BCH3120 Lecture 1: Metabolism Overview PDF

lOMoARcPSD|18519470 BCH3120 Lecture 1: Metabolism overview Why study metabolism? - To better understand how our own body works - To better understand socioeconomic aspects of our society (obesity crisis, rise in diabetes etc.) - To better understand medical discoveries (eg. ge...

lOMoARcPSD|18519470 BCH3120 Lecture 1: Metabolism overview Why study metabolism? - To better understand how our own body works - To better understand socioeconomic aspects of our society (obesity crisis, rise in diabetes etc.) - To better understand medical discoveries (eg. genetic metabolic diseases – inherited from parents and affect metabolic processes) Metabolism is the sum of biochemical processes involved in the synthesis (anabolism), breakdown (catabolism), and inter-conversion of constituents in cells and organisms - Both biochemical processes use electron carriers to generate energy - Catabolism and anabolism use different metabolic pathways (need specific energy levels and enzyme regulation in each path – must differ from each other) Catabolism: breakdown of energy-containing nutrients (carbs, fats, proteins) into energy- depleted end products (CO2, H2O, NH3 etc.) - Generates ATP/energy Anabolism: synthesis of cell macromolecules (proteins, polysaccharides, lipids, nucleic acids) from precursor molecules (amino acids, sugars, fatty acids, nitrogenous bases) - Consumes ATP/energy Metabolic pathway: a series of enzymatic reactions leading to a metabolite product - Irreversible once pathway is past the committing step (step that “locks” metabolite within specific metabolic pathway) - Compartmentalization: occurs in specific intracellular sites (also occurs at level of organ/tissue) for effective regulation Metabolism requires building blocks (nutrients from food), workers (enzymes and cofactors) and traffic control (compartmentalization, enzymatic control, thermodynamics) - All constantly work together to convert energy in nutrients into useful purpose - Requires large amount of control to regulate use of resources Control measures of metabolism: 1. Thermodynamics Downloaded by Aa Ab ([email protected])  lOMoARcPSD|18519470 - Energy is provided via electron flow - Energy is released to form a bond - Energy is absorbed to break a bond - Stronger stable bonds have higher chemical energy than weaker unstable bonds - Weaker bonds have higher potential energy (once energy is absorbed to break bond, atoms can reorganize themselves to form stronger bonds with lower potential energy) Glucose has high potential energy (products have lower deltaG and are stronger than reactants) deltaG (change in total Free Gibbs energy) = deltaH (change in total enthalpy) – T deltaS (change in entropy) - deltaS = change in randomness of a reaction of system - deltaG = the measure of total free energy capable of doing work for a reaction at constant temperature and pressure Change in total enthalpy = sum of enthalpy of broken bonds (reactants) – sum of enthalpy of formed bonds (products) - Number and kinds of bonds involved in reaction - Negative value = exothermic (energy is released – favourable reaction) - Positive value = endothermic (energy is absorbed – unfavourable reaction) Endergonic reaction: deltaG > 0 (requires energy – unfavourable reaction) - Anabolic reactions (synthesis of complex molecules) - Non-spontaneous unless coupled with exergonic reactions (energy released must be greater than energy consumed by the endergonic reaction – sum of deltaG must be negative) Exergonic reaction: deltaG < 0 (releases energy – favourable reaction) - Catabolic reactions (breakdown of complex molecules) - Spontaneous (can be coupled to endergonic reactions to help drive them forward) - Eg. occurs in 1st step of glycolysis (formation of glucose-6-phosphate is coupled with ATP hydrolysis) Eg. high energy intermediates (organic phosphate compounds) commonly coupled with endergonic reactions (provides link between catabolism and anabolism) - Used as “energy currencies” - Eg. ATP hydrolysis - Our body makes 70 kg ATP each day (necessary for survival) - Must be coupled to another reaction to prevent energy loss as heat (group transfer reaction – inorganic phosphate from ATP transferred to enzyme or substrate) Standard free energy change (deltaGº’) Downloaded by Aa Ab ([email protected])  lOMoARcPSD|18519470 - initial concentration of each component is 1.0 M, at 25oC, pH 7.0 and constant pressure Actual deltaG = deltaGº’ + RT ln ([C][D])/([A][B]) Standard deltaG doesn’t usually equal actual deltaG - Standard deltaG of ATP hydrolysis is -30.5 kJ/mol - Actual deltaG is -52 kJ/mol Rate-limiting step: slowest step in reaction (determines entire reaction speed) - May be the same step as the committing step (step that “locks” metabolite in pathway) 2. Cell-cell communication and compartmentalization Metabolic pathways are localized - Segregation: separates metabolites/enzymes from other paths - Selective transport: Allows membrane receptors or proteins to control certain steps of a metabolic pathway by letting in or pumping out metabolites or substrates selectively - Metabolite sensing: Control of metabolic pathways by enzymes or receptors or osmotic gradients sensing, abundance of nutrients, metabolites and substrates 3. Inhibition and allosteric control used to control metabolic rate Enzymes lower the activation energy required to carry out a metabolic pathway - Allows reaction to proceed faster than without an enzyme - Enzyme lower activation barrier by distorting structure of macromolecule Macromolecules are kinetically stable and provides a barrier for biochemical reactions to occur - Breakdown products are more thermodynamically stable Competitive inhibition: Inhibitor ligand binds to and saturates catalytic pocket (blocks substrate from binding – enzymatic activity cannot occur) - Endogenous inhibitor: found within the cell (eg. end-products of reaction) - Exogenous inhibitor: found outside of cell (eg. poisons) Non-competitive (allosteric) inhibition/activation: ligand binds to a site different from substrate catalytic pocket (alters enzyme structure – affects substrate ability to bind in that pocket) - Inhibitors decrease substrate’s ability to bind (switches enzyme from R to T state) - Activators increase substrate’s ability to bind (switches enzyme from T to R state) Downloaded by Aa Ab ([email protected])  lOMoARcPSD|18519470 - T state = tense state (low/no affinity for substrate) - R state = relaxed state (high affinity for substrate) 4. Post-translational modifications control the activities of already-synthesized enzymes - Acute control of enzyme activities - Phosphorylation, methylation and acetylation (reversible modifications – cause conformational changes in enzymes) Transcriptional control: long-term control of the level of mRNA expression Translational control: controlling conversion of mRNA to protein at each step of translation 5. Enzyme Turnover controls half-life of enzymes - Lower half-life = shorter duration of activity - Don’t always want enzyme around constantly (may produce too much of a particular compound) - Eg. PEST motif in ubiquitin pathway lowers half-life of enzyme Lecture 2: Carbohydrates and Glycolysis Carbohydrates are the most abundant biomolecule on earth - Carb oxidation is the principal source of energy in non-photosynthetic cells - Human brain gets most of its energy from glucose metabolism - Made up of at least one carbonyl group and OH group attached to a carbon - Carbonyl at the end of carbon chain: Aldose - Carbonyl within carbon chain: Ketose Types of carbs: - Monosaccharides: formed of a single unit (ex: glucose) - Disaccharides: formed of 2 units (ex: saccharose = glucose + fructose) - Oligosaccharides: a short chain of monosaccharide (from 3 to 10 units), their structure is non-repetitive and complex, they are often bonded to non-carbohydrate molecules (ex: glycoproteins or glycolipids) - Polysaccharides: long monosaccharide chains, their structure is repetitive and simple, they are linear (ex: cellulose) or branched (ex: glycogen) - Proteoglycans: long chain of monosaccharide units, bonded to proteins Downloaded by Aa Ab ([email protected])  lOMoARcPSD|18519470 - Peptidoglycans: long chain of monosaccharide units bonded to each other by small peptides Monosaccharides: CnH2nOn (N is at least 3 and < 8) Classified based on: - Number of carbon atoms (3 = triose, 7 = heptose etc.) - Position of carbonyl group (aldose or ketose) - Chirality of molecule (D or L configuration) Monosaccharide chirality: contains at least one asymmetric (chiral) carbon - Chiral carbon: carbon bound to 4 different substituents/groups - D configuration: OH group is right of the chiral carbon - L configuration: OH is left of chiral carbon - Structure with > 3 carbons: look at the bottom chiral carbon (chiral carbon farthest from carbonyl group) - Most monosaccharides found in nature are in the D configuration Enantiomers: compounds that are mirror images of each other - Differ in light refraction properties (light reflected on right = D configuration, light reflected on left = L configuration) - D and L configurations are enantiomers of each other Diastereomers: stereoisomer (different configurations) that are not enantiomers Monosaccharide cyclic structure: most pentoses/hexoses are cyclic - Very few monosaccharides have a linear structure at neutral pH - Hemiacetal linkage formed between carbonyl at C1/C2 (depending on if aldose or ketose) and OH group at C5 Forms alpha and beta stereoisomers - Alpha anomer: OH from C1 and distal CH2OH are on opposite sides (easier to break bonds – found in glucose) - Beta anomer: OH from C1 and distal CH2OH are on the same side (harder to break bonds – found in cellulose) Disaccharides: 2 monosaccharides held together by an O-glycosidic bond Downloaded by Aa Ab ([email protected])  lOMoARcPSD|18519470 - Forms between hydroxyl groups of two monosaccharides - Occurs via condensation reaction (water molecule released as a product) Polysaccharides: made of multiple monosaccharide units linked together via O-glycosidic bonds - Can be linear (eg. cellulose) or branched (eg. glycogen or starch) - Can be made up of only one type of monosaccharide unit (homopolysaccharide) or 2+ different types of units (heteropolysaccharide) Physiological Roles of Carbohydrates: 1. Provide support and protection to biological structures (eg. plants, body parts) 2. Principal source of fuel/energy which can also be stored (eg. glycogen) 3. Metabolic role – can be changed into other types of molecules (amino acids, fatty acids and nucleotides) for increased nutrient and energy source Glycolysis: breakdown of glucose (6-carbon sugar) into pyruvate (3-carbon compound) via Pentose Phosphate Pathway (PPP) - Glucose can instead be stored in the form of glycogen for future use - Entering the PPP is the committing step of glycolysis - Breakdown of glucose releases potential energy (used to synthesize ATP from ADP for energy) - Can occur under aerobic or anaerobic conditions 10 total reactions in glycolysis - First 5: Energy Investment Phase (allows the rest of the reactions to take place) - Last 5: Energy Generation Phase (uses up energy generated from investment phase) Sources of glucose: - Diet: polysaccharides (from starch and glycogen) and disaccharides (from sucrose, maltose and lactose) - Breakdown of stored glycogen - Metabolic conversion of non-carb precursors into carbs via gluconeogenesis in the liver and kidneys Downloaded by Aa Ab ([email protected])  lOMoARcPSD|18519470 Glycolysis Occurs mainly in cytoplasm of muscles and brain Energy Investment Phase: Reactions 1-5 - 2 ATP molecules consumed to convert one molecules of glucose into 2 molecules of GAP Reaction 1: a-D-Glucose is phosphorylated to form a-D-Glucose-6-phosphate (G6P) by the hexokinase - Hydrolysis/consumption of ATP allows reaction to take place (highly favourable) - Standard deltaG = -18.4 kJ/mol - Irreversible reaction - Mg2+ is present to increase phosphate’s affinity for OH on glucose - Catalyzed by hexokinases 1-4 (found in mammals) Hexokinase 1, 2 and 3 - Mainly located in skeletal muscles - Not specific to glucose - Low Km (low concentration of glucose needed to reach Vmax/maximum velocity – Vmax is reached almost instantly) - Strongly inhibited by large amounts of G6P product Hexokinase 4 (Glucokinase) - Found in liver and pancreas - Glucose specific - High Km (high concentrations of glucose needed to reach Vmax – Vmax is reached slowly with increase in blood glucose entering cell) - More responsive to glucose concentration changes (allows liver to adjust rate of glucose usage to variations in blood glucose levels) - Does not get inhibited by G6P GLUT (Glucose transporter) moves glucose molecules across plasma membrane - > 12 GLUTs in various tissues in body GLUT2: found in liver, pancreas and kidney - Insulin-independent - Quickly equilibrates glucose concentration across plasma membrane (more glucose in blood = more glucose transported into cell) - Allows hexokinase 4 to adjust transport rate to glucose concentration in blood GLUT4: found in skeletal muscles, adipose tissues and heart Downloaded by Aa Ab ([email protected])  lOMoARcPSD|18519470 - Insulin-dependent - Only active/released in presence of sugar/insulin (sequestered without glucose – remains bound within plasma membrane) Reaction 2: a-D-Glucose-6-phosphate (G6P) is isomerized into D-Fructose-6-phosphate (F6P) by the phosphohexose isomerase - Isomerization: rearrangement of bonds in current molecule (nothing new added) - Reversible (standard deltaG = +1.7 kJ/mol) - Can shift concentrations of reactants/products to push reaction forward (adding more reactants and less products) Reaction 3: D-Fructose-6-phosphate (F6P) is phosphorylated at C-1 by the phosphofructokinase 1 (PFK-1) to generate D-Fructose-1,6-bisphosphate (FBP) - Consumes 1 ATP to power reaction - Coupled reaction with ATP makes reaction highly favourable (standard deltaG = -15.9 kJ/mol) and irreversible 2 phosphates on F6P activate the molecule for cleavage Reaction 4: D-Fructose-1,6-bisphosphate (FBP) is cleaved to generate two 3-carbon (3C) molecules: Glyceraldehyde-3-phosphate (GAP) and Dihydroxyacetone phosphate (DHAP) - Cleavage occurs using fructose-1,6-bisphosphate aldolase - Reaction is strongly unfavourable (endergonic) and reversible - Standard deltaG = 23.9 kJ/mol - Actual deltaG in cell = -1.3 kJ/mol - Reaction is favourable in cell conditions if more reactant or less product is added Reaction 5: Isomerization of the Dihydroxyacetone phosphate (DHAP) to Glyceraldehyde-3- phosphate (GAP) by the Triose phosphate isomerase (TPI) - Weakly unfavourable (endergonic) and reversible - Standard deltaG = 7.6 kJ/mol - Actual deltaG = 0 kJ/mol (system is at equilibrium – can occur in either direction) Energy Generation Phase: - Each reaction step occurs twice (once for every GAP molecule) - 2 ATP, 1 NADH and 1 pyruvate are generated for every GAP molecule Downloaded by Aa Ab ([email protected])  lOMoARcPSD|18519470 Reaction 6: Oxidation and phosphorylation of Glyceraldehyde-3-phophate (GAP) to generate 1,3- bisphosphoglycerate (BPG) - Catalyzed by Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and NAD + electron acceptor coenzyme - Standard deltaG = 6.3 kJ/mol - Reversible (reaction moves forward because of reaction 7) Reaction 7: Synthesis of an ATP by the transfer of a phosphoryl group from 1,3- bisphosphoglycerate (BPG) to form 3-phosphoglycerate (3PG) - Catalyzed by the Phosphoglycerate kinase - Standard deltaG = -17.2 kJ/mol - Actual deltaG is around 0 kJ/mol - Reversible - Consumption of BPG powers reaction 6 forward (less product available – shifts reaction to right) - Formation of 3PG powers reaction 8 forward (more reactant available – shifts reaction right) Reaction 8: Isomerization of 3-phosphoglycerate (3PG) into 2-phosphoglycerate (2PG) by the phosphoglycerate mutase - Standard deltaG = 4.4 kJ/mol - Reversible (reaction moves forward because of reaction 6 and 7) Reaction 9: Dehydration of 2-phosphoglycerate (2PG) into phosphoenolpyruvate (PEP) by the enolase - OH removed from 2PG to make water as a product - Standard deltaG = -3.2 kJ/mol - Reversible (PEP concentrations kept low by pyruvate kinase in next step – pushes reaction forward) Reaction 10: Synthesis of an ATP by the transfer of a phosphoryl group from phosphoenolpyruvate (PEP) by the pyruvate kinase to produce pyruvate - Standard deltaG = -29.7 kJ/mol - Irreversible Net reaction: 2 pyruvate, 2 ATP and 2 NADH are generated in total Aerobic glycolysis (including Kreb’s Cycle and oxidative phosphorylation) produces 30-32 ATP molecules for every molecule of glucose Downloaded by Aa Ab ([email protected])  lOMoARcPSD|18519470 - CO2 and H2O are produced as final products Anerobic Glycolysis Glycolysis was probably the first energy yielding pathway utilized by the earliest known organisms, when the atmosphere was still anaerobic - Anerobic glycolysis only produces 2 ATP in total without consuming oxygen Anerobic glycolysis occurs using fermentation - Fermentation: Anaerobic degradation of a nutrient (glucose), with no net change in the oxidation state, to produce energy - Occurs always in red blood cells (they lack mitochondria – no available oxygen to use) Lactic fermentation: Reduction of pyruvate to lactate - 2 ATP generated from the production of FBP from glucose - Regenerates NAD+ for further glycolysis under anaerobic conditions (NADH donates electrons to convert pyruvate into lactate) - 2 NADH produced and used (no net change in oxidation state) During exercise: lactate builds up in muscles (amount of oxygen in body doesn’t match what is needed for aerobic glycolysis) - During exercise: lactate travels to liver to be catabolized in aerobic glycolysis or converted back into glucose (Cori Cycle) Cori Cycle: lactate is converted into glucose which is used for gluconeogenesis (requires consumption of 6 ATP) - Highly energy-expensive (only 2 ATP produced in anaerobic glycolysis) Alcoholic fermentation: Reduction of pyruvate to ethanol - 2 ATP generated and used - Pyruvate is converted into acetaldehyde (via pyruvate decarboxylase) which is converted into ethanol (via alcohol dehydrogenase) - 2 NADH generated and used during ethanol step Downloaded by Aa Ab ([email protected])  lOMoARcPSD|18519470 Actual deltaG is different depending on cell type and time period (why it is shown as a range) Downloaded by Aa Ab ([email protected])  lOMoARcPSD|18519470 Gluconeogenesis Opposite of glycolysis (pyruvate is made back into glucose) - Occurs in kidney cortex and mainly liver (mainly in cytoplasm – 1st reaction is in the mitochondrial matrix) Three irreversible reactions of glycolysis must be bypassed using different enzymes than in glycolysis - These reactions can’t go in reverse because of how highly favourable they are Brain requires majority of the body’s glucose (120g/day out of total 160g) - Why glucose must be synthesized from other sources when levels are low - Brain can’t function without adequate glucose levels Glycogen: glucose reserves stored in muscles and liver (can provide 190g of glucose) - Liver produces glucose and shares it with rest of body - Muscles consume glucose and don’t share it with rest of body Gluconeogenesis occurs almost all the time throughout the day Gluconeogenesis precursors Lactate – made from pyruvate during anerobic glycolysis (not enough oxygen available to begin TCA cycle during exercise) - Made in the red blood cells and muscles - Makes up 1/3 of precursors - Travels to liver to be converted back into pyruvate via Cori Cycle (oxygen levels in liver are more plentiful than in active muscles) - Newly made pyruvate can now be made into glucose Alanine – made from ammonia via protein breakdown (ammonia is toxic and must be converted) - Made in the muscles - 1/3 of precursors Glycerol – made in the breakdown of triglycerides in adipose tissue, lipids from food and lipoproteins - Triglyceride breakdown also produces fatty acids (used for energy) - 1/12 of precursors Downloaded by Aa Ab ([email protected])  lOMoARcPSD|18519470 Amino acids (except leucine and lysine) – found in food/tissue proteins - 1/8 of precursors Propionyl-CoA – made from the breakdown of odd-numbered fatty acids Gluconeogenesis reaction 1: occurs in mitochondrial matrix - 1st part of reverse of glycolysis reaction 10 (1st reaction needed to bypass) - Split into 2 reactions 1st reaction: carboxylation (addition of a carboxyl group) of pyruvate to form oxaloacetate - Requires one ATP - Carboxyl group taken from bicarbonate (reacts with pyruvate) - Carboxyl group is only added to form a good substrate for 2nd part of reaction - Biotin added as a prosthetic group (helps attach carboxyl group) Pyruvate enters mitochondrial matrix via Mitochondrial Pyruvate Carrier (MPC – made of MPC1 and MPC2 heterodimers) - MPC also used to convert pyruvate into Acetyl-CoA (TCA cycle substrate) 1st reaction catalyzed by pyruvate carboxylase (only found in mitochondrial matrix) - Catalyst helps to replenish oxaloacetate to carry out TCA cycle (oxaloacetate is an intermediate of TCA cycle) 2nd reaction: oxaloacetate is reduced (removal of carboxyl group – electron gain) to malate using NADH and mitochondrial Malate Dehydrogenase (MDH2) - Oxaloacetate cannot leave mitochondrial matrix unless reduced - Once in cytosol: malate re-oxidized to form oxaloacetate again using NAD+ and cytosolic Malate Dehydrogenase (MDH1) - High concentration of NADH in mitochondria compared to cytosol (oxaloacetate reduced by NADH here) - Mitochondrial membrane is impermeable to NADH/NAD+ so electrons (reducing equivalents) are given to an enzyme to cross membrane and oxidize malate Reaction 2: phosphorylation and decarboxylation of oxaloacetate to form phosphoenolpyruvate (PEP) in cytosol 2nd part of reverse of glycolysis reaction 10 - Catalyzed by phosphoenolpyruvate carboxykinase (PEPCK) Downloaded by Aa Ab ([email protected])  lOMoARcPSD|18519470 - Carboxyl group added in reaction 1 removed in the form of CO2 - Carboxyl removal provides electrons necessary for phosphorylation - GTP hydrolysis provides energy to carry out reaction 2 high energy phosphates must be invested for synthesis of one phosphoenolpyruvate - Overall: 2 ATP and 2 GTP invested to make glucose Alternative pathway for first bypass: used if lactate is the precursor/substrate - Lactate dehydrogenase generates cytosolic NADH (oxidizes lactate to pyruvate) - PEP formed in mitochondria instead of cytosol Reactions 3-8: same as glycolysis reactions 8-4 but in reverse - Can adjust concentrations (less glycolysis reactant and more glycolysis product) to push reaction toward gluconeogenesis end Reaction 9: hydrolysis of FBP into F6P - Reverse of glycolysis reaction 3 (2nd reaction to bypass) - Uses 1 ATP Catalyzed by FBPase-1 (removes phosphate from FBP) - Temperature-sensitive enzyme (helps regulate metabolic rate of hibernating animals – less active in the cold and inhibits gluconeogenesis) Reaction 10: same as glycolysis reaction 2 but in reverse Reaction 11: hydrolysis of G6P into glucose - Reverse of glycolysis reaction 1 (3rd reaction to bypass) - Catalyzed by G6Pase Glycolysis can’t directly occur in reverse due to irreversible reaction 1, 3 and 10 - Highly unfavourable reactions in reverse Gluconeogenesis is energetically costly (requires 4 ATP, 2 GTP and 2 NADH) but physiologically necessary - Brain, nervous system, and RBCs generate ATP mostly from glucose Downloaded by Aa Ab ([email protected])  lOMoARcPSD|18519470 - Necessary for producing more glucose between meals (after diet glucose has been used up) Gluconeogenesis: glucose is made from other substrates (lactate, glycerol, proteins etc.) - Allows for a constant supply of blood glucose - Fats/proteins used as energy for rest of body (glucose conserved for brain) Lactate produced during anerobic glycolysis in RBCs and muscles - Converted into pyruvate in liver via lactate dehydrogenase (Cori cycle) - Eventually made into glucose (requires 6 ATP) - No change in oxidation state (reduced to lactate and re-oxidized to glucose) Triglycerides are broken down into glycerol (found in adipose tissue) and 3 fatty acid molecules - Fatty acids used as principal metabolic fuel in TCA cycle when glucose and glycogen levels are depleted - Glycerol is phosphorylated and oxidized in the liver into dihydroxyacetone phosphate (further converted into GAP – used in reaction 7 of gluconeogenesis) Muscle proteins are broken down into individual amino acids when carb and fatty acid levels are low to make glucose - High rates of protein breakdown only seen in hyperproteic diets, long-term fasting (starvation) and Type 1 Diabetes - All amino acids except for Leucine and Lysine are glucogenic (can be converted into glucose) - Some amino acids are glucogenic but can also be converted into ketone bodies for fuel - Many amino acids can be made into oxaloacetate (either made into glucose or goes through TCA cycle) Regulation of glycolysis and gluconeogenesis Pasteur effect: when anerobic yeast are exposed to oxygen, the rate of glucose utilisation decreases greatly - Anerobic glycolysis only makes 2 ATP while aerobic glycolysis makes 30-32 ATP - Less glucose needs to be used for energy in aerobic glycolysis because more energy is being made through the formation of glucose - Lots of glucose, G6P and F6P are made but there are only small amounts of the other product Reaction 1, 3 and 10 (irreversible reactions) are the most regulated reactions of glycolysis Downloaded by Aa Ab ([email protected])  lOMoARcPSD|18519470 - All three are the rate-limiting steps of glycolysis (regulation will change the rate of glycolysis) - Reaction 3 is the most regulated out of the 3 - Regulated allosterically (binding of a ligand at a site other than protein’s catalytic site) to increase (activate) or decrease (inhibit) activity - Allosteric activation makes catalytic pocket more suitable for substrate to bind - Allosteric inhibition makes catalytic pocket more inaccessible to substrate Glycolysis reaction 1 is regulated by the hexokinase 1 (in muscles) and hexokinase 4 (in liver and pancreas) - Hexokinase 1-3 is inhibited by the product of reaction 1 (G6P) - Hexokinase 4 is NOT inhibited by G6P (can be used in glycogen metabolism to store glucose or the pentose phosphate pathway to make glucose) GLUT2 transporter equilibrates glucose between the blood and liver cells - High concentrations of glucose inhibit the glucokinase regulatory protein (GKRP) and releases glucokinase into the cytosol (allows more glucose to be taken up) - Low concentrations of glucose (< 5mM) activates GKRP activity via F6P (sequesters glucokinase in nucleus so no glucose can be taken up) - F6P in low glucose concentrations is made via gluconeogenesis in the liver - F6P in high glucose concentrations (glycolysis) is a reactant during reaction 3 (irreversible – converted very quickly into product) Gluconeogenesis reaction 11 is regulated by G6P - G6Pase Km > intracellular concentration of G6P - Activates G6Pase in high G6P concentrations (allows more glucose to be made) Glycolysis reaction 3 is regulated in multiple ways PFK inhibited in high levels of ATP and citrate (first intermediate of TCA cycle) - AKA low levels of ADP (glycolysis blocked because ATP is not needed) - Citrate is made during first reaction of TCA cycle (accumulation of citrate means TCA cycle is already running at full speed and cannot cope – glycolysis is inhibited) - Takes more substrate (F6P) to increase PFK activity (need sufficient amount of ATP – too much causes inhibition) PFK activated in high levels of ADP - AKA low levels of ATP (glycolysis stimulated to make more ATP) - Takes less substrate to increase PFK activity (low levels of ATP makes enzyme more active to produce more ATP – more substrate used up) PFK is a dimer (4 subunits – 1 + 2 go together and 3 + 4 go together) Downloaded by Aa Ab ([email protected])  lOMoARcPSD|18519470 - 2 + 3 are the same, 1 + 4 are the same - 2 catalytic sites for FBP on each dimer part - 2 regulatory sites for ATP on each dimer site PFK is activated by fructose-2,6-bisphosphate (glycolysis activated) - Produced from F6P via PFK-2/FBPase-2 enzyme (FBPase-1 used in gluconeogenesis reaction 9 and PFK-1 used to glycolysis reaction 3) - Differs from fructose-1,6-bisphosphate by placement of phosphate on which carbon - PFK-2 and FBPase-2 are the same enzyme (PFK-1 and FBPase-1 are different enzymes) - Less substrate needed to reach Km FBPase is inhibited by fructose-2,6-bisphosphate (gluconeogenesis inhibited) - More substrate needed to reach Km Insulin is released during high glucose levels - Stimulates glycolysis via phospho-protein phosphatase (dephosphorylates PFK/FBPase-2 enzyme) - Dephosphorylation activates the PFK-2 part (F6P phosphorylated to make FBP) Glucagon is released during low glucose levels - Stimulates gluconeogenesis via cAMP-dependent protein kinase (phosphorylates PFK- 2/FBPase-2 enzyme) - Phosphorylation activates the FBPase-2 part (removes phosphate from FBP to make F6P) Glycolysis reaction 10 is regulated through allosteric control of the protein kinase - Inhibited by Acetyl-CoA and ATP (glycolysis is no longer needed to produce more ATP) - Activated by FBP - Pyruvate kinase inactivated in the presence of glucagon (low glucose levels – prevents breakdown of glucose via glycolysis) Gluconeogenesis reaction 1 is regulated through allosteric control of the pyruvate carboxylase - Activated by Acetyl-CoA (can also inhibit its own production from pyruvate through pyruvate dehydrogenase complex) - Acetyl-CoA part of TCA cycle - Acetyl-CoA stimulates gluconeogenesis through the breakdown of fatty acids (produces Acetyl-CoA) Downloaded by Aa Ab ([email protected])  lOMoARcPSD|18519470 Lecture 5: Glycogen Glycogen is a glucose polymer made of 100,000s of glucose (no defined number of glucose) - D-glucose molecules linked together via O-glycosidic bonds - Intra-chains: alpha bonds between carbon 1 and 4 - Inter-chains: alpha bonds between carbon 1 and 6 Glucose is a reducing agent (can oxidize/donate 2 electrons from each aldehyde/ketone to an oxidizing agent) - Contains an aldehyde/ketone that can react to form a hemiacetal/ketal - Hemiacetal/ketal: attached to ether bond and an alcohol (derived from aldehydes or ketones) - Anomeric carbons are the carbon at the centre of the hemiacetal/ketal Maltose is a reducing sugar (both compounds in disaccharide form hemiacetal/ketals in cyclic form) - Sucrose is not (beta-D-fructose in disaccharide does not form a hemiacetal/ketal in cyclic form) Glycogen only has one reducing sugar end (all other ends are non-reducing) - Why is glucose not stored in the cell? Glucose has a high osmotic pressure and accumulation in the cell would result in a hypotonic solution (water would invade the cell and cause it to burst) - Cell doesn’t recognize glycogen as having a large concentration of glucose (why it is stored instead of glucose) Glycogen is highly branched so metabolism can occur faster (each end is non-reducing and can be reduced into its linear form of glucose) - Enzymes cleave from non-reducing towards reducing end Glycogen in the liver: stored for public use (majority goes to brain) - Used when there is a large drop in blood glucose concentrations - Makes up 1/3 of glycogen stored in body (1/8 of liver weight) - Used up within a day - Stored as granules Glycogen in the muscles: stored for private use (muscles only) Downloaded by Aa Ab ([email protected])  lOMoARcPSD|18519470 - Used as a fuel for muscle cells (during contractions) - Glycogen is muscles is used up faster than glycogen in liver - Makes up 2/3 of glycogen stored in body (0.8% of total muscle weight) Glycogen metabolism occurs mainly in the gut post-prandial (post-meal), liver between meals and muscles when they are being used - Gut breaks down muscle and starch from diet (produces lots of glycogen – used for storage or energy source for muscles) - Liver stores glucose post-meal in glycogen form and breaks down glycogen between meals to provide energy for tissues and organs - Liver helps equilibrate blood glucose concentrations via glucokinase activity (acts as a “glucostat”) - Glycogen synthesized when muscles are at rest and broken down when muscles are contracting Process of metabolism: glucose is stored, broken down when needed by organs, and enters the muscles/liver/other organs to be used up and excess is stored again If we didn’t eat for several days: this would occur (depending on organism’s nutritional state and need for energy) - First: glucose from diet would be used up - Second: stored glycogen would be broken down into glucose to be used - Third: new glucose is made via gluconeogenesis - Body will also breakdown fats and proteins for energy Anabolism of glycogen is known as glycogenesis - Occurs in liver and muscles Glycogenesis reaction 1: glucose is phosphorylated by hexokinase 1, 2 and 3 (in muscles) or glucokinase (in liver) to form G6P - Same reaction as glycolysis reaction 1 - Requires 1 ATP Reaction 2: G6P isomerized to G1P (phosphate moved to a different carbon) via phosphoglucomutase - Reversible Reaction 3: G1P activated by the addition of UDP by UDP-glucose pyrophosphorylase Downloaded by Aa Ab ([email protected])  lOMoARcPSD|18519470 - UDP addition is used to make reactant into a good substrate for next reaction (high- energy molecule when cleaved – thermodynamically powers next reaction forward) - UDP is removed from UTP and added onto glucose to form UDP-glucose and phosphate Reaction 4: Linear chains synthesized (binding of carbon 1 to carbon 4 alpha bonds) - Catalyzed by glycogen synthase - Glucose transferred to non-reducing end of a glycogen primer (initiates main strand) - Primer is a small chain of glucose residues assembled by glycogenin protein - UDP released in process (makes reaction exergonic due to high energy) Reaction 5: side chains are formed (binding of carbon 1 to carbon 6 alpha bonds) - After 11 molecules are formed in intra-chain: last 6-7 glucose molecules are removed from non-reducing end and transferred closer to reducing end - Carried out via amylo-(1,4-1,6)-transglycosylase Reaction 6: extension of side-branches and formation of new intra-chains Each glucose unit added onto glycogen costs 2 ATP - 1 ATP from step 1 and 1 ATP from step 3 (convert UDP into UTP) - 30-32 ATP made in aerobic glycolysis (investment pays off) Catabolism of glycogen is known as glycogenolysis - O-glycosidic intra-chain bonds hydrolyzed by alpha-amylase from saliva and pancreas (digestive fluids) - Produces maltose and alpha-dextrin - Maltose: disaccharide held by an o-glycosidic intra-chain bond (bond hydrolysed by maltase to form glucose) - Alpha-dextrin: polymer mixtures of at least 6 D-glucose units held in 1-4 bonds and at least one 1-6 inter-chain bond - Alpha-dextrin hydrolysed by alpha-dextrinase to remove inter-chains and alpha-amylase to remove intra-chains to form maltose Reaction 1: terminal glucose molecule from non-reducing end hydrolyzed and phosphorylated (phosphorolysed) by glycogen phosphorylase to form G1P - Removal of terminal glucoses stops 4 glucose units before branch point (1-6 glycosidic bond) of inter-chain - Multiple G1Ps formed for every phosphorolysis reaction Downloaded by Aa Ab ([email protected])  lOMoARcPSD|18519470 Each G1P made in step 1 is isomerised to form G6P via phosphoglucomutase - Muscles: G6P directly enters glycolysis (glucose not shared with anyone else) - Liver: phosphate removed from G6P to form glucose via G6Pase (exported into blood) - G6Pase only found in liver Glycogen storage disease 1 (GSD1): deficiency of G6Pase activity - Phosphate can’t be removed to form glucose (must rely on gluconeogenesis to make glucose or glycolysis to break down small pre-existing glucose amounts) - G6P is made into glycogen again via glycogenesis (abnormal glycogen accumulation) - Requires diet of complex carbs (allows for a slow breakdown – constant glucose release into blood) Reaction 2: 3 remaining glucose molecules on branched chain transferred onto the non- reducing end of the main intra-chain or another intra-chain - Occurs via glycogen debranching enzyme Reaction 3: Interchain bond that the 3 glucose molecules were previously removed from is hydrolyzed via glycogen debranching enzyme Glycogen catabolism in the muscles: glucose is instantly converted to G6P due to the low Km of the hexokinase - Hexokinase is almost always active - Glucose is quickly broken down via glycolysis or used in the TCA cycle Glycogen catabolism in the liver: glucose exported directly to bloodstream due to high Km of glucokinase - Little G6P is formed due to low activity from glucokinase - Glucose used up by organs in blood Other glycogen metabolic defects: Hypoglycemia: no breakdown of glycogen to maintain blood glucose (seen in people with GSD1) - People with G6Pase deficiency have enlarged livers due to glycogen build-up Lactic acidosis: impairment in gluconeogenesis due to issues with G6Pase - Lactate is not converted back to glucose due to accumulation of G6P - Builds up in muscles as a result and causes pain Downloaded by Aa Ab ([email protected])  lOMoARcPSD|18519470 Regulation of glycogen anabolism occurs through the regulation of glycogen synthase - Regulated allosterically and covalently (via phosphorylation) - Glycogen synthase activated through dephosphorylation - Synthesis process is the same in both muscles and liver Positive regulation: activation of glycogen synthase (from synthase b to synthase a) Allosterically: glucose and G6P bind to synthase and induce a confirmational change that favours dephosphorylation by PP1 Covalently: dephosphorylation occurs via PP1 (phosphoprotein phosphatase-1) - PP1 is activated in response to insulin release (high blood glucose levels) - Insulin-sensitive kinase phosphorylates glycogen targeting protein (Gm) on 1st site - Gm activates PP1 and keeps it in close contact with glycogen synthase Both regulations occur simultaneously Negative regulation Covalently: PKA phosphorylates Inhibitor-1 and Gm (on its 2nd site) - Doubly phosphorylated Gm no longer in close contact with PP1 - Phosphorylated Inhibitor-1 can now bind to and inactivate lone PP1 - PKA induced by binding of glucagon and epinephrine to its receptors (affects multiple enzymes in response) Glucagon released in low glucose levels - Need glucose to replenish blood glucose levels (why glycogen synthase is inhibited) - Epinephrine released in stressful periods (glucose needed for energy to power SNS – why glycogen synthase is inhibited) Covalently (part 2): glycogen synthase phosphorylated on three different serine sites by glycogen synthase kinase 3 (GSK3) - Synthase must be primed by casein kinase 3 (CK3) first before being phosphorylated - CK3 phosphorylates synthase and unfolds molecule to make it accessible for GSK3 - GSK3 activity inhibited by release of insulin Regulation of glycogen catabolism occurs through the regulation of glycogen phosphorylase - Regulated allosterically and covalently - Activated through phosphorylation via phosphorylase b kinase Downloaded by Aa Ab ([email protected])  lOMoARcPSD|18519470 - Breakdown is different depending on whether in muscles or liver Activated via glucagon release (low blood glucose levels) - Occurs via activated PKA (carries out phosphorylation cascade of different proteins) - Eventually leads to phosphorylation of phosphorylase B into A to break down glycogen Allosterically (muscles): calcium release from working muscles and AMP accumulation from muscle contraction stimulates phosphorylation of phosphorylase B into A - Calcium acts on kinase to cause confirmation change of phosphorylase B AMP binds to phosphorylase to cause confirmation change and make it a better substrate for phosphorylation - AMP accumulates when there is low energy in muscles (need to stimulate glucose breakdown to produce more ATP) - ATP allosterically inhibits AMP (competes with AMP for site – causes confirmation change and makes phosphorylase B a worse substrate for phosphorylation) Covalently (muscles): epinephrine stimulates glycogen breakdown in muscles to power energy for SNS activation - Favours phosphorylase B phosphorylation through the PKA pathway Allosterically (liver): high glucose concentrations bind to allosteric sites on phosphorylase A - Exposes A’s phosphorylation site to PP1 - PP1 can dephosphorylate A to B and this reduces glycogen breakdown Covalently (liver): - Glucagon stimulates phosphorylase B kinase to phosphorylate B into A (activates glycogen breakdown via PKA pathway) - Insulin stimulates PP1 to dephosphorylate A into B (inhibits glycogen breakdown) Insulin can stimulate PP1 to regulate both glycogen synthase (increase) and phosphorylase (decrease) at the same time Glucagon can stimulate PKA to regulate both glycogen synthase (decrease) and phosphorylase (increase) at the same time Insulin and glucagon work reciprocally to regulate and maintain glucose levels within a narrow range Pentose Phosphate Pathway: Downloaded by Aa Ab ([email protected])  lOMoARcPSD|18519470 G6P is the first substrate for reaction (integrated with glycolysis, gluconeogenesis and glycogenesis/genolysis) - Generated by glucokinase in liver (PPP most active in high glucose concentrations) - Excess glucose: some glucose stored as glycogen, some broken down via glycolysis, some converted into new molecules via PPP 2 phases of PPP: oxidative (reduction of NAPD+ into NADPH) and non-oxidative Not designed to produce energy - Produces NADPH (provides electrons for reactions requiring reducing steps – steroid synthesis, repair oxidative damage etc.) - Produces ribose-5-phosphate (essential for nucleic acid synthesis and other co-enzymes) Pathway is mainly active in liver (allows synthesis of fatty acid and cholesterol) - Activity of PPP in liver is greater than that of glycolysis (beta-oxidation of lipids generates majority of liver’s energy) - Also active in adipose tissue and mammary gland (fatty acid synthesis) - Active in steroidogenic tissues (steroid hormone production) - Active in RBCs (oxidative damage repair) 2 NADPH produced during PPP (oxidative phase) 1st production is coupled with glutathione reductase reaction (used for oxidative damage) - OH on G6P oxidized to form carboxyl - Cyclic G6P broken (hydrolyzed) to form linear molecule 2nd production used for reductive biosynthesis - Carboxyl group released as CO2 (forms Ribulose 5-Phosphate) - Ketone converted into aldehyde (forms ribose-5-phosphate) Non-oxidative phase: F6P formed from ribose-5-phosphate + xylulose-5-phosphate (catalyzed by transketolase) can enter glycolysis or gluconeogenesis, produce more G6P and go through PPP again - Can’t occur forever (lose one CO2 molecule for every PPP carried out – carbon lost and eventually there will be none left to carry out oxidative PPP) Overall summary of the three steps of cellular respiration: Downloaded by Aa Ab ([email protected])  lOMoARcPSD|18519470 Stage 1: Oxidation of fatty acids, glucose and some amino acids yields acetyl CoA and reducing equivalents (e- ) Stage 2: Oxidation of acetyl- CoA in the TCA cycle has four steps where reducing equivalents are further produced. Stage 3: Reducing equivalents carried by NADH and FADH2 are funneled into the electron transfer chain and oxidative phosphorylation (producing H2O and ATP) Cellular respiration: Process in which cellular energy is generated through the oxidation of nutrient molecules with O2 as the ultimate electron acceptor - Where majority (90%) of body’s energy is generated from Involves 3 steps: 1. Carbon from metabolic fuels (pyruvate from glycolysis, amino and fatty acids) is incorporated into acetyl-CoA - Step 1 and 2 both occur in mitochondrial matrix - Pyruvate transformed into acetyl-CoA via pyruvate dehydrogenase - Lipids transformed via beta-oxidation - Amino acids either converted to pyruvate first or directly converted into acetyl-CoA 2. The citric acid cycle oxidizes acetyl-CoA to produce CO2, reduced electron carriers (NADH, FADH2), and a small amount of ATP 3. The reduced electron carriers (NADH, FADH2) are reoxidized, providing energy for the synthesis of additional ATP - Occurs in inner mitochondrial membrane Coenzyme A has several subunits - Beta-Mercapto-ethylamine subunit has a reactive thiol group (where acetyl group is added onto) - Addition of acetyl forms a thiol ester (bond cleavage is very exergonic and favourable due to instability of bond) Pyruvate enters mitochondrial matrix via MPC protein - Converted into acetyl-Coa via pyruvate dehydrogenase (PDH) - Oxidative decarboxylation: PDH removes carboxyl group in the form of CO2 and electrons (get stored on NADH) - Multiple enzymes and cofactors involved Downloaded by Aa Ab ([email protected])  lOMoARcPSD|18519470 - Irreversible reaction (deltaG = -33.4 kJ/mol) Can also form oxaloacetate via pyruvate carboxylase (undergoes gluconeogenesis or anaplerosis) PDH: multienzyme complex (3 enzymes and 5 cofactors to allow channeling of substrates) - E2 makes up core (60 monomers) - E3 fills in gaps of core (12 dimers) - E1 forms outer shell (30 tetramers) Enzyme carries out 5 different reactions 1. Carbon 1 on pyruvate is removed to form CO2 - Bond formed between pyruvate carbon 2 and TPP coenzyme 2. Hydroxyethyl group is transferred to lipoic acid of lipoic domain on enzyme along with 2 enzymes 3. Transesterification and reduction of lipoic domain - Acetyl group added onto coenzyme A 4. Lipoic domain is reoxidized (electrons transferred to disulphide bond on enzyme) 5a. Cysteines on enzyme oxidized (electrons transferred to FAD to make FADH2) - Flavin nucleotides tightly bound to flavoproteins like E3 (act as cofactors) 5b. Electrons transferred to NAD+ to form NADH Regulation of PDH E2 inhibited by acetyl-CoA (more is bound to enzyme so less needs to be made – reaction doesn’t proceed forward) E3 inhibited by NADH Covalent: E1 inhibited by PDH kinase phosphorylation - Kinase activated by high amounts of ATP, NADH and acetyl-CoA (products of reaction – less need to be formed if already in high amounts) - Kinase inhibited by pyruvate and ADP (reactants of reaction – more ATP needed to be produced for fuel) Activated by PDH phosphatase dephosphorylation - Phosphatase activated by calcium and magnesium in abundance - Calcium is an important signalling molecule for muscle contraction (high amounts means high amounts of ATP needed) Downloaded by Aa Ab ([email protected])  lOMoARcPSD|18519470 - Magnesium binds strongly to ATP (more free magnesium = less ATP in cell – more needed to be made) - Activated by insulin (stimulates glycolysis, glycogenesis and lipogenesis – acetyl-CoA is a precursor of lipogenesis so PDH is activated to help produce it in adipocytes and hepatocytes) TCA cycle used to oxidize (remove hydrogen from) all metabolic fuels - Oxygen is ultimate acceptor of electrons in electron transport chain (reoxidizes coenzymes that were reduced in the Krebs cycle and is necessary for cycle to move forward) TCA cycle is made of 8 reactions (3 are irreversible) - Produces 1 GTP, 3 NADH, 1 FADH2 and 2 CO2 for every acetyl-CoA 1. Acetyl-CoA reacts with oxaloacetate in citrate (hydrolysis of thiolester bond pushes reaction forward – irreversible) - Catalyzed by citrate synthase 2. Isomerization of citrate into isocitrate - Catalyzed by aconitase - Reversible and occurs in 2 steps 3. Decarboxylation of isocitrate into alpha-ketoglutarate via isocitrate dehydrogenase with NAD+ - One CO2 and one NADH produced - Irreversible reaction - Done in 2 steps 4. Oxidative decarboxylation of alpha-ketoglutarate into succinyl-CoA - Via alpha-ketoglutarate dehydrogenase - One CoA consumed - One CO2 and one NADH generated - Irreversible reaction 5. GDP phosphorylation by transformation of succinyl-CoA into succinate - Via succinyl-CoA synthetase - Produces one GTP - Reversible 6. Dehydrogenation of succinate into fumarate - Via succinate dehydrogenase and FAD as cofactor - FADH2 is produced - Reversible reaction 7. Fumarate rehydrated into L-malate via fumarase Downloaded by Aa Ab ([email protected])  lOMoARcPSD|18519470 - One H2O consumed - Reversible 8. Dehydrogenation of L-malate into oxaloacetate via malate dehydrogenase with NAD+ as coenzyme - Produces one NADH from oxidation of malate - Reversible (oxaloacetate in low concentration so citrate can be produced from reaction with acetyl-CoA) - High oxaloacetate concentrations: reaction moves the opposite way and is converted into malate (malate eventually converted back into oxaloacetate) TCA cycle is regulated through - Speed at which PDH converts pyruvate into acetyl-CoA - Speed of the 3 irreversible reactions (determined by concentrations of oxaloacetate and acetyl- CoA – regulates activity of citrate synthase) High accumulation of citrate inhibits citrate synthase by blocking the catalytic pocket - Slows down pyruvate dehydrogenase activity so less acetyl-CoA is made and reaction decreases - High amounts of succinyl-CoA have the same effect on citrate synthase and a-ketoglutarate dehydrogenase - Citrate accumulation also allosterically inhibits PFK in glycolysis (increases effect of ATP – citrate acts as intracellular signal once energetic needs of cell are satisfied) Calcium is an allosteric activator - Produced during muscle contractions (energy is needed to contract muscles) - Activates the TCA cycle to produce more ATP for energy Amphibolic pathway involves catabolic and anabolic processes - Anaplerotic: reactions that replenish TCA cycle intermediates - Caraplerotic: reactions that use up TCA intermediates to form amino acids and other biomolecules (eg. glucose) Majority of energy generated from TCA cycle actually becomes available to the cell through reoxidation of coenzymes FADH2 and NADH via the electron transport chain - 1 NADH produces about 2.5 ATP (10 protons pumped out in total) - 1 FADH2 produces about 1.5 ATP (6 protons pumped out in total) Only 4 ATP/glucose molecule, 2 from glycolysis and 2 from the TCA cycle - 90% of free energy from glucose is stored in the reduced coenzymes - Reoxidization of these coenzymes through oxidative phosphorylation is used to synthesize ATP Downloaded by Aa Ab ([email protected])  lOMoARcPSD|18519470 Electrons transferred to a series of electron carriers from the electron transport chain (ETC) found in the mitochondrial inner membrane - Energy generated from the movement of electrons used to pump protons into the intermembrane space - Protons moved back into the matrix through the ATP synthase (phosphorylates ADP to produce ATP in process) Energy is thought to be generated this way via the chemiosmotic theory - Chemical energy through oxidation is converted into osmotic energy through transmembrane protein complexes (proton pumps) in the mitochondria - Osmotic energy used to create a proton gradient between the intermembrane space and matrix - Osmotic energy is converted back into chemical energy through ATP synthase Reduction potential values of the ETC electron carriers increases in a sequence that corresponds to their position in the chain - Enthalpy decreases (becomes more negative) with each carrier - Binding affinity increases with each carrier - Oxygen is the final electron acceptor and gets reduced to water in the process Complex 1: NADH-dehydrogenase 2 electrons received from NADH and transferred to coenzyme Q via the FMN (flavin mononucleotide) coenzyme and 8 iron-sulfur clusters - 4 protons are pumped into the intermembrane space using this energy - Coenzyme Q is a mobile transporter and can carry electrons to different complexes - Fe-S cluster can only carry one electron at a time - Ubiquinone (fully oxidized Q) converted into semiquinone radical intermediate (only carries one electron) which is converted into ubiquinol (fully reduced Q) - Ubiquinol can diffuse to complex 3 Complex 2: succinate dehydrogenase (same reaction that catalyses TCA reaction #6) - Direct link between TCA cycle and ETC - Electrons transferred from FADH2 through the Fe-S clusters to coenzyme Q - No proton pump in complex 2 (no protons get pumped into inner space – all electrons moved to complex 3) Complex 3: 2 electrons transferred from ubiquinol to cytochrome c via cytochrome c1 and Fe-S proteins - 2 protons pumped for every electron into the intermembrane space - 2 reduced cytochrome c are produced for every ubiquinol (cytochrome c can only carry one electron at a time) - 1 electron stored on one cytochrome c (produces semiquinone Q) and 2 nd cytochrome c comes to accept 2nd electron (produces fully oxidized ubiquinone Q) Downloaded by Aa Ab ([email protected])  lOMoARcPSD|18519470 - Ubiquinone reused to donate more electrons in next round of cellular respiration Complex 4: cytochrome c oxidase (final stage of ETC) Homodimer with 3 subunits - 1st subunit: 2 heme groups (a and a3) and 1 copper ion (CuB) - 2nd subunit: 2 copper ions Cytochrome c transfers 2 electrons to oxygen (oxygen reduction produces water) - 1 proton pumped for every electron into the intermembrane space via proton channel Complex 5: ATP synthase Made of F0F1 complex F0 is composed of roughly 14 integral membrane proteins forming the base and the peripheral stalk of the ATP synthase (acts as proton “turbine”) - 8-15 c subunits, one a subunit (where protons enter) and two b subunits - Protons entering A unit bind to one of the c rings (1 proton for every c ring) and causes its rotation - After 1 full turn: protons leave the synthase through a different part of the A unit - P side = intermembrane side, N side = matrix side F1 is composed of 5 proteins, designated a, b, d, e and g, forming the knob and the central stalk. - 3 alphabeta, 1 gamma, 1 delta and 1 epsilon - ATP synthase activity All parts of synthase rotate except for A, B and alphabeta subunits (stationary) Rotation of gamma subunit due to protons moving through F0 causes confirmation changes in the alpha beta dimers that alters their substrate-binding abilities - 3 different confirmations: open (empty – available for ADP to bind after ATP release), loose (loosely bound to ADP and Pi) and tight (ATP formation) The inner mitochondrial membrane is impermeable to NADH - Electrons must be carried through the membrane through a different carrier Malate-aspartate shuttle used in liver, heart and kidney mitochondria - Electrons received reduces oxaloacetate into malate via malate dehydrogenase - Malate transported into matrix via malate-a-ketoglutarate transporter - Once in matrix: malate reoxidized into oxaloacetate Downloaded by Aa Ab ([email protected])  lOMoARcPSD|18519470 - Oxaloacetate transaminated into aspartate via aspartate aminotransferase - Aspartate transported out of matrix via glutamate-aspartate transporter - Aspartate transaminated into oxaloacetate Glycerol-3-phosphate shuttle used in the skeletal muscles and brain - Receiving electrons reduces DHAP to glycerol-3-phosphate via cytosolic glycerol-3-phosphate dehydrogenase - Glycerol-3-phosphate enters intermembrane space - Glycerol-3-phosphate reoxidized into DHAP - Electrons from oxidation stored on FAD and transferred to ubiquinone to make ubiquinol Shuttle bypasses complex 1 and 2 of the ETC Oxidative phosphorylation uses a chemiosmotic coupling mechanism - Uncoupling occurs when protons are returned to the matrix other then by the ATP synthase - Chemical uncouplers/endogenous uncoupling proteins allow protons to bypass ATP synthase and return to the matrix by other routes - Eg. thermogenin in inner mitochondrial membrane of brown adipose tissue allows heat production through proton movement – produces energy for heat regulation in infants and mammals during hibernation - Thermogenin keeps hibernating animal brains stimulated in case of predators or increased outdoor temperature (signals end of hibernation) Chemical uncoupling: DNP (2,4-dinitrophenol) - Generates heat like UCP1 aka thermogenin does - Done through the transport of protons across the membrane DNP was used in the 1930s as a weight loss supplement, unfortunately, it had major side effects; causing blindness (development of cataract), hyperthermia and even death. Inhibitors of oxidative phosphorylation affect certain cofactors involved in each complex 1. Complex I inhibitor: Rotenone (pesticide, insecticide and nonselective piscicide) - Blocks all electrons from NADH from moving through ETC 2. Complex III inhibitor: Antimycin A (Streptomyces antibiotic) 3. Complex IV inhibitors: Carbon monoxide and cyanide - Backs up entire system (ubiquinol fully saturated – can’t accept anymore electrons) - No proton gradient gets made (ubiquinol can’t be reoxidized to accept new electrons – no ATP gets made and cell eventually dies) Downloaded by Aa Ab ([email protected])