Metabolism II - Biochemistry PDF

15 Khaled Samer Leen Qasim Diala Abu-Hassan Gluconeogenesis - Gluconeogenesis occurs in specific sites, highest percentage in liver with some contribution of the kidneys. - In this pathway, we are in an emergency state, so we don’t specify one precursor to use in gluconeogenesis, in fact we use seve...

15 Khaled Samer Leen Qasim Diala Abu-Hassan Gluconeogenesis - Gluconeogenesis occurs in specific sites, highest percentage in liver with some contribution of the kidneys. - In this pathway, we are in an emergency state, so we don’t specify one precursor to use in gluconeogenesis, in fact we use several precursors. ▪ The first one is lactate that comes out of anaerobic respiration that happens in RBCs and exercising muscles, it’s converted to pyruvate by lactate dehydrogenase. ▪ Another precursor is glucogenic amino acids (the majority of aa are glucogenic) and the easiest one to enter into this pathway is alanine because it can be transaminated (remove amino group) so it becomes pyruvate (the keto acid of alanine) *Where does this happen? It happens by degrading the proteins in the muscles releasing their amino acids and these amino acids can be used as precursors for gluconeogenesis. ▪ The third precursor is glycerol which is going to be released once the fatty acids in the adipocytes are hydrolyzed, move through the bloodstream on albumin and it’ll be distributed to different cell types, these cells are going to use fatty acids as a source of energy. (except the brain, medulla and RBCs) → Where and when does gluconeogenesis occur? - During an overnight fast (one day) ~ 90% of gluconeogenesis occurs in the liver and 10% by the kidneys - During prolonged fasting (for weeks, starvation) kidneys become more glucose-producing organs (40% of total glucose production), the liver still the major contributor. → How can these different precursors enter into the pathway? They can enter at different stages: ▪ Lactate and alanine can be converted to pyruvate in the very first step of pathway. ▪ Some amino acids like aspartate can be degraded to oxaloacetate which is the 2nd intermediate in the pathway. ▪ Glycerol that has three carbons is expected to enter at step 3 where 3 carbon molecules appear, it enters as a triose with phosphate, specifically dihydroxyacetone phosphate. These are the main substrates of gluconeogenesis, but we can use other carbs and convert them to glucose, but we don’t consider this as gluconeogenesis. Why? Because the definition of gluconeogenesis is the synthesis of glucose out of non-carb precursors. - Other monosaccharides can be dealt with by converging to some intermediates in the glycolytic pathway. - fructose enters into the glycolytic pathway as triose phosphate. - Galactose enters as glucose directly, because of similar Isomerization. Let’s take an overview to the pathway, as mentioned before, it’s the opposite of glycolysis. Glycolysis is composed of 10 steps, seven of which are reversible and three are irreversible, all the reversible steps are going to be reversed by the same enzymes, whereas the 3 irreversible steps will be reversed by different enzymes. - The step of glucose phosphorylation to Glucose 6-phosphate that was catalyzed by glucokinase in glycolysis is reversed (dephosphorylation) by phosphatase. - Whereas the phosphorylation of Fructose 6-phosphate to Fructose 1,6- bisphosphate that was catalyzed by phosphofructokinase-1 in glycolysis, is reversed by phosphatase in gluconeogenesis. - The last step in glycolysis (which is the first step of gluconeogenesis) is converting Phosphoenolpyruvate to pyruvate by pyruvate kinase in glycolysis. - In gluconeogenesis it is reversed in two steps, the first one converts pyruvate to oxaloacetate and then oxaloacetate is converted to Phosphoenolpyruvate, so overall, we have 11 steps in gluconeogenesis versus 10 steps in glycolysis. ❖ Reversing the irreversible steps: 1. From pyruvate to phosphoenolpyruvate (PEP) - In glycolysis, pyruvate is formed in the cytosol. whereas the pyruvate which will enter gluconeogenesis exists in either cytosol or mitochondria. - In mitochondria, the first reaction is the carboxylation of pyruvate to oxaloacetate by pyruvate carboxylase. - And this needs ATP, CO2 and biotin, oxaloacetate have to exit mitochondria because the enzyme of step #3 (#9 in glycolysis) is cytosolic, but oxaloacetate cannot cross the mitochondrial membrane, so it’s reduced to malate which can cross it. Oxaloacetate (OAA) to phosphoenolpyruvate (PEP) - As you can see, pyruvate carboxylase is carrying biotin and biotin’s function here is to carry CO2 that will be added to pyruvate. - Pyruvate carboxylase can be activated by acetyl CoA, WHY acetyl CoA? Gluconeogenesis occurs when the cell is in fasting state, under this condition, these cells get their energy from fatty acids, when fatty acids are degraded, this produces big amounts of acetyl CoA that cannot be managed by kreb’s cycle, so some of it stay as acetyl CoA and it will activate this process. - When malate arrives at the cytosol, it will be oxidized into oxaloacetate and NAD+ will be reduced to NADH, then oxaloacetate is converted to Phosphoenolpyruvate by removing carboxyl group and adding phosphate this is done by PEP carboxykinase enzyme. - This Enzyme is found in both cytosol and mitochondria. o The generated PEP in the mitochondria is transported to the cytosol by a specific transporter o The PEP that is generated in the cytosol requires the transport of OAA from the mitochondria to the cytosol Notice that Fructose 1,6 bisphosphatase is the opposite of phosphofructokinase 1. 2. From fructose-1,6-bisphosphate to fructose-6-phosphate Fructose 1,6-bisphosphatase removes phosphate from carbon #1 as inorganic phosphate, this enzyme is regulated negatively by these allosteric regulators: 1) AMP 2) Fructose 2,6 bis-phosphate Which are the positive regulators for phosphofructokinase 1 3. From glucose-6-phosphate to glucose - Dephosphorylation of glucose 6- phosphate by Glucose 6phosphatase which is present inside the Endoplasmic Reticulum. - Once Glucose 6-phosphate is formed in cytosol it is going to move into ER via glucose 6-phosphate Translocase. Notice that Glucose 6-phosphatase is the opposite of Hexokinase/Glucokinase - Glucose that is formed in the ER is transported by the glute 7 (which exists on ER membrane) to the cytosol of the hepatocyte/ kidney cell, then they exit the cell through other types of glutes (e.g. glute 2) present on the cell membrane, then glucose arrives in the blood stream, from there it can maintain its levels and also supply the tissues that’s exclusively dependent on glucose as a source of energy. - Muscle lacks glucose 6-phosphatase, and therefore muscle glycogen cannot be used to maintain blood glucose levels. - Only occurs in kidney and liver ❖ Comparison between enzymes in glycolysis versus in gluconeogenesis: - In glycolysis, phosphorylation of glucose by hexokinase used ATP to provide energy and phosphate group, notice that ΔG in phosphorylation is positive that’s why we have to couple this reaction with ATP hydrolysis to make the net ΔG negative. - By logic, the opposite reaction (dephosphorylation of Glucose 6-phosphate) that occurs by the phosphatase, already has a negative ΔG, that’s why we didn’t use ATP. → Energy requirements of gluconeogenesis ▪ In the converting of 1,3 bisphosphophoglycerate to glyceraldehyde 3-P, inorganic phosphate is released and 2 NADH are oxidized into 2 NAD+ ▪ In the converting of 3-phosphoglycerate to 1,3 bis-phosphoglycerate we use 2 ATP that are hydrolyzed to 2 ADP. ▪ in the converting of pyruvate to oxaloacetate we consume energy due to the carboxylase and we have to repeat the reaction twice, that’s why 2 ATP are consumed. ▪ from oxaloacetate to phosphoenolpyruvate we consumed 2 GTP. To sum up: 6 ATPs were hydrolyzed to ADP to make 1 molecule of glucose Remember: When gluconeogenesis occurs, the body relies on fatty acids as a source of energy. ❖ Regulation of gluconeogenesis: Generally, the allosteric regulators of gluconeogenesis are opposite of glycolysis, to guarantee that the two opposite pathways are not work at the same time. Mainly by: 1. The circulating level of glucagon. Hormonal regulation mainly depends on glucagon, because it’s the dominating hormone while fasting. So it would be high in concentration, then it attaches to GPCR activating cAMP synthesis, then protein kinase A. - Glucagon lowers the level of fructose 2,6bisphosphate, resulting in activation of fructose 1,6bisphosphatase and inhibition of PFK-1 - Inhibition of pyruvate kinase - Glucagon increases the transcription of the gene for PEP-carboxykinase 2. The availability of gluconeogenic substrates. 3. Slow adaptive changes in enzyme activity due to an alteration in the rate of enzyme synthesis or degradation, or both ‫ال تنسوا أهلنا يف غزة من دعائكم‬ THE END OF SHEET #15