CAROHYDRATE METABOLISM PART 2 BY PROF.DR. SOUAD ABOAZMA OXIDATION OF GLUCOSE The pathways for oxidation of glucose are classified into two main groups: a- The major pathways for complete oxidation of glucose into CO2, H2O and energy are: 1- Glycolysis → convert one molecule of glucose into 2 mol of pyruvic acid + 2 NADH.H+. 2- Oxidative decarboxylation of pyruvic to acetyl CoA + NADH.H++CO2 3- Complete oxidation of acetyl CoA in Kerb’s cycle into CO2, H2O and energy . b- The minor pathways for oxidation, which are not for energy production. 1- Hexose monophosphate pathway (HMP). 2- Uronic acid pathway. GLYCOLYSIS EMBDEN-MEYERHOF PATHWAY Def.: oxidation of glucose to give pyruvic acid in presence of O2 and lactic acid in absence of mitochondria (RBCs) and in absence of O2 . Site: Cytoplasm of all cells especially muscles and RBCs. Steps: H – C = O H – C = O H C – OH H – C – OH Hexokinase, glucokinase OH – C – H OH – C – H H – C – OH Mg H – C – OH H – C – OH ATP ADP H – C – OH CH2OH CH2O-P D-Glucose G-6-P Mechanism of oxidation of glyceraldehydes 3-phosphate. Enz: glyceraldehydes 3-P dehydrogenase which is inhibited by the –SH poison iodoacetate, thus able to inhibit glycolysis. ENERGY PRODUCTION FROM GLYCOLYSIS: A. glycolysis in presence of O2 (Aerobic glycolysis): Reaction catalyzed by ATP production Stage I 1. Hexokinase/Glucokinase reaction (for -1 ATP phosphorylation) 2. Phosphofrutokinase-1 (for phosphorylation) -1 ATP Stage III 3. Glyceraldehyde-3-P dehydrogenase (oxidation of + 6 or +4 ATP 2 NADH in electron transport chain) 4. Phosphoglycerate kinase (substrate level +2 ATP phosphorylation) Stage IV 5. Pyruvate kinase (substrate level phosphorlyation) +2 ATP Net gain = 10 or 8 - 2 = 8 or 6ATP B. Glycolysis in Absence of O2 (Anaerobic glycolysis): •In absence of O2 re-oxidation of NADH at glyceraldehyde-3-P- dehydrogenase stage cannot take place in electron-transport chain. But the cells have limited coenzyme. Hence to continue the glycolytic pathway NADH must be oxidized to NAD+. This is achieved by reoxidation of NADH by conversion of pyruvate to lactate (without producing ATP) by the enzyme lactate dehydrogenase. Occurs in cells with no mitochondria as RBCs (mature) ,or under low O2 supply as intensive muscular exercise. In anaerobic glycolysis per molecule of glucose oxidation 4 - 2 = 2 ATP will be produced. REGULATION OF GLYCOLYSIS There are 3 types of mechanisms responsible for regulation of the enzyme activity which are: 1- Changed in rate of enzyme synthesis that affect the quantity of enzyme as: * Induction →↑ rate of enzyme synthesis at gene expression →↑ mRNA synthesis →increase enzyme concentration. * Repression →↓ rate of enzyme synthesis at gene expression →↓ mRNA synthesis →decrease enzyme concentration. 2- Covalent modification by reversible phosphorylation dephosphorylation. 3- Allosteric regulation by allosteric activator or inhibitor that affect the quality of the enzymes. A- Allosteric regulation of glycolysis: There are 4 regulatory enzymes which responsible for 3 irreversible reaction in glycolysis. Hexokinase 1.It is found in most tissues to give G-6-P when blood glucose level is low. 2.Acts on glucose and other hexoses to give hexose-6-P. 3.It has low km and Vmax→ acts maximally at fasting bl. glucose level. 4.It is inhibited by its products, which is G-6-P → allosteric feedback inhibition. Glucokinase 1.It is found in liver and acts maximally after meal. 2.Acts only on glucose. 3.It has a high km and high Vmax → so it is active when bl. glucose level is high (after meal). 4.It is induced (↑its rate of synthesis) by insulin. 5.It is not inhibited by G-6-P. Phosphofructokinase 1.It is the major regulatory enzyme in most tissues. 2.It is allosterically activated by F-6-P, AMP and inhibited by ATP, citrate, and H+. Pyruvate kinase 1.It is allosterically inhibited by ATP, fatty acids, alanine, and acetyl CoA. And activated by F-1-6 diphosphate. 2. It is phosphorylated by cAMP dependent protein kinase, which becomes inactive and dephosphorylated by phosphatase enzyme, which becomes active. B- Hormonal regulation:• Insulin/glucagons ratio is the main hormonal regulation of glucose utilization; it increases during glucose feeding and decreases during fasting. A.Glucagons: it is secreted in case of carbohydrates deficiency or in response to low blood glucose level (hypoglycemia). It affects liver cells mainly as follows: 1.It acts as repressor of glycolytic key enzymes except hexokinase. 2.Through cAMP-dependent protein kinase A, it produces phosphorylation of specific protein enzymes that lead to inactivation of glycolytic key enzymes( only for pyruvate kinase). B.Insulin: it is secreted after feeding of carbohydrate or in response to high blood glucose level (hyperglycemia). It stimulates all pathways of glucose utilization. Insulin binds to a specific cell membrane receptors and produces certain signal cascade, which results in the following: 1.It acts as inducer for glycolytic key enzymes. 2.It activats phosphodiesterase enzyme(decreases cAMP that leads to inhibition of protein kinase A). 3.It activats protein phosphatase-1 that produces dephosphorylation of glycolytic key enzymes and their activation. INHIBITORS OF GLYCOLYSIS: 1- Aresnate : which used in oxidative step insted of Pi→ so glycolysis proceeds in presence of arsenate but ATP, which formed from 1-3 diphosphoglycerate is lost. 2- Iodoacetate produces inhibition of glyceraldehydes-3-P dehydrogenase (inhibitor of SH group). 3- Flouride inhibits enolase →↓↓ glycolysis in bacteria →no production of lactic acid produced by bacteria, which cause dental caries. It used as anticoagulant in blood sample used for estimation of blood glucose →↓↓ glycolysis in RBCs . FORMATION OF 2,3 DIPHOSPHOGLYCERATE IN RBCS: 2:3 diphosphoglycerate has an effect on O2 binding power of haemoglobin→ It lowers O2 affinity by haemoglobin →↑ dissociation of O2 to the peripheral tissues as in cases of high altitude. CLINICAL SIGNIFICANCE OF 2,3 DIPHSOPHOGLYCERATE: 1- Persons who live at high altitude undergo state of low O2 affinity for HB due to simultaneous increase of 2,3 diphosphoglycerate. This increase can be reversed on returning to sea level. 2- Fetal HB has less 2,3 diphosphoglycerate than adult HB, so fetal HB has high O2 affinity. 3- During storage of blood in blood banks, there is decrease in 2,3 diphosphoglycerate so, stored blood has high O2 affinity, which is not suitable for blood transfusion especially to ill patients. If 2,3 diphosphoglycerate is added to stored blood, it can’t penetrate RBCs wall. So, it is advisable to add insoine, which is a substance that can penetrate RBCs wall and change it into 2,3 diphosphoglycerate through HMP shunt. DIFFERENCES BETWEEN AEROBIC AND ANAEROBIC GLYCOLYSIS Aerobic glycolysis Anaerobic glycolysis - Site Cytoplasm of all RBCs and skeletal muscle tissues during muscular ex. - End products Pyruvic acid + Lactic acid + NAD+ NADH.H+ - Energy production 6 OR, 8 ATP 2 ATP - Lactate dehdyrogenase Not needed Needed DISEASES ASSOCIATED WITH IMPAIRED GLYCOLYSIS 1- Hexokinase deficiency : •In patients with inherited defects of hexokinase activity, the red blood cells contain low concentrations of the glycolytic intermediates including the precursor of 2,3-DPG. •In consequence, the hemoglobin of these patients has an abnormally high oxygen affinity. •The oxygen saturation curves of red blood cells from a patient with hexokinase deficiency are shifted to the left, which indicates that oxygen is less available for the tissues. 2- Pyruvate kinase deficiency (hemolytic anemia): •All red blood cells are completely dependent upon glycolytic activity for ATP production. •Failure of the pyruvate kinase reaction, the production of ATP will decrease leading to hemolysis of red cells. •Inadequate production of ATP reduces the activity of the Na+ - and K+ -stimulated ATPase ion pump. 3- Lactic acidosis:- •Blood levels of lactic acid are normally less than 1.2 mM. In lactic acidosis, the values for blood lactate may be 5 mM or more. •The high concentration of lactate results in lowered blood pH and bicarbonate levels. •High blood lactate levels can result from increased formation or decreased utilization of lactate. •Common cause of hyperlacticidemia is anoxia. •Tissue anoxia may occur in shock and other conditions that impair blood flow, in respiratory disorders, and in severe anemia. AEROBIC AND ANAEROBIC EXERCISE USE DIFFERENT FUELS Aerobic exercise is exemplified by long-distance running, while anaerobic exercise by sprinting or weight lifting. During anaerobic exercise there is really very little inter-organ cooperation. The vessels within the muscles are compressed during peak contraction, thus their cells are isolated from the rest of the body. Muscle largely relies on its own stored glycogen and phosphocreatine. Phosphocreatine serves as a source of high-energy phosphate for ATP synthesis for first 4-5 seconds until glycogenolysis and glycolysis are stimulated. Glycolysis becomes the primary source of ATP for want of oxygen. Aerobic exercise is metabolically more interesting. For moderate exercise, much of thet energy is derived from glycolysis of muscle glycogen.. However, a well-fed individual doesn't store enough glucose and glycogen to provide the energy needed for running long distances. The respiratory quotient, the ratio of carbon dioxide exhaled to oxygen consumed, falls during distance running. This indicates the progressive switch from glycogen to fatty acid oxidation during a race. Lipolysis gradually increases as glucose stores are exhausted, and, as in the fast state, muscles oxidize fatty acids in preference to glucose as the former become available. Marathon runner (42.2 KM or 26 Miles) •In the marathon runner predominantly red fibres (oxidative) are used. •Red fibres contain myoglobin and mitochondria. •The major sources of energy in marathon runner are:- •Aerobic metabolism is the principal source of ATP. •Blood glucose. •Hepatic glycogen is degraded to maintain the level of blood glucose. •Muscle glycogen is also a source of fuel but it is degraded slowly than in sprinter .