2-Glycolysis Fall 2024 Biochemistry II (PDF)

Biochemistry II (PB 503) Dr. Mahmoud Senousy Associate Professor of Biochemistry 1  Outline Transport of glucose into cells. Overview of glucose metabolism. Differences of aerobic and anaerobic glycolysis....

Biochemistry II (PB 503) Dr. Mahmoud Senousy Associate Professor of Biochemistry 1  Outline Transport of glucose into cells. Overview of glucose metabolism. Differences of aerobic and anaerobic glycolysis. Steps of the pathway and energy yield. Regulation of glycolytic pathway. Physiological lactate formation and utilization. Pathological lactic acidosis. Pyruvate kinase deficiency. 2  Transport of Glucose into Cells Glucose enters the cell by one of two transport mechanisms: 1. Sodium- and ATP-independent transport system: “facilitated diffusion” ▪ This system is mediated by a family of at least 14 glucose transporters in cell membranes, designated GLUT-1 to GLUT-14 (glucose transporter isoforms 1-14). ▪ These transporters exist in the membrane in two conformational states. 3  GLUTs are tissue specific: GLUT Site Function GLUT1 erythrocytes, brain Glucose uptake (from blood to cells) GLUT2 liver, kidney, β-cells of ✓ Glucose uptake (from blood to cells) pancreas, small intestine. ✓ Release of glucose to blood from the liver and kidneys during fasting. ✓ Transport of monosaccharides from intestinal mucosal cells into portal blood during absorption. GLUT3 neuron cells Glucose uptake (from blood to cells) GLUT4 adipose tissue, skeletal Glucose uptake (from blood to cells) muscles (insulin-dependent) GLUT5 intestinal mucosal cells, specific for fructose transport testes ✓ absorption from intestinal lumen to intestinal mucosal cells. ✓ uptake from blood to testes. 4 2. Sodium- and ATP-dependent co-transport system: “Active transport” ❑ Requires Na+-dependent glucose cotransporter (SGLT), energy, and Na+ ions. ❑ Na+ is transported down its concentration gradient and drags glucose against its concentration gradient. ❑ This type of transport occurs in the epithelial cells of the intestine, renal tubules, and choroid plexus (part of the blood brain barrier). 5  Overview of Glucose Metabolism Sources Liver glycogen Non-carbohydrates Other hexoses Dietary CHOs e.g., fructose and galactose Glycogenolysis Digestion & Gluconeogenesis absorption Blood glucose Glycolysis Pentose Fate phosphate Lipogenesis Glycogenesis Pyruvate pathway Ribose-5-P Glycogen Fats and NADPH mainly in liver Acetyl CoA stored in adipose tissue and skeletal Krebs cycle muscles CO2 + H2O + Energy 6  Glycolysis The glycolytic pathway is the breakdown of glucose to pyruvate or lactate to provide energy (in the form of ATP) and intermediates for other metabolic pathways. When: fed state (insulin) Where: Location (cellular tissue): all tissues Intracellular location (cellular organelle): cytosol Initial substrate: D-glucose Final product: pyruvate (aerobic conditions) / lactate (anaerobic conditions). i.e. Glycolysis does not require oxygen and can occur under aerobic and anaerobic conditions. 7  Types of Glycolysis Aerobic glycolysis Anaerobic glycolysis Cells Have mitochondria and adequate oxygen Lack mitochondria (RBCs) or are supply poorly vascularized (lens and cornea of the eye, kidney medulla) or deprived of sufficient oxygen e.g., (skeletal muscles during intense exercise) Steps 10 steps 11 steps End product Pyruvate Lactate Net equation D-glucose + 2 NAD+ + 2 ADP + 2 Pi→ D-glucose + 2 ADP + 2 Pi→ 2 pyruvate + 2 NADH.H+ + 2 ATP 2 lactate + 2 ATP Net ATP 8 ATP 2 ATP 8  4 1 2 3 Aldol cleavage Phosphorylation Aldose to ketose Phosphorylation isomerization (DHAP) 5 Mg2+ Mg2+ Aldolase Ketose to aldose or Glucokinase -1 isomerization (PFK-1) D- (GAP) (2) 6 Coupled 2 2 oxidation and (GAPDH) phosphorylation 2 Fluoride 2 2 2 2 - (2) (2) (2) (2) (2) ~ 10 9 8 7 (1,3-BPG) Aerobic Dehydration Phosphate Substrate level Substrate level phosphorylation group shift phosphorylation 9  Notes on Glycolysis 1-There are ▪ 3 irreversible steps: hexokinase/glucokinase, PFK-1 and pyruvate kinase. ▪ 4 kinases in glycolysis: 3 are irreversible, whereas phosphoglycerate kinase is the only reversible kinase in glycolysis. ▪ 2 substrate level phosphorylation: catalyzed by phosphoglycerate kinase and pyruvate kinase. ▪ One reaction producing NADH (oxidative phosphorylation): Glyceraldehyde 3- phosphate dehydrogenase. 2- The aim of the first step (phosphorylation) is to ▪ effectively traps glucose (as glucose 6-phosphate is negatively charged and can’t pass the cell membrane and also has no specific transmembrane carrier). ▪ activate glucose to proceed in glycolysis 3- Mg2+ is a metal cofactor of kinases as they utilize ATP complexed with Mg2+. 4- Fluoride inhibits enolase, so water fluoridation reduces dental caries. 10  Exercise Glyceraldehyde 3-phosphate X1 Q. Name the enzymes X1 to X4. 1,3-BPG Q. Mention the inhibitor (I) of X2 enzyme X4. 3-Phosphoglycerate Q. Which enzyme does catalyze X3 substrate level phosphorylation 2-Phosphoglycerate reaction? I X4 PEP Glycolysis Fall 2020 11 5-Anaerobic glycolysis 1 Step 11: Reduction of ADP 2 pyruvate to lactate by lactate dehydrogenase (LDH) 3 ADP DHAP LDH reoxidizes NADH to 4 5 NAD+ so glycolysis can GAPDH 6 proceed under anaerobic 2 ADP conditions and pyruvate is 7 reduced to lactate. 8 No ATP formation from NADH. 9 2 ADP Mg2+ 10 11 Anaerobic (LDH) 12 Q. What is the significance of LDH reaction under anaerobic condition Because there is only a limited amount of NAD+ in the cell, the NADH formed by glyceraldehyde 3-phosphate dehydrogenase must ? be reoxidized to NAD+ for glycolysis to continue. In aerobic glycolysis, oxidation of NADH to NAD+ occurs via the respiratory chain, whereas under anaerobic conditions LDH accomplishes the reoxidation of NADH to NAD+ while reducing pyruvate to lactate. 13 6-Energy yield of glycolysis The glycolytic pathway occurs in two stages: 1-Energy investment phase: constitutes the first 5 reactions ending with 2 molecules of glyceraldehyde 3-phohphate. 2- Energy generation phase: constitutes the subsequent 5 reactions. 14 7- Differences between glucokinase and hexokinase Item Hexokinase Glucokinase Specificity Acts on glucose, fructose and Acts on glucose ONLY galactose Site Most tissues Predominant in liver parenchymal cells and β- cells of pancreas. Affinity to glucose high low Low: permits efficient High: functions only when the intracellular phosphorylation even when concentration of glucose in the hepatocyte is Km tissue concentrations of elevated following consumption of a CHO-rich glucose are low. meal. Low: cannot phosphorylate High: allows the liver to effectively remove the Vmax more sugars than the cell can flood of glucose delivered by the portal blood use. and thus minimizes hyperglycemia during the absorptive period. Inhibition by Yes NO glucose-6-P (product) Effect of insulin No effect Induced by insulin 15  Regulation of Glycolysis There are 3 Key enzymes (regulatory enzymes): 3 irreversible steps 1-Hexokinase/Glucokinase. 2-PFK-1 (rate limiting step). 3-Pyruvate kinase. Types of regulation on the glycolytic pathway: 1- Hormonal regulation (long-term). 2- Allosteric regulation (short-term). 3- Chemical regulation of glucokinase (short-term). 4- Covalent modification of pyruvate kinase (short-term). 16 1- 17 2- Allosteric regulation of glycolysis Short-term mechanism Hexokinase PFK-1 Pyruvate kinase Allosteric --- AMP , Fructose 1,6- activator Fructose 2,6-bisphosphate bisphosphate (liver) (Feed forward activation) Allosteric Glucose 6- ATP, citrate ATP inhibitor phosphate (Product inhibition) 18 Formation of Fructose 2,6-bisphosphate Formed from F-6-P by phosphofructokinase-2 (PFK-2) (side reaction). Fructose 2,6-bisphosphate is the most potent activator of PFK-1. 19 3- Chemical regulation of glucokinase in the liver (short-term): ✓ Glucokinase is indirectly activated by glucose and indirectly inhibited by F-6-P. ▪ Glucose causes release of glucokinase from glucokinase regulatory protein (GKRP) that exists in the nucleus of hepatocytes. The enzyme enters the cytosol where it phosphorylates glucose to glucose 6- phosphate, thus activating the enzyme activity within minutes to hours (indirect activator). ▪ Fructose 6-phosphate causes translocation of glucokinase back into the nucleus, where it binds tightly to the GKRP, thus rendering the enzyme inactive (indirect inhibitor). 20 4- Covalent modification of pyruvate kinase in the liver (short-term): ▪ Covalent modification of hepatic pyruvate kinase enzyme occurs through phosphorylation by a cAMP- dependent protein kinase via glucagon hormone during fasting. ▪ Pyruvate kinase is inhibited by phosphorylation. 21  Physiological Lactate Formation 1- Lactate formation is the major fate of pyruvate in RBCs that lack mitochondria or in tissues that are poorly vascularized (lens & cornea of the eye, kidney medulla). 2- Lactate formation in muscle: in intense exercising skeletal muscles, NADH production from glycolysis exceeds the oxidative capacity of the ETC (due to insufficient O2 supply)→ elevated NADH/NAD+ ratio →reduction of pyruvate to lactate→ lactate accumulates in muscle→ drop in the intracellular pH, potentially resulting in cramps. 22  Lactate Utilization ❖ Much of this lactate eventually diffuses into the blood stream and can be used by the: 1- Liver which has low NADH/NAD+ ratio, thus oxidizes lactate to pyruvate, which is either converted to glucose by gluconeogenesis or converted to acetyl CoA that is oxidized to CO2 and H2O in Krebs cycle. 2- Heart also has low NADH/NAD+ ratio and oxidizes lactate to pyruvate which is exclusively converted to acetyl CoA that is oxidized to CO2 and H2O in Krebs cycle. Liver CO2 + H2O or Glucose (via gluconeogenesis) Heart 23 CO2 + H2O  Pathological 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. ❑ To survive, the cells use anaerobic glycolysis as a backup system for generating ATP, producing lactic acid as the end product. 24  Pyruvate Kinase Deficiency ❑ The normal mature erythrocyte lacks mitochondria & is therefore, completely dependent on glycolysis for production of ATP. ❑ The 2 ATP molecules meet the metabolic needs of the RBC & the maintenance of the biconcave, flexible shape of the cell, which allows it to squeeze through narrow capillaries. ❑ Pyruvate kinase deficiency → decreased ATP production → alteration in the RBC membrane → changes in cell shape → premature death and lysis of RBCs → mild to severe hemolytic anemia. ❑ The severity of the disease depends on: ❖ Degree of enzyme deficiency (generally 5 to 35% of normal levels). ❖ Extent to which the individual's RBCs compensate by synthesizing increased levels of 2,3-BPG. 25 26