CBI Chemistry Notes PDF

CBI 1: Fundamentals of Chemistry Atom – smallest particle with properties of the given element - Contains protons, neutrons and electrons Unified mass unit: 1/12 mass of a atom of carbon-12 - Also can be in the form of dalton (Da) - e.g. 1H or 1.008 Da Periodic Table – ar...

CBI 1: Fundamentals of Chemistry Atom – smallest particle with properties of the given element - Contains protons, neutrons and electrons Unified mass unit: 1/12 mass of a atom of carbon-12 - Also can be in the form of dalton (Da) - e.g. 1H or 1.008 Da Periodic Table – arrangement of elements by atomic number (Z) Ionization energy: energy required to remove valence electron Electron Affinity: energy change when addition electron is attached Electronegativity: ability of atom to attract electrons - Estimated by taking average of ionization energy and electron affinity Atomic radius: distance from center of nucleus to outermost shell of electron Isotopes: same element different mass number (different number of neutrons) - Carbon has two stable isotopes (carbon 12 & 13) Nuclide Symbols Mass number X Atomic number Rutherford-Bohr model: positively charge nucleus surrounded by negatively charged electrons in specific orbits - Not accurate for atoms with more than one electron Quantum mechanical model - Electrons have wave-like properties - Behavior of these waves follow the Schrodinger wave equation (solution of wave equation is wave function)  - Wave function describes probability of electron found at a particular point in space in/around the atom - Solving wave equation will give different probability densities depending on energy of the atom Atomic orbitals: regions of space in an atom where there is high probability of finding an electron Quantum numbers: the size, shape, orientation and properties of the electron - Principal (n) - shell (larger value means higher energy and further away from nucleus) - Orbital (l) - s, p, d, f (0,1,2...) o Orbitals that have same energy can be described as degenerate - Magnetic (m1) - orientation of orbital (e.g. 1 for s orbital, 3 sorts of p orbital : -1, 0,1) - Spin (ms) - follows pauli exclusion principle where electrons in the same orbital spin in opposite directions Orbital occupancy: - Electrons occupy lowest energy orbital first (aufbau principle) - Electrons fill seperate before pairing (Hund’s rule) Ionic Bond – transfer of electron to more electronegative atom Covalent Bond – sharing electrons (less than 1 difference in electronegativity) Polar electronegative bond – electrons not equally shared Structural formula – order of chemical groups (writing each chemical group one after another) VSEPR theory: valence, shell, electron, pair, repulsion e.g. linear, triganol planar, tetrahedral Valence Bond theory - Overlap of atomic orbitals Or - Hybridisation Atomic orbital overlap: - Sigma bonds formed end to end overlap - Pi bonds form sideways overlap above and below bond axis Hybridisation: new set of orbitals by combining Molecular Orbital theory: describes molecular orbitals as regions of space where electrons are likely to be found in molecules CBI 2: Fundamentals of Chemistry Moles – number of discrete particles/entities it contains - Avogadro's number: 6.022 * 1023 Molar mass – mass of one mole of a substance - Number of moles = mass/ molar mass 1 g mol-1 = 1 dalton = 1 Da = 1/12 of the mass of a carbon-12 atom Molar concentration – amount of solute divided by volume of solution - number of moles / volume of solution c=n/V When you substitute n, you get c = m / (Mr*V) Mass concentration – mass of solute divided by volume of solution Volume concentration – volume of solute divided by volume of solution 1 M = 1 mol / L = 1 mol / dm3 = 1 mol dm-3 c1V1 = c2V2 c1 = Concentration of the stock solution (we have already) V1 = Volume of the stock solution (to be used in the dilution) c2 = Concentration of the dilute solution (that we desire) V2 = Volume of the dilute solution (that we desire) Acids and Bases: Water: K = rate constant At equilibrium, the rate of association and the rate of dissociation are equal. Therefore we can equate both rate equations to form: This can be rearranged to find the dissociation constant You can assume in equilibrium that h+ and A- are same value and equate it both to H+^2 This can be transferred to what we know about the dissociation of water to find the acid dissociation constant of water at equilibrium: Ionic product of water: - We get rid of the concentration of water as it is effectively constant and very high in comparison to the concentration of the ions Bronsted Lowry Acids and Bases: Acid – proton donor Base – proton acceptor high ka = low pka and vice versa Strong acid -> high dissociation -> high ka -> low pka Weak Acid -> low dissociation -> low ka -> high pka - Concentration of the undissociated acid and its conjugate base in solution will be equal Strong acid: A substance that completely dissociates to form a hydrogen ion and a conjugate base ion when placed in aqueous solution Weak acid: A substance that only partially dissociates to form a hydrogen ion and a conjugate base ion when placed in aqueous solution pH: concentration of hydrogen ions in solution Because we know the ionic product of water is a constant value, we can use it to figure out concentration of H+ and OH- ions. Kw = 1 x 10-14 mol2 dm-6 [H+][OH-] = 1 x 10-14 mol2 dm-6 pH + pOH = 14 Buffers: allow biological fluids to remain within a pH range that can be tolerated by other biological molecules present. PH buffer solution contains a weak acid in excess and conjugate base (and vice versa) - Addition of acid or base to buffer solution will cause little change to the pH - Buffers perform most effectively around their pH unit which is why we need different buffers for different applications How to prepare buffer: - Solution of weak acid (which will partially dissociate in conjugate base) and solution of salt of the same acid (which will completely dissociate added to available conjugate base) The Henderson-Hasselbach Equation: Factors that affect rate of absorption: - Route of administration: IV (intravenously), oral (tablets) etc - Dosage: concentration - Lipid solubility: Weak acid are absorbed primarily across the stomach Weak base are absorbed primarily across the intestine CBI 3: Biomolecular Bonding Structural isomerism: - Same molecular formula, different order Stereoisomerism: differ in three-dimensional arrangement - E/Z isomerism - Optical isomerism (enantiomer) : non-superimposable mirror images o Stereocenter – 4 different functional groups attached around center atom o Enantiomers: non-superimposable mirror images of each other ▪ have identical properties when in a symmetric environment, apart from their interaction with plane polarized light - Diastereomers: stereoisomers that differ in the absolute configuration of one or more but not al stereocenters o non-mirror image, non-superimposable o Differ by one stereocenter (epimer) o Differ by more than one (diastereomer) Anomer – specific type of epimer (forms at an anomeric carbon of a cyclic saccharide as a result of acetyl and hemiacetyl formation) ** you can seperate enantiomers using chiral coloum with stationary phase that contains chiral group Or turn them into diastereomers “react racemic mixture with a single enantiomer of a chiral compound” Cahn-Ingold-Prelog rules – R/S Corn Rules: Intramolecular bonding: bonding within molecule Intermolecular bonding: between molecules Dipoles: - Charge is distributed across molecule - Difference in electronegativity can cause polarity of a molecule Permanent dipole interaction: non-symmetric distribution of charge across molecule London dispersion forces: transient dipoles that lead to attraction within and between molecules - electron distribution can induce dipole which causes domino affect to induce dipole on surrounding molecules - Larger molecule = greater LDF Hydrogen Bonds: very electronegative atom attracts electron deficient hydrogen Ionic bond: electrostatic force of attraction between positively and negatively charged groups Melting points of molecules: - Higher melting points has to do with better packing in the solid - More symmetry has increased order which increases melting points CBI 4: Biomolecular Interactions Thermodynamics: the relative energies between reactants and products, and the exchange of energy between system and surroundings System – reactants and products in the reaction Surroundings – everything else outside system Boundary – where the two meet ** total energy in an isolated system does not change ** total energy of system and surroundings does not change Thermodynamic temperature: absolute measure of the average total internal energy (Kelvin, K) Heat: energy per mol (kJ mol^-1) Enthalpy: total amount of energy that a chemical system possesses - Kinetic energy --> can come in the form of moving electrons, vibration of atoms connected by bonds, rotation and translation of molecules etc. - Chemical potential energy --> covalent or ionic bonds between atoms/ions, intermolecular forces between molecules Bond energy: average amount of energy required (in gas phase and standard conditions) to break 1 mol of all bonds of same type within same chemical species as the bond energy (kJ mol^-1) ** why do we care? Tells us how much energy is required to break them but also how much energy is released when they are formed. By calculating difference between products and reactants we determind if energy is released or taken in. ΔH > 0 = endothermic reaction where products have greater chemical potential energy than reactants, so input of energy is required for reaction to proceed ΔH < 0 = exothermic reaction where reactants have greater chemical potential energy than products, so reactants give out energy in the form of heat Entropy: randomness of a system (J mol-1 K-1), measure of energy dispersal at a given temperature ΔS(total) = ΔS(system) + ΔS(surroundings) ΔS >0 indicates that the reaction increases the dispersal of energy in the system ΔS one product 2. Elimination reaction: two or more components (functional groups or atoms) are lost 3. Substitution reaction: one atom or functional group is replaced by another a. SN1 i. Rate determining step depends on 1 reactant b. SN2 i. Rate determining step depends on 2 species ii. One group becoming attached to central atom, while another is detaching Reaction profile for SN1 reaction^^ Reaction profile for SN2 reaction  4. Rearrangement reaction: internal connectivity of atoms in a molecule is changed 5. Redox reaction: electrons are transferred from one reactant to another a. Oxidation is loss of electrons and increase in oxidation state b. Reduction is gain in electrons and decrease in oxidation state Nicotinamide adenine dinucleotide (NAD) is a major redox agent in biological reactions: the nicotinamide ring of NAD+ acts as the hydride/electron acceptor. Flavin adenine dinucleotide (FAD) is a common redox reagent in biological reactions: the ring system of FAD acts as a hydrogen/electron acceptor CBI 5: Structure and Function of Biomolecules Carbohydrates Empirical formula - (CH2O)n Monosaccharide  Aldose: end in C=O  Ketose: in the middle C=O Fischer projection: horizontal lines towards you, vertical lines away from you Monosaccharide nomenclature: Locate the carbonyl group Draw fischer projection Identify stereocenter furthest away from carbonyl group If the –OH group is on the left side of the stereocenter then its L-isomer or right side is D-isomer Cyclisation: when a carbonyl carbon of an open chain reacts via nucleophilic attack from one of the –OH groups in the chain - Forms a circle: from aldose = hemiacetal, from ketose = hemiketal Cyclisation creates a new stereocenter which forms new anomers, which are identified by α- (hydroxyl group other side) and β- (hydroxyl group same side) - to the substituent on the last carbon How to identify alpha or beta: 1) Identify anomeric carbon (carbon that has o in side chain and connected to main o) 2) Then check if both OH groups are pointing same direction or not Disaccharide: when two monosaccharides react to form a glycosidic bond in a condensation reaction - Naming of bond: α,β-1,2 glycosidic bond. Polysaccharide: long chain Important ones – cellulose (β-D-glucose via β-1,4 glycosidic bonds), starch and glycogen (α-D-glucose connected via α-1,4 glycosidic bonds) - If you connect 1,6 glycosidic bond to amylose it creates amylopectin - Starch contains both amylose and amylopectin, glycogen is amylopectin Structure: cellulose, chitin Post-translational modification: e.g. ABO blood groups Energy storage: glycogen, starch Lipids: long hydrocarbon chains with a carboxylic acid group at the end - Most importantly known for their amphiphilic properties Fatty acids: No C=C bond is saturated, C=C bond is unsaturated Trans/E opposite side, Cis/Z same side (leads to bend or kink in the chain) ** A fatty acid containing a total of 16 carbon atoms (in its hydrocarbon chain) and zero double bonds would be described as: 16:0 How to state the position of the double bond: 1. The ω- or n- notation starts counting at the methyl group (-CH3): ω-7 or n- 7 2. The IUPAC or C- notation starts counting at the carboxylic acid carbon (- COOH): 16:1(9) or C-9 Triacylglycerol: forms by carboxylic acid from fatty acid reacting with hydroxyl group from glycerol forming ester bond - Function: chemical energy storage molecules Glycerophospholipid: two fatty acids and the remaining OH bond to phosphate that bonds to glycerol to form a phospholipid - Amphilic molecules: hydrophobic tail and hydrophilic head Sphingolipid: sphingoid backbone (amino acid serine and long chain fatty acyl CoA) and fatty acid and head group  - Sphingomyelins contain a phosphate group that is most commonly bound to a choline or ethanolamine group. They are mainly found in myelin sheaths. - Cerebrosides are the simplest sphingolipids and contain a monosaccharide as a head group and are found in the membranes of neurons. - Gangliosides are complex sphingolipids that contain an oligosaccharide as head groups with a minimum of one sialic acid unit; they make up around 6% of the lipids in the brain. Sterol Lipids: has a ring system that is rigid and hydrophobic, while the polar hydroxyl group is hydrophilic Lipid aggregation: - Lipids spontaneously aggregate so that their hydrophobic tails interact and their hydrophilic heads face out towards water - This is driven by the hydrophobic effect, where breaking hydrogen bonds in water is energetically unfavorable so the hydrophobic tails face towards each other - This maximizes LDF between fatty acid tails and head groups form electrostatic and hydrogen bonds with water molecules Main properties: They are cooperative structures They can extend and fuse with themselves They are self-repairing They are impermeable for ions and polar molecules Nucleic Acids: Nucleotides: linear polymers that contain coded information that is transferred from one generation to the next - Nucleoside is without phosphate Guanine and ctyosine have 3 hydrogen bonds between Adenine and Thymine/uracil have 2 hydrogen bonds between Monomeric nucleotides: molecules with other functions like energy metabolism Proteins: Amino acids: α-carbon atom that is bound to an amino group (-NH2), a carboxylic acid group (-COOH), and a side chain (-R) that determines its individual and specific physicochemical properties. Primary sequence: chain of amino acids linked by peptide bonds Secondary sequence: hydrogen bonds between the backbone carbonyl and nitrogen atoms - This forms β-sheets, α-helices and hairpin bends/β-turns Tertiary sequence: covalent and non-covalent interactions between amino acid subunits - e.g. hydrogen bonds, ionic interactions, dipole interactions, disulfide bonds etc Quaternary sequence: multiple polypeptide chains form together **proteins that facilitate correct folding are called chaperones Protein purification: 1. Lysis: releasing proteins by disrupting/destroying plasma membrane 2. Isolate specific protein a. Size-exclusion chromatography: sample applied to coloumn that contains beads with pores, small molecules penetrate. b. Order of elution: from large to small c. Ion-exchange chromatography: amino acids separating by charge (occurs due amino acid side chain) by interacting with beads via ionic forces i. Isoelectric point: the pH where protein has overall net charge of zero d. Affinity Chromatography: specific amino acids or whole protein have tags. These tags load onto a column and interact with column matrix, non-tagged items will flow through. Protein then eluted with high concentrations of small molecules that interact with column matrix or tag itself. e. SDS- PAGE: biomolecules given negative charge by denaturing the proteins. Then current is applied and the proteins go towards the positive anode. Smaller proteins travel farther also. Bacterium were modified to express protein with His-tag 1. Affinity chromatography with Ni-NTA column 2. SDS page Both methods were done to separate the protein of interest from the rest 3D Protein structure: 1. Protein function 2. Protein interaction with other ligands 3. Drug design and development 4. Disease mechanisms 5. Application in other fields SEC-purified His-tagged protein --> functional assays for protein characterisation --> protein structure determination = x-ray crystallography, cryo-electron microscopy, NMR -- > 3D protein structure CBI 6: Enzymes and Kinetics of Biocatalysis Enzymes: catalyze biochemical reactions by lowering the energy required to reach the transition state Enzymes bind specific substrates at their active site: - The substrate binds to the active site of the enzyme, forming a reaction intermediate called the enzyme-substrate complex  - This interaction between the substrate and the functional group in the active site of the enzyme lowers the activation energy by providing an alternative reaction pathway - Following the formation of the enzyme-substrate complex, the reaction mechanism will give rise to product and free enzyme - The enzyme is released and ready to bind more substrate - At low substrate concentration: rate of reaction is directly proportional to concentration of substrate (first-order kinetics) - At high substrate concentration: rate of reaction is unaffected by increase in substrate concentration (zero-order kinetics) - The maximum velocity of the enzyme = all enzyme molecules are occupied, reaction equilibrium is reached The Michaelis-Menten equation:  k1 = rate constant for the formation of enzyme-substrate (ES) complex from the enzyme (E) and the substrate (S). k-1 = rate constant for the reverse reaction, the dissociation of the enzyme- substrate complex (ES) to form enzyme (E) and substrate (S). k2 = rate constant for the formation of product (P) from the enzyme-substrate complex (ES). This is also commonly known as kcat. **Km is specifc to each enzyme and independent of enzyme concentration ** low Km means low dissociation constant which represents high affinity of the enzyme to its substrate  - Km is the substrate concentration at half of Vmax The Lineweaver-Burk plot: - Bc Vmax can never be reached only approached, this means we must find another way to find Km kcat = k2: max number of chemical conversions of substrate molecules per second that a single catalytic site will execute for a given enzyme that is saturated by its substrate. - the smaller the KM and the larger the kcat, the more efficient an enzyme will be KM The Michaelis-Menten constant, defined as the substrate concentration at half the maximum velocity. Vmax The rate of reaction when the enzyme is saturated with substrate is the maximum rate of reaction. Kcat A measure of the number of substrate molecules "turned over" by enzyme per second. Reversible inhibitor: Competitive inhibitor   Inhibitor resembles substrate and binds to active site  Degree of inhibition depends of concentration of substrate  The effects of competitive inhibitor can be overcome by sufficient substrate  Km is increased, Vmax remains the same (more substrate is needed to obtain same reaction rate) Uncompetitive inhibitor  Binds to ES complex to prevent release of product  Cannot be overcome by addition of sufficient substrate  Vmax is reduced, Km is reduced by the same amount  ES is depleted by this inhibitor so due to reaction equilibrium, K1 increases and more substrate binds to enzyme  If K1 increases, Km decrease because lower substrate concentration is required to form half the maximal concentration of ES Non-competitive inhibitor  Binds at another site, does not prevent substrate molecule from binding  Vmax is decrease, Km is unchanged bc inhibitor lowers concentration of available functional enzyme but does not affect the binding of the substrate  - In general, warm conditions = higher KE = optimal enzyme activity - However high temperatures can inactivate enzymes bc molecules vibrate and twist that non-covalent bonds breaj - Proteins loses secondary and/or tertiary structure and becomes inactive Cofactors (non-protein chemical or metal compounds that enzymes require in order to function) Allosteric regulation of enzymes: - Allosteric binding causes conformational change in the enzyme that can result in any type of inhibition - Allosteric effects can also result in activation of an enzyme Allosteric effects in multi-subunit proteins: At low substrate concentration, the reaction rate increases as substrate concentration increases. After the substrate binds to the first active site of the enzyme, there is a change in the enzyme’s quaternary structure such that the other sites become more likely to bind substrate, so the reaction speeds up. Once all the sites are saturated with substrate the reaction rate reaches a plateau. What affects activity of an enzyme (recap): - PH - Temperature - Inhibitors - Covalent modifications (like phosphorylation of critical amino acid side chain can either activate or inactivate an enzyme) CBI 7: Glycolysis and Gluconeogenesis C6H12O6 + 6 O2→ 6 CO2 + 6 H2O Glycolysis: Glucose + 2 NAD+ + 2 ADP + 2 Pi → 2 Pyruvate + 2 NADH + 2 H+ + 2 ATP + 2 H2O Step 1: trapping glucose and preparing it to be cleaved - investment phase: Steps in glycolysis that require the input of energy, provided by the hydrolysis of ATP 1.1 Glucose to glucose-6-phosphate - Phosphorlyation is catalysed by hexokinase - ATP to ADP - Reaction is irreversible (glucose cannot leave cell now) - bc it cannot diffuse out by facilitated diffusion o This is because the phosphate has a negative charge which prevents it from diffusing through the membrane 1.2 glucose-6-phosphate to fructose-6-phosphate - This isomerism creates a resonance structure which has more stability, and makes the beta bond more reactive - This leads to break of C-C bond due to beta bond reactivity which is needed to cleave glucose in step 2  - It allows production of two/three carbon metabolites rather than one/two and one/four 1.3 fructose-6-phosphate to fructose-1,6-bisphosphate - Second phosphorylation in the first carbon - ATP to ADP - Irreversible - Catalyzed by allosteric enzyme phosphofructokinase - Can be inhibited by citrate: high levels of citrate signal that enough energy is present in the cell, thus it acts as an allosteric inhibitor for the phosphofructokinase Step 2: Lysis into 2 C3 units 2.1 fructose-1,6-bisphosphate to dihydroxyacetone phosphate and glyceraldehyde-3- phosphate - Reversible reaction - Catalyzed by enzyme aldolase 2.2 dihydroxyacetone phosphate to glyceraldehyde-3-phosphate - From a ketose carb to an aldose carb - Reversible reaction - As glyceraldehyde is used up in the following steps, the equilibrium shifts to the right - From now on all reactions will take place twice per glucose molecule Step 3: The payoff phase – because it produces energy in form of ATP and NADH ** these reactions all take place twice 3.1 gylceraldehyde-3-phosphate to 1,3bisphosphogylcerate - contains high energy intermediate form - First reaction step very high negative G, while second step very endergonic - Using the enzyme, the low energy intermediate is avoided (by formation of thioester intermediate) and reaction is catalyzed 3.2 1,3bisphosphoglycerate to 3-phosphoglycerate - ADP to ATP - This kind of reaction is known as substrate level phosphorylation as the phosphate donor has a very high phosphorylation potential 3.3 3-phosphogylcerate --> pyruvate  - First phosphate gets moved to second position - Second step enolase catalyzes a dehydration reaction - Third step substrate level phosphorylation occurs, ADP to ATP (irreversible) Substrate level phosphorlyation: transfer of phosphate group from a molecule to ADP to form ATP Depending on presence of oxygen, pyruvate can be converted into different substances Gluconeogenesis: synthesis of glucose 2 Pyruvate + 4ATP + 2GTP + 2NADH + 6H2O --> Glucose + 2NAD+ + 4ADP + 2GDP + 6Pi + 2H+ Occurs in: liver and adrenal cortex (kidney) An alternative set of enzymes is used to bypass the high gibbs free energy in the unidirectional reaction of glycolysis. Glycolysis Step 1.1 (last step of gluconeogenesis) - Reverse glucose-6-phosphate to glucose - Catalysed by glucose-6-phosphotase, a membrane bound protein in the ER - This reaction takes place in the ER lumen Glycolysis Step 1.3 - Reverse fructose-1,6-bisphosphate to fructose-6-phosphate - Catalyzed by fructose-1,6-bisphosphotase Last step of glycolysis step 3.3 - catalyzed by pyruvate carboxylase (PC) and phosphoenolpyruvate carboxykinase (PEP-CK)  - Pyruvate --> oxaloacetate (this reaction takes place in mitochondrial matrix) - Oxaloacetate --> malate (so that it can be transported into cytoplasm - In cytoplasm malate converted back to oxaloacetate and then decarboxylated and phosphorylated by phosphoenolpyruvate carboxykinase Alternative GNG precursors: - Lactate - Glucogenic amino acids - Glycerol Control of blood glucose concentration: Normoglycemia - The condition in which there is a normal concentration of glucose in the blood Hypoglycemia - The condition in which there is a concentration of glucose in the blood lower than the range considered normal Hyperglycemia - The condition in which there is a concentration of glucose in the blood higher than the range considered normal After a meal, blood plasma glucose levels increase and then decrease as glucose is subsequently up taken by different cells/organs. The glucose taken up by cells is used as substrate for glycolysis. Glycolysis is a catabolic pathway that is switched on when we have high levels of glucose in our blood or need it for other processes. When glucose levels are low glycolysis is switched off, so if glucose is still needed other processes like (gluconeogenesis) are switched on. This allows glucose to be produced and released to the blood to maintain concentration within normal range. Fast regulation of glucose - Inhibiting or activating the enzymes involved in glycolysis and gluconeogenesis Slow regulation of glucose  - When blood glucose levels fall, glucagon is released form alpha cells in pancreas - Glucagon activated gluconeogenesis and the conversion of stored glycogen to into glucose - When blood glucose levels are high, beta cells of pancreas release insulin that activates glycolysis - These hormones work by altering expression of the genes that code for the enzymes in glycolysis and gluconeogenesis. Thus this regulation is much slower CBI 8: Krebs Cycle and Oxidative Phosphorylation Oxidative Decarboxylation: Reaction that simultaneously reduces a compound and removes carbon dioxide. An example is the conversion of pyruvate to acetyl-CoA that reduces NAD+ to NADH while releasing one molecule of CO2. Pyruvate + NAD+ + CoA -> Acetyl-CoA + NADH + CO2 + H+ ** pyruvate is converted into acetyl-CoA by pyruvate dehydrogenase TCA/Krebs Cycle: Acetyl-CoA + 3NAD+ + FAD + ADP + Pi + 2H2O -> CoA + 3NADH + FADH2 + 3H+ + ATP + 2CO2 1) C4 and C2 molecules form together to make C6 a. This reaction is driven by hydrolysis of phosphodiester bond 2) Isocitrate is formed by dehydration step followed by hydration step 3) Oxidative decarboxylation which forms NADH and CO2 4) Oxidative decarboxylation released NADH and CO2 5) Energy transfer reaction a. Hydrolysis of energy rich phosphodiester powers substrate level phosphorlyation b. Formation of ATP (when high energy needed) or GTP  6) Oxidation to fumerate a. FAD covalently bound to enzyme b. Enzyme is embedded in the inner mitochondrial membrane and part of ETC 7) Hydration reaction (stereospecific) 8) Oxidation reaction which is unfavourable with high Gibbs free energy a. Driven by the use of its product NADH – consists of adenine, two ribose sugars, a diphosphate group and a nicotimadine group - Protons and electrons are added to nicotimadine ring when NAD+ is reduced to NADH FAD – comprised of ADP unit that is connected via a sugar moiety to an isoalloxazine ring. This ring is the active electron carrier part and can accept two electrons and protons. Acetyl-CoA: formed by beta oxidation of a fatty acid Ketone bodies: - Under conditions of starvation, acetyl-CoA may be converted into ketone bodies to act as alternative fuel source - Too high concentration of ketone bodies can be life-threatening as it accumulates in the blood which leads to drop in pH. The result is an acidosis that can impair tissue function and the central nervous system. Four enzyme complexes involved in ETC: - Complex I: NADH-Q oxidoreductase - Complex II: Succinate-Q reductase - Complex III: Q-cytochrome c oxidoreductase - Complex IV: Cytochrome c oxidase Ubiquinone: can be reduced by two electrons, highly hydrophobic and thus shuttles electrons through the inner mitochondrial membrane Cytochrome C: small water soluble protein with a heme center and a iron inside the protein. Cyt C can carry one electron at a time. ETC: A series of enzyme complexes that perform redox reactions, coupling the transfer of electrons - from electron donors (NADH and FADH2) to the terminal electron acceptor (oxygen) - to the movement of hydrogen ions across the mitochondrial inner membrane into the mitochondrial intermembrane space. - NADH binds to complex I, it gets oxidised to NAD+ and proton and 2e - The two electrons formed are transported via internal metal clusters to ubiquinone which takes up two protons from the matrix to be reduced - This electron transfer allows complex I to transfer 4 protons from the matrix into intermembrane space  - Complex II catalyses reaction from succinate to fumerate in kreb cycle, which leads to reduction in FAD - FAD covalently bound to enzyme complex where it transfers two electrons and protons to complex II - Electrons and protons are taken up by ubiquinone to form QH2 - Ubiquinone gives the electrons to complex III, this leads to release of 4 protons to intermembrane space, while another 2 protons are taken from matrix - The electrons are then transferred to cytochrome C (located in intermembrane space), two cytochrome c molecules are necessary to take both electrons from ubiquinone. - Cytochrome C shuttles the electrons to complex IV (happens 4 times) o Cyt C uses iron atom, coordinated as part of a haem complex, which can alter redox state from Fe(II) to Fe(III) - Complex IV catalyzes the reduction of molecular oxygen to water by taking up four protons from the matrix, it also pumps 4 protons across inner mitochondrial membrane - These protons generates proton gradient Proton-motive force: the energy that is created by a chemical gradient due to the difference in concentration of protons on each side, and a charge gradient due to the positive charge of these protons Proton motive force = chemical gradient + charge gradient ATP synthase: - Proton flows through the intermembrane channel from the a subunit to the c subunit - Proton neutralizes the charge of glutamate inside the C unit due to the high pH - C unit rotates clockwise - Low pH on matrix side leads to release of protons in the matrix - The loose state in the beta subunit allows ADP and phosphate to bind to catalytic side for ATP synthesis - T state supports formation of ATP by tightly binding ATP - Open (o) state, amino acids rearrange in the active side so ATP can leave the enzyme  - When this happens the conformational stage of all the three beta subunits changes. The loose conformation becomes tight, catalysing the formation of ATP, the tight conformation becomes open, being able to release ATP and the open conformation becomes loose, ready to bind ADP and inorganic phosphate. - Another rotation again causes the conformational change, closing the catalytic cycle of the subunits. Thus, the role of the proton gradient is not to drive the reaction but to release ATP from the synthase. ** rotation of the c ring leads to 120 degree rotation of gamma unit Malate/aspartate shuttle: NADH reduction, net yield of 2.5 ATP Glycerol phosphate shuttle: FADH reduction, net yield of 1.5 ATP