Glycolysis Lesson 1 PDF

GLYCOLYSIS Dr. Dinusha Balasooriya BAMS(Hons), MSc Clinical Biochemistry(Reading), UOP overview Glycolysis, the major pathway for glucose oxidation, occurs in the cytosol of all cells. It is unique, in that it can function either aerobically or anaerobically, dependi...

GLYCOLYSIS Dr. Dinusha Balasooriya BAMS(Hons), MSc Clinical Biochemistry(Reading), UOP overview Glycolysis, the major pathway for glucose oxidation, occurs in the cytosol of all cells. It is unique, in that it can function either aerobically or anaerobically, depending on the availability of oxygen and intact mitochondria. It allows tissues to survive in presence or absence of oxygen, e.g., skeletal muscle. RBCs, which lack mitochondria, are completely reliant on glucose as their metabolic fuel, and metabolize it by anaerobic glycolysis. Overview This is the conversion of one glucose into two pyruvate molecules The free energy released is used to make ATP and NADH. The sequence of 10 reactions is highly conserved. The pathway does not depend on oxygen. It is thought that it evolved before mitochondria and the associated e¯ transport chain. The enzymes of glycolysis are all located in the cytoplasm  Definition  The glycolytic pathway is employed by all tissues for the breakdown of glucose to provide energy (in the form of ATP) and intermediates for other metabolic pathways.  Glycolysis is at the hub of carbohydrate metabolism because virtually all sugars—whether arising from the diet or from catabolic reactions in the body can ultimately be converted to glucose. Glycolysis  Glucose Transport into Cells Glucose cannot diffuse directly into cells, but enters by one of two transport mechanisms: 1. Na+ -independent, facilitated diffusion transport system 2. Na+ -monosaccharide cotransporter system.  1. Na+ -independent facilitated diffusion transport  This system is mediated by a family of 14 glucose transporters in cell membranes.  They are designated GLUT-1 to GLUT-14 (glucose transporter isoforms 1–14).  These transporters exist in the membrane in two conformational states.  Extracellular glucose binds to the transporter, which then alters its conformation, transporting glucose across the cell membrane.  The glucose transporters display a tissue-specific pattern of expression. GLUT-1 Erythrocytes and blood brain barrier, but is low in adult muscle, GLUT-2 Liver and kidney (GLUT-2 is also found in pancreatic β cells) GLUT-3 Neurons. GLUT-4 Adipose tissue and skeletal muscle. Glut proteins have specialized functions  In facilitated diffusion, glucose movement follows a concentration gradient, that is, from a high glucose concentration to a lower one.  For example, GLUT-1, GLUT-3, and GLUT-4 are primarily involved in glucose uptake from the blood.  In contrast, GLUT-2, which is found in the liver and kidney, can either transport glucose into these cells when blood glucose levels are high, or transport glucose from these cells when blood glucose levels are low (for example, during fasting).  GLUT-5 is unusual in that it is the primary transporter for fructose (instead of glucose) in the small intestine and the testes.  2. Na+-monosaccharide cotransporter system  This is an energy-requiring process that transports glucose “against” a concentration gradient—that is, from low glucose concentrations outside the cell to higher concentrations within the cell.  This system is a carrier-mediated process in which the movement of glucose is coupled to the concentration gradient of Na+, which is transported into the cell at the same time.  The carrier is a sodium-dependent–glucose transporter or SGLT.  This type of transport occurs in the epithelial cells of the intestine and renal tubules  Reactions of Glycolysis The conversion of glucose to pyruvate occurs in two Stages 1. Energy investment phase The first five reactions of glycolysis correspond to an energy investment phase in which the phosphorylated forms of intermediates are synthesized at the expense of ATP. 2. Energy generation phase The subsequent reactions of glycolysis constitute an energy generation phase in which a net of two molecules of ATP are formed by substrate-level phosphorylation per glucose molecule metabolized.  Energy Investment Phases Step 1- Phosphorylation of glucose (Activation step) The irreversible phosphorylation of glucose, therefore, effectively traps the sugar as cytosolic glucose 6-phosphate, thus committing it to further metabolism in the cell. Mammals have several isozymes of the enzyme hexokinase that catalyze the phosphorylation of glucose to glucose 6-phosphate.  Hexokinase and Glucokinase 1. Hexokinase: In most tissues, the phosphorylation of glucose is catalyzed by hexokinase, one of three regulatory enzymes of glycolysis (others phosphofructokinase and pyruvate kinase). Hexokinase has broad substrate specificity and is able to phosphorylate several hexoses in addition to glucose. Hexokinase is inhibited by the reaction product, glucose 6-phosphate, which accumulates when further metabolism of this hexose phosphate is reduced. Hexokinase has a low Km (and, therefore, a high affinity) for glucose. This permits the efficient phosphorylation and subsequent metabolism of glucose even when tissue concentrations of glucose are low. 2. Glucokinase: In liver parenchymal cells and β cells of the pancreas, glucokinase is the predominant enzyme responsible for the phosphorylation of glucose. In β cells, glucokinase functions as the glucose sensor, determining the threshold for insulin secretion. In the liver, the enzyme facilitates glucose phosphorylation during hyperglycemia. Despite the popular but misleading name glucokinase, the sugar specificity of the enzyme is similar to that of other hexokinase isozymes. Step 2 - Isomerization of glucose 6-phosphate The isomerization of glucose 6-phosphate to fructose 6-phosphate is catalyzed by phosphoglucose isomerase. The reaction is readily reversible and is not a rate limiting or regulated step. Step 3 - Phosphorylation of fructose 6-phosphate The irreversible phosphorylation reaction catalyzed by phospho- fructokinase- 1 (PFK-1) is the most important control point and the rate-limiting and committed step of glycolysis. PFK-1 is controlled by the available concentrations of the substrates ATP and fructose 6-phosphate. Step 4 - Cleavage of fructose 1,6-bisphosphate Aldolase cleaves fructose 1,6-bisphosphate to dihydroxyacetone phosphate, glyceraldehyde 3-phosphate. The reaction is reversible and not regulated Step 5 - Isomerization of dihydroxyacetone phosphate Triose phosphate isomerase interconverts dihydroxyacetone phosphate and glyceraldehyde 3-phosphate. Dihydroxyacetone phosphate must be isomerized to glyceraldehyde 3- phosphate for further metabolism by the glycolytic pathway. This isomerization results in the net production of two molecules of glyceraldehyde 3-phosphate from the cleavage products of fructose 1,6- bisphosphate.  Energy Generating Phases Step 6 - Oxidation of glyceraldehyde 3-phosphate The conversion of glyceraldehyde 3-phosphate to 1,3-bisphosphoglycerate by glyceraldehyde 3-phosphate dehydrogenase is the first oxidation-reduction reaction of glycolysis aldehyde end of the molecule is oxidized and phosphorylated by a dehydrogenase enzyme and NAD+, this produces 1,3-bisphospho-glycerate and NADH. Step 7 - Synthesis of 3-phosphoglycerate producing ATP When 1,3-BPG is converted to 3-phosphoglycerate, the high energy phosphate group of 1,3- BPG is used to synthesize ATP from ADP. This reaction is catalyzed by phosphoglycerate kinase, which, unlike most other kinases, is physiologically reversible. Because two molecules of 1,3-BPG are formed from each glucose molecule, this kinase reaction replaces the two ATP molecules consumed by the earlier formation of glucose 6- phosphate and fructose 1,6-bisphosphate. This is an example of substrate-level phosphorylation, in which the energy needed for the production of a high- energy phosphate comes from a substrate rather than from the electron transport chain. Step 8 - Shift of the phosphate group from carbon 3 to carbon 2 The shift of the phosphate group from carbon 3 to carbon 2 of phosphor glycerate by phospho glycerate mutase is freely reversible. Step 9 - Dehydration of 2-phosphoglycerate The dehydration of 2-phosphoglycerate by enolase redistributes the energy within the 2-phosphoglycerate molecule, resulting in the formation of phosphoenolpyruvate (PEP), which contains a high- energy enol phosphate. The reaction is reversible despite the high-energy nature of the product. Step 10 - Formation of pyruvate producing ATP The conversion of PEP to pyruvate is catalyzed by pyruvate kinase, the third irreversible reaction of glycolysis. The equilibrium of the pyruvate kinase reaction favors the formation of ATP. The 2 glyceraldehyde 3-phosphate units are converted into 2 pyruvate units in phase two of glycolysis. This is another example of substrate-level phosphorylation. 2 ATPs are used in phase one of glycolysis, and 4 ATPs are made in phase two of glycolysis. The net result is the synthesis of 2 ATPs from glycolysis. The 2 NADHs formed are made in the cytoplasm and must be transported to the mitochondria to join the electron transport chain and make ATP. Reduction of pyruvate to lactate Lactate, formed by the action of lactate dehydrogenase, is the final product of anaerobic glycolysis in eukaryotic cells. The formation of lactate is the major fate for pyruvate in lens and cornea of the eye, kidney medulla, testes, leukocytes and red blood cells, because these are all poorly vascularized and/or lack mitochondria. 1. Lactate formation in muscle: In exercising skeletal muscle, NADH production (by glyceraldehyde 3-phosphate dehydrogenase and by the three NAD+-linked dehydrogenases of the citric acid cycle) exceeds the oxidative capacity of the respiratory chain. This results in an elevated NADH/NAD+ ratio, favoring reduction of pyruvate to lactate. Therefore, during intense exercise, lactate accumulates in muscle, causing a drop in the intracellular pH, potentially resulting in cramps. Much of this lactate eventually diffuses into the bloodstream, and can be used by the liver to make glucose. 2. Lactate consumption: The direction of the lactate dehydrogenase reaction depends on the relative intracellular concentrations of pyruvate and lactate, and on the ratio of NADH/NAD+ in the cell. For example, in liver and heart, the ratio of NADH/NAD+ is lower than in exercising muscle. These tissues oxidize lactate (obtained from the blood) to pyruvate. In the liver, pyruvate is either converted to glucose by gluconeogenesis or oxidized in the TCA cycle. Heart muscle exclusively oxidizes lactate to CO2 and H2O via the citric acid cycle. 3. Lactic acidosis: Elevated concentrations of lactate in the plasma, termed lactic acidosis, occur when there is a collapse of the circulatory system, such as in myocardial infarction, pulmonary embolism, and uncontrolled hemorrhage, or when an individual is in shock. The failure to bring adequate amounts of oxygen to the tissues results in impaired oxidative phosphorylation and decreased ATP synthesis. To survive, the cells use anaerobic glycolysis as a backup system for generating ATP, producing lactic acid as the end product. Hormonal regulation Regular consumption of meals rich in carbohydrate or administration of insulin initiates an increase in the amount of glucokinase, phosphofructokinase, and pyruvate kinase in liver. These changes reflect an increase in gene transcription, resulting in increased enzyme synthesis. High activity of these three enzymes favors the conversion of glucose to pyruvate, a characteristic of the well-fed state.