Cheat sheet - metabolic biochem - 2021 notes HD PDF

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full lectures notes on metabolic biochemistry - 2021 HD...

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LECTURE 1: METABOLIC BIOCHEM - CATABOLISM (exergonic)– breakdown of molecules to release energy or used in other reactions - ANABOLISM (endergonic)– synthesis of compounds and requires energy - METABOLITES – substrates, intermediates FREE ENERGY – energy available to do work Sources: Carbohydrates - Yield glucose upon digestion Simple Carbs – immediate sources of energy - Mono  glucose, fructose, galactose - Disaccharides  sucrose, lactose and maltose Complex carbs - Starch (amylum) – glucose units joined by glycosidic bonds - Cellulose – composed of beta glucose units rotates at 180 degrees o Humans cannot digest cellulose - Glycogen – main storage form of glucose

ATP (Adenosine Triphosphate) 1. 3 Phosphate group 2. Adenine base 3. Ribose sugar

Sources: Lipids & fats - Concentrated forms of energy - Yield fatty acids and acetyl CoA - Acetyl CoA further digested to produce energy

NAD (Nicotinamide Adenine Dinucleotide) - Carrier of energy in the form of hydrogens and electrons

Cell Membranes – plasma cell membrane, lysosomal, nuclear and endoplasmic reticulum Sources: Proteins - Building block of tissues and cells - Yield amino acids and nitrogen upon digestion - Amino acids linked by peptide bonds – consume energy when these peptide bonds are broken

REDUCED MOLECULES HAVE MORE ENERGY - Have accepted electrons which can be passed on to release energy - Saturated – more energy, unsaturated (less energy)

METABOLOMICS - Study of the unique chemical fingerprints that specific cellular processes leave behind - Metabolome – collection of all metabolites which are the end products of cellular processes - Metabolic profiling – complete overview of what’s happening in the cell at that point in time under different conditions

LAWS OF THERMODYNAMICS - Isolated System – no exchange of matter and energy - Closed System – no exchange of matter, may exchange energy - Open System – may exchange energy or matter - Equilibrium - forwards and reverse rates are equal concentrations 1. Conservation of Energy - Can change or be transported but cannot be created or destroyed 2. The universe tends towards increasing disorder - All natural processes, the entropy increases

LECTURE 2: BIOENERGETICS AND ENZYMES - Reaction Coupling - using energy from an exergonic reaction to activate an endergonic reaction o G3 = G1 (+ve) + G2 (-ve) o ADP  ATP reaction Metabolic Coupling Reactions: Redox reactions - Key role in the flow of energy - Electrons carry energy from one molecule to another - Storing energy to later couple another reaction - E.g. cellular respiration, photosynthesis

Rate constant (k) - Rate of reaction – speed of conversion of reactants into products - How fast concentration of A is decreasing and how fast B is increasing Equilibrium constant (Keq) - Ratio of concentration of products to reactants - [B]/[A] Velocity – V= K.[S] - Reaction rate - Amount of substrate converted per unit of time - Substrate – either product or reactant - [A]  [B] – forward reaction Two-step enzyme reactions

Gibbs Free Energy (G) –spontaneity of a reaction - Positive (unfavourable) – non-spontaneous, endergonic reaction - Negative (favourable) – spontaneous, exergonic

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G – change in free energy H – enthalpy  heat (exo/endothermic) J/mol S – entropy  disorder J/mol/K T = 298K (25) R = 8.315 J/mol/K

ENZYMES - Catalyse reactions – allow reactions to reach equilibrium faster DO NOT alter equilibrium endpoint - Lower activation energy but DO NOT change G Specificity - Size & shape, repulson & attraction, hydrophobicity and hydrophilicity Stereospecificity – only bind to one isomer - Isomers – same chemical formula but different spatial arrangement - Enantiomers – mirror image molecules Types of enzymes - Oxidoreductases – oxidation/reduction reactions - Transferases – transfer functional groups - Hydrolases – hydrolytic cleavage (add H2O) - Ligases and synthases – bond formation - Lyases – removal of a group or addition of group to a double bond - Isomerases – intramolecular rearrangement

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Kcat (step 2) – number of substrate molecules converted to a product Steady state enzyme kinetics: Concentration of enzyme substrate complex is constant o Linear section of graph – where velocity calculated

Michaelis-Menten Equation - [S] vs velocity

Acetaldehyde Dehydrogenase Reaction

LECTURE 3: GLYCOLYSIS - Breakdown of glucose, converted into 2 molecules of pyruvate - First step of oxidation reaction via cellular respiration Glucose  (glycolysis)  2 pyruvate Cellular Respiration -

High vs low Km High – cytosol - Higher conc of substrate to start conversion - Slower to reach half Vmax Low – mitochondria - Lower conc of substrate to start conversion - Faster to reach half Vmax

Conversion of energy from the chemical bonds of glucose to the phosphate bonds in ATP Energy from oxidation is used to power synthesis of ATP (coupled reaction)

End products from pyruvate: - CO2 – 32 ATP via oxidation (aerobic conditions) - Ethanol or lactic acids – 2 ATP via reduction (anaerobic) More carbon-hydrogen bonds broke the more energy!

Lineweaver-Burk Equation

ENZYME INHIBITORS (reversible) Irreversible reactions – inhibitor prevents enzyme from catalysing the reaction - Competitive – bind to enzyme so substrate cannot bind Vmax unchanged, Km increased - Non-competitive – bind to allosteric site so product can’t be formed - Vmax decreased, Km unchanged - Uncompetitive – bind to substrate complex – Vmax & Km decreased

Anabolic phase – 2 ATP in - Use blood glucose – when just ate - Use liver glucose – when haven’t eaten

Catabolic phase (produces energy) – 4 ATP out and 2 NADH out

GLUCONEOGENESIS – synthesis of glucose - Generation of glucose from non-carb sources - Mainly occurs in liver - Doesn’t use all the same enzymes in glycolysis (uses 7) o 3 of the reactions in glyco have negative G (irreversible) = OTHER REACTIONS NEEDED Bypass 1 - Produce oxaloacetate to produce phosphoenolpyruvate - Pyruvate carboxylase – MITOCHONDRIA (regeneration of NADH) - PEP carboxykinase – CYTOPLASM (rich in NAD+) o Produced in cytosol and mitochondria TOTAL ENERGY AND REGULATION Glycolysis produces = 2 pyruvate + 2 NADH + 2 ATP Gluconeogenesis requires = 2 pyruvate + 2 NADH + 4 ATP + 2 GTP - Pathways can only occur 1 at a time Enzyme regulation Only irreversible pathways can use inhibitors and activators because using inhibitors and activators in reversible pathways will affect the whole process of both gluconeogenesis and glycolysis - Hexokinase – glucokinase - Transcription – high blood glucose conc

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(hexokinase active = activate glycolysis), low blood glucose conc (G6Pase active) = activate gluconeogenesis Pyruvate kinase – inhibitors – Acetyl CoA, ATP  activators – AMP, fructose 1 Pyruvate carboxylase – activators – Acetyl CoA, ATP  Inhibitors – fructose, AMP

Pentose phosphate pathway – when glucose 6-phosphate is oxidised and donates electrons to NADPH resulting in a pentose sugar - NADPH and pentose sugars can: decrease reactive oxygen species toxicity, synthesis of fatty acids, cell proliferation and growth

LECTURE 4: PYRUVATE DEHYDROGENASE COMPLEX Pyruvate  (pyruvate dehydrogenase)  AcetylCoA  Aerobic phase  Occurs in mitochondria – CAC  CAC – engine that feeds the oxidative phosphorylation process

Pyruvate Dehydrogenase Complex - 3 enzymes - Acts as a channel – control flow of substrates and compact - Generates NADH - Under anaerobic conditions, pyruvate converted to lactic acid - Highly exergonic (irreversible) Coenzymes: - TPP – E1 - Lipoamide – E2 - CoA - FAD – E3 - NAD+

PROCESS THIAMINE PYROPHOSPHATE (TPP) – E1 1. Pyruvate - Enzyme: pyruvate dehydrogenase is decarboxylated and product (acetyl binds tomiddle TPP carbon - Acidic carbongroup) interacts with - Sourced from VitB1 group oxidised to acetate and electrons 2. Acetyl E2 LIPOAMIDE (lipollysine) are transferred to thiol groups in lipoamide - Enzyme: dihydrolipoyl 3. Acetate binds transacetylase Coenzyme A to make acetyl-CoA - Dithiol group (enters gets oxidised the citric andacid reduced cycle), co-enzymes need E3 to bedinucleotide recycled FAD – flavin adenine - Enzyme: dihydrolipoyl 4. Two H- ions dehydrogenase removed from reduced lipoamide (recycled) to FAD - Accept 1-2 hydride ions and (H-)transferred  1-2 electrons 5. Electrons - Sourced from VitB2 transferred to NAD+ COENZYME A - Not bound in PDH complex - Binds acetate to make acetyl-coA NAD+ (nicotinamide adenine dinucleotide) - Carries 1 hydride ion = 1 electron - Sourced from VitB3

LECTURE 4: CITRIC ACID CYCLE (CAC) Phase 1: Acetyl-CoA catabolism – 2 CO2 & 2 NADH

Phase 2: Regeneration of oxaloacetate – 1 GTP, FADH & NADH

ANAPLEROTIC REACTIONS – reactions that replenish substrates needed for other pathways

TOTAL ENERGY OUTPUT/CYCLE - 1 GTP – quickly converted to ATP - 3 NADH – step 3,4,8 - 1 FADH2 – 6 Regulation of CAC - Inhibitors – high energy molecules e.g. ATP, acetyl-CoA, NADH - Activators – low energy molecules e.g. ADP, CoA, Ca2+ - Steps 1, 3 and 4 are regulated - Rate-limiting steps - Rapid response to produce energy

Regulation of PDH - Pyruvate allosterically regulated by dehydrogenase complex o Regulation of enzyme by binding an effector molecule at a site other than the enzyme’s binding site

LEC 6: OXIDATIVE PHOSPHORYLATION AND ETC Redox reactions - Electrons are transferred as H-(hydride) ions - Electrons are bound to electron carriers e.g. NAD+/NADH, FAD/FADH2 - Oxidation (OIL) – loses electrons - Reduction (RIG) – gains electrons

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O2 – readily gains electrons, NADH - readily gives up electrons

Inner Mitochondria membrane - Impermeable (entry/exit of molecules via membrane channels)– site of proton gradient OXIDATIVE PHOSPHORLYATION - Energy of oxidation drives ATP synthesis - Involves the flow of electrons through a chain of membrane-bound carriers in mitochondria - Begins  electron entry into respiratory chain 1. Electrons (from NADH and FADH) provide energy for the proton pump to pump H+ ions into intermembrane space 2. H+ ions diffuse back into the mitochondrial matrix through ATP synthase and energy is released to convert ADP to ATP ELECTRON TRANSPORT CHAIN - Transfer of electrons through carriers, from NADH/FADH2 to membrane bound enzymes - Results in the production of a proton gradient across the inner mitochondrial membrane - ETC is the final electron transport to the terminal electron acceptor, O2, which is reduced to H2O

Electron Transport Chain - NAD must be recycled from NADH - Flow of electrons to the ETC – allows H+ ions (protons) to cross inner membrane against its gradient - Results: regeneration of NAD, transfer of electrons to form water, charge separation

DISEASE: Ischemia - Lack of O2 due to lack of blood flow – leads to no ETC/oxidative phosphorylation  proton flow stops  ATP production stops - Cell relies on anaerobic glycolysis – increases lactic acid levels - ATP dehydrogenase instead of synthase

Electron carrier: Ubiquinone (Q) - Small and Hydrophobic - Transfers electrons from I & II to III - Double bonded oxygen on ring Electron carrier: Cytochrome C - Hydrophilic - Transfers electrons from complex III to IV - Heme group with Fe ion to stabilise e transfer Iron-sulfur (Fe-S) centres - Electron transferral – found in 1, 2 and 3 complex - Complex 4 has a copper-sulfur centre

COMPLEX REGULATION: Inhibitors - Inhibits complex IV – cyanide/carbon monoxide - Rotenone – inhibits complex I - Antimycin A – inhibits complex III

ETC COMPLEXES: 1. Complex I - NADH to ubiquinone (Q) - Ubiquinone oxidoreductase, NADH dehydrogenase - Contains flavin mononucleotide (FMN) – pick up 2 H- ions - 6 Fe-S centres, L shaped - Ubiquinone becomes ubiquinol to diffuse through membrane into complex III - Catalyses 2 reactions:  Exergonic transfer of H- from NADH to ubiquinone to form ubiquinol  Endergonic transfer of 4H+ from matrix to intermembrane space 2. Complex II - Succinate to ubiquinone - Succinate is oxidised to fumarate - Succinate dehydrogenase and FADH2, part of the TCA cycle - 4 different polypeptide chains – SDHA & SHHB and SDHC & SDHD (contain heme group – prevent escape of electrons from protein) 3. Complex III (Cytochrome bc1)– electron shuffle - Ubiquinone  Cytochrome C oxidoreductase - Swaps from a 2 electron carrier (QH) to a 1 electron carrier (Cyt C) - Three environments – top (P side +ve), middle (intra-membrane), bottom (N side -ve) - Q has 2 binding sites: o P side – 2x QH2 oxidised to release 4H+ in total o N side – Q is reduced to Q4. Complex IV (cytochrome oxidase) - Cyt c  oxygen - Contains copper and heme groups to facilitate transfer - 4 electrons from Cyt c. and 4H+ from matrix to convert to water

Uncoupling: Heat Production - Uncoupled mitochondria in brown adipose tissue produce heat  fuels oxidation Chemiosmotic gradient – proton motive force - Powers synthesis of ATP from ADP and inorganic phosphate - Two components:  Chemical potential energy – difference in H+ concentration  Electrical potential energy – H+ move across without a counter ion - Transfer of electrons from NADH to O2 is highly exergonic - Electron transport and ATP synthesis are coupled Complex V: ATP synthase - Readily reversible - H+ travels through ATP synthase to get into matrix - F0 – transmembrane channel (Rotor) – H+ pore  In the membrane - F1 – catalytic head – catalyses ATP hydrolysis  Made up of 9 subunits Binding-change model 3 conformations: - Beta-ADP  loose-binding ADP & P  change - Beta-ATP  binds ATP tightly - B-empty  low affinity for ATP  ATP leaves - ATP only released when both ADP and P bind to another segment - Y-stalk only rotates - +ve feedback – more ADP, faster rotation = ATP

ATP synthesis: goes to the right - ATP synthase stabilises ATP relative to ADP + Pi by binding ATP more tightly, releasing enough energy to counterbalance cost of making ATP - Binding energy drives equilibrium right REGULATION: depends on energy needs - Low energy and high ADP/NAD+ turns ON energy production - High energy and ATP/NADH – shuts down energy production

LECTURE 7: GLYCOGEN METABOLISM GLYCOGEN - Glucose is stored as glycogen in the body - Found in the liver and skeletal muscle - Glycogen is a branched homopolysaccharide of glucose - Linked by alpha carbons 14 but branched at 16 GLYCOGEN STORAGE - Stored as cytosolic granules – contain enzymes that can synthesise and degrade glycogen - Muscle glycogen – quick source of energy - Liver glycogen – reservoir of glucose when dietary glucose is unavailable GLYCOGENOLYSIS – BREAKDOWN 1. Glycogen phosphorylase - Catalyses the cleavage of the a(14) yielding glucose-1-phosphate - Acts repetitively on the non-reducing ends of glycogen branches Branch points a(16)? - Cannot remove glucose from branch points – stops 4 glucose units away from a(16) - Further degradation can only occur by enzyme if glycogen debranching enzyme transfers the branches 2. Glycogen debranching enzyme 2 active sites and catalyses 2 reactions: Reaction 1: Transferase reaction - Transfers 3 glucose residues from a 4-residue limit branch to the end of another branch Reaction 2: Glycosidase reaction - The a(16) glucosidase of debranching enzyme catalyses the hydrolysis of a(16) yielding 1 free glucose

SYNTHESIS ENZYMES UDP-glucose phosphorylase - Glucose-1-phos + UDP  UDP-glucose (sugar nucleotide) - UDP-glucose  substrate for polymerisation of mono (glucose) into di/complex poly (glycogen) - UDP-glucose – immediate donor of glucose Glycogen synthase (opp to glycogen phosphorylas) - UDP-glucose adds glucose units to glycogen via enzyme - Transfer of UDP-glucose to the hydroxyl at C4 to form a a(14) linkage 3. Phosphoglucomutase Glucose-1-phosphate  glucose-6-phosphate - Favours the forward direction – beneficial when we need energy quickly from muscles - Reverse direction – too much glucose-6-phos Glucose-6-phosphate - Muscle – enter glycolysis - Liver – may be dephosphorylated by glucose-6phosphatase for release of glucose into blood GLYCOGENESIS - SYNTHESIS 1. Production of glucose-6-phosphate - Muscle: catalysed by hexokinase I and hexokinase II - Liver: catalysed by hexokinase IV 2. Glucose-6-phosphate converted to glucose1-phosphate 3. Synthesis of UDP-glucose - Catalysed by UDP-glucose phosphorylase

Glycogen branching enzyme - Branching – allows glycogen to be more compact and soluble - Transfers segment (7 glucose residues) from the end of a glycogen chain to the C6 hydroxyl to create the a(16) branch Glycogenin - Initiates new glycogen chains – primer - Catalyses the assembly - Glycosidic bonds are formed between C1 of UDP (primary glucose molecule) and hydroxyl of tyrosine 194 on glycogenin - Repeated until linear glucose polymer with a(14) is formed - Once chain contains >10 glucose residues, glycogen synthase catalyses elongation of glycogen chains

REGULATION OF GLYCOGEN METABOLISM

Glycogen synthase and phosphorylase reciprocally regulated by: 1. Allosteric effectors Phosphorylase - AMP (activates) – enhance relax conform - ATP and glucose-6-phosphate (inhibits) – enhance tense (T) conformation Synthase - Activated by glucose-6-phosphate – synthase active when glucose-6 levels are high 2. Reversible phosphorylation - Covalent attachment of a phosphoryl group alters the catalytic properties - Phosphoryl groups usually attached at specific serine, threonine or tyrosine amino acid residues - ATP – phosphoryl donor Phosphorylase - Kinase - makes phosphorylase a more active - Phosphatase – phosphorylase b less active Synthase (opposite to phos) 3. Hormones (insulin, glucagon, epinephrine) - INSULIN - induces the synthesis of glycogen (to lower blood glucose levels) – upregulate glycogen synthase  Activation of phosphoprotein phosphatase - GLUCAGON AND EPINEPHRINE – breakdown  Hormones trigger formation of second messengers (cAMP) - phosphorylase...