Basic concept and design of metabolism The glycolytic pathway Oxidative decarboxylation of pyruvate and of other 2-oxocarboxylic acids Biochemistry I Lecture 3 2009 (J.S.) Living organisms require a continual input of free energy for three major purposes: - the performance of mechanical work in cellular movements, - the active transport of molecules and ions across membranes, - the synthesis of macromolecules and other biomolecules from simple precursors. The free energy used in these processes, which maintain an organism in a state that is far from equilibrium, is derived from the environment. 2 Metabolism is essentially a series of chemical reactions that provides energy transformations: Energy is being extracted from fuels (nutriments) and used to power biosynthetic processes. Catabolism (catabolic reactions) converts chemical energy by decomposing foodstuffs into biologically useful forms. Anabolism (anabolic reactions) requires energy - useful forms of energy are employed to generate complex structures from simple ones, or energy-rich states from energy-poor ones. 3 Types of chemical reactions in metabolism Type of reaction Description Oxidation—reduction Electron transfer Ligation requiring ATP Formation of covalent bonds (i.e., carbon- cleavage carbon bonds) Isomerization Rearrangement of atoms to form isomers Group transfer Transfer of a functional group from one molecule to another Hydrolytic Cleavage of bonds by the addition of water Addition or removal of Addition of functional groups to double functional groups bonds or their removal to form double bonds 4 Reactions can occur spontaneously only if they are exergonic (if aG, the change in free energy, is negative). The Gibbs free-energy change AG The maximal amount of useful energy that can be gained in the reaction (at constant temperature and pressure). a A + b B — c C + d D AG = Ga+b - Gc+d ag = ag + rT In [c]C [d]d ag° = _ rt In K The AG of a reaction depends on the nature of the reactants (expressed by the AG0 term) and on their concentrations (expressed by the second term). 5 An endergonic reaction cannot proceed spontaneously, but such a thermodynamically unfavourable reaction can be driven by an exergonic reaction to which it is coupled. Energetic coupling occurs because the two reactions share a common reactant or intermediate. Example: Malate H20 AG°1 = + 2 kJ/mol Fumarate NH3 Aspartate AG°2 = -15.4 kJ/mol The overall net free energy change is negative (AG0 = - 13.4 kJ/mol), the conversion of malate to aspartate is exergonic. 6 The reaction which is used to drive endergonic ones is very oft the hydrolysis of ATP. Example: Glucose ATP ADP ■Glucose 6-phosphate AG0 = + 13.8 kJ mol-1 AG0' = -30.5 kJ moM = -16.7kJ moM adenine HO OH O H9C h ov 6 o OH HO^_/ OY OH Glucose 6-phosphate (G-6P) HO OH ADP 7 Adenosine triphosphate (ATP) is a high-energy compound that serves as the "universal currency" of free energy in biological systems. ATP hydrolysis drives metabolism by shifting the equilibrium of coupled reactions. ATP ADP ATP + H2O -> ADP + Pi AG°' (at pH 7) = - 30,5 kJ mol-1 8 The metabolic interplay of living organisms in our biosphere Living organisms can be divided into two large groups according to the chemical form of carbon they require from the environment. Autotrophic cells ("self-feeding" cells) - green leaf cells of plants and photosynthetic bacteria - utilize CO2 from the atmosphere as the sole source of carbon for construction of all their carbon-containing biomolecules. They absorb radiant energy of the sun. The synthesis of organic compounds is essentially the reduction (hydrogenation) of CO2 by means of hydrogen atoms, produced by the photolysis of water (generated dioxygen O2 is released). Heterotrophic cells - cells of higher animals and most microorganisms - must obtain carbon in the form of relatively complex organic molecules (nutrients such as glucose) formed by other cells. They obtain their energy from the oxidative (mostly aerobic) degradation of organic nutrients made by autotrophs and return CO2 to the atmosphere. Carbon and oxygen are constantly cycled between the animal and plant worlds, solar energy ultimately providing the driving force for this massive process. 9 (dehydrogenations) 10 Most of the Gibbs' free energy in the body originates in the exergonic synthesis of water (2H2 + O2 ® 2H2O, 25 °C): AG° = - 474.3 kJ mol-1 Fatty acids of fats are a more efficient fuel source than saccharides such as glucose because the carbon in fatty acids is more reduced 11 Stages in the extraction of energy from foodstuffs The first stage of catabolism Large molecules in food are broken down into smaller units Stage II Degradation to a few amphibolic intermediates Stage III The final common pathways - most of the ATP is produced from the complete oxidation of the acetyl unit of acetyl CoA 12 High-energy compounds Adenosine monophosphate (AMP) is not a high-energy compound (no anhydride bond) GTP, CTP, UTP, TTP are quite analogous to ATP. as well as GDP, CDP, UDP, TDP are analogous to ADP. Different types of high-energy compounds Anhydrides di- and triphosphates ATP, ADP, UTP, CTP etc. phosphosulfate phosphoadenosyl-phosphosulfate (PAPS) acylphosphates 1,3-bisphosphoglycerate o-po3?-CH-OH COO-| CH2-O- PO32- Ester phosphoenolpyruvate C-O-PO3 II CH2 Thioesters acyl coenzymes A Amides phosphocreatine HN=C^ xNH-PO32-^N-CH 2COOH CH3 14 Factors contributing to the large change in AG0 of hydrolysis: 1 Electrostatic repulsion of negatively charged groups 2~ o - o - o •i 11 11 R 6 6 6 2 Products of hydrolysis are more stable than the reactant because of greater resonance possibilities 2~ O 2~ O 0^P^ ^-co-r -► + _ Vr 0 3 and the groups in the products are more prone to isomerization or they exhibit more states of ionization phosphoenolpyruvate- -► Hydrogenphosphate2- + pyruvate- More negative el. charges and tautomerization enolpyruvate to the ketoform 15 Synthesis of ATP by phosphorylation of ADP in the cell 1 Oxidative phosphorylation in mitochondria accounts for more than 90 % of ATP generated in animals. The synthesis of ATP from ADP and Pi is driven by the electrochemical potential of proton gradient across the inner mitochondrial membrane. This gradient is generated by the terminal respiratory chain, in which hydrogen atoms, as NADH + H+ and FADH2 produced by the oxidation of carbon fuels, are oxidized to water. The oxidation of hydrogen by O2 is coupled to ATP synthesis. 16 2 Phosphorylations of ADP on the substrate level are provided by few reactions, in which a nucleoside triphosphate is synthesized by utilization of the free energy of hydrolysis of a soluble energy-rich compound. - Energy released by certain carbon oxidations can be converted into high phosphoryl-transfer potential and so the favourable oxidation is coupled with the unfavourable synthesis (phosphorylation) of ATP. - The high phosphoryl-transfer potential of phosphoenolpyruvate arises primarily from the large driving force of the subsequent enol-ketone conversion. Dehydration of 2-phosphoglycerate "traps" the molecule of the product in its unstable enol form. 17 Examples of substrate-level phosphorylations In glycolysis phosphoglycerate kinase 1,3-bisphosphoglycerate ' 3-phosphoglycerate ADP ATP pyruvate kinase phosphoenolpyruvate L--^ ' pyruvate ADP ATP In the citrate cycle thiokinase succinyl coenzyme A —^—succinate + CoA GDP + Pi GTP In skeletal muscle phosphocreatine serves as a reservoir of high-potential phosphoryl groups that can be readily transferred to ATP: creatine kinase phosphocreatine creatine ADP + H+ ATP 18 Control of metabolism Metabolism is regulated by controlling ■ catalytic activity of enzymes allosteric and cooperative effects, reversible covalent modification, substrate concentration ■ the amount of enzymes synthesis of adaptable enzymes ■ the accessibility of substrates compartmentalization segregates biosynthetic and degradative pathways, the flux of substrates depends on controlled transfer from one compartment of a cell to another ■ the energy status of the cell of which the energy charge or the phosphorylation potential are used as indexes ■ communication between cells hormones, neurotransmitters, and other extracellular molecular signals often regulate the reversible modification of key enzymes [ATP] + 1/2[ADP] Energy charge = - [ATP] + [ADP] + [AMP] can have a value ranging from 0 (all AMP) to 1 (all ATP). Catabolic (ATP-generating) pathways are inhibited by an energy charge, whereas anabolic (ATP-utilizing) pathways are stimulated by a high-energy charge. The energy charge of most cells ranges from 0.80 to 0.95. Phosphorylation potential = —- [ADP] x [Pi] is an alternative index of the energy status of a cell. In contrast with the energy charge, it depends on the concentration of Pi and is directly related to the free energy storage available from ATP. 20 The glycolytic pathway 21 Glucose is an important and common nutrient for most organisms. In mammals glucose is the only fuel that the brain uses under non-starvation conditions and the only fuel that red blood cells can use at all. Some fates of glucose: C6H12°6 Glucose Glycolysis ANAEROBIC FERMENTATION CH3— CH2OH Ethanol COMPLETE OXIDATION C02 + H20 ANAEROBIC GLYCOLYSIS 0 HO —C — 1 - H 1 CH3 Lactate _J 22 Glucose transporters mediate the thermodynamically downhill movement of glucose across the plasma membranes of animal cells. The members of the family of transporters have distinctive roles. Family of glucose transporters Name Tissue location Comments GLUT! A\] mammalian tissues 1 mM Basal glucose uptake Li L U I _ Liver and pancreatic /3 cell* 1 o JÜ ill \ 1 In the pancreas, plays a role in refutation of insulin In the liver, removes excess glucose from the blood GLUT3 AH maminfilian tissues 1 mM BasaE glucose uptake GIJ. "M \ lns:i:le ;md c<-1!s S m \ 1 Amount in musele plasma membrane endurance training GLUT5 Small intestine Primarily a fructose transporter 23 Glucose transporter GLUT4 transports glucose into muscle and fat cells. The presence of insulin, which signals the fed state leads to a rapid increase in the number of GLUT4 transporters in the plasma membrane. Hence, insulin promotes the uptake of glucose by muscle and adipose tissue. GLUT4 in the membranes of endosomes 24 The glycolytic pathway (also known as the Embden-Meyerhof pathway) The conversion of glucose into two molecules of pyruvate is anaerobic with the concomitant net production of two molecules of ATP. Under anaerobic conditions, pyruvate can be processed to lactate. Under aerobic conditions, pyruvate can be decarboxylated to acetyl CoA and completely oxidized to CO2, generating much more ATP. Glycolysis is common to all types of cells. In eukaryotic cells, glycolysis takes place in the cytosol. Reactions of glycolysis are catalyzed by enzymes. Three of them are irreversible. (In gluconeogenesis, pyruvate is converted to glucose: those three reactions differ and are catalyzed by different enzymes.) Fructose and galactose also enter into glycolysis. 25 The glycolysis can be thought of as comprising three stages: Trapping the glucose in the cell and destabilization by phosphorylation. Cleavage into two three-carbon units. Oxidative stage in which new molecules of ATP are formed by substrate-level phosphorylation of ADP. 26 The phosphorylation of glucose by ATP: CH2OH —O i OH HO^— + ATP Hexokinase OH CH2OP032-Q OH * + ADP + H+ OH Glucose OH Glucose 6-phosphate Glc-6-P Hexokinase reaction traps glucose in the cell, Glc-6-P cannot diffuse through the membrane, because of its negative charges. Conversion of Glc-6-P to glucose catalysed by glucose 6-phosphatase takes place only in the liver (and to a lesser extent in the kidney). The addition of the phosphoryl group begins to destabilize glucose, thus facilitating its further metabolism: - through further reactions of glycolysis, but also through reactions starting - synthesis of glycogen (glycogenesis) - the pentose phosphate pathway (supplying NADPH), - synthesis of other saccharides (e.g. mannose, galactose, amino sugars, glucuronic acid). 27 The phosphorylation of glucose in the cytosol accelerates the entry of glucose into the cell. On the contrary to other tissues, the liver cells (and the pancreatic (3-cells) comprise a specialized isoenzyme of hexokinase called glucokinase. The enzyme is very efficient, but its affinity for glucose is low (value of Michaelis constant is high, Km = 10 mmol/l). It means that the uptake of glucose by the liver cells (as well as (3-cells of pancreatic islets secreting insulin) shall predominate, if there is a steep rise in blood glucose. The role of glucokinase is to provide glucose for the synthesis of glycogen and for the formation of fatty acids. Glucose will not be wasted in other tissues when it is abundant. Hexokinases present in the other tissues are inhibited by glucose 6-phosphate, the reaction product. High concentration of this molecule signal that the cell no longer requires glucose for energy, for storage in the form of glycogen, or as a source of biosynthetic precursors, and the glucose will be left in the blood. High affinities of hexokinases for glucose (Michaelis constant Km < 0,1 mmol/l) will ensure the constant and preferential flow of glucose into the extrahepatic tissues, if the blood glucose level is low.. Glucokinase In the liver, specific for glucose Not inhibited by Glc-6-P Low affinity for glucose Inducible (in the liver) by insulin Hexokinases In extrahepatic tissues, broad specifity for hexoses Inhibited by Glc-6-P High affinity for glucose Not inducible by insulin 28 The isomerization of Glc-6-P to fructose 6-phosphate catalysed by phosphoglucose isomerase: 29 The second phosphorylation catalysed by phosphofructokinase is the rate-limiting step and a major control point of glycolysis : Common features of the rate-limiting step of a metabolic pathway: - The molar activity (turnover number, kcat) of the particular enzyme is smaller than those of other enzymes taking part in the metabolic pathway. - The reaction rate does not usually depend on substrate concentration [S] because it reaches the maximal value Vmax. - The reaction is practically irreversible. The process can be reversed only by the catalytic action of a separate enzyme. 2-03POH2C CH2OH .O. 2- 03POH2C + ATP Phosphofructokinase O HO CH2OP032- OH + ADP + H Fructose 6-phosphate (Fru-6-P) OH Fructose 1,6-bisphosphate (Fru-1,6-P2) Allosteric control of phosphofructokinase: • allosteric inhibition by ATP and citrate, • allosteric activation by AMP, ADP, and in the liver by fructose 2,6-bisphosphate 30 Stage 1 - summary Glucose Hexokinase ATP ADP Glucose-6-phosphate Phosphoglucose isomerase Fructose-6-phosphate ATP Phospho- f fructokinase V^^Qp Fructose-1,6-bisphosphate CH2OH —O (OH HO ^— OH HO OH CH2OP032" —O OH OH OH 2-03POH2C CH2OH XL OH 2-03POH2C CH2OP032- OH OH 31 Stage 2 The splitting of fructose 1,6-bisphosphate into two triose phosphates catalysed by aldolase: HO-CH CH OH I CH -OH I CH20PO32- Fmctose 1 ,6-bisphosphate The possible loss due to redox shuttle transport Oxidative decarboxylation of two pyruvates: 2 molecules NADH => Decomposition of 2 acetyl CoA in the citrate cycle: => the overall yield Net yield: 2 molecules ATP 6 molecules ATP -2 molecules ATP 6 molecules ATP 24 molecules ATP 36 - 38 molecules ATP / 1 molecule glucose * Supposing that reoxidation of NADH will give 3 ATP and FADH2 2 ATP (in spite of the lower values are referred to in recent literature). 46 The control of glycolysis Three control points are the three irreversible reactions of glycolysis catalysed by 1 hexokinase, 2 phosphofructokinase 1, 3 pyruvate kinase. c Glucose Hexokinase GLYCOLYSIS F-2,6-BP © AMP 0 ATP0 Citrate 0 H+0 F-1,6-BP 0 ATP 0 Alanine 0 1 Fructose 6-phosphate GLUCONEOGENESIS , 0 F-2.6-BP Fructose l £C 1, 6-bisphosphatase ] Kz) AMP 0 Citrate ^Fructose 1,6-bisphosphate Several steps Phosphoenolpyruvate Phosphoenol- pyruvate carboxy kinase 0 ADP Oxaloacetate Pyruvate carboxylase Pyruvate 0 Acetyl CoA 0 ADP T I 1 Hexokinase(s) present in the extrahepatic tissues are inhibited by glucose 6-phosphate, the reaction product. High concentration of this molecule signal that the cell no longer requires glucose for energy, for storage in the form of glycogen, or as a source of biosynthetic precursors, and the glucose will be left in the blood. 48 2 Phosphofructokinase is the key enzyme in the control of glycolysis Phosphofructokinase (PFK) in the liver is a tetramer of four identical subunits. The positions of catalytic and allosteric sites are indicated. 49 Allosteric inhibition of PFK by ATP ATP as a substrate of the PFK catalyzed reaction binds to the catalytic site. At high concentration of ATP it also binds to a specific regulatory site that is distinct from the catalytic site and allosterically inhibits the PFK activity. AMP reverses the inhibitory action of ATP - glycolysis is stimulated as the energy charge falls. [Fructose 6-phosphate] A fall in pH value also inhibits PFK activity - inhibition by H+ prevents excessive formation of lactic acid and a drop in blood pH. 50 Allosteric activation of phosphofructokinase by fructose 2,6-bisphosphate 51 (A) Allosteric activation of PFK by Fru-2,6-P2 (B) The inhibitory effect of ATP is reversed by Fru-2,6-P2 52 The concentration of Fru-2,6-P2 is controlled by a regulated bifunctional enzyme. Fru-2,6-P2 is formed in a reaction catalyzed by phosphofructokinase 2, and hydrolyzed to Fru-6-P by a specific phosphatase fructose bisphosphatase 2. Both activities are present in a single polypeptide chain: Kinase domain Phosphatase domain 1 32 250 470 Regulatory region 53 Control of the bifunctional enzyme by phosphorylation and dephosphorylation 54 3 Control of pyruvate kinase activity - by phosphorylation and dephosphorylation - by allosteric effectors Insulin HIGH BLOOD GLUCOSE LEVEL ♦A Phosphorylated pyruvate kinase (less active) Dephosphorylated pyruvate kinase (more active) LOW BLOOD GLUCOSE LEVEL Glucagon ADP 4? ATP Phosphoenolpyruvate + ADP + H Fructose ATP 1,6-bisphosphate Alanine > Pyruvate + ATP 55 Oxidative decarboxylation of pyruvate and of other 2-oxocarboxylic acids 56 Glucose The synthesis of acetyl-CoA by the pyruvate dehydrogenase complex Is a key irreversible step in the metabolism of glucose. The oxidative decarboxylation of pyruvate takes place within the matrix of mitochondrion. Under aerobic conditions, the pyruvate is transported into mitochondria in exchange for OH- by the pyruvate carrier, an antiporter. Pyruvate + CoA + NAD+ ® ® acetyl CoA + CO2 + NADH Pyruvate Pyruvate dehydrogenase complex Acetyl CoA CO Lipids 57 Matrix Inner mitochondrial membrane Outer mitochondrial membrane Oxidative decarboxylation of pyruvate represents the link between glycolysis and the citric acid cycle. GTP Pyruvate CO-2 e Acetyl CoA Pyruvate produced by glycolysis is converted into acetyl CoA, the substrate (fuel) for the citric acid cycle. 2 co2 8 e 59 Pyruvate dehydrogenase complex - schematic representation The three enzymes of the complex: E1 - the decarboxylating component of the dehydrogenase E2-the transacetylase core E3 - dihydrolipoyl dehydrogenase 61 Pyruvate dehydrogenase complex of E. coli Enzyme Abbreviation Number of chains Prosthetic group Reaction catalyzed Pyruvate dehydrogenase Ei 24 TPP Oxidative decarboxylation component of pyruvate Dihydrolipoyl transacetylase E2 24 Lipoamide Transfer of the acetyl group to CoA Dihydrolipoyl dehydrogenase E3 12 FAD Regeneration of the oxidized form of lipoamide The enzyme complex requires the participation of five coenzymes: Thiamine diphosphate Lipoamide (lipoate attached to the E2 by an amide linkage to lysyl) Coenzyme A FAD (flavin adenine dinucleotide) NAD+ 62 Steps in the oxidative decarboxylation of pyruvate o CO; o o o 2 CoA O Pyruvate -► H3C - Decarboxylation -> hhC Oxidation by lipoamide Acetaldehyde Acetyl (as hydroxy ethyl (bound to bound to TDP) DHlipoamide) ■* H3C Transfer to CoA CoA Acetyl CoA Decarboxylating component Transacetylase E1 E2 Reoxidation of dihydrolipoamide to lipoamide (2 hydrogen atoms accepted by FAD and then by NAD+ resulting in NADH + H+) v-----____^ Dihydrolipoyl dehydrogenase 63 Decarboxylating component of pyruvate dehydrogenase E1 contains bound thiamine diphosphate (TDP): A A T á o o' o The thiazole ring of the coenzyme TDP binds pyruvate. The product of decarboxylation is acetaldehyde bound onto TDP in the form of a-hydroxyethyl: E1 catalyses the transfer of a-hydroxyethyl to the lipoyl arm of transacetylase Ez 64 Transacetylase E2 contains bound lipoic acid that is attached to the amino group of the side chain of certain lysyl residue. That is why it is named lipoamide. Lipoamide (oxidized form, a disulfide) acts as an arm that accepts the hydroxyethyl group from TDP. Hydroxyethyl group ("activated acetaldehyde") reduces lipoamide to dihydrolipoamide and thus is oxidized to acetyl bound as a thioester - 6-acetyllipoamide. The acetyl is then transferred to coenzyme A : Lysine side chain of the transacetylase E j 65 NH2 Coenzyme A O CH3 O O II N O C-CH-C-CH2-O-P-O-P-O-CH2 / 1 1 1 1 _ HS CH^CH^HN /C-CH2-CH2-HN HO CH3 O° O 0 Cysteamine ß-Alanine Pantoic acid Pantothenic acid Acyls are attached to the sulfanyl group by means of a thioester bond. N 0 O o-p=o 3'-phospho ADP 66 hUC R R' N7+ -c - + CH Hydroxyethyl TDP (ionized torm) H "R" Lipoamide H,C. R' HS + H^C H R" TDP 67 The dihydrolipoyl arm then swings to E3, where it is reoxidized. Dihydrolipoyl dehydrogenase E3 contains bound coenzyme FAD that accepts two hydrogen atoms which are passed on to NAD+.. Dihydrolipoyl dehydrogenase 68 In the citrate cycle, the oxidative decarboxylation of 2-oxoglutarate (to succinyl CoA) closely resembles that of pyruvate: The 2-oxoglutarate dehydrogenase complex consists of E1 (decarboxylating 2-oxoglutarate) and E2 (transsuccinylase) components different from but homologous to the corresponding enzymes in the pyruvate dehydrogenase complex, whereas E3 (dihydrolipoyl dehydrogenase) components of the two complexes are identical. 69 Regulation of the pyruvate dehydrogenation complex Ca+2 Inhibition - by the immediate products NADH and acetyl-CoA, - by ATP, and - by phosphorylation (depending e.g. on glucagon) Activation by dephosphorylation (depending on insulin) 70