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Carbohydrates Carbohydrates • Named so because many have formula Cn(H2O)n • Produced from CO2 and H2O via photosynthesis in plants • Range from as small as glyceraldehyde (Mw = 90 g/mol) to as large as amylopectin (Mw = 200,000,000 g/mol) • Fulfill a variety of functions including: – energy source and energy storage – structural component of cell walls and exoskeleton – informational molecules in cell-cell signaling • Often covalently linked with proteins or lipids to form glycoconjugates – glycoproteins, proteoglycans, glycolipids Aldoses and Ketoses Two principal groups An aldose contains an aldehyde functionality A ketose contains a ketone functionality The simplest carbohydrates – aldehydes or ketones with two hydroxyl groups Aldose - from triose to hexose Asymmetric centre in monosaccharides • Enantiomers: stereoisomers that are nonsuperimposable mirror images Enantiomers • Sugars may contain many chiral centers, only the most distant from the carbonyl carbon is designated as D or L • D and L isomers of a sugar are enantiomers Enantiomers • Enantiomers: stereoisomers that are non-superimposable mirror images • In sugars that contain many chiral centers, only the one that is most distant from the carbonyl carbon is designated as D or L • D and L isomers of a sugar are enantiomers – e.g. L and D glucose have the same water solubility • Most hexoses in living organisms are D stereoisomers • Some simple sugars occur in the L-form, such as Larabinose Cyclic structure of monosaccharides Hemiacetals and Hemiketals • Aldehyde and ketone carbons are electrophilic • Alcohol oxygen atom is a nucleophilic Aldehydes form hemiacetals with alcohols Ketones form hemiketals with alcohols Cyclization of Monosaccharides • Pentoses and hexoses undergo intramolecular cyclization Cyclization of Monosaccharides • The former carbonyl carbon becomes a new chiral center, called the anomeric carbon • The former carbonyl oxygen becomes a hydroxyl group; the position of this group determines if the anomer is or • If the hydroxyl group is on the opposite side (trans) of the ring as the CH2OH moiety the configuration is • In the hydroxyl group is on the same side (cis) of the ring as the CH2OH moiety, the configuration is The ring forms exist in equilibrium with the open-chain forms Important Hexose Derivatives The Glycosidic Bond • Reaction of anomeric carbon with hydroxyl or amino group forms O-glycosidic or N-glycosidic bond • Two sugar molecules can be joined via a glycosidic bond between an anomeric carbon and a hydroxyl carbon disacharides • The glycosidic bond between monomers (an acetal bond) is less reactive than the hemiacetal (at the second monomer) Maltose • The disaccharide formed upon condensation of two glucose molecules via 1 4 bond is called maltose Polysaccharides Polysaccharides • Natural carbohydrates are usually found as polymers • These polysaccharides (glycans) can be – homopolysaccharides – heteropolysaccharides • Polysaccharides do not have a defined molecular weight. – This is in contrast to proteins because unlike proteins, no template is used to make polysaccharides Storage function of polysaccharides Homopolysaccharides – how to effectively store the cellular fuel Glycogen – in animals Starch – in plants Heavily hydrated – hydrogen bonding with water Glycogen • Glycogen is a branched homopolysaccharide of glucose – Glucose monomers form (1 4) linked chains extensively branched – Branch-points with (1 6) linkers every 8-12 residues – Molecular weight reaches several millions – Functions as the main storage polysaccharide in animals Glycogen Starch • Starch is a mixture of two homopolysaccharides of glucose • Amylose is unbranched polymer of (1 4) linked residues • Amylopectin is branched like glycogen but the branch-points with (1 6) linkers occur every 24-30 residues • Molecular weight of amylopectin is up to 200 million • Starch is the main storage homopolysaccharide in plants Starch Metabolism of Glycogen and Starch • Glycogen and starch often form granules in cells • Granules contain enzymes that synthesize and degrade these polymers • Glycogen and amylopectin have one reducing end but many non-reducing ends • Enzymatic processing occurs simultaneously in many non-reducing ends Structural function of polysaccharides Both homo- and heteropolysaccharides Cellulose – in plants Agar – in marine algae Chitin – in arthropods - insects, lobsters, crabs and fungi (mushroom) Glycosaminoglycans – extracellular matrix Cellulose • Cellulose is a homopolysaccharide of glucose – Most abundant polysaccharide in nature – Glucose monomers form (1 4) linked linear chains – Hydrogen bonds form between adjacent monomers – Additional H-bonds between chains – Structure is tough and water-insoluble – Cotton is nearly pure fibrous cellulose Additional H-bonds between chains Cellulose Metabolism • The fibrous structure, and water-insolubility makes cellulose a difficult substrate to act on • Fungi, bacteria, and protozoa secrete cellulase, which allows them to use wood as source of glucose • Most animals cannot use cellulose as a fuel source because they lack the enzyme to hydrolyze (1 4) linkages • Ruminants and termites live symbiotically with a microorganisms that produces cellulase • Cellulases hold promise in the fermentation of biomass into biofuels Chitin • Chitin is a homopolysaccharide of N-acetylglucosamine – N-acetylglucosamine monomers form (1 4) linked linear chains – Forms extended fibers that are similar to those of cellulose – Hard, insoluble, cannot be digested by vertebrates – Structure is tough but flexible, and water-insoluble – Found in cell walls in mushrooms, and in exoskeletons of insects, spiders, crabs, and other arthropods Glycoconjugates Glycoconjugates 1. Glycoproteins – proteins with small oligosaccharides attached 2. Proteoglycans – large polychaccharides bound to peptides or proteins 3. Glycolipids – conjugate of oligosacharides and lipids The sugar code • Variety of monosacharides linked in linear or branched chains • Bilions of combinations Glycoproteins • A protein with small oligosaccharides attached – Carbohydrate attached via its anomeric carbon to serin, asparagin or threonin – About half of mammalian proteins are glycoproteins – Very variable – Function as enzymes, transport, receptors, hormones, structural proteins – Carbohydrates play role in protein-protein recognition Oligosaccharide linkages in glycoproteins Glycolipids • A lipid with covalently bound oligosaccharide – Parts of plant and animal cell membranes – Recognition function on membrane surfaces Bacterial lipopolysaccharide Proteoglycans • Different polysacharides - glucosaminoglycans linked to the core protein • Many are secreted into extracellular matrix • Some are membrane bound Glycosaminoglycans • Linear polymers of repeating disaccharide units • Various amino saccharides (on C2) – N-acetyl-glucosamine or – N-acetyl-galactosamine • Negatively charged groups (on C6) – Uronic acids (C6 oxidation) – Sulfate esters • Extended hydrated molecule, minimal charge repulsion • Forms meshwork with fibrous proteins to form extracellular matrix – Connective tissue, cartilage, tendon, cornea, bones – Lubrication of joints Repeating units of some common glycosaminoglycans of extracellular matrix Proteoglycan Aggregates of Extracellular Matrix • Core proteins e.g. hyaluronan or aggrecan with covalently bound glycosaminoglycans form huge (Mr > 2•108) non-covalent aggregates • Hold lots of water (1000 X its weight); provides lubrication • Very low friction or elastic material • Covers joint surfaces: articular cartilage – Reduced friction – Load balancing Proteoglycan aggregate of the extracellular matrix Proteoglycans as the structural polysaccharides of extracellular matrix Extracellular Matrix (ECM) • Material outside the cell • Strength, elasticity, and physical barrier in tissues • Main components: – Proteoglycan aggregates (Heparan, Chondroitin, Keratan) – Polysaccharides (Hyaluronic acid) – Fibrous proteins (Collagen, Elastin) Glucose Catabolism Glycogen Metabolism Gluconeogenesis Central Importance of Glucose • Glucose is an excellent fuel – Yields good amount of energy upon oxidation – Can be efficiently stored in the polymeric form – Many organisms and tissues can meet their energy needs on glucose only • Glucose is a versatile biochemical precursor – Bacteria can use glucose to build the carbon skeletons of: • • • • All the amino acids Membrane lipids Nucleotides in DNA and RNA Cofactors needed for the metabolism Major Pathways of Glucose Utilization • When there’s plenty of excess energy, glucose can be stored in the polymeric form (starch, glycogen) • Short-term energy needs are met by oxidation of glucose via glycolysis to ATP and NADH • Pentose phosphate pathway generates NADPH that is used for detoxification, and for the biosynthesis of lipids and nucleotides • Structural polysaccharides (e.g. in cell walls of bacteria, fungi, and plants) and ribose are derived from glucose Glycolysis Almost universal central pathway Glycolysis: Importance • Glycolysis is a sequence of enzymecatalyzed reactions by which glucose is converted/oxidized into pyruvate • Pyruvate can be further aerobically oxidized • Pyruvate can be used as a precursor in biosynthesis • In this process, some of the oxidation free energy is captured by the synthesis of ATP and NADH Glycolysis: Overview • In the evolution of life, glycolysis was probably one of the earliest energy-yielding pathways • It developed before photosynthesis, when the atmosphere was still anaerobic • Thus, the task upon early organisms was how to extract free energy from glucose anaerobically? • The solution – Activate it first by transferring couple of phosphates to it – Collect energy later form the high-energy metabolites of the activated glucose The two phases of glycolysis the preparatory phase the payoff phase Phosphorylated intermediates: Each of the nine glycolytic intermediates is phosphorylated 1. release of energy upon hydrolysis of phosphoester bonds, substrate phosphorylation 2. lack of membrane transporters of phosphorylated sugars, no need to spend energy to keep them inside the cell Glycolysis: The Preparatory Phase 1) The Hexokinase Reaction • The first step, of glucose, is catalyzed by hexokinase in eukaryotes, and by glucokinase in prokaryotes • This process uses the energy of ATP 2) Phosphohexose Isomerization • An aldose isomerize into ketose • Rearangement of hydroxyl and carbonyl groups at C1 and C2 cardons • Carbonyl at C2 serves for further phophorylation 3) The Second Priming Reaction; The First Commitment • ATP is the donor of the second phosphate group • From this point the product, fructose 1,6bisphosphate is committed to become pyruvate and yield energy • This is an irreversible step 4) Cleavage of 6-Carbon Sugars Reaction products: glyceraldehyde 3-phosphate (GAP) dihydroxyacetone phosphate (DAP) • Strongly positive DG’o favors the reverse reaction • Low concentration of products makes the reaction reversible 5) Triose Phosphate Interconversion • Two triose phosphates: DAP and GAP • Only GAP is the substrate for the next enzyme • DAP is converted enzymatically to GAP by triose phosphate isomerase • C1, C2, C3 indistinguishable from C4, C5, C6 • Reversible reaction driven by consumption of GAP During preparatory phase of glycolysis glucose has been phosphorylated at C1 and C6 and then cleaved into two identical molecules of glyceraldehyde 3-phosphate Glycolysis: The Payoff Phase 6) Glyceraldehyde 3-Phosphate Dehydrogenase Reaction • First energy-yielding step in glycolysis • Oxidation of aldehyde with NAD+ gives NADH • Phosphorylation yields an high-energy reaction product 7) First Substrate-Level Phosphorylation • 1,3-bisphosphoglycerate is a high-energy compound • Substrate level phosphorylation • The reaction is reversible • Kinases - enzymes that transfer phosphate groups from ATP to various substrates The overall oxidation of glyceraldehyde 3phosphate to 3-phosphoglycerate is energy coupling process with 1,3-bisphosphoglyceraldehyde as an intermediate The enegry of aldehyde oxidation is here conserved in NADH and ATP Reversible reaction with DG’o = - 12.2 kJ/mol 8) Conversion of 3-Phosphoglycerate to 2-Phosphoglycerate • Reversible isomerization reaction • Enzymes that shift functional groups around are called mutases 9) Dehydration of 2-Phosphoglycerate • The goal of this step is to create a better phosphoryl donor • Loss of phosphate from 2-phosphoglycerate would give a secondary alcohol with no further stabilization … 10) Second Substrate-Level Phosphorylation • … but loss of phosphate from phosphoenolpyruvate yields to an enol that spontaneously tautomerizes into ketone • The tautomerization effectively lowers the concentration of the reaction product and drives the reaction toward ATP formation Feeder Pathways for Glycolysis Feeder Pathways for Glycolysis • Dietary dichaccharides and polysaccharides (sucrose, lactose, starch, glycogen) - hydrolytic digestion to monosaccharides - transport to target cells • Endogenous glycogen or starch - phosphorolytic generation of glucose 1-phosphate by a Pi attach on the 1-4 glycosidic bond of last residues by phosphorylase - phosphorylated monosaccharide can not be transferred through membrane - glucose 1-phosphate is converget to glucose 6phosphate by phosphoglucomutase Konec prvni casti Ruzne tezky test pro chemiky a pro biofyziky a bioinformatiky The two phases of glycolysis the preparatory phase the payoff phase Fate of Pyruvate Pyruvate metabolism under aerobic and anaerobic conditions • Aerobic conditions – pyruvate is oxidised to acetylCoA • NADH is further oxidised to NAD+ in electron transport chain by oxygen • Under anaerobic condition NADH cannot be reoxidized • Energy yielding glycolysis would stop due to the lack of NAD+ Under Anaerobic Conditions, Animals Reduce Pyruvate to Lactate • During long exercise (demand for ATP exceeds the oxidation capacity – not enough of ATP produced in NADH oxidation) • Organism regenerates more NAD+ to produce ATP in glycolysis lactate builds up in the muscle Under Anaerobic Conditions, Animals Reduce Pyruvate to Lactate • The acidification of muscle prevents its continuous intensive work • The lactate can be transported to liver and converted to glucose there The Cory Cycle • Lactate produced after exercise in muscles returns to the liver • Lactate in liver is converted to glucose which moves back to muscles to be converted to glycogen Under Anaerobic Conditions, Yeast Ferments Glucose to Ethanol • Both steps require cofactors – pyruvate decarboxylase Mg++ and thiamine pyrophosphate – alcohol dehydrogenase Zn++ and NAD+ in Pyruvate metabolism under aerobic conditions Conversion of Pyruvate to Acetyl-CoA • the reaction: oxidative decarboxylation of pyruvate • acetyl-CoA can then enter the citric acid cycle and yield energy • acetyl-CoA can be used to synthesize storage lipids • catalyzed by the pyruvate decarboxylase complex • requires five coenzymes and three enzymes Overall reaction catalyzed by the pyruvate dehydrogenase complex - oxidative decarboxylation - Five coenzymes Thiamine pyrophosphate – TPP Flavin adenine dinucleotide – FAD Coenzyme A – CoA, CoA-SH Nicotine adenine dinucleotide – NAD Lipoic acid – Lipoate Pyruvate Dehydrogenase Complex (PDC) PDC is a large (Mr = 7.8 × 106 Da) multienzyme complex - pyruvate dehydrogenase (E1) - dihydrolipoyl transacetylase (E2) - dihydrolipoyl dehydrogenase (E3) short distance between catalytic sites allows channeling of substrates from one catalytic site to another channeling minimizes side reactions Three-dimensional Reconstruction from Cryo-EM data Gluconeogenesis Gluconeogenesis: Precursors for Carbohydrates • • • • • • Glucose – central role in metabolism Universal in living organisms Some tissues depends only on glucose Brain requires about 120 g of glucose pear day Glucose supply varies over a day Need for glucose synthesis from noncarbohydrate precursors Carbohydrate synthesis from simple precursors Mammals (vertebrates) cannot convert Fatty acids (Acetyl-CoA) to Sugars (Glucose) Glycolysis vs. Gluconeogenesis • Glycolysis occurs mainly in the muscle and brain • Gluconeogenesis occurs mainly in the liver • They are not identical pathways • 7 of the 10 are reversible glycolytic reactions 1. Conversion of glucose to Glc 6-phosphate 2. Phosphorylation of fructose 6phosphate 3. Conversion of phosphoenolpyruvate to pyruvate Large negative DG The three reactions are bypassed by a separate set of enzymatic reactions Both pathways occurs largely in cytosol Synthesis of Oxaloacetate • Conversion of pyruvate to energy-rich phosphoenolpyruvate requires two energyconsuming steps • In the first step, pyruvate is transported into mitochondria and converted into oxaloacetate by pyruvate carboxylase • Pyruvate carboxylase reaction is the first site of regulation of gluconeogenetic pathway • It is positively effected by Acetyl-CoA (product of fatty acid catabolism) • Pyruvate carboxylase reaction can replenish intermediates in other metabolic pathways Oxalacetate is converted to phosphoenolpyruvate In the second step, oxaloacetate is converted to phosphoenolpyruvate Oxaloacetate picks up phosphate from GTP The phosphoenolpyruvate carboxykinase reaction occurs either in the cytosol or the mitochondria From Pyruvate to Phosphoenolpyruvate Mitochondrial membrane has no transporters for oxaloacetate In mitochondria: Oxaloacetate has to be first reduced to malate by malate dehydrogenase Malate is transported from mitochondria and reoxidized to oxaloacetate From Pyruvate to Phosphoenolpyruvate Transport of malate from mitochondria is accompanied by the transport of reducing “power” of NADH from mitochondria to cytosol [NADH]/[NAD+] ratio in cytosol is about 105 times lower than in mitochondria NADH is consumed in gluconeogenesis – need for NADH in cytosol Second Bypasse • Conversion of Fructose 1,6-bisphosphate to Fructose 6-phosphate by Fructose 1,6bisphosphatase (FBPase-1) • Release of an inorganic phosphate • Resonance stabilization of phosphate DG’o = - 16.3 kJ/mol 1. Conversion of glucose to Glc 6-phosphate 2. Phosphorylation of fructose 6phosphate 3. Conversion of phosphoenolpyruvate to pyruvate Large negative DG The three reactions are bypassed by a separate set of enzymatic reactions Both pathways occurs largely in cytosol Third Bypasse • Conversion of Glucose 6-phosphate to Glucose • Reaction does not synthesis ATP - simple hydrolysis of phosphate ester to inorganic phosphate • Resonance stabilization of phosphate DG’o = - 13.8 kJ/mol Gluconeogenesis – energetically expensive Requires 4ATP, 2GTP, 2NADH for one molecule of Glucose Only 2ATP and 2NADH are produced in glycolysis Glycolysis and Gluconeogenesis are reciprocally regulated - 16,7 kJ/mol - 13.8 kJ/mol - 14,2 kJ/mol - 16.3 kJ/mol - 31,4 kJ/mol 0,9 kJ/mol Pentose Phosphate Pathway Pentose Phosphate Pathway • The main goals are to produce NADPH for anabolic reactions and ribose 5-phosphate for nucleotides In rapidly dividing cells - high demand for ribose 5-phosphate Cells with high reductive biosynthesis demand for NADPH Demand of NADPH in oxygen exposed cells NAD and NADP in metabolism NAD+/NADH - catabolism, further in ATP production NADP+/NADPH – anabolism, biosynthetic reactions Pentose Phosphate Pathway Oxidative phase 1. Oxidation of Glucose 6-phosphate by glucose 6phosphate dehydrogenase to 6-phophogluconate 2. Oxidation and decarboxylation of 6-phophogluconate by 6-phophogluconate dehydrogenase to Ribulose 5-phosphate 3. Isomerisation of Ribulose 5-phosphate to Ribose 5-phosphate by phosphopentose isomeraze Pentose Phosphate Pathway Nonoxidative phase In tissues requiring only NADPH Ribulose 5-phosphate is regenerated to Glucose 6-phosphate in nonoxidative paths In a series of reactions catalysed by transketolases and transaldolases 6 pentoses are transformed in to 5 hexoses 6 x C5 = 5 x C6 NADPH Regulates Pentose Phosphate Pathway NADPH inhibits glucose-6phosphate dehydrogenase Learning objectives – Carbohydrates, their chemistry and function – Glycoconjugates, glycoproteins, peptidoglycans, glycolipids – Glycolysis preparatory and payoff phase principles of energy extraction – Pyruvate, anaerobic and aerobic metabolism – Regeneration of NAD+ – Acetyl-CoA production – Gluconeogenesis – Importance of the first bypass reaction in metabolism – Pentose phosphate cycle – Production od NADPH and ribose