Glycolysis

GENERAL OVERVIEW Principle route of metabolism for Glucose, Fructose, Galactose & other dietary carbohydrates Enzyme/transporters demonstrate Stereospecificity for naturally occurring D-isomers Glycolysis can take place WITHOUT O2 * Red blood cells (RBCs), which lack mitochondria, are nearly completely reliant on glycolysis as a source of energy. As such, many of the deficiencies of glycolytic enzymes have a profound effect on RBC function. TRANSPORT OF GLUCOSE INTO CELLS - NEEDS a transport protein - Insulin stimulates glucose transport into muscle and adipose cells by causing glucose transport proteins (GLUT-4) within cells to move to the cell membrane. - Insulin does not significantly stimulate the transport of glucose into tissues such as liver, brain, and RBCs. * Hereditary deficiency of GLUT-1, an insulin-independent transporter, results in decreased glucose in the cerebrospinal fluid. Patients manifest with intractable seizures in infancy and developmental delay. REACTIONS OF GLYCOLYSIS - Unlike glucose, which can diffuse through the transporters on the cell membranes, glucose 6-phosphate is obligated to the intracellular compartment. - Fructose-6-phosphateisphosphorylated by ATP,forming fructose 1,6-bisphosphate and ADP. 1. Enzyme:phosphofructokinase -1(PFK-1) 2. The first committed step of glycolysis. - Fructose 1,6-bisphosphate is cleaved—with the enzyme aldolase—to form the triose phosphates, glyceraldehyde 3-phosphate and dihydroxyacetone phosphate (DHAP). - Absence of the A isoform of aldolase (found in RBCs and muscle) has been reported. The disorder presents with a nonspherocytic hemolytic anemia. Patients also have episodes of rhabdomyolysis (destruction of muscle cells) following febrile illness. * Note: Two moles of glyceraldehyde 3-phosphateare formed from 1 mole of glucose. * Patients with triose phosphate isomerase (TPI) deficiency have neonatal-onset hemolytic anemia as well as progressive neurologic involvement. Children have progressive hypotonia with eventual diaphragm paralysis that requires ventilation, as well as cardiomyopathy. - Glyceraldehyde 3-phosphate is oxidized by NAD+ and reacts with inorganic phosphate (Pi) to form 1,3-bisphosphoglycerate and NADH + H+.1. Enzyme: glyceraldehyde3-phosphatedehydrogenase 2. The aldehyde group of glyceraldehyde 3-phosphate is oxidized to a carboxylic acid, which forms a high-energy anhydride with Pi. - 1,3-Bisphosphoglycerate reacts with ADP—using the enzyme phosphoglycerate kinase—to produce 3-phosphoglycerate and ATP. - 2 - Phosphoglycerateisdehydrated—using the enzyme enolase—to phosphoenolpyruvate (PEP), which contains a high-energy enol phosphate * The enzyme enolase is inhibited by fluoride. To prevent ongoing glycolysis in a patient’s blood samples collected for sensitive glucose tolerance tests, blood is collected in tubes containing fluoride. - Phosphoenolpyruvate reacts with ADP to form pyruvate and ATP in the last reaction of glycolysis. 1. Enzyme: pyruvatekinase 2. Pyruvatekinase is more active in the fed state than in the fasting state. SPECIAL REACTIONS IN RED BLOOD CELLS - In RBCs, 1,3-bisphosphoglycerate can be converted to 2,3-bisphosphoglycerate (BPG), a com- pound that decreases the affinity of hemoglobin for oxygen. - BPG is dephosphorylated to form inorganic phosphate and 3-phosphoglycerate, an intermediate that reenters the glycolytic pathway. - Fetal hemoglobin (HbF), composed of two a subunits and two g subunits, has a lower affinity for BPG than does HbA, and therefore, HbF has a higher affinity for O2. This difference in maternal and fetal hemoglobin facilitates the unloading of O2 at the maternal–fetal interface (i.e., the placenta). REGULATORY ENZYMES OF GLYCOLYSIS Hexokinase is found in most tissues and is geared to provide glucose 6-phosphate for ATP production even when blood glucose is low. - Hexokinase has a low Km (Michaelis constant) for glucose (about 0.1 mM). Therefore, it is working near its maximum rate (Vmax), even at fasting blood glucose levels (about 5 mM). - Hexokinase is inhibited by its product, glucose 6-phosphate. Therefore, it is most active when glucose 6-phosphate is being rapidly used. Glucokinase is found in the liver and actively functions at a significant rate only after a meal (when blood glucose is high). - Glucokinase has a high Km for glucose (about 6 mM). Therefore, it is very active after a meal, when glucose levels in the hepatic portal vein are high, and it is inactive during the post- absorptive state or fasting, when glucose levels are low. - Glucokinase is induced when insulin levels are high. * Maturity-onset diabetes of the young (MODY) type 2 is an autosomal dominant disorder involving mutations in the glucokinase (GCK) gene. Patients have nonprogressive hyperglycemia that is usually asymptomatic at diagnosis and is usually managed with diet alone. - PFK-1 is an allosteric enzyme regulated by several factors. It functions at a rapid rate in the liver when blood glucose is high or in cells such as muscle when there is a need for ATP. * Phosphofructokinase deficiency (a form of glycogen storage disease [type VII] in which glycogen accumulates in muscles) results in inefficient use of glucose stores by RBCs and muscles. Patients experience hemolytic anemia as well as muscle cramping. - PFK-1 is activated by fructose 2,6-bisphosphate (F-2,6-BP), an important regulatory mechanism in the liver - PFK-2 acts as a kinase (in the fed state when it is dephosphorylated) and as a phosphatase (in the fasting state when it is phosphorylated). PFK-2 catalyzes two different reactions. 1. PFK-1 is activated by adenosine monophosphate (AMP), an important regulatory mechanism in muscle 2. In muscle during exercise AMP levels are high and ATP levels are low 3. Glycolysis is promoted by a more active PFK-1, and ATP is generated. 4. PFK-1 is inhibited by ATP and citrate which are important regulatory mechanisms in muscle 5. When ATP is high the cell does not need ATP, and glycolysis is inhibited. 6. High levels of citrate indicate that adequate amounts of substrate are entering the tricarboxylic acid (TCA) cycle. Therefore, glycolysis slows down. Pyruvate kinase 1. Pyruvate kinase is activated by fructose 1,6-bisphosphate and inhibited by alanine and by phosphorylation in the liver during fasting when glucagon levels are high 2. Pyruvate kinase is activated in the fed state. Insulin stimulates phosphatases that dephosphorylate and activate pyruvate kinase in the liver. * Deficiency of pyruvate kinase causes decreased production of ATP from glycolysis. RBCs have insufficient ATP for their membrane pumps, and a hemolytic anemia results. THE FATE OF PYRUVATE Conversion to lactate - Pyruvate can be reduced in the cytosol by NADH, forming lactate and regenerating NAD+. - NADH, which is produced by glycolysis, must be reconverted to NAD+ so that carbons of glucose can continue to flow through glycolysis. - Lactate dehydrogenase (LDH) converts pyruvate to lactate. LDH consists of four subunits that can be either of the muscle (M) or the heart (H) type. - Lactate is released by tissues (e.g., RBCs or exercising muscle) and is used by the liver for glu- coneogenesis or by tissues such as the heart and kidney, where it is converted to pyruvate and oxidized for energy. * The LDH reaction is reversible. Conversion to acetyl coenzyme A (CoA) 1. Pyruvate can enter mitochondria. 2. There it can be converted by pyruvate dehydrogenase to acetyl CoA, which can enter the TCA cycle. Conversion to oxaloacetate (OAA) 1. Pyruvate can be converted to OAA by pyruvate carboxylase. - This reaction serves to replenish intermediates of the TCA cycle as well as provide substrates for gluconeogenesis. - The enzyme is activated by acetyl CoA. Conversion to alanine - Pyruvate can be transaminated to form the amino acid alanine. - The enzyme involved is alanine aminotransferase, which requires pyridoxal phosphate as a cofactor. Glucose 6-Phosphate ATP ---> ADP From glucose Within a cell, glucose 6-phosphate is produced by phosphorylation of glucose on the sixth carbon. This is catalyzed by the enzyme hexokinase in most cells, Glucokinase in certain cells, most notably liver cells - One molecule of ATP is consumed in this reaction - Once Phosphorylated its stuck in the cell - The phosphorylation adds a charged phosphate group so the glucose 6-phosphate cannot easily cross the cell membrane. Hexokinase (HK) Deficiency - Low 2,3-diphosphoglycerate of the red cells causes high oxygen affinity of hemoglobin for oxygen. At the same time it makes unloading of oxygen to tissues very difficult. This leads to left shift of the hemoglobin oxygen dissociation curve and tissue anoxia (or tissue hypoxia) - Lack of ATP formation due to failure of glycolytic pathways cause hemolytic anemia Generalization Glycolysis deficiency in enzymes = - All glycolytic enzymes share one aspect in common; their deficiency is associated with hemolytic anemia Hexokinase/Glucokinase - They differ in Km and Vmax, they are located in different tissues and they are regulated Hexokinase - All other tissues to include Skeletal muscles, Cardiac muscles - Km= 0.05mM - Low Vmax = Low Capacity - Mutations to the enzyme can cause Hyper or Hypoglycemia Glucokinase - Liver - Beta cells in the Pancreas - Km= 5mM - High Vmax = High Capacity -Mutations to the enzyme can cause Hyper or Hypoglycemia Phosphofructokinase - Rate limiting enzyme for glycolysis and is highly regulated. Phosphofructokinase DeficiencyATP----> ADP - Glycogen storage disease - The mutation impairs the ability of phosphofructokinase to phosphorylate fructose-6-phosphate prior to its cleavage into glyceraldehyde-3-phosphate which enters the energy generation phase of glycolysis, effectively limiting energy production - Impairs the ability of cells such as erythrocytes and rhabdomyocytes (skeletal muscle cells) to use carbohydrates (such as glucose) for energy Phosphofructokinase Deficiency Diagnosis A diagnosis can be made through a muscle biopsy that shows excess glycogen and then further tests that will show the levels of phosphofructokinase enzyme. Glyceraldehyde 3-phosphate dehydrogenase NADH + H+ NAD+ Pi (Inorganic Phosphate) - It catalyzes an oxidation-reduction reaction, only oxidation-reduction reaction in glycolysis and produces energy rich NADH+H - Arsenic metal ion competes with iP for this enzyme and inhibit the energy yield from this reaction - The sixth step of glycolysis and thus serves to break down glucose for energy and carbon molecules - Converts Glyceraldehyde 3-phosphate to D-glycerate 1,3-bisphosphate - Releasing NADH + H+, NAD+ & Inorganic Phosphate Glyceraldehyde 3 phosphate dehydrogenase deficiency - GAPDH has been described to exhibit higher order multifunctionality in the context of maintaining cellular iron homeostasis - GAPDH can also be inhibited by arsenate, inhibiting glycolysis in red blood cells and causing hemolytic anemia Phosphorglycerate kinaseADP à ATP - It catalyzes the substrate-level phosphorylation resulting in ATP production at substrate level Phosphorglycerate kinase deficiency - Phosphoglycerate kinase deficiency is a genetic disorder that affects the body's ability to break down the simple sugar glucose - Most people with phosphoglycerate kinase deficiency have either the hemolytic form or the myopathic form - Is caused by mutations in the PGK1 gene - Mutations in the PGK1 gene reduce the activity of phosphoglycerate kinase, which disrupts energy production and leads to cell damage or cell death Pyruvate kinase ADP ---> ATP - It catalyzes the substrate level phosphorylation resulting in ATP production at substrate level and is one of the regulated enzyme Deficiency of this enzyme leads to hereditary hemolytic anemia. Regulation of pyruvate kinase: It is activated by fructose 1,6, BP (feed forward activation). It is inhibited by ATP, acetyl CoA, long chain fatty acids and alanine. Insulin activates this enzyme by keeping it in dephosphorylated state whereas glucagon keeps this enzyme inactive by phosphorylating it. Pyruvate kinase deficiency - Pyruvate kinase deficiency is an inherited disorder that affects red blood cells, which carry oxygen to the body's tissues - People with this disorder have a condition known as chronic hemolytic anemia, in which red blood cells are broken down (undergo hemolysis) prematurely, resulting in a shortage of red blood cells (anemia) - Pyruvate kinase deficiency is a common cause of a type of inherited hemolytic anemia called hereditary nonspherocytic hemolytic anemia. In hereditary nonspherocytic hemolytic anemia, the red blood cells do not assume a spherical shape as they do in some other forms of hemolytic anemia. - Pyruvate kinase deficiency is caused by mutations in the PKLR gene. The PKLR gene is active in the liver and in red blood cells, where it provides instructions for making an enzyme called pyruvate kinaseGlucokinase Acts like a sensor for the Beta cells of the Pancreas - Is present in hepatocytes and beta-cells of pancreas - Present in liver and beta-cells of pancreas and has high Km and high Vmax for glucose. - This indicates, the enzyme will be active only at high glucose concentration (like after food) and is a high capacity enzyme - It is not feedback inhibited by its end product glucose 6-phosphate, but it is induced by insulin Glucokinase mutations - Mutations cause mild fasting hyperglycemia, the hallmark of GCK-MODY; homozygous inactivating mutations result in more severe hyperglycemia presenting as permanent neonatal diabetes mellitus (PNDM) - Other GCK mutations result in over-secretion of insulin and present with hyperinsulinemic hypoglycemia - Leads to a rare form of disease called maturity-onset diabetes of the young (MODY) - All due to ineffective insulin production or release by pancreatic beta cells Phosphofructokinase-1 PFK-1 - Rate limiting enzyme - Control point for Glycolysis - Is the master regulator of glycolysis and is the most important enzyme for glycolysis as it determines if the glucose enters into glycolysis or some other pathway, hence it is called rate limiting enzyme - PFK-1 is inhibited by ATP and citrate, activated by AMP and fructose 2,6-bis-phosphate - PFK is allosterically inhibited by ATP, so glycolysis is slowed when cellular ATP concentrations are high. ATP binds to a site on PFK distinct from the active site, causing a conformational change resulting in rotation of the positions of Arg162 and Glu161Insulin Stimulates and Glucagon Inhibits ATP --> ADP --> AMP AMP= Hunger signal for the cell, positively regulate PFK-1 there by increase in glycolysis and hence the ATP generation When cellular energy is limited, glycolysis should be unregulated. PFK is allosterically activated by high levels of AMP. AMP overcomes the inhibitory effect of ATP.Aldolase enzyme Three forms of it A, B & C - Cleaves six carbon fructose 1,6-bisphosphate into three carbon intermediates glyceraldehyde 3-phosphate and dihydroxyacetone phosphate Deficiency of Aldolase B leads to disease called hereditary fructose intolerance Aldoses - Present in the liver and kidney - Unlike fructose malabsorption, there are little or no gastrointestinal symptoms like bloating and diarrhea. - This is due to the problem lying with fructose breakdown once it is absorbed into the body whereas fructose malabsorption is a problem within the gut - Symptoms of hypoglycemia may also be present in hereditary fructose intolerance due to the problem with aldolase B impairing gluconeogenesis Glyceraldehyde 3-phosphate dehydrogenase - Catalyze oxidation-reduction reaction, produce NADH+H - NADH+H is oxidized by two major mechanisms to replenish NAD+ required for glycolysis to continue - - Under anaerobic conditions pyruvate is diverted towards lactate formation, the reaction is catalyzed by lactate dehydrogenase (LDH), which oxidizes NADH+H produced during glycolysis step (glyceraldehyde 3-phosphate dehydrogenase) to provide NAD+ needed to continue glycolysis. - Formation of lactate is the major fate of pyruvate in lens and cornea of the eye, kidney medulla, testes, leukocytes and red blood cells (these tissues are poorly vascularized and/or lack mitochondria) - In exercising skeletal muscle, NADH production exceeds the oxidative capacity of respiratory chain, resulting in elevation of NADH/NAD+ ratio, favoring conversion of lactate from pyruvate - During intense exercise, lactate (lactic acid) accumulates in muscle, causing a drop in intracellular pH, potentially resulting in muscle cramps Two major mechanisms for oxidizing NADH - The NADH linked conversion of pyruvate to lactate (anaerobic glycolysis). - Oxidation of NADH via respiratory chain (aerobic glycolysis). This requires NADH+H shuttle mechanism to enter into mitochondria, as mitochondria is impermeable to NADH+H Mechanisms of arsenic poisoning Arsenate can replace inorganic phosphate in the step of glycolysis that produces 1,3-bisphosphoglycerate from glyceraldehyde 3-phosphate. This yields 1-arseno-3-phosphoglycerate instead, which is unstable and quickly hydrolyzes, forming the next intermediate in the pathway - Glycolysis proceeds but not ATP that was suppose to be generated by 1,3 Biphosphoglycerate - In Arsenic poisoning 8 ATP's are lost Bisphosphoglycerate mutase in RBCs synthesize 2,3-BPG - 2,3-BPG binds beta-chains of hemoglobin A (HbA) and decreases its affinity for oxygen - Helping with the unloading of O2 in the body- 2,3-BPG shifts the oxygen dissociation curve towards right and increase the release of oxygen from HbA in peripheral tissues, but still allows 100% saturation in the lungs -One of the changes while adopting to high altitude is increased synthesis of 2,3-BPG to maintain tissue oxygenation. - RBC's Use 2 ATP's Diseases related to 2,3-BPG - Hyperthyroidism = 2,3-BPG content in erythrocytes by changes in the expression of phosphoglycerate mutase (PGM) and 2,3-BPG synthase - Iron deficiency anemia This illness is characterized by a lack of iron, and as 2,3-BPG needs this chemical element to be synthesized, BPG concentration decreases and hemoglobin binds tightly to oxygen. As a result, oxygen release to tissue is reduced. - Chronic respiratory disease with hypoxia Recently, scientists have found similarities between low amounts of 2,3-BPG with the occurrence of high altitude pulmonary edema at high altitudes. Three enzymes that are irreversible in Glycolysis - Glucokinase/hexokinase - Phosphofructokinase - Pyruvate kinaseEnzymes are induced by insulin and repressed by Glucagon Glucokinase, phosphofructokinase, pyruvate kinase Substrate-level phosphorylation 2 of them in Glycolysis - A type of metabolic reaction that results in the formation of adenosine triphosphate (ATP) by the direct transfer and donation of a phosphoryl (PO3) group to adenosine diphosphate (ADP) from a phosphorylated reactive intermediate How does Glucagon in hepatocytes stimulate gluconeogenesis? - Glucagon inhibits this enzyme by phosphorylating it via G protein - cAMP mediated activation of protein kinase A - This leads to accumulation of phosphoenolpyruvate which takes a reverse reaction towards glucose In RBC's how does Glycolysis occur? - No mitochondria - Anaerobic 2 Pyruvates --> 2 Lactates --> 2 ATP's - RBC's use up glucose - RBC's Consume NADH + H+ and their by product is NAD+ which is regenerated for Glycolysis and used to produce ATP Fluoride inhibits which enzyme of Glycolysis? A. Hexokinase B. Pyruvate kinase C. Enolase D. Phosphofructokinase Fluoride acts primarily by inhibiting enolase in the glycolytic pathway. Fluoride strongly inhibits the enzyme in the presence of inorganic phosphate. The inhibitory species is the fluorophosphate ion, which when bound to magnesium forms a complex with enolase and inactivates the enzyme - If fluoride does not enter blood cells rapidly, then it cannot rapidly inhibit the production of lactate, which is produced from pyruvate, the final product of glycolysis