Introduction to Metabolism & Glycolysis 2023-2024 PDF

Introduction to Metabolism & Glycolysis Dr Lynn O’Connor, 2023-2024 1  Metabolism Enzymatic reactions are not isolated Occur in pathways Product of one rxn is the substrate for the next – assembly line Differ...

Introduction to Metabolism & Glycolysis Dr Lynn O’Connor, 2023-2024 1  Metabolism Enzymatic reactions are not isolated Occur in pathways Product of one rxn is the substrate for the next – assembly line Different pathways intersect COLLECTIVELY CALLED METABOLISM CATABOLIC –break down complex molecules to simpler components ANABOLIC – Synthesize complex molecules 2 Metabolic Map Each pathway composed of multienzyme sequences Each enzyme may have catalytic or regulatory features Note: consequences of blockage in the flow e.g. by drugs by genetic/metabolic disorders 3  Catabolic pathways Functions 1. Capture ATP from energy rich molecules 2. Convert molecules to building blocks of other components 4 3 stages of catabolism 5  Anabolic pathways Combine Combine simple components into complex ones e.g. amino acids to form complex proteins Require energy (endergonic)-usually ATP which is converted to ADP and Pi Often needs reducing power NADPH Catabolism is a convergent process Anabolism is a divergent process 6  Regulation of Metabolism Intracellular communication Intercellular communication Second messenger systems Adenylyl cyclase a) GTP-dependent regulatory proteins b) Protein kinases c) Dephosphorylation of proteins d) Hydrolysis of cAMP 7  Overview of glycolysis All tissues use this pathway Function a) Oxidize glucose to provide energy in the form of ATP b) Provides intermediates for other metabolic pathways Glycolysis is at the hub of CHO metabolism -virtually all sugars are converted to glucose 8 Aerobic Glycolysis: Occurs in cells with mitochondria and with an adequate supply of oxygen – pyruvate is the end product Anaerobic glycolysis: Pyruvate is converted to lactate as NADH is oxidized to NAD+. Occurs without the need for oxygen - Important in tissue with no mitochondria e.g. red blood cells and also in anoxic tissues 9  Transport of Glucose into Cells A. Sodium –independent facilitated diffusion transport system Facilitated by a family of 14 glucose transporters found in the cell membrane GLUT-1 to GLUT-14. Exist in 2 conformational state a) Tissue specificity of glucose transporter gene expression: -GLUTs display a tissue specific pattern of expression GLUT-1 is abundant in erythrocytes; also transports glucose across the blood brain barrier GLUT-2 in liver, kidney and  cells of the pancreas GLUT-3 in Neurons GLUT-4 is abundant in muscle and adipose tissue (insulin dependent) GLUT-5 – primary transporter for fructose in the SI and the testes 10 a) Tissue specificity of glucose transporter gene expression: b) Specialized functions of glucose transporter isoforms In facilitated diffusion, transporter-mediated glucose movement is DOWN a concentration gradient GLUT1, 3 and 4 mainly take up glucose from the blood GLUT-2 (liver and kidney) can either transport glucose into the cells when blood glucose levels are high Or Transport glucose from these cells when blood glucose levels are low (important in fasting) GLUT-5 is the primary transporter for Fructose in the small intestine and the testes 11 B. Sodium-monosaccharide cotransport system Energy requiring process Transports glucose AGAINST a conc gradient Transporter mediated process in which the movement of glucose is coupled to the conc gradient of Na+, which is transported into the cell at the same time Called sodium-dependent glucose transporter (SGLT) Occurs in the epithelial cells of the intestine, the renal tubules and choroid plexus 12 Glucose transport in intestinal epithelial cells 13  Reactions of Glycolysis Phase 1: Energy investment phase (first 5 reactions) Phosphorylated intermediates formed using ATP Phase 2: Energy –generation phase – a net of 2 molecules of ATP are formed by substrate-level phosphorylation per glucose molecule metabolized 14  1. Phosphorylation of Glucose Phosphorylated sugars do not penetrate membranes easily. Phosphorylating the glucose traps it inside the cell 1st irreversible step in glycolysis Hexokinase 1-111 – broad specificity of substrate Inhibited by end product Low Michaelis constant (Km) – so efficient phosphorylation even when glucose conc is low Hexokinase IV (Glucokinase) Found in liver parenchyma &  cells pancreas In  cells serves as a glucose sensor –determines threshold for insulin secretion In hypothalmus serves as a glucose sensor, key role in adrenergic response to hypoglycemia Higher Km than Hexokinase 1-111 Indirectly inhibited by fructose 6-phosphate 15  Regulation of glucokinase activity by glucokinase regulatory protein GKRP in the liver regulates the activity of glucokinase through reversible binding In the presence of F 6-P, glucokinase is translocated into the nucleus and binds tightly to the regulatory protein – making it inactive When glucose conc increases glucokinase is released from the regulatory protein Enzyme reenters the cytosol – phosphorylates G to G 6-P Glucokinase functions as a glucose sensor in the maintenance of blood glucose homeostasis 16  2. Isomerization of glucose 6- phosphate Catalyzed by phosphoglucose isomerase Reaction is reversible Not a rate limiting or regulated step 17  3. Phosphorylation of fructose 6 phosphate The most important control point in glycolysis – catalyzed by phospho-fructokinase -1 (PFK-1) a) Regulation by energy levels within the cell - inhibited by high concs ATP - inhibited by high conc citrate - activated allosterically by high conc AMP b) Regulation by fructose 2,6-bisphosphate -2,6-bisphophate is potent activator of PFK-1 - can override inhibition of high ATP conc i) During the well-fed state ii) During Fasting 18 4. Cleavage of fructose 1,6-bisphosphate Aldolase cleaves F 1,6-diP to DHAP and Glyceraldehyde 3-P Reaction is reversible and not regulated 5. Isomerization of DHAP Trios phosphate isomerase interconverts DHAP and glyceraldehyde 3-phosphate 19 6. Oxidation of glyceraldehyde 3-phosphate This is the first oxidation-reduction rxn of glycolysis - by glyceraldehyde 3-phosphate dehydrogenase (GPDH) Note: limited NAD+ in the cell To maintain glycolysis the cell must constantly regenerate it by oxidizing NADH 2 mechanisms 1. Conversion of pyruvate to lactate (NADH-linked) (anaerobic glycolysis) 2. Oxidation of NADH via respiratory chain (aerobic glycolysis) Pentavalent arsenic (arsenate) poisoning – competes with Pi as a substrate for GPDH –prevents net ATP/NADH production 7. Synthesis of 3-phosphoglycerate, producing ATP 8. Shift of the phosphate group 9. Dehydration of 2-phosphoglycerate 10. Formation of Pyruvate 20 10. Formation of Pyruvate Catalyzed by pyruvate kinase (PK) and is the 3rd irreversible rxn in glycolysis Feedforward regulation: Activated by fructose 1,6 bisphosphate Regulated by covalent modification: phosphorylation by a cAMP-dependent protein kinase leads to inactivation of the hepatic isozyme of PK. Dephosphorylation of the enzyme reactivates it PK deficiency : Mature RBC lack mitochondria – glycolysis only for ATP production. -hemolytic anemia Individuals heterozygous for PK deficiency have resistance to the most severe form of malaria 21  Reduction of pyruvate to lactate Lactate Dehydrogenase (LDH) forms lactate from pyruvate Final product of anaerobic glycolysis in eukaryotes Is the major fate for pyruvate in the lens and cornea of the eye, kidney medulla, testes, leukocytes and RBCs because they are poorly vascularized and/or lack mitochondria Lactate formation in Muscle -when oxidative capacity of ETC exceeded lactate is formed Lactic Acidosis: Elevated conc of lactate in plasma when there is a collapse of Lactate utilization: circulatory system - Can be taken up by the liver and heart e.g. Myocardial infarction, pulmonary and converted to pyruvate embolism and uncontrolled hemorrhage, or shock 22  Energy yield from Glycolysis Anaerobic Glycolysis 2 molecules of ATP/glucose No net change in NADH Aerobic Glycolysis 2 molecules of ATP/glucose 2 molecules of NADH 23  Hormonal Regulation of Glycolysis Reciprocal regulation by insulin and glucagon At 3 major control points 3 major regulatory enzymes are transcriptionally upregulated by insulin and downregulated by glucagon Hormonal regulation is coupled with the quick allosteric inhibition and activation Covalent regulation (phosphorylation/dephosphorylation) 3 major types of regulation 1. Hormonal 2. Allosteric 3. Covalent regulation 24  Summary 3 irreversible reactions shown – very important 25 Why is CHO metabolism of interest to you? Changes in cancer cells 26 27 Bu et al., 2018, Cell Metabolism 27, 1– 14 June 5, 2018 ª 2018 Elsevier Inc. https://doi.org/10.1016/j.cmet.2018.0 4.003 28  What you should know at the end of this lecture Good understanding of anabolism and catabolism –functions and links What is glycolysis Why is glycolysis a central pathway for all CHO metabolism? How does glucose get into the cell? How does this differ in different tissues. Why are there so many different glucose transporter? How do they differ? What are the 3 regulatory steps of glycolysis? How are these steps regulated? What’s the difference in aerobic and anerobic glycolysis? Under what conditions is anerobic glycolysis useful? What is the energy yield from anerobic vs aerobic glycolysis? If both aerobic and anerobic glycolysis yield the same amount of ATP why do we consider aerobic the more efficient pathway? 29