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Dr. Abir Alghanouchi Biochemistry department Sciences college All carbohydrates to be catabolized must enter the glycolytic pathway Glycolysis is degradation of glucose to generate energy (ATP) and to provide pyruvate (in the presence of oxygen) or lactate (in the absence of oxygen) Glycolysis is central in generating both energy and metabolic intermediates. Site: Glycolysis takes place in the cytoplasm of all cells in the body but it is of physiological importance in: 1. Tissues with no mitochondria: mature RBCs, cornea and lens 2. Tissues with few mitochondria: Testis, leucocytes, medulla of the kidney, retina, skin and gastrointestinal tract 3. Tissues undergo frequent oxygen lack: skeletal muscles especially during exercise Biological importance of glycolysis: glycolysis: 1. Energy production: Under anaerobic conditions: glycolysis gives 2 ATP Under aerobic glycolysis: glycolysis gives 8 ATP 2. Oxygenation of tissues: Through formation of 2,3 bisphosphoglycerate, which decreases the affinity of Hemoglobin to O2. 3. Provides important intermediates: Dihydroxyacetone phosphate: can give glycerol3phosphate, which is used for synthesis of TGs and PLs (lipogenesis). 3 Phosphoglycerate: which can be used for synthesis of amino acid serine. Pyruvate: which can be used in synthesis of amino acid alanine. 4. Aerobic glycolysis provides the mitochondria with pyruvate, which gives acetyl CoA Krebs' cycle. Steps: There are 10 enzyme-catalyzed reactions There are two stages : Stage 1: (Reactions 1-5) A preparatory stage in which glucose is phosphorylated, converted to fructose which is again phosphorylated and cleaved into two molecules of glyceraldehyde-3phosphate. In this phase there is an investment of two molecules of ATP Steps: Stage 2: (reactions 6-10) The two molecules of glyceraldehyde-3-phosphate are converted to pyruvate with concomitant generation of four ATP molecules and two molecules of NADH. Thus there is a net gain of two ATP molecules per molecule of Glucose in glycolysis. 6 CH2OH 5 H 4 OH O H OH H 2 3 H OH glucose 6 CH OPO 2− 2 3 ATP ADP H H 1 OH 5 4 Mg2+ OH Hexokinase O H OH 3 H H 2 H 1 OH OH glucose-6-phosphate 1. Phosphorylation of glucose: Hexokinase catalyzes: Glucose + ATP glucose-6-P + ADP ATP binds to the enzyme as a complex with Mg++ A phosphoanhydride bond of ATP (~P) is cleaved ADP 6 CH2OH 5 H 4 OH O H OH H 2 3 H OH glucose 6 CH OPO 2− 2 3 5 O ATP ADP H H 1 OH 4 Mg2+ OH H OH 3 H 2 H 1 OH Hexokinase H OH glucose-6-phosphate The reaction catalyzed by Hexokinase is irreversible (glucose-6-P can not diffuse out of the cell because there are no specific carriers for phosphorylated sugars) This reaction is catalyzed by several isoenzymes of hexokinase and glucokinase: both requires Mg2+ as a cofactor Comparison between glucokinase and hexokinase enzymes: Glucokinase Hexokinase Site Liver only All tissue cells Affinity to glucose Low affinity (high km) i.e. it acts only in the presence of high blood glucose concentration. High affinity (low km) i.e. it acts even in the presence of low blood glucose concentration. Substrate Glucose only Glucose, galactose and fructose Effect of insulin Induces synthesis of glucokinase. No effect Effect of glucose-6-p No effect Allosterically inhibits hexokinase Function Acts in liver after meals. It removes glucose coming in portal circulation, converting it into glucose -6-phosphate. It phosphorylates glucose inside the body cells. This makes glucose concentration more in blood than inside the cells. This leads to continuous supply of glucose for the tissues even in the presence of low blood glucose concentration. 6 CH OPO 2− 2 3 5 O H 4 OH H OH 3 H H 2 OH H 1 OH 6 CH OPO 2− 2 3 1CH2OH O 5 H H 4 OH HO 2 3 OH H Phosphoglucose Isomerase glucose-6-phosphate fructose-6-phosphate 2. Isomerization of glucose-6-P: Phosphoglucose Isomerase catalyzes: glucose-6-P (aldose) fructose-6-P (ketose) It is not rate-limiting or regulated step Phosphofructokinase 6 CH OPO 2− 2 3 O 5 H H 4 OH 6 CH OPO 2− 2 3 1CH2OH O ATP ADP HO 2 3 OH H fructose-6-phosphate 5 Mg2+ 1CH2OPO32− H H 4 OH HO 2 3 OH H fructose-1,6-bisphosphate 3. Phosphorylation of fructose-6-P Phosphofructokinase 1 (PFK-1) catalyzes: fructose-6-P + ATP fructose-1,6-bisP + ADP Rate-limiting step PFK-1 is an allosteric enzyme, it is inhibited allosterically by elevated levels of ATP 2− 1CH2OPO3 2C O HO 3C H 4C H H H 2− CH OPO 2 3 3 Aldolase OH 2C OH 1CH2OH 2− OPO CH 2 3 6 dihydroxyacetone phosphate 5 C fructose-1,6bisphosphate O + O 1C H 2C OH 2− 3 CH2OPO3 glyceraldehyde-3phosphate Triosephosphate Isomerase 4. Cleavage of fructose-1,6-bisP: Aldolase catalyzes: fructose-1,6-bisphosphate dihydroxyacetone-P + glyceraldehyde-3-P The reaction is reversible Aldase A occurs in most tissues Aldase B occurs in liver and kidney 1CH2OPO3 2C O HO 3C H 4C H H 2− H Aldolase 2− CH OPO 2 3 3 OH 2C OH 1CH2OH 2− CH OPO 2 3 6 dihydroxyacetone phosphate 5 C fructose-1,6bisphosphate O + O 1C H 2C OH 2− CH OPO 3 2 3 glyceraldehyde-3phosphate Triosephosphate Isomerase 5. Isomerization of dihydroxyacetone phosphate: Triose Phosphate Isomerase (TIM) interconverts: dihydroxyacetone-P glyceraldehyde-3-P Two molecules of glyceraldehyde-3-P bare produced for each glucose Summary of Second Stage of Glycolysis (Energy Investment) Glucose + 2 ATP ——> 2 GA3P + 2 ADP + 2 H+ Recall that there are 2 GAP per glucose Glyceraldehyde-3-phosphate Dehydrogenase H O 1C H 2 C OH OPO32− + H+ O NAD+ NADH 1C + Pi H C OH 2− CH OPO 2 3 3 glyceraldehyde3-phosphate 2 2− OPO CH 2 3 3 1,3-bisphosphoglycerate 6. Oxidation of glyceraldehyde-3-phosphate Glyceraldehyde-3-phosphate Dehydrogenase catalyzes: glyceraldehyde-3-P + NAD+ + Pi 1,3-bisphosphoglycerate + NADH +H+ High energy compound Glyceraldehyde-3-phosphate Dehydrogenase H O 1C H 2 C OH OPO32− + H+ O NAD+ NADH 1C + Pi H C OH 2− OPO CH 2 3 3 glyceraldehyde3-phosphate 2 2− CH OPO 2 3 3 1,3-bisphosphoglycerate This is the only step in Glycolysis in which NAD+ is reduced to NADH NAD+ is the cofactor in this reaction which acts as an oxidizing agent Glyceraldehyde-3-P Dehydrogenase is a tetrameric enzyme with 4SH group on each polypeptide chain (one SH in its active site) Glyceraldehyde-3-P Dehydrogenase is inhibited by iodoacetate Phosphoglycerate Kinase O− OPO32− ADP ATP O O 1C H 2C OH 2− 3 CH2OPO3 1,3-bisphosphoglycerate C 1 Mg 2+ H 2C OH 2− 3 CH2OPO3 3-phosphoglycerate 7. Formation of ATP from 1,3 BPG and ADP Phosphoglycerate Kinase catalyzes the Transfer of phosphoryl group fron 1,3 bisphosphoglycerate to ADP generating ATP: 1,3-bisphosphoglycerate + ADP 3-phosphoglycerate + ATP phosphate transfer is reversible, since one ~P bond is cleaved & another synthesized This Phosphoglycerate Kinase 1C H 2C OH 2− 3 CH2OPO3 1,3-bisphosphoglycerate O− OPO32− ADP ATP O O C 1 Mg 2+ H 2C OH 2− 3 CH2OPO3 3-phosphoglycerate 2 molecules of ATP are produced (by Substrate-level phosphorylation) Recall every molecule of glucose gives rise to 2 trioses!!! Substrate level phosphorylation This means phosphorylation of ADP to ATP at the reaction itself In glycolysis there are 2 examples: o 1.3 Bisphosphoglycerate + o ADP 3 Phosphoglycerate + ATP Phospho-enol pyruvate + ADP Enolpyruvate + ATP Phosphoglycerate Mutase O− O O− O C C 1 1 H 2C OH 2− 3 CH2OPO3 H 2C OPO32− 3 CH2OH 3-phosphoglycerate 2-phosphoglycerate 8. Shift of the P group from C3 to C2 Phosphoglycerate Mutase catalyzes the Conversion of 3-phosphoglycerate to 2-phosphoglycerate (2-PG). 3-phosphoglycerate 2-phosphoglycerate It is a freely reversible reaction Enolase − O O C 2 C − − O O C 1 H H+ OPO32− 3 CH 2OH C OH − O O − 1C OPO32− CH 2OH 2C O PO32− 3 CH 2 2-phosphoglycerate enolate intermediate phosphoenolpyruvate 9. Dehydration of 2-P-glycerate to phosphoenolpyruvate Enolase catalyzes: 2-phosphoglycerate phosphoenolpyruvate + H2O High energy compound dehydration reaction is Mg++-dependent and reversible Enolase is inhibited by fluoride To measure glucose level in blood, fluoride is added to inhibit Enolase and stop glycolysis This Pyruvate Kinase O− O C 1 C 2 ADP ATP O− O C 1 OPO32− 3 CH2 phosphoenolpyruvate C 2 O 3 CH3 pyruvate 10. Formation of pyruvate Pyruvate Kinase catalyzes the transfer of phosphoryl group from PEP to ADP generating ATP and Pyruvate phosphoenolpyruvate + ADP pyruvate + ATP This enzyme requires Mg++ and K+ Irreversible reaction Pyruvate Kinase O− O C 1 C 2 ADP ATP O− O C 1 OPO32− 3 CH2 phosphoenolpyruvate C 2 O 3 CH3 pyruvate This phosphate transfer from PEP to ADP is spontaneous (the free energy of PEP hydrolysis is coupled to the synthesis of ATP) This is the second substrate level phosphorylation reaction of glycolysis Summary of Second Stage of Glycolysis 2 GA3P + 2 NAD+ + 4 ADP + 2 Pi 2 Pyruvate + 2 NADH + 2 H+ + 4 ATP Summary of Glycolysis Glucose + 2 NAD+ + 2 ADP + 2 Pi 2 Pyruvate + 2 NADH + 2 H+ + 2 ATP can directly be used for doing work or synthesis NOTE: NAD+ must be regenerated for glycolysis to proceed! Balance sheet for ~P bonds of ATP: How many ATP ~P bonds expended? ________ How many ~P bonds of ATP produced? (Remember there are two 3C fragments from glucose.) ________ production of ~P bonds of ATP per glucose: ________ Net What happens next? Under the aerobic condition: pyruvate is catabolized further in mitochondria through pyruvate dehydrogenase and citric acid cycle where all the carbon atoms are oxidized to CO2. The free energy released is used in the synthesis of ATP, NADH and FADH2. Under anaerobic condition: In absence of oxygen, NADH+ H+ is not oxidized by the respiratory chain. Pyruvate is converted to Lactate in homolactic fermentation or in ethanol in alcoholic fermentation to regenerate NAD+. This helps continuity of glycolysis, as the generated NAD+ will be used once more for oxidation of another glucose molecule (step6). Homolactic Fermentation: Lactate Dehydrogenase O− O C C NADH + H+ NAD+ O O− O C HC OH CH3 CH3 pyruvate lactate Skeletal muscles ferment glucose to lactate during exercise, when the exertion is brief and intense. Lactate dehydrogenase (LDH) reduces pyruvate to lactate using NADH and thereby oxidizing it to NAD+ NAD+ is regenerated by lactic fermentation to carry out GAPDH reaction of glycolysis Cell membranes contain carrier proteins that facilitate transport of lactate Lactate Dehydrogenase O− O C C NADH + H+ NAD+ O O− O C HC OH CH3 CH3 pyruvate lactate Lactate released to the blood may be taken up by other tissues, or by skeletal muscle after exercise, and converted via Lactate Dehydrogenase back to pyruvate, which may be oxidized in Krebs Cycle or (in liver) converted to back to glucose via gluconeogenesis Lactate Dehydrogenase O− O C C NADH + H+ NAD+ O O− O C HC OH CH3 CH3 pyruvate lactate Lactate serves as a fuel source for cardiac muscle as well as brain neurons. Astrocytes, which surround and protect neurons in the brain, ferment glucose to lactate and release it. Alcoholic fermentation Pyruvate Decarboxylase Alcohol Dehydrogenase CO2 NADH + H+ NAD+ O− O C C O CH3 pyruvate H O C CH3 acetaldehyde H H C OH CH3 ethanol Microorganisms and yeast convert pyruvate to ethanol, which is excreted as a waste product, and carbon dioxide to regenerate NAD+ for glycolysis NADH is converted to NAD+ in the reaction catalyzed by Alcohol Dehydrogenase. Pyruvate Decarboxylase Alcohol Dehydrogenase CO2 NADH + H+ NAD+ O− O C C H C O CH3 CH3 pyruvate O acetaldehyde H H C OH CH3 ethanol It is a two step process: 1. Pyruvate decarboxylase (PDC) reaction: reaction: This enzyme is Mg++dependent and requires an enzyme-bound cofactor, thiamine pyrophosphate (TPP). In this reaction a molecule of CO2 is released producing acetaldehyde. 2. Alcohol dehydrogenase reaction: Acetaldehyde is reduced to ethanol using NADH as reducing power, thus regenerating NAD+ Cytosol Mitochondrion Glycolysis Glucose 2 Pyruvic acid 2 AcetylCoA Krebs Cycle Electron Transport Maximum per glucose: by direct synthesis by direct synthesis by ATP synthase Energy production of glycolysis: glycolysis: ATP produced ATP consumed Net energy In absence of oxygen (anaerobic glycolysis) 4 ATP: (Substrate level phosphorylation) 2ATP from 1,3 DPG. 2ATP from phosphoenol pyruvate 2ATP 2 ATP/ Glucose Molecule From glucose to glucose -6-p. From fructose -6-p to fructose 1,6 p. In presence of oxygen (aerobic glycolysis) 4 ATP (substrate level phosphorylati on) 2ATP from 1,3 BPG. 2ATP from phosphoenol pyruvate. + 6ATP (from oxidation of 2 NADH + H+ in mitochondria). 2ATP -From glucose to glucose -6-p. -From fructose -6-p to fructose 1,6 p. 8 ATP/ Glucose Molecule Differences between aerobic and anaerobic glycolysis Aerobic Anaerobic End product Pyruvate Lactate/ethanol Energy 8 ATP 2 ATP Regeneration of NAD+ Through respiration chain in mitochondria Availability to TCA in mitochondria Through Lactate /ethanol formation Available and 2 Pyruvate Not available as lactate is cytoplasmic substrate can oxidize to give 30 ATP Special features of glycolysis in RBCs Mature RBCs contain no mitochondria, thus: o They depend only upon glycolysis for energy production (=2 ATP). o Lactate is always the end product. Glucose uptake by RBCs is independent on insulin hormone. Reduction of met-hemoglobin: Glycolysis produces NADH+H+, which used for reduction of met-hemoglobin in red cells. In most cells 2,3 bisphosphoglycerate is present in trace amount, but in erythrocytes it is present in significant amount: In red cells 1,3 BPG is converted to 2,3BPG which unites with oxy Hb and helps release of oxygen at tissues. Regulation of Glycolysis There are three steps in glycolysis that have enzymes which regulate the flux of glycolysis These enzymes catalyzes irreversible reactions of glycolysis The hexokinase (HK) II. The phoshofructokinase (PFK) III. The pyruvate kinase I. They are regulatory enzymes which are regulated by the level of ATP in the cell I- PhosphofructokinasePhosphofructokinase-1 (PFK(PFK-1): It is the most important regulatory enzyme which catalyzes the first irreversible reaction unique to the glycolytic pathway (the committed step) It is an allosteric enzyme inhibited by elevated level of ATP, which: is the end product of glycolysis as well as it is substrate for PFK-1 o At high [ATP], PFK has lower affinity for the other substrate, fructose-6-P. ATP binds to inhibition site of PFK, and thereby decreases the activity of enzyme. o Sigmoidal dependence of reaction rate on[fructose-6-P] is seen. AMP, present at significant levels only when there is extensive ATP hydrolysis, antagonizes effects of high ATP. AMP, ADP and Fructose 2, 6 biphosphate act as allosteric activators of PFK-1. II II-- Hexokinase It is allosterically inhibited by its product Glucose 6 phosphate. In liver, glucokinase is inhibited by Fructose 6P and ATP (acts as a competitive inhibitor of this enzyme) III-- Pyruvate Kinase III It is allosterically inhibited by ATP. ATP binding to the inhibitor site of PK decreases its ability to bind to PEP the substrate. It is also inhibited by Acetyl Coenzyme A and long chain fatty acid because they are source rich ATP which inhibits PK. Hormonal regulation of glycolysis Insulin and Glucagon (secreted by the pancreas) are the main endocrine that modulate blood glucose levels and they act antagonistically Insulin is secreted in hyperglycemia and after carbohydrates feeding, it causes: 1. Induction for synthesis of glycolytic key enzyme 2. Activation of protein phosphatase 1 producing dephosphorylation and activation of glycolytic key enzymes Glucagon is secreted in hypoglycemia or in CHO deficiency and it affects liver cells mainly as follows: 1. It acts as repressor of glycolytic key enzymes (PFK1, Pyruvate kinase, glucokinase) 2. It produces phosphorylation of specific enzymes leading to inactivation of glycolytic key enzymes 2-deoxyglucose: inhibits hexokinase Mercury and iodoacetate: inhibit glyceraldehyde-3-P dehydrogenase Fluoride: inhibits enolase by removal of Mg2+ as Mg fluoride Arsenate: is uncoupler of oxidation and phosphorylation, it forms 1-arseno-3-phosphoglycerate which interferes with ATP formation at substrate level It is the inhibition of glycolysis by the presence of oxygen Explanation: Aerobic oxidation of glucose produces increased amount of ATP and citrate. Those inhibit PFK1