Glycogenesis Glycogenolysis - Enzymes involved: o Glycogen phosphorylase o Glycogen debranching enzyme o Phosphoglucomutase Glycogenesis - Starting point of glycogen synthesis is the production of glucose-6-phosphate Glycogen Branching - Existing glycogen chains are elongated by the action of glycogen synthase - Elongated glycogen chains can have branches - Branching increases polymer solubility - Branching creates many ends for enzyme attack for rapid release of glucose Lecture 8 – Fatty Acid Metabolism Biological Roles of Lipids - Highly concentrated energy stores - Fuel molecules - Signal molecules - Components of membranes - Serve as insulation Fatty Acids

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Constituents of lipids Hydrocarbon chains o Of various lengths o Of various degrees of unsaturation o Terminate with carboxylate groups o Fatty acids in biological systems usually contain an even number of carbon atoms (16 & 18 b...