Course 2 Metabolism PDF

Stanislav Skamene, Svatava Vyhnánková, Filip Otepka, Dominika Kubátová Course 2 Metabolism Translated by Kamila Kočí  Table of contents 2.1 What fuels our cells? …………………………………………………...….………………………………………….……………………….. 1 2.2 Chemical reactions...

Stanislav Skamene, Svatava Vyhnánková, Filip Otepka, Dominika Kubátová Course 2 Metabolism Translated by Kamila Kočí  Table of contents 2.1 What fuels our cells? …………………………………………………...….………………………………………….……………………….. 1 2.2 Chemical reactions in metabolism..…………………………………………….…………………..…….….…………………………. 3 2.3 Enzymes …………………………………………………………………….…………………………………………….………………………….. 7 2.4 Coenzymes, vitamins, trace elements..…………………………………………………………….…………………….……………. 9 2.5 Inhibition of enzymes, role in pharmacology …………………….……………………………………………………………….. 13 2.6 Epithelial tissues II - Glandular epithelia ……………………………………………………………...……………………………… 15 2.7 Structure and origin of mitochondria ………………………………………………………………………………………………….. 18 2.8 Electron transport chain and ATP formation ………..……………….………………………………………….………………… 21 2.9 Krebs cycle ……………………………………………………………………………………………….…………………….………………….. 23 2.10 Epithelial tissues III - Barriers and transport ……………………………………………………………………………………… 27 2.11 Degradation and synthesis of Glucose ………………………………………………………………………………………………. 31 2.12 Pentose phosphate cycle, metabolism of Fructose and Galactose ……………………………………………….…… 35 2.13 Lipid breakdown, ketone bodies ………………………………………………………………………………………………….…… 38 2.14 Energy storage in cells ……………………………………………………………………………………………….……….….………… 42 2.15 Muscle tissue ………………………………………………………………………………………………………..…………………………. 45 2.16 Cell motility and molecular motors ………………………………………………………………………………………………….. 48 2.17 Metabolism of amino acids ……………………………………………………………….……….…………………………………….. 51 2.18 Muscle metabolism, muscle contraction ………………………………………………………………………………………….. 58 2.19 Blood - morphology of blood elements …………………………………………………………….……………………………… 61 2.20 Hemopoiesis ……………………………………………………………………………………………….………………………….………… 65 2.21 Hemocoagulation, biochemistry of thrombocytes and leukocytes ………….………………………………………… 69 2.22 Iron and heme metabolism ………………………………………………………………………….…………………………………… 74 2.23 Biochemistry of erythrocytes ……………………………………………………………………….……..……………………………. 77 2.24 Connective tissue ……………………………………………………………………………………………………………………………… 80 2.25 Metabolism of connective tissue ………………………………………………………………………………………….………….. 86 2.26 Components of blood plasma and their functions …………………………….……………………………………………… 90 2.27 Regulation of metabolic pathways at cell level ………………………………………….……………………………………… 96 2.28 Energy metabolism - the overview …………………………………………………………………………………………………… 99 2.29 Cartilage, bone and ossification …………………………………………………………………………….……………………….. 104 2.30 Enzymopathy - heritable disorders of metabolism …………………………………………………………………………. 109 2.31 Biochemical correlations of monogenic diseases ………………………………………………………………………….… 112 2.32 Newborn screening …………………………………………………………………….………………………………………………….. 115 2.33 Extreme situations in cell ……………………………………………………………………………………………………………….. 116 2.1 WHAT FUELS OUR CELLS? doc. MUDr. Jan Trnka, Ph.D. Energy - = the ability of the system to perform work - types of energy transfer - heat and work o Heat - disordered movement of energy from place to place o Work - an ordered movement of energy from place to place - internal energy of a system - U = all energy (kinetic and potential) of all particles of the system o its value cannot be measured (too complex to calculate) - we can only measure its changes = ΔU First law of thermodynamics – law of conservation of energy = the internal energy of an isolated system never changes - it is not possible to produce / consume energy - energy in an isolated system is neither produced nor disappears, but only transformed from one form to another - we cannot construct a perpetual mobile (of first type) - a machine that generates energy from nothing - ΔU = W + Q o ΔU - change of internal energy of the system o W - work received by the system o Q - heat received by the system Enthalpy - H - It is an artificial definition created by maths which helps with calculations on internal energy - is defined as the sum of the internal energy of the system + the system pressure * the volume of the system o H = U + pV - at constant pressure, the enthalpy change is equal to the heat received by the system: ΔHp = Q o if the system (for example, a mixture of solutions in a vessel) undergoes chemical reactions in which it releases heat into the environment (= exothermic reactions), Q will be less than 0 and the enthalpy of these reactions will be negative Entropy - S - The notion of entropy was introduced by the German physicist Rudolf Clausius in the context of thermodynamics in order to explain why some processes are reversible and others are not - ΔS = Q/T o the change in entropy is equal to the heat “given out/donated”divided by the thermodynamic temperature at which the “giving/ donation” occurred Second law of thermodynamics - originates from knowledge on entropy - determines the natural direction in which natural processes take place - in an isolated system, the entropy must remain the same or increase (it can never decrease) o for reversible processes, their S = 0 o in order for the process to run over and over in one direction without power supply (perpetuum mobile of the second type), the entropy of this process would have to be equal to 0 ▪ however, since T does not drop to 0 (ie absolute zero), each action will increase entropy, and therefore the perpetuum mobile of the second type cannot be assembled - virtually all processes taking place in nature can be considered irreversible - because a part of the energy is always converted into waste heat, which increases entropy - however, entropy can be decreased locally - e.g cells constantly decrease their entropy but increase entropy of their surroundings 1 Gibbs energy - G - another fabricated quantity - this time to simplify the assessment of whether the process can run spontaneously or not - is defined as enthalpy minus temperature multiplied by entropy: - G = H - TS => ΔG = ΔH - T * ΔS => ΔG = - T * ΔSsurrounding - T * ΔSsystem => ΔG = - T * Δ(Ssurrounding + ΔSsystem) => ΔG = - T * ΔSuniverse - change of Gibbs energy (ΔG) = the heat supplied minus the heat wasted to the surroundings o The change in Gibbs energy tells us the maximum amount of work you can get from that reaction o The change in Gibbs energy of both direct and feedback reactions has the same absolute value, with only a different sign o Negative change in Gibbs energy = positive change in entropy – spontaneous process ▪ Not always – for example, if an obstacle needs to be overcome ATP - ATP drives our cells because the concentration of ATP / ADP is far from equilibrium = the potential for work is great o the more the reaction is from its equilibrium, the more work it can do = what determines the ability to do the work is only the distance from the equilibrium of the reaction - the process of decomposing ATP to ADP is thermodynamically spontaneous 2 2.2 CHEMICAL REACTIONS IN METABOLISM doc. RNDr. Ing. Petr Tůma, Ph.D. Metabolism of macronutrients - macronutrients = carbohydrates, lipids, proteins - carbohydrates and lipids break down to CO2 and H2O - proteins break down into CO2, H2O and NH3 o NH3 is toxic to the body (mainly the brain) and is therefore converted to urea and excreted through urine - all macronutrients have their own catabolic and anabolic pathways and some pathways in common (e.g Krebs cycle) o their pathways are linked through carboxylic acids Main metabolic reactions Catabolic reactions - glycolysis - degradation of carbohydrates to pyruvate / lactate - glycogenolysis - degradation of glycogen to glucose - lipolysis - degradation of triglycerides to glycerol + fatty acids - beta-oxidation - degradation of fatty acids to acetyl-CoA - ketone body breakdown – in case of starvation, liver produces ketone bodies which are send off to other tissues for degradation - degradation of proteins and amino acids Anabolic reactions - gluconeogenesis – glucose synthesis - glycogenesis – glycogen synthesis - Fatty acid synthesis - lipogenesis – synthesis of TAG from fatty acids and glycerol - ketogenesis – production of ketone bodies in liver - proteosynthesis – protein synthesis - ornithine cycle – urea formation Amphibolic reactions (some anabolic and some catabolic) - pyruvate dehydrogenase reaction – converts pyruvate to Acetyl-CoA - Krebs or Citrate cycle - consumes Acetyl-CoA to produce CO2, NADH and FADH2 o note. NADH should be correctly written as NADH + H+ - electron transport chain - consumes NADH and FADH2 to produce ATP and H2O 3 Carboxylic acids - have a dominant position in the whole metabolism - dissociate in the body (due to pH) –hydrogen is cleaved from -COOH group and anions are formed in a resonant structure, where two oxygen atoms exchange one electron o e.g. acetic acid ▪ under normal conditions - CH3COOH = acetic acid ▪ in the body - CH3COO- = acetate ▪ the carboxylic acid anion names are derived from the Latin names of their respective acids ▪ acetic acid in latin is acidum aceticum -> its anion is called acetate - acyls are still very common in metabolism; these are carboxylic acids that lose OH Hydroxy acids x keto acids - in the body, hydroxy acids are constantly converted to keto acids and keto acids to hydroxy acids - a classic example is lactate (hydroxyacid) and pyruvate (keto acid) - conversion of hydroxy acid to keto acid is oxidation –hydrogen is removed o NAD+ is needed to take both hydrogens and becomes a NADH + H + - Conversion of keto acid to hydroxy acid is reduction – hydrogen is added o NADH + H+ is needed, which donates both of its hydrogens and becomes a NAD+ 4 Detoxification of ammonia - transamination -> hydrolytic deamination / oxidative deamination -> urea synthesis - everything takes place in the liver, transamination can take place elsewhere, urea synthesis also takes place in the kidneys - amino acids are degraded to CO2, H2O and NH3 - however, ammonia is toxic to the body because it messes up the membrane potential and causes swelling o diseased liver loses (among other things) the function of detoxifying ammonia, causing brain swelling = liver encephalopathy - literally the only difference between an amino acid and a keto acid is that one has NH 3 and the other has oxygen - transamination - amino acid and keto acid exchange NH3 and oxygen o AMK1 + KK2 -> KK1 + AMK2 o Coenzyme of transamination is pyridoxal phosphate o alanine + α-ketoglutarate -> pyruvate + glutamate o aspartate + α-ketoglutarate -> oxaloacetate + glutamate ▪ ALT (alanine transaminase) and AST (aspartate transaminase) enzymes are responsible for these two transaminations) ▪ ALT and AST are also markers of liver function - their increased plasma concentration means that liver cells are breaking down - hydrolytic deamination - glutamine -> glutamate + NH3 - oxidative deamination - glutamate -> α-ketoglutarate + NH3 - urea synthesis (ornithine cycle) - CO2 + 2x NH3 -> urea Decarboxylation - removal of the carboxyl group in the form of CO 2 - amino acids become biogenic amines o histidine -> histamine, hormone and neurotransmitter o glutamate -> GABA (gamma-Aminobutyric Acid), inhibitory neurotransmitter o tryptophan -> 5-hydroxytryptofan -> serotonin, one of the hormones of happiness Carboxylation - opposite of decarboxylation - CO2 is added - for example, in the synthesis of fatty acids 5 Hydrogen as a source of energy - very big respiratory chain simplification - 2H2 + O2 → 2H2O + energy (in the forms of ATP) Dehydrogenation - oxidation of single bond to double bond o -CH2-CH2- -> -CH=CH- + 2H+ + 2e- o very frequent reaction in metabolism - for example in β-oxidation of fatty acids, Krebs cycle and synthesis of unsaturated fatty acids - dehydrogenation of 2-keto acids – it proceeds simultaneously o R-CO-COOH + HCoA -> R-CO-CoA + CO2 + 2H+ + 2e- - dehydrogenation of alcohols o primary alcohols ▪ alcohol -> aldehyde -> carboxylic acids methanol -> formaldehyde -> formic acid ethanol -> acetaldehyde -> acetic acid ▪ methanol itself is completely harmless, only after its metabolism to formic acid it becomes extremely toxic 10 ml is enough to completely destroy the optic nerve (and cause permanent blindness), 15 ml is deadly ▪ all alcohols are metabolized by one enzyme (alcohol dehydrogenase), therefore ethanol is given as a treatment for methanol poisoning - the enzyme will have more work and metabolize methanol slowler, giving the liver and kidneys time to remove formic acid from the blood o secondary alcohols ▪ alcohol -> ketone o tertiary alcohols – dehydrogenation does not occur Reactions of monosaccharides - esterification o glucose could theoretically leave at the moment of entry into the cell, therefore in all cells immediately after entry it is esterified with phosphate to glucose-6-phosphate, which no longer crosses the membrane o Only hepatocytes have an enzyme that can separate the phosphate again - oxidation o oxidation of glucose on 6th carbon -> glucuronic acid o oxidation of glucose on 1st carbon -> gluconic acid - reduction o glucose reduction produces an alcohol  glucitol - epimerization o fructose, mannose and galactose can be converted to one another though glucose 6 2.3 ENZYMES doc. RNDr. Ing. Petr Tůma, Ph.D. 2.3.1 Thermodynamics and kinetics - If a reaction can occur or not is determined by Gibbs energy - Only reactions with negative Gibbs energy can take place o At the same time, however, they need enough energy to surpass the activation barrier – this is where enzymes help out Speed of chemical reaction - Change in the mass of reactant or products per unit of time - example - reaction 3H2 + N2 -> 2NH3 o the formula is quite intuitive- the rate of chemical reaction is measured as the amount converted by the reaction over time ▪ v = concentration of product / time o also works the other way around – the rate of reactants lost over time ▪ v = - concentration of reactants / time o however, we must be careful about the stoichiometric coefficients – for H the formula needs to be adjusted by multiplying it by 1/3, for the NH3 by multiplying by 1/2 Dependence of reaction rate on concentration - A + B -> products - Particles in the system fly at great speed – by this movement the particles collide together; in order for a reaction to occur, particles A and B ,must collide, creating new bonds between the atoms and the old ones disappearing  product is formed - Collision theory – the rate of reaction is proportional to the number of active collisions between molecules A and B per second - Kinetic equation v = k * [A] * [B] // k is coefficient, [A] is concentration A, [B] is concentration B o Applies for reactions of I. and II. order o Reaction of I. order are monomolecular reactions – one molecule spontaneously disintegrates into 2 molecules ▪ v = k * [A] ▪ N2O4 -> NO2 + NO2 ▪ v = k * [N2O4] o reaction of II. order are bimolecular – two molecules react ▪ v = k * [A] * [B] ▪ a collision of a maximum 2 particles is assumed, a more complex reaction takes place via intermediates ▪ example reaction - 4HBr + O2 -> 2H2O + 2Br2 does not proceed directly but through intermediates a) HBr + O2 -> HOOBr - slow b) HBr + HOOBr -> 2 HOBr c) HBr + HOBr -> H2O + Br2 v = k · [HBr] · [O2] ▪ the speed of the whole system is controlled by the speed of the slowest intermediate step Activation energy and temperature - v = k * [A] * [B] o k = proportionality constant = k(-EA/RT) – the so- called Boltzmann factor ▪ expresses the fraction of molecules in the system with energy higher than the activation energy (EA) - Boltzmann factor calculates which molecules have enough temperature to overcome the activation energy - Effect of temperature – increasing the temperature by 10 °C increases the reaction by 2-3 x 7 Principle of catalysis - In the human body we cannot heat substances too much – we need another way to increase the rate of chemical reaction - catalysis = reduction of activation energy o enzymes are catalytic proteins – they reduce activation energy without changing the ratio of reactants and products ▪ captures reactant molecules on its surface, stabilizes them and allows them to orient themselves appropriately - the catalyst may be a metal or an enzyme 2.3.2 Enzymes - high efficiency catalysts which increase the rate of chemical reactions by several orders of magnitude - they are proteins, so they are easily regulated - e.g. acetylcholinesterase- protein capable of cleaving 25 000 molecules of acetylcholine in one second - allows reactions to proceed at low temperatures, neutral pH and atmospheric pressure - enzymes have an active site on their surface where substrates can bind o shape of the active site corresponds to the shape of the substrate o enzyme stabilizes the substrate – a product can be formed – reaction takes place and the product is released from the active site - isoenzymes - enzymes having the same function but different structure (and hence physical and chemical properties) o if two different isoenzymes catalyse the dame reaction, same product is formed Rate of enzyme catalysed reaction - determined using the Michaelis-Menten equation - KM is Michaelis constant - expresses the concentration of the substrate at which the reaction will proceed at half maximum speed; that is, how much substrate is needed to feed 50% of the enzymes o determines the affinity of the substrate to the enzyme - the higher the constant, the lower the affinity o units - mol/l (because it expresses concentration) - it is inversely proportional -> the Michaelis-Menten equation is hyperbolic o low substrate concentration - [S] < KM ▪ at low substrate concentration, the velocity is almost proportional to the substrate concentration ▪ v = vmax * [S] / KM o substrate concentration equal KM - [S] = KM ▪ follows from the definition of Michaelis constant that in this case v = vmax / 2 o very high substrate concentration - [S] >>> KM ▪ in this case, the enzyme is completely saturated with the substrate and everything is controlled only by the ability of the enzyme ▪ v = vmax Enzyme activity - qualitative evaluation of enzyme - amount of substrate converted by enzyme per unit of time - unit catal [cat] = mol.s-1 - concerts 1 mole of substrate per second - unit [U] = micromol.min-1 - concerts 1 micromole per minute - enzyme activity is dependent on pH, temperature, concentration of activator and inhibitors in the reaction mixture … Allosteric enzymes - besides the active site, they have another site- allosteric (from Greek, allos = other), where modulators (activators or inhibitors) attach, which influences the affinity of the enzyme to its substrate - most allosteric enzymes tend to be oligomeric (composed of multiple subunits) - homotrophic regulation of allosteric enzymes substrate is also a modulator o eg. haemoglobin - has 4 sites for O2, sites as soon as one oxygen connects, others connect more easily o the reaction rate curve is sigmoidal - nothing, nothing, nothing and suddenly shoots up to the maximum o function as regulatory enzymes of metabolic pathways - heterotrophic regulation of allosteric enzymes - modulator and substrate are different molecules o in the case of haemoglobin, the heterotrophic regulator would be CO 2 Specificity of enzymes - enzymes are specific both by their substrate (sugar, alcohol…) and by their action (deamination, oxidation…) 8 2.4 COENZYMES, VITAMINS, TRACE ELEMENTS doc. RNDr. Ing. Petr Tůma, Ph.D. 2.4.1 Enzymes and coenzymes - proteins with catalytic activity (accelerate reactions) o catalytically active RNA is not an enzyme but a ribozyme - the name of the enzyme is usually its substrate + reaction type which is catalyses + ase o e.g. enzyme catalysing dehydrogenation of lactate is called lactate dehydrogenase - enzymes have several specificities o binding – what substrate the enzyme works with ▪ absolute - the enzyme processes only one molecule (eg urease) ▪ relative - the enzyme processes one group of molecules (eg Hexokinase - phosphorylates all 6C sugars) o effect - what type of reaction does the enzyme catalyse ▪ dehydrogenation / oxidation / decarboxylation / transamination… o stereospecificity - enzymes can only process one chiral type ▪ monosaccharides must be D, amino acids L - enzymes can be pure protein (so-called simple enzymes) or composed of protein and non-protein parts (complex enzymes) o protein part of a complex enzyme is apoenzyme, non-protein part is cofactor o cofactors are ▪ organic – e.g. haeme ▪ inorganic - metal ion - Zn, Cu, Fe, Mn stabilize the active centre, aid redox reactions and polarize bonds o division of cofactors ▪ prosthetic groups - firmly bound to the enzyme, part of the stable structures ▪ coenzymes - only weakly bound to the enzyme, can completely detach Enzyme classes - divided into 6 basic classes, depending on the type of catalysed reaction Oxidoreductases - Aox + Bred -> Ared + Box - catalyse intermolecular oxidation-reduction reactions; transfer of hydrogen, electrons, or reaction with oxygen - types of enzymes - dehydrogenases, oxidases, peroxidases, oxygenases - eg alcohol dehydrogenase, lactate dehydrogenase (lactate pyruvate), phenylalanine hydroxylase (phenylalanine -> tyrosine) - coenzymes of oxidoreductases are o NAD (nicotinamidadenine dinucleotide) and NADP ((nicotinamidadenine dinucleotide phosphate) ▪ participate in the transfer of hydrogen and electrons ▪ NADP+, NAD+ - oxidized forms ▪ NADPH + H+, NADH + H+ - reduced form ▪ derived from nicotinamide o FAD a FADH2 - flavinadenine dinucleotide ▪ also participates in hydrogen and electron transfer, but in different reactions than NAD (usually in the formation/destruction of double bonds) ▪ FAD - oxidized forms ▪ FADF2 – reduced form ▪ derived from riboflavin (vitamin B2) o coenzyme Q - ubiquinone/ubiquinol ▪ part of the respiratory chain ▪ prosthetic group – haem 9 Transferases - A-x + B -> A + B-x - transfer groups (-CH 3, -NH 2, phosphate) from the donor to the acceptor - types of enzymes - C-transferases, glycosyltransferases, aminotransferases, phosphotransferases (or kinases) - the coenzymes of transferases are o ATP (adenosine triphosphate), GTP (guanosine triphosphate) ▪ carry phosphate groups o CoA - Coenzyme A ▪ transmits acyls o TDP - thiamine diphosphate (also TPP - thiaminpyrophosphate) ▪ carries carbon groups ▪ precursor - thiamine (vitamin B1) o PALP - pyridoxal phosphate ▪ carries the NH2 group ▪ precursor - pyridoxine (vitamin B6) o THF - tetrahydrofolate ▪ carries single-carbon residues; precursor: folic acid Hydrolases - A-B + H2O -> A-H + B-OH - catalyse hydrolytic cleavage of the substrate = cleaves bonds with the help of water - we divide them into groups o Proteases - cleaves peptide bonds in protein and peptide molecules o glucosidases - cleaves glycosidic bonds o lipases - cleaves ester bonds in lipids o phosphatases - remove the phosphate group o amylases - cleaves bonds between glucose molecules in polysaccharides - hey are mostly simple enzymes, or they have a metal ion - anyways, no coenzymes Lyases - A-B -> A + B - enzymes catalysing bond decomposition in a different way than hydrolysis or oxidation - double bonds or cyclic compounds are often formed - can cleave (or introduce) small molecules, e.g. H2O, CO2 or NH3 - coenzymes similar to transferases Isomerases - A -> A‘ - catalyse reactions within a molecule of one substrate, move atoms (groups) from one carbon to another - e.g cis-trans-isomerase, ribose phosphate-isomerase, epimerase (changes the orientation of OH groups) - most often they do not contain coenzymes Ligases - A-b + C -> A-C + b - they catalyse the synthesis of simple molecules to complex molecules - often energy-intensive bonds with simultaneous energy consumption (mostly ATP -> ADP + Pi) - often contain coenzymes of transferases o biotin (vitamin H) - carboxylation 2.4.2 Trace elements 10 2.4.3 Vitamins - organic substances that we cannot synthesize - mostly act as cofactors Water soluble vitamins Vitamin B1 - thiamine - involved in carbohydrate metabolism - active form - coenzyme TPP (thiaminpyrophosphate) - metabolic function - transfer of hydroxy-alkyl residues = oxidative decarboxylation o eg, oxidative decarboxylation of pyruvate and α-ketoglutarate - symptoms of deficiency - fatigue, convulsions, digestive disorders, nerve disorders, beri-beri nerve disease - sources - cereals, yeast, lentils, offal, yolk, pork - RDI - 1,5 mg Vitamin B2 - riboflavin - components of flavoproteins, enzymes involved in oxidative reduction processes (respiratory chain) - active form – coenzyme FAD - symptoms of deficiency - nflammation of oral corners, lips, damage to mucous membranes and skin, growth arrest - sources - meat, milk, eggs, liver, yeast, beer - RDI - 1,7 mg Vitamin B3 - nicotinic acid - Also known as niacin form NIcotinic ACid vitamIN - Active form – nicotinamide nucleotides NAD and NADP - symptoms of deficiency - convulsions, nervous disorders, pellagra disease - sources - meat, fish, yeast, multigrain cereal, lentils - RDI - 10 mg Vitamin B5 – pantothenic acid - basis of coenzyme A, contribution to protein synthesis and oxidation reduction processes - symptoms of deficiency - nervous disorders, convulsions - sources - meat, cheese, eggs, liver, yeast, lentils - DDD - 6-8 mg Vitamin B6 - pyridoxine - part of enzymes involved in amino acid metabolism (transaminases) o pyridoxal phosphate -> transamination and decarboxylation of AA - symptoms of deficiency - disorders of haemoglobin production, inflammation of skin and mucous membranes, epileptic inflammation - sources - liver, whole grain cereal products, egg yolk, yeast - RDI - 1,5-2 mg Vitamin B7 = Vitamin H = coenzyme R = Biotin - has a bunch of names because it was discovered by many scientists in different areas of metabolism and each gave it their own name before it was discovered that it was only one and the same molecule - significant coenzyme, promotes cell growth and division - symptoms of deficiency - skin diseases, anorexia, fatigue - sources - eggs, liver, vegetables, yeast, formed by intestinal bacteria - RDI - 0,5-1 mg Vitamin B9 – Folic acid - t is often called folate from Latin acidum folicum - affects the amino acid metabolism necessary for the formation of red blood cells - active form - tetrahydrofolate(THF) - transfer of monocarbon residues o carries methyl residues and alters deoxyuridine phosphate - symptoms of deficiency: disorders of protein synthesis, anaemia - sources - eggs, leafy vegetables, yeast - RDI - 0,4 mg 11 Vitamin B12 - cobalamin - does not occur in plants, it is formed only in animals - ensures normal haematopoiesis - consists of a tetrapyrrole skeleton (similar to haemoglobin) with a cobalt atom attached inside - metabolic function - transport of methyl groups - symptoms of deficiency - anaemia, degeneration of spinal nerves - sources - liver, meat, intestinal bacteria - RDI - 3-31 μg Vitamin C - Ascorbic acid - Allows for iron absorption, formation of collagen and erythrocytes, promotes blood clotting, production of antibodies, is an antioxidant - oxidoreductase cofactor (electron donor) - symptoms of deficiency - gingivitis, bleeding, decreased resistance to infections, scurvy - the most severe stage of avitaminosis - sources - vegetables (Brussels sprouts, peppers), fruits (blackcurrants, strawberries), potatoes, internal organs of animals - RDI - 60-200 mg - should be higher in smokers, the elderly and women using oral contraceptives Fat soluble vitamins Vitamin A - retinol - component of visual pigment, important for epithelial formation, antioxidant - active forms o retinal - vision, carbohydrate transport o retinoic acid - signalling molecule, ensures development, differentiation, growth - symptoms of deficiency - night blindness, drying of cornea and conjunctiva, rough skin, stopping growth - sources - liver, egg yolk, butter, cheese, sea fish fat, provitamin (β-carotene) in plant foods (carrots) - RDI - 1 mg Vitamin D – calciferol - group of vitamins, most important - D3 (cholecalciferol) and D2 (ergocalciferol) - controls the metabolism of calcium and phosphorus, promotes their absorption from the small intestine and bone deposition - effective form - hormone calcitriol - hypervitaminosis - increase of calcium absorption, its deposition in tissues and formation of kidney stones - symptoms of deficiency - softening and deformation of bones - rickets (rachitis) - sources - fat of sea fish, butter, liver, egg yolk, also due to UV radiation of the skin - RDI – 0.02mg Vitamin E - tocopherol - antioxidant, supports the activity of the gonads - protection of the organism against cancer (together with A and C) - symptoms of deficiency - muscle weakness, vascular system disorders - sources - vegetable oils, cereal sprouts - RDI - 10-15 mg Vitamin K - phylloquinone - supports the process of blood clotting, promoting the synthesis of prothrombin in the liver - active form - phylohidroquinone - symptoms of deficiency - impaired blood clotting - sources - leafy vegetables, made up of intestinal bacteria - RDI - 0.08 mg 12 2.5 INHIBITION OF ENZYMES, ROLE IN PHARMACOLOGY doc. MUDr. Jan Trnka, Ph.D. 2.5.1 Inhibition of enzymes Michaelis-Menten equation - how fast the reaction will proceed is described by the Michaelis-Menten model - it suggests that the reaction rate depends on the substrate concentration (higher concentration = higher speed) ▪ however, this does not work indefinitely; there is a finite maximum speed 𝑣𝑚𝑎𝑥 ∗ [𝑆] - 𝑣= 𝐾𝑀 + [𝑆] - [S] = substrate concentration, written in brackets because it is an equilibrium quantity - Enzyme + Substrate Enzyme-Substrate complex -> Enzyme + Product o The reaction to produce a product is also reversible in some cases, but we do not account for it for simplicity - Maximum, speed is achieved when the enzyme is saturated - at an infinite substrate concentration o in practice this speed can be achieved, it is a real value - Michaelis constant (KM) = substrate concentration at which half of the maximum reaction rate is reached o the concentration of the enzyme does not matter, only the substrate concentration o we can imagine that the constant describes the affinity of the enzyme to the substrate - the lower the constant, the higher the affinity Competitive inhibition - competitive inhibitors “compete” with substrate molecules for the active site of the enzyme o Successful binding results in a dysfunctional enzyme-inhibitor complex o these inhibitors often resemble the substrate - they must fit in the same place as the substrate, so they must look similar o E + I EI - it is a reversible action - by increasing the substrate concentration, it is possible to decrease the efficiency of inhibition- by increasing the substrate concentration we decrease the chance that the enzyme will pair with the inhibitor o Vmax will remain the same – at infinite concentration of substrate competitive inhibitors will not be effective - the action of a competitive inhibitor increases KM, because we need more substrate for the same reaction rate Uncompetitive inhibition - the uncompetitive inhibitor is in all cases allosteric (= another binding site on the enzyme other than the active site) o binding of the inhibitor causes a conformational change in the enzyme which is then unable to bind its substrate - the inhibitor binds to the enzyme completely regardless of whether or not the substrate is bound to the enzyme= the concentration of the reactants does not affect the potency of the inhibition - vmax decreases - we have less functional enzyme molecules o the inhibitor does not look at the substrate concentration at all - even if we increase the substrate concentration, there will always be some of the enzyme molecules in the solution that will be inhibited - KM stays the same- the inhibitor only takes out several molecules of enzyme, it does not change its affinity to the substrate Non-competitive inhibition - Inhibitors that bind only to the enzyme-substrate complex o they are often allosteric o E + S + I -> ES + I ESI - this inhibition is often observed with enzymes that bind multiple substrates - it works by binding the first substrate to the enzyme, causing a change in the conformation of the enzyme and revealing an additional binding site to which the inhibitor subsequently binds - the inhibitor is very poor at low substrate concentration - it does not have enough ES complexes to bind to o high substrate = lots of ES complexes= a lot of opportunities for the inhibitor to bind to an enzyme - substrate concentration does not affect the binding of the inhibitor to the enzyme-substrate complex - vmax and KM decrease (enzyme appears to have higher affinity for substrate) 13 2.5.2 The role of inhibitors in pharmacology Acetylcholinesterase (AChE) - AChE is an enzyme responsible for the hydrolytic cleavage (degradation) of acetylcholine in the synaptic cleft between two neurons o is a hydrolase and contains serine in its active site - acetylcholine is a minor but very important neurotransmitter found on neuromuscular plates, in the CNS and in the PNS o It plays a role in maintaining consciousness, attention, memory formation and muscle signaling o it is an ester of acetic acid and choline - AChE causes cleavage of the ester bond between acetyl and choline - acetic acid and choline is formed - if the neuron is stimulated and the acetylcholine is released into the synaptic cleft, it must also be removed - acetylcholine cannot act permanently, it would cause hyper stimulation o for this reason, we directly breakdown acetylcholine via AChE ▪ the enzyme attacks the carbonyl carbon (C=O) Inhibitory acetylcholinesterase - amplify signal on synapses that use acetylcholine as a neurotransmitter (cholinergic synapse) - irreversible inhibitors o carbachol - similar to acetylcholine, has many mechanisms of action, including inhibition of acetylcholinesterase ▪ contains carbamoyl group -NH2-CO- o sarin – nerve gas ▪ banned gas that is currently being used in the war in Syria ▪ irreversible inhibition of AChE -> increase in acetylcholine -> uncoordinated muscle contractions -> muscle paralysis, including respiratory ones -> suffocation death ▪ works by serine (which is located in the active site of AChE) attacks sarin -> fluorine is cleaved from sarin and the rest of the sarin then binds to serine and inhibits AChE ▪ belongs to organophosphate compounds - substances that are not used in medicine, but its modified forms are used as pesticides and insecticides in agriculture - reversible inhibitors o edrophonium - competitive inhibitor ▪ it is very fast and reversible inhibitor (its effect is only in a matter of minutes), therefore it is used for diagnostics ▪ myasthenia gravis - an autoimmune disease attacking acetylcholine receptors he main symptom is muscle weakness - after delivery of edrophonium to patients this symptom disappears temporarily (for a few minutes) and then reappears o donepezil - a substance used to improve the cognitive abilities of people with Alzheimer disease ▪ blocking of AChE basically amplifies the signal that acetylcholine sends - muscle is shouting instead of contracting ▪ there are fewer neurons and thus less acetylcholine in people with Alzheimer's disease - inhibition of acetylcholinesterase thus amplifies the signal that this neuron residue still emits 14 2.6 EPITHELIAL TISSUES II - GLANDULAR EPITHELIA MUDr. Eva Maňáková, Ph.D. - glands are sets of cells or individual cells producing a substance with biological function - proteins, lipids, complex compounds of sugars and proteins = secrete o goblet cells - separate gland cells (technically it is a gland consisting of one cell) ▪ forms mucus mucin ▪ are in the GIT, the airway, the conjunctiva in the upper eyelid… Division of glands - exocrine- secretion is produced on the body surface either directly or through the duct o secretion exits the cell through its apical surface o sweat glands, mammary glands, sebaceous glands, salivary glands… - endocrine- secretion remains in the body, most often goes from the cell to the surrounding connective tissue and then into the bloodstream o secretion from the cell via basal surface o pancreas, pituitary, adrenal gland, thyroid gland… - amphicrine- combination of exocrine and endocrine glands 2.6.1 Exocrine glands - we distinguish several qualities of exocrine glands o mechanism of excretion of secrete o position of glandular cells in relation to superficial epithelium o structure of the secretory portion o architecture of the gland o type of secrete Mechanism of secrete excretion - merocrine secretion o secretory granules are excreted by exocytosis o secretion synthesis is continuous but its secretion is not - secretion is stored inside the cell o The merocrine cell has a lot of RER, GA and secretory granules - apocrine secretion („reverse phagocytosis “) o the cell secretes secretions by cleaving the portion of the apical cytoplasm in which the secretions are located o typical for the mammary gland - fat droplets accumulate at the apical pole and the whole piece of the cell breaks apart o frequent in lipid secreting cells - Holocrine secretion o the cell secretes secretion so in a way that it disintegrates completely and disappears by apoptosis o typical for sebaceous glands - eccrine secretion o secretion is secreted by individual molecules through the cell membrane (either by itself or via carriers) and is passively followed by water on the basis of osmosis o Hydrophilic and ionic secretion needs transporters to get through the cell membrane o the result of eccrine cell activity is mucus - solution of secrete, water and ions isotonic with cytoplasm ▪ from this solution, the ions are gradually pumped back into the cells o Cystic fibrosis - caused by a malfunction of the membrane transporter for Cl- ▪ the consequence of a higher concentration of chloride ions leads to excessive reabsorption of sodium from mucus ▪ because water follows sodium, mucus dehydrates and increases its viscosity ▪ too thick mucus explains the symptoms of cystic fibrosis - thin tubes (pulmonary alveoli, vas deferens) clog and excessive mucus density interferes with the ability of antimicrobial peptides, leading to frequent infections (mainly respiratory) 15 Position of glandular cells - intraepithelial glands o are built directly into the epithelium o they do not have an outlet/ duct system- they open directly to the surface of the epithelium o goblet cells- mucin mucus production; is oppressed by other cells, hence the shape of the cup ▪ have RER, large GA, apical cytoplasm filled with secretory granules o in some organs, the whole surface epithelium is composed of mucin producing cells (stomach, uterus) - extraepithelial glands o are placed under the epithelium of origin o consist of a secretory section and a duct ▪ if they are far from the epithelial surface, they have a long and branched duct system Structure of the secretory portion - one layer of secretory epithelial cells which sit on the lamina basalis and surround the lumen where the secretion is drained - simple types of secretion o tubular - tube shape, most common, mucinous glands (see below) o acinar -round shape, narrow lumen ▪ for these glands, serous secretion is typical - proteins, enzymes o alveolar - shape of a bladder, have a wide, well recognizable lumen - compound type of secretory portion o tuboacinous- the secretory compartment has the shape of a tube with a round end o e.g. submandibular and sublingual o tuboalveolar- there is a wide lumen at the end of the tube ▪ e.g. mammary glands in lactation, sweat glands Architecture of glands - simple tubular glands - do not need a duct, they open directly to the surface (e.g crypts in the stomach) - compound glands have a branching duct system that gathers secretions from multiple sites and gathers them into one o they look like a river - small springs run down to bigger and eventually run into the sea (=the surface of the epithelium) o types of ducts in compound glands ▪ intralobular- surrounded by epithelial structures, inside the lobule, leads directly from acinus to interlobular duct intercalated duct – forms part of the intralobular duct striated- connects the intercalated duct to the interlobular duct. Also, part of interlobular ducts ▪ interlobular- outlet between lobule ▪ main - connects to the interlobular ducts, it is the last duct before the secretion exits the gland - function of ducts o the removal of the secrete o secretion modification - so-called primary secretions can enrich or otherwise change to a secondary secretion that is secreted to the surface Properties of secretions - exocrine gland secretions (except sebaceous glands) are watery - serous secretion - is produced by alveolar and acinous cells o is basophilic - most often it is peptide hormones, proteins and enzymes o Serous secreting cell (aka serous cell) has round nucleus, RER, GA and secretory granules - mucinous secretion - is produced by tubular cells o goblet cell - Produces mucus rich in mucin o mucinous cell has an active nucleus, GER, GA and a secretory granule o mucin secretory granules are well stainable by PAS dye, which is sensitive to polysaccharides - mixed secretion - seromucinous o both types of secretory compartments occur side by side and also in direct combination - e.g. tuboalveolar o Serous semilumen - a cap from serous cells at the end of the mucinous tubule 16 Myoepithelial („basket“) cells - something between smooth muscle and epithelium - contractile epithelial cells used to expel secretions from secretory compartments and into ducts - slender star-shaped cells (basket cells) which are found in glandular epithelium as a thin layer above the basement membrane but generally beneath the luminal cells - they contain cytokeratin’s as epithelial cells and actin and myosin as smooth muscle cells Regulation of secretion - the secretory activity of the exocrine glands is controlled by the nervous vegetative system and sometimes hormonal - under pathological conditions, other substances, such as inflammatory mediators, may affect the quality and quantity of secretion 2.6.2 Endocrine glands - reverse polarization - the cell secretes secretion towards the basalis lamina - construction of endocrine glands - always trabecula of cells, only in the thyroid gland we find follicles (due to storage of iodine) - DNES - Diffusion Neuro Endocrine System - endocrine cells scattered everywhere along the GIT, secrete signalling molecules to regulate digestive system function - types of endocrine glands according to hormones produced o polypeptides and proteins - adenohypophysis, parathyroids, islets of Langerhans… o catecholamines - adrenal medulla o steroid hormones - adrenal cortex, testis, ovary o thyroxine – thyroid gland Thyroid Gland - Cuboidal epithelium arranged around the follicle - inside the follicle is colloid - thyroglobulin - a storage form of thyroid hormones Liver - characteristic arrangement - amphicrine character o Exocrine function - forms bile o endocrine function - involved in the metabolism and production of plasma proteins - the basic morphological unit of the liver is the lobule of the central vein - the hexagonal prism consists of the plates of hepatocytes o The central vein sits right in the middle of the whole prism o fenestrated capillaries and bile ducts lead from the central vein towards the periphery of the lobules - at the point of contact of the three adjacent lobules there is a triad - 1 vessel, 1 vein and 1 bile duct 17 2.7 STRUCTURE AND ORIGIN OF MITOCHONDRIA MUDr. Eva Maňáková, Ph.D. 2.7.1 Structure - mitochondria are a membrane semi-autonomous (semi-independent) organelles - two membranes - inner (bent) and outer (smooth), intermembrane space between them o inside the inner membrane is the mitochondrial matrix - size - 0,5-1 μm wide and 3-5 μm long o membrane thickness 6-8 nm and intermembrane space is +- 20 nm - microtubules move through towards where they are needed - to the surrounding places with high ATP consumption o usually are concentrated around the nucleus or basolateral labyrinth - active cells have the most mitochondria - hepatocytes 800, oocytes up to 100 000, cardiomyocytes up to 30 - 40% of their volume Outer membrane - contains many porins - channels that allow molecules to pass through the membrane o as a result, the outer membrane non-selectively permits all molecules smaller than 10 kDa - Bcl proteins - part of outer mitochondrial membrane - important proteins in the regulation of apoptosis - Contains a few enzymes of fatty acid metabolism and phospholipids - unlike the cell membrane, it contains almost no cholesterol Intermembrane space - similar composition to cytosol (much less protein than matrix) - there are proapoptotic proteins, cytochrome C and a lot of protons Inner membrane - the original membrane of the former prokaryotic cell - almost impermeable and very selective - everything except for small uncharged molecules needs a carrier - it incorporates the respiratory chain complexes o ATP synthase forms elemental bodies on the inner mitochondrial membrane - folds (cristae) - sometimes they are modified and form tubules (in cells producing steroid hormones) - is composed of 75% proteins and 25% phospholipids o contains specific phospholipids cardiolipins that help with membrane elasticity Mitochondrial matrix - has a gel like consistency because of its high protein concentration (500 g protein per 1L matrix) - contains enzymes of Krebs cycle, β-oxidation of fatty acids and ornithine cycle, various nucleotide coenzymes, inorganic ions (Ca), mtDNA, relevant tRNA and mRNA, mitochondrial ribosomes, chaperones and chaperonins Import of proteins into mitochondria - most proteins are imported from the cytoplasm - they are synthesized on ribosomes in the cytosol - chaperones ensure that proteins remain unpacked so that they can be transported through the mitochondrial membrane - on the inner and outer membrane are translocators for proteins - Tom and Sam complexes external, Tim complexes internal o Tom (transporter of outer membrane) complex gets protein into intermembrane space o Sam complex is for proteins to remain integrated into the outer mitochondrial membrane o Tim (transporter of inner membrane) 23 complex pushes proteins from the intermembrane space into the matrix o Tim 22 complex integrates proteins from the intermembrane space into the inner mitochondrial membrane Mitochondrial DNA = mtDNA - small (16.5 kbp) circular double helix without introns - encodes 2 rRNA, 22 tRNA and 13 proteins o most of the genome has moved into the nucleus of the host cell, making the mitochondria nucleus dependent and unable to live independently - 600-1000 mitochondrial proteins are encoded by nuclear DNA - in the matrix of mitochondria there is also a proteosynthetic apparatus and 70S ribosomes that can produce molecules encoded by mitochondrial DNA o mitochondrial ribosomes resemble bacterial ribosomes, which means that some antibiotics targeting bacterial ribosomes may also affect mitochondria 18 2.7.2 Mitochondrial function - production of ATP by oxidative phosphorylation - Krebs cycle, β-oxidation, ketogenesis, steroid synthesis, ornithine cycle, gluconeogenesis, iron and calcium metabolism (regulation of intracellular calcium, triggering of apoptosis or necrosis, synaptic plasticity and thermogenesis - basolateral labyrinth - transfer of water and ions using Na / K ATPase with high consumption of ATP o Insufficient activity of mitochondria leads to transmission disorders and thus to cell oedema Apoptosis - programmed cell death requiring gene expression, proteosynthesis, and ATP - triggers in response to apoptotic signal, stress, cell damage… - mitochondria contribute by releasing proapoptotic factors from the intermembrane space, interrupting the supply of energy to the rest of the cell, and overproducing ROS (reactive oxygen species) Defects in membrane permeability - membrane damage is one of the main causes of cell death - the initial phase of cell damage is disruption of the membranes - their ability to selectively permeability is impaired o this harms ATP production, leading to ATP deficiency, non-functional membrane pumps and cell swelling Mitochondrial theory of aging - mitochondria naturally form oxygen radicals that may damage mtDNA or host cell DNA - accumulation of mutations in mtDNA with age gradually decreases the function of mitochondria and respiratory complexes - mitochondrial function impairment affects long-lived cells - neurons, cardiomyocytes, muscle cells - consequence - heart failure, muscle weakness, diabetes mellitus, dementia, neurodegeneration… 2.7.3 Mitochondrial diseases - heteroplasmia - very variable symptoms - different distribution of mutant mitochondria throughout the body, symptoms/consequences of disease will depend on where the mutant MIT is located - mutation in MIT are classified according to where they occur Mutations in mitochondrial DNA - maternal inheritance - inherited along the maternal line because paternal mitochondria are not passed on to the offspring - most variable - there are several tens of copies of mitochondrial DNA and at the same time we have tens of thousands of copies of mitochondrial DNA in the cell - DAD - diabetes and deafness o Mutated leucine gene synthesizes abnormal proteins o damage of hair cells and pancreatic β cells - LHON - Leber's hereditary optical neuropathy o begins in middle age with optic nerve dying and ends with complete blindness o can be caused by multiple mutations in complex 1 - mitochondria do not work well and their production of ATP is not enough for powerful and energy-intensive nerves Mutations in nuclear DNA - onset of the disease in prenatal development or just after delivery - inherited by Mendelian inheritance, because the mutated genes are found in the nucleus and not in the mitochondria - Leigh syndrome - progressive degenerative brain disease Therapy - with the help of classic medicines very difficult, practically at all - possibility of so-called mitochondrial transplantation - the child then has 3 parents, from one egg, from the second sperm and from the third mitochondria o Legal only in UK - Unfortunately, the CRISPR method does not work on mitochondria because the DNA can no longer cross the membrane Division and fusion - there is a balance in the cell between fission and fusion mitochondria - fusion - content mixing, protein replenishment, mtDNA repair, distribution of metabolic intermediates - division - increase of mitochondria 19 Mitochondrial inheritance - mitochondria reproduce exclusively asexually - mtDNA is inherited maternally - all mitochondrial embryo genetic information comes from the egg and not from the sperm - mtDNA mutates faster than nuclear genome - more susceptible to mutation accidents o heart and nerve tissue (brain, retina) - most often mitochondrial diseases o the cell is able to recognize and destroy mitochondria with defective mtDNA or trigger apoptosis - mitochondria in the cell can exchange mtDNA but do not combine it 2.7.4 Evolutionary origin of mitochondria - mitochondria have evolved from independent bacteria (rickettsia), which have entered other bacteria (archebacteria) o The bacteria contribute the ability to synthesize ATP and the host cell delivers the substrates - two hypotheses as to how this connection occurred 1) Eukaryotes developed first and then symbiosis occurred with mitochondria 2) entry of mitochondria into the cell resulted in eukaryotes ▪ some eukaryotic organisms do not have mitochondria, or just residues - most of the genome has moved into the nucleus of the host cell, making the mitochondria dependent on it and unable to live independently of the organelle - all eukaryotic cells are descendants of a single cell that has absorbed bacteria with oxidative phosphorylation capacity 20 2.8 ELECTRON TRANSPORT CHAIN AND ATP FORMATION doc. MUDr. Jan Trnka, Ph.D. - summary of glucose metabolism = C6H12O6 + O2 -> H2O + CO2 + Energy o during this time, the oxygen is reduced from 0 to -2 and carbon is oxidized from 0 to +4 o glucose degradation can conveniently be divided into two phases ▪ the first phase is the transfer of electrons to the transmitters (NAD, FAD) and the formation of CO 2 – ie. glycolysis, Krebs cycle ▪ the second phase is the transfer of electrons to the final acceptor (oxygen) and the utilization of the energy we get Only in the second phase we need oxygen The process of the second phase of glucose metabolism is the so-called Respiratory Chain 2.8.1 Respiratory chain - „mitochondrial electron transport chain “ - is located in the inner mitochondrial membrane - consists of 4 large enzymes (complexes), coenzyme Q and cytochrome C - the respiratory chain is explained from the end, for better understanding Complex IV (cytochrome C oxidase; ferrocytochrome) - the last respiratory chain complex - takes electrons from cytochrome C and passes them to oxygen o oxygen is reduced from 0 to -2 = 4 electrons are needed for each O2 - cytochrome C - a relatively small protein containing one molecule of heme o heme contains an iron atom that can pass between Fe 2+ and Fe3+ - this is how electron transfer functions o the whole cytochrome C can therefore carry only one electron at a time o is electrostatically attached to the inner membrane from outside (not part of the membrane) - complex IV is inhibited it cyanide Complex III (ubiquinol-ferricytochrome C oxidoreduktase) - transfers electrons from coenzyme Q to cytochrome C - contains FeS (iron-sulphur) centre - an evolutionary ancient electron carrier - coenzyme Q (ubiquinone) - electron transporter inside the inner mitochondrial membrane o it's not a protein, it's just a small molecule (on picture) o has two forms - ubiquinone (oxidized form) and ubiquinol (reduced form) o there are many other types of coenzyme Q - mammals have coenzyme Q10 ▪ 10 because it contains 10 isoprene units o contains two oxygen in its structure, which can be reduced and then re-oxidized - it can carry two electrons at the same time ▪ these two electrons can be obtained from many different sources - complex I, complex II, ETF dehydrogenase… Complex I (NADH oxidase; NADH-ubiquinone oxidoreductase) - a giant complex composed of 45 subunits - it is much larger than the membrane itself - oxidizes NADH + H+ to NAD+ o 2 electrons are released which travel to the coenzyme Q through complex I Complex II (succinate dehydrogenase) - embedded in the inner mitochondrial membrane, but not completely - partially communicating directly with the mitochondrial matrix - active also in Krebs cycle – oxidizes succinate to fumarate o In this reaction, 2 electrons are released and go through FAD to coenzyme Q o FAD (unlike NAD) is not a separate molecule, it is just a part of enzymes (like a human hand) Proton pumping - Complexes I, III and IV are large proteins that cross the entire inner membrane and pump protons from the mitochondria matrix into the intermembrane space during electron transfer, creating a strong proton gradient 21 Complementary proteins of the respiratory chain - serves mainly as an alternative pathway of electron transport to coenzyme Q - ETFDH (ETF dehydrogenase, electron-transferring-flavoprotein-dehydrogenase) o ETF dehydrogenase contains FAD in its structure, thanks to which it can take electrons from ETF (electron transfer flavoprotein) and then pass them on to coenzyme Q - GPDH (glycerol- phosphate dehydrogenase) o NADH, that is formed outside the mitochondria cannot spontaneously cross the mitochondrial membrane → it is transmitted by GPDH and subsequently reaches the respiratory chain where it donates its two electrons 2.8.2 Creating ATP ATP synthase - an enzyme not part of the respiratory chain but also in the inner mitochondrial membrane - whole protein works like a turbine o has a proton channel that allows protons to pass from the intermembrane space to the matrix according to the concentration gradient o As it passes through the proton channel, it turns ATP synthase - like water flowing through a hydroelectric power station - rotation of ATP synthase connects ADP and Pi into ATP - oligomycin - bacterial product, ATP synthase inhibitor Uncouplers - uncoupling proteins (UCP) - eg. thermogenin - transfers protons from the intermembrane space directly across the membrane and bypasses ATP synthase o uncouplers can be thought of as a channel that passes through the inner mitochondrial membrane, and allow protons to flow without any resistance - heat is a by-product - in this way brown adipose tissue allows mammals to warm up or bears to survive during hybernation - they are called uncouplers because normally oxygen consumption and ATP production are interconnected o when production of ATP is stopped, oxygen production stops - proton gradient will accumulate in the intermembrane space until it is so strong that respiratory chain complexes simply no longer push protons there o uncouplers will disturb this - protons will still flow, the respiratory chain will work, but ATP will not be produced - there are other substances that can act as uncouplers o dinitrophenol - once used as a very effective weight loss medicine, but it was virtually impossible to prescribe the correct dosage and very often the use of dinitrophenol led to extreme weight loss and death ▪ Nowadays we are beginning to experiment with dinitrophenol again 22 2.9 KREBS CYCLE doc. RNDr. Ing. Petr Tůma, Ph.D. - also, known as citric acid cycle, citrate cycle and tricarboxylic acid cycle - occurs in the matrix of mitochondria - 'crossroad of metabolic pathways' - the interconnection of carbohydrate, lipid and protein metabolism - amphibolic pathway (ie anabolic and catabolic pathway) o catabolic function - energy source, ATP synthesis o Anabolic function - formation of precursors for the synthesis of glucose, lipids, amino acids, porphyrins - burns nutrients to water, CO2 and reduced coenzymes that continue into the respiratory chain 2.9.1 Krebs cycle 1) synthesis of citrate (starting substance, hence citrate cycle)) - oxaloacetate + acetyl-coA -> citrate + CoA 2) dehydrogenation and hydrogenation -> formation of isocitrate (so called citrate activation) 3) dehydrogenation and decarboxylation -> formation of 2-oxoglutarate (or α-ketoglutarate) ▪ here CO2 is split off and NADH if formed 4) dehydrogenation, decarboxylation and binding of CoA -> formation of succinyl-CoA ▪ CO2 is slip off again and NADH is formed 5) cleavage of CoA -> formation of succinate ▪ GTP is formed 6) dehydrogenation -> formation of fumarate ▪ FADH is formed 7) hydration -> formation of malate 8) dehydrogenation -> formation of oxaloacetate ▪ NADH is formed - total yield from 1 acetyl-coA = 2 CO2, 1 GTP, 3 NADH and 1 FADH2 o the use of cofactors in the respiratory chain is about 10 ATP 23 Enzymes of Krebs cycle o - regulatory enzymes - citrate synthase, isocitrate dehydrogenase and α-ketoglutarate dehydrogenase - enzymes that are inhibited by high concentrations of ATP, preventing excessive consumption of acetyl-CoA - pyruvate dehydrogenase or pyruvate dehydrogenase complex - an enzymatic complex that catalyses the oxidative decarboxylation of pyruvate, which produces acetyl-CoA, which subsequently enters the Krebs cycle a number of coenzymes participate in this reaction along with the complex ▪ pyruvate + NAD+ + CoA -> acetyl-CoA + NADH + H+ + CO2 ▪ pyruvate dehydrogenase reaction is irreversible o citrate synthase - catalyses the first reaction of the Krebs cycle – condensation of oxaloacetate and acetyl CoA o ATP is not required for the condensation of oxaloacetate and acetyl-CoA under citrate synthase, on the contrary - if ATP is present, the Krebs cycle is generally inhibited (with sufficient energy, the cycle is not necessary) o aconitase - converts citrate into cis-aconitate (dehydration) and then cis-aconitate to isocitrate (hydration) o Isocitrate dehydrogenase - dehydrogenates isocitrate to α-ketoglutarate (= 2-oxoglutarate) ▪ the rate of reaction depends on the concentration of ATP and NADH -> excess of ATP or NADH means that the reaction will proceed slowly (again the excess of ATP and NADH indicates enough energy and therefore no need to produce more) ▪ this reaction generates NADH + H o α-ketoglutarate dehydrogenase- α-ketoglutarate -> succinyl-CoA + NADH + H+ ▪ catalyses oxidative decarboxylation with simultaneous binding of α-keto carbon to CoA and formation of NADH + H + ▪ the same mechanism as the oxidative decarboxylation of pyruvate ▪ coenzyme thiamine pyrophosphate is required o succinyl -CoA-synthetase – cleaves attached CoA and at the same time generates GTP (GDP+Pi -> GTP) ▪ stupid nomenclature of the enzyme, because it does not synthesize succinyl-CoA, but breaks it down o succinate dehydrogenase - Flavin enzyme with firmly bound zFAD and non-heme iron ▪ succinate dehydrogenase produces fumarate ▪ the reaction is carried out with simultaneous transfer of hydrogen to FAD, which is reduced to FADH2 ▪ succinate dehydrogenase is also complex II in the respiratory chain o fumarate hydratase - hydrates fumarate into malate o malate dehydrogenase - dehydrogenates malate into oxaloacetate ▪ oxaloacetate and fumarate link the citrate cycle to the urea cycle ▪ oxaloacetate can also be used for glucose synthesis and aspartate (aspartic acid) synthesis 24 2.9.2 Involvement in intermediate metabolism Sources of acetyl-coA - Coenzyme A is only a carrier of acetyl, it does not degrade during metabolism and can occur freely - source of acetyl-CoA may be, for example, glycolysis occurring in the cytoplasm o 1 glucose -> 2 pyruvates that enter the mitochondria and pyruvate dehydrogenase create acetyl-coA from them (see above) - β-oxidation of fatty acids occurring in mitochondria o carries much more energy than glycolysis - amino acids - degradation of some AA produces pyruvate, others produce directly Krebs cycle intermediates o alanine -> pyruvate; aspartate -> oxaloacetate… Cataplerotic reactions - these are reactions that deplete the Krebs cycle intermediates - if ATP is not needed, the Krebs cycle intermediates will begin to convert to other useful compounds o citrate - exits from mitochondria into the cytoplasm where it cleaves to acetyl-CoA + oxaloacetate ▪ acetyl-CoA is used for the synthesis of fatty acids and steroids ▪ oxaloacetate returns to mitochondria or is transaminated to aspartate o α-ketoglutarate -> its transamination forms glutamate (also the formation of glutamine, histidine...etc.) o succinyl-CoA -> serves for porphyrin synthesis (=cyclic organic compound consisting of 4 pyrrole nuclei) o malate, oxaloacetate -> either converted to AA (aspartate), or gluconeogenesis produces glucose Anaplerotic reactions - anaplerotic reactions are reactions that produce citrate cycle intermediates o typical example is the synthesis of oxaloacetate from pyruvate - pyruvate + CO2 + ATP -> oxaloacetate + ADP + Pi ▪ catalysed by the enzyme pyruvate carboxylase with the participation of biotin cofactor o metabolism of amino acids ▪ aspartic acid (Asp), asparagine (Asn) -> oxaloacetate ▪ glutamic acid (Glu), glutamine (Gln), histidine (His), proline (Pro), arginine (Arg) -> α-ketoglutarate ▪ valine (Val), threonine (Thr), methionine (Met) -> succinyl-CoA ▪ phenylalanine (Phe), tyrosine (Tyr) -> fumarate + acetylCoA ▪ alanine (Ala), serine (Ser), cysteine (Cys), glycine (Gly) -> pyruvate o Fatty acids with an odd number of carbons -> after β-oxidation remains propionyl-CoA, which is converted to succinyl-CoA Regulation of Krebs cycle - is closely related to the respiratory chain, the consumption of ATP and the consumption of reduced cofactors - activation - low ATP/ADP or NADH/NAD - inhibition - high ATP/ADP or NADH/NAD - Krebs cycle regulatory enzymes = citrate synthase, isocitrate dehydrogenase and α-ketoglutarate dehydrogenase 25 2.9.3 Transport of metabolites - outer membrane of mitochondria has a lot of pores, which makes it quite permeable o inner membrane tightly regulates transport of substances and is almost impermeable -> carriers are used for transport of larger molecules - matrix is charged negatively (OH groups), intermembrane space charged positively (proteins) ▪ the membrane potential ranges from 180 to 200 mV o molecules that can cross the membrane ▪ free diffusion - O2, CO2 a H2O ▪ antiport - pyruvate and H2PO4 for OH-, ATP for ADP, Ca2+ for 2H+ Shuttles - specific mechanism by which substances that would not normally pass through the inner mitochondrial membrane are transferred Malate-aspartate shuttle - ensures the transfer of NADH from the cytoplasm to the mitochondria 1) in the cytoplasm NADH transfers hydrogen to oxaloacetate, resulting in malate formation 2) malate enters the matrix by antiport with α-ketoglutarate 3) malate passes hydrogen to NAD, oxaloacetate is formed again 4) transamination → oxaloacetate becomes aspartate and glutamate becomes α-ketoglutarate 5) aspartate enters the cytoplasm with the glutamate antiport 6) second transamination → aspartate becomes oxaloacetate and α- ketoglutarate becomes glutamate - result = in the cytosol less NADH and in the matrix more NADH Glycerol phosphate shuttle - the way by which cytosol rapidly regenerates NADH to NAD in glycolysis 1) NADH in cytoplasm converts dihydroxyacetone phosphate to glycerol-3-phosphate 2) glycerol-3-phosphate transfers hydrogen to mGPD (mitochondrial glycerol-3-phosphate dehydrogenase) o mGPDH has FAD as a prosthetic group, which then reduces coenzyme Q in the respiratory chain - result = oxidized NAD in the cytoplasm and reduced coenzyme Q in the respiratory chain 26 2.10 EPITHELIAL TISSUES III - BARRIERS AND TRANSPORT MUDr. Eva Maňáková, Ph.D. Characteristics of epithelial tissue o Predominantly cells over extracellular matrix o high adhesivety of cells o polarization of cells o avascular tissue - tissue nourishes by diffusion from the connective tissue under the basement membrane o Strong nerve innervation o high regenerative ability due to stem cells in the basement membrane o originates from all 3 germ layers - ectodermal epidermis, endodermal glands, mesodermal endothelium Diffusion barrier o one of the most important functions of the epithelium is to create compartments in the body and prevent free diffusion o examples - intestine, kidneys, exocrine glands, brain capillaries (haematoencephalic barrier), plexus choroideus (barrier blood-liquor), thymus, testes Junctional complex o zonula occludens (tight junction) cells leave no space between themselves o serves for compartmentalization of surface plasmalema in resorptive epithelial cells o zonula adherens (adhesive connection) - strong and mechanically resistant band connection using cell actin fibres o macula adherens (desmosome) - strong and mechanically resistant point connection using intermediate filament cells Zonula occludens o proteins claudins and occludins via adapter proteins connected to actin filaments of neighbouring cells o prevents the paracellular passage of substances => all substances must travel transcellularly o permeability varies among different epitheliums o impermeable - collecting ducts of kidneys, urothelium, capillaries in the brain o almost permeable - small intestine, proximal tubulus of the kidney Basement membrane o mediates interaction between cell and matrix o anchors cells o attached to connective tissue via anchor fibrils o consists of proteins and proteoglycans o lamina basalis ensures attachment of cells to the basement ▪ lamina rara (lucida) - transmembrane proteins syndecan or integrins connect cell and rara lamin ▪ lamina densa - it consists of collagen IV, laminin, perlecan o lamina fibroreticularis - contains microfibrils and anchor fibers (fibrillin and collagen VI and VII), which fixate the basal lamina to the fibres of collagen III (connective tissue) under the basement membrane o special basement membranes are made of two basement membranes (kidneys, lungs) 27 2.10.1 Diffusion barriers Types of epithelial transmission o simple diffusion - O2, CO2, NO o passive transport (facilitated diffusion) - carriers, ion channels, aquaporins (for water) o active transport - pumps (ATPases), the most important is Na / K ATPase o for larger molecules - endocytosis (pinocytosis, phagocytosis), transcytosis (transport of substances from the apical membrane to the basal or vice versa) and exocytosis o aquaporins - transmembrane proteins with a hydrophilic tunnel that facilitate diffusion of water o example in collecting duct in kidney, large intestine Diffusion barrier in transporting epithelium o impermeable junctions - ileum and colon, water and ions must pass transcellularly (or controlled) o permeable junctions - duodenum and jejunum, there is an absorption of ions and nutrients o water passes paracellularly according to the concentration gradient Pinocytosis o runs through pinocytic vesicles -> the cytoplasmic membrane in invaginates and then gets excised o some proteins trigger invagination of the plasmalema and the protein dynamin closes the sac o clathrin-mediated endocytosis o Clathrin coat - adapter protein o the ingested molecule (LDL, transferrin) binds to the receptor o Clathrin-independent endocytosis o caveola vesicles - numerous on endothelial cells o protein caveolin envelops the membrane o functions - endocytosis of viruses, transcytosis, start of signalling cascades (eg insulin) Transcytosis o transfer of macromolecules across barriers - endothelium o transmission of antibodies across the placenta into the fetus - transplacental transmission, passively from the mother's body o transmission of IgA antibodies through mucous membranes Endothelium o vascular lining - barrier between blood and tissues o simple squamous epithelium o it has different permeability - from very permeable in bone marrow to impermeable in brain o endothelial function o gas exchange (diffusion), transport of substances, synthesis of vasoactive substances (mainly NO), vascular growth control, regulation of immune responses (facilitate the transfer of white blood cells to the site of inflammation) and participation in haemostasis Transfer of substances through the endothelium o transcellular - blood-brain barrier o paracellular - intestinal epithelium o types of capillaries o continuous - almost or completely impermeable o fenestrated - the endothelium has proteins that create holes and allow selective passage of substances o sinusoid there are large holes in both the endothelium and its basement membrane that allow free passage of virtually all substances in the blood 28 Glykokalyx o glycoprotein and glycolipid layer, which covers the plasma membrane of some epithelium and other cells o prevents blood clotting and white blood cells sticking o allows cells of the immune system to pass to the sites of inflammation 2.10.2 Blood Barriers Blood - primary urine barrier o on one side is a fenestrated endothelium o on the other hand, there are cytoplasmic processes of podocytes o basement membranes of both are pivoted and pressed together - one thick basement membrane is formed, which forms most of the barrier o there is alot of heparan sulphate in this membrane, which is negatively charged, which repels proteins (as they are also negatively charged) and helps keep them in the blood o the gaps between the protuberances of the podocytes are quite small and the vast majority of proteins have no chance of getting through o albumin is just below the permeability limit - if the kidneys do not work as they should, albumin will get through and can be detected in the urine - albumin thus acts as an excellent marker of kidney function Blood-air barrier o thin-walled barrier - two epithelial cells (alveoli epithelium + endothelium) attached to each other, connected by basement membranes o alveoli epithelium - single-layered flat epithelium (type I pneumocytes) o continuous capillaries The blood-brain barrier o BBB (Blood-Brain Barrier) - a barrier between the blood and the internal brain environment o several layers o endothelium- continuous capillary o basement membrane ▪ there may be pericytes in the basement membrane - a special cell type only for this occasion, helping to maintain BBB functionality and brain homeostasis o astrocyte processes- supporting glial cells, play a role in BBB and neuronal sheath formation o only water, a few gases and fat-soluble molecules can pass through the BBB, everything else needs carriers o is due to well-formed zonulae occludentes and a small number of caveol 29 2.10.3 Multilayered epithelia as a barrier o multiple layers - basal, intermediate and superficial o gradual cell differentiation - in the basal layer there are stem cells that proliferate and differentiate towards the surface o tight junctions are present between the cells of the upper third of the epithelium (when they are up, they are not needed below) o thick basement membrane o the basal layer is of cubic or cylindrical cells o there is a fibrous layer under the basement membrane - either lamina propria or (in the skin) dermis Layered squamous epithelium o keratinised - epidermis skin o stratum corneum - lucidum - granulosum - spinosum - basale o keratin is insoluble in water -> prevents water loss -> only lipophilic substances pass through the skin ▪ in the stratum granulosum highest keratinization takes place and there is also a large number of zonulae occludentes ▪ cytokeratin filaments and profilaggrin are involved in the production of keratin o non keratinized oral cavity, oesophagus, vagina, cornea, conjunctiva, larynx o stratum basale - parabasale - intermedium - superficiale o high glycogen content in superficial cells bacteria metabolize glucose anaerobically to lactate and ensure low pH Melanocytes o protects against damage from UV radiation by melanin synthesis o The melanocytes are present in the epidermis of the pars basalis o melanin-containing granules are passed to cells in the stratum spinosum in which the granules disintegrate, and the melanin is released into the cytoplasm o melanosomes (organelles) - from GER and GA - start of melanin formation o synthesis - dopa (enzyme tyrosinase) is synthesized from tyrosine, which polymerizes to melanin Transient epithelium - urothelium o location - urinary tract o lots of tight junctions o surface cells (umbrelocytes) are the largest cells in the urothelium, they have differently bent membrane o the rest of the epithelium - low cubic cells o Special protein in tight junctions - uroplakin - even increases their effectiveness o The surface of the cells (umbrelocytes) becomes more stained due to the presence of cytokeratin filaments o uroplakin - in the membrane o takes part in sealing as well 30 2.11 DEGRADATION AND SYNTHESIS OF GLUCOSE MUDr. Josef Fontana 2.11.1 Glucose - Central position in carbohydrate metabolism – all carbohydrates can be converted to glucose and vice versa - Energy can be obtained from it even in the absence of O2 - All of our cells are able to use it and some tissues are even strictly dependent on it o erythrocytes – because they do not have mitochondria they therefore do not use Krebs cycle or the respiratory chain o cells of CNS - however, during long-term starvation they adapt and 50% of their consumption is covered by ketone bodies Glycaemia = blood glucose concentration - normal fasting value is 3,3-5,6 mmol/l o while after a meal it can be up to 7,1 mmol/l even in a healthy person - regulation of blood glucose o insulin – lowers glycaemia- glucose from blood enters cells ▪ cells use up glucose through glycolysis, pentose formation or glycogen storage o glucagon, adrenalin, growth hormone, cortisol – increase glycaemia – glucose goes from liver to blood ▪ in hepatocytes, glucose is formed de novo by gluconeogenesis or by degradation of glycogen storage - sources of glucose - exogenous (food), glycogen breakdown and gluconeogenesis (production of glucose from other metabolites) Glucose transport across membranes - two different mechanisms o secondary active transport using SGLT-1,2 (sodium-glucose transporter) ▪ glucose enters the cell by symport with sodium ▪ secondary active because the cell must expend energy to get rid of the sodium that came with glucose ▪ glucose is absorbed in the intestines and the proximal kidney tubule in this way o facilitated diffusion through GLUT 1-7 transporters ▪ used in the transfer of glucose between blood and cells ▪ GLUT are channels in the cytoplasmic membrane that can open and allow glucose to pass freely across the membrane ▪ GLUT 1 - erythrocytes, blood-brain barrier ▪ GLUT 2 - liver, kidney, pancreatic β-cells, enterocytes ▪ GLUT 3 - brain ▪ GLUT 4 - adipose tissue, skeletal muscle, heart Insulin in the blood increases the amount of GLUT 4 transporters Glucose phosphorylating and dephosphorylating enzymes - as soon as glucose enters the cell, it is activated by enzymes and ATP through the phosphorylation from Glc to Glc-6-P - irreversible reaction - Activation of glucose is the first step in its metabolism, while ensuring that glucose does not flush out of the cell (phosphate cannot cross the membrane) - two isoenzymes phosphorylate glucose o glucokinase - in hepatocytes and pancreatic β-cells ▪ active at higher glycaemia (KM = 10 mM) ▪ β- cells respond to higher blood glucose levels via insulin secretion o hexokinase - everywhere except hepatocytes and pancreatic β-cells ▪ active at much lower concentrations than glucokinase (KM = 0.1 mM) ▪ is inhibited by its product (Glc-6-P) Dephosphorisation of glucose - converts Glc-6-P back to glucose by cleavage of inorganic phosphate - is present only in the liver, kidneys and enterocytes - is in smooth ER - Glc-6-P gets here via translocase o o is in this cell compartment so that newly formed glucose does not immediately phosphorylate in the cytosol 31 2.11.2 Glycolysis - catabolic reaction - conversion of glucose to 2 molecules of pyruvate (or lactate if we do not have enough oxygen) - occurs in the cytoplasm of all cells with inorganic phosphate - has two functions o energy production under anaerobic conditions - during glycolysis in addition to pyruvate, ATP is produced directly o source of acetylCoA - it can be used for everything possible Course of glycolysis - can be divided into 3 phases o First phase - glucose will be converted to fructose-1,6-bisphosphate (Fru-1,6-PP) with the investment of two ATPs o 1. step – glucose activation - glucokinase or hexokinase enzyme, 1 ATP is consumed 2. step - isomerization - enzyme isomerase converts Glc-6-P to Fru-6-P 3. step - phosphorylation - 6-phosphofructo-1-kinase enzyme phosphorylates Fru-6-P to Fru-1,6-PP using 1 ATP o Second phase - Fru-6-P breaks down into two identical three-carbon monosaccharides 4. step - cleavage - the enzyme aldolase A divides Fru-1,6-PP into glyceraldehyde-3-phosphate (Gra-3-P) and dihydroxyacetone phosphate (DHAP) DHAP is produced much more than Gra-3-P 5. step - isomerization - Triose phosphate isomerase enzyme converts DHAP to Gra-3-P o Third phase - Gra-3-P is converted to pyruvate, from which the cell obtains 4 ATP and 1 NADH ▪ since we now have two Gra-3-P molecules, the following reactions will run twice each 6. step - phosphorylation - enzyme glyceraldehyde phosphate dehydrogenase phosphorylates Gra-3-P to 1,3-bisphosphoglycerate (1,3-PP-Gly), inorganic phosphate (Pi) enters the reaction and NAD is reduced to NADH 7. step - dephosphorylation - phosphoglycerate kinase enzyme cleaves one phosphate from 1,3-PP-Gly to form 3-phosphoglycerate (3-P-Gly) and one ATP 8. step - isomerization - phosphoglyceromutase enzyme converts 3-P-Gly to 2-P-Gly 9. step - dehydration - enzyme enolase from 2-P-Gly cleaves water, phosphoenolpyruvate (PEP) is formed 10. step - dephosphorylation - the pyruvate kinase enzyme cleaves the remaining phosphate, producing pyruvate and one ATP - total 1 NADH and 2 ATP are extracted from glycolysis - two ATPs were invested in the first stage and two ATPs were obtained from each glyceraldehyde-3-phosphate in the third stage = -2 + 2 * 2 = +2 32 Metabolic fate of pyruvate - branch point of glycolysis - fate of pyruvate depends on oxidative and redox state of the cell (sufficient O 2 and NAD) o aerobic conditions - pyruvate enters the MIT matrix, where it is converted to acetylCoA o anaerobic conditions- pyruvate is converted to lactate (via enzyme lactate dehydrogenase) and released into the blood ▪ this happens because under aerobic conditions NADH is reduced in mitochondria, becomes NAD and can again help with glycolysis ▪ under anaerobic conditions, however, NADH accumulates until finally there is no NAD and glycolysis stops ▪ when pyruvate is converted to lactate, NADH is converted to NAD and can immediately return to glycolysis Pyruvate + NADH + H+ Lactate + NAD+ ▪ there are no mitochondria in erythrocytes and so anaerobic glycolysis takes place there even under aerobic conditions Regulation of glycolysis - the regulatory points are 3 enzymes o glucokinase/hexokinase – activates glucose to Glc-6-P through using ATP o 6-fosfofrukto-1-kináza (PFK-1) - phosphorylatesFru-6-P to Fru-1,6-PP through using ATP ▪ the main regulatory enzyme ▪ allosteric enzyme - the activator is Fru-2,6-PP (this molecule is produced by insulin) ▪ is inhibited if the cell has enough energy = high concentration of ATP or citrate in the cytoplasm, acidic pH in the presence of counter-regulatory hormones o pyruvate kinase - dephosphorylates PEP to form pyruvate and ATP 2.11.3 Glycogenosis - apart of the regulatory points, gluconeogenesis is the same as glycolysis, but reverse o glycolysis regulatory points are irreversible - gluconeogenesis bypasses them (bypass 1, 2 and 3) - gluconeogenesis precursors - pyruvate, lactate, glycerol, oxaloacetate, propionate and glucogenic AMK (Ala, Gln…) - takes place in the kidneys, liver and a little in enterocytes - begins in mitochondria and then moves to the cytoplasm - energetically consuming o 2 Pyruvates + 4 ATP + 2 GTP + 2 NADH + 2 H+ + 4 H2O → glucose + 4 ADP + 2 GDP + 6 Pi + 2 NAD Bypass 1 - bypasses pyruvate kinase catalysed reaction (10th step of glycolysis) - transport of pyruvate to mitochondria -> carboxylation of pyruvate (enzyme pyruvate carboxylase, cofactor biotin, consumption of one ATP) -> formation of oxaloacetate (OAA) -> transfer of OAA to cytosol -> conversion of OAA to PEP by PEP carboxykinase (consumption of GTP) Bypass 2 - bypasses PFK-1 catalysed reaction (3rd step of glycolysis) - the enzyme fructose-1,6-bisphosphatase is used - simply phosphate cleavage, no ATP formed, only inorganic phosphates Bypass 3 - bypasses the reaction catalysed by hexokinase (1st step of glycolysis) - enzyme Glc-6-phosphatase (not found in sk