GLYCOLYSIS Dr Vivek Joshi,MD Fall 2015

Learning Objectives  Introduction  Bio Medical Importance  Site  Reactions of the pathway  Energetic of Glycolysis  Regulation of the pathway  Clinical Significance

Introduction Glycolysis was one of the first metabolic pathways studied and is also one of the best understood Also known as the Embden-Meyerhof pathway

Introduction Major pathway for oxidation of Glucose Glucose Pyruvate/Lactate Glycolysis: Aerobic /Anaerobic Aerobic –Glucose Pyruvate Anaerobic Glucose Pyruvate Lactate NAD+ NADH + H+ ETC-ATP NADH + H+ NAD+

Bio Medical Importance Provision of energy Anaerobic Glycolysis-Skeletal muscle during exercise Aerobic Glycolysis- Heart muscles -Poor survival under ischemic conditions Hemolytic anemia- Pyruvate kinase deficiency

Sites of Glycolysis Sub cellular site : In the cytoplasm of eukaryotic Cells Tissue site : Occurs in practically all living cells Aerobic glycolysis   In the presence of oxygen   In the Cells with mitochondria Anaerobic glycolysis   In the absence of oxygen   In the RBCs & contracting skeletal muscle

Reactions of the pathway

1 st Irreversible Step Glucose to Glucose 6-phosphate by Hexokinase/Glucokinase -Nonionic glucose is converted into an anion that is trapped in the cell, since cells lack transport systems for phosphorylated sugar. -Biologically inert glucose becomes activated into a labile form capable of being further metabolized

Hexokinase v/s Glucokinase Uptake of Glucose and phosphorylation Uptake of Glucose by the tissue-Glucose transporters(GLUT) Phosphorylation of Glucose   Hexokinase   Wide tissue distribution   Low Km (High affinity for glucose)   Low Vmax   Inhibited by Glucose-6-P   Utilization of glucose to provide energy even when [glucose] is low

 Glucokinase  Liver & islet cells of pancreas  High Km- Low affinity for the substrate  High Vmax -Can effectively phosphorylate Glucose in the cell.  Induced by Insulin  At high blood glucose levels (After meals)-Glucose uptake by GLUT 2 in the liver and pancreas with phosphorylation by Glucokinase  Acts as ‘glucose sensor’ in islets cells of pancreas–determines threshold for insulin secretion

Hexokinase v/s Glucokinase

Conversion of Glucose 6-phosphate to Fructose 6-phosphate

2 nd Irreversible step- Fructose 6 phosphate to Fructose 1,6 bisphosphate by Phosphofructokinase -1 PFK-1 is the most regulated enzyme.

Cleavage of (6C) Fructose 1,6 biphosphate to 2 triose sugars

Phosphotriose Isomerase converts dihydroxyacetone phosphate to Glyceraldehyde 3-phosphate

Glyceraldehyde 3-phosphate Dehydrogenase catalyzes the only step where NADH is synthesized

Phosphoglycerate kinase catalyzes the first substrate-level phosphorylation step in glycolysis

A mutase moves the phosphate from a 3- to a 2- position on glycerate

Enolase results in generation of the high-energy bond in Phosphoenolpyruvate Enolase is inhibited by Fluoride (used in blood collection tubes for blood glucose estimations)

Pyruvate kinase catalyzes the second substrate- level phosphorylation step in glycolysis

Glucose Glucose PYRUVATE PYRUVATE ACETYL Co A OXALOACETATE CITRIC ACID CYCLE (TCA) GLUCONEOGENESIS Aerobic Glycolysis LDH Alanine Transaminase Pyruvate Carboxylase (BIOTIN) Pyruvate Dehydrogenase ATP ADP CO2 NAD + NADH + H+ Lactate CoASH Metabolic Fate of Pyruvate

 Aerobic conditions (Presence of oxygen)  Pyruvate can be converted to acetyl-CoA and enter the Krebs Cycle  Acetyl-CoA is completely oxidized during Krebs cycle and generates ATP through oxidative phosphorylation  Anerobic conditions (Absence of oxygen)/Absence of mitochondria  Seen mainly in RBCs and the contracting skeletal muscle  Pyruvate is converted to lactate Metabolic Fate of Pyruvate

Conversion of Pyruvate to Lactate by Lactate Dehydrogenase (LDH) PyruvateLactate NADH + H + NAD + Lactate Dehydrogenase (LDH)  Lactate is the final product of anerobic glycolysis  Is formed mainly in the RBC, lens and cornea of the eye, kidney medulla, testes and leukocytes  Is also formed in the intensely exercising skeletal muscle ( accumulation causes cramps)

Energy yield of Glycolysis Aerobic Glycolysis (Glucose to pyruvate) = 8 ATP/mol of glucose Glucose + 2 Pi + 2 ADP + 2 NAD 2 Pyruvate + 2 ATP + 2 NADH Anaerobic Glycolysis (Glucose to lactate) = 2 ATP/mol of glucose Glucose + 2 Pi + 2 ADP 2 Lactate + 2 ATP

  Irreversible and Regulated steps catalyzed by:   Glucokinase- Induction and Repression   Hexokinase- Product Inhibition   Phosphofructokinase-1- Allosteric Regulation ( Most regulated enzyme)   Pyruvate Kinase- Covalent Modification Regulation of Glycolysis PFK-2 (Controls PFK-1 of Glycolysis in the liver)-Covalent Modification

Product Inhibition –Inhibited by Glucose -6-P(Product) HEXOKINASE

  Induced by Insulin   Near complete Deficiency-Neonatal Type I Diabetes   Mutation of Glucokinase gene Decrease Km- Hyperinsulinemia and Hypoglycemia Increase Km-Maturity Onset Diabetes of the Young(MODY) GLUCOKINASE

  Most Regulated Enzyme   Positive Allosteric Effector- High Fructose -2,6 bis phosphate (Liver) High ADP (Liver)/ High AMP(Muscle )   Negative Allosteric Effector –Citrate (Liver) Phosphofructokinase-1(PFK-1)

F2,6 BP Phosphofructokinase-1(PFK-1) Fructose 2,6 bisphosphate (F2,6BP) Potent activator -Fructose 2,6 bisphosphate (F2,6BP)

Fructose-6 Fructose-1,6 BP PFK-1 PFK-1 + Fructose-6 Fructose-2,6 BP PFK-2 PFK-2 Fructose 2,6 bisphosphate (F2,6BP)

Synthesis of F2,6BP is catalyzed by the bifunctional enzyme Phosphofructokinase-2/fructose-2,6-bisphosphatase (PFK-2/F-2,6-BPase) Phosphofructokinase-2(PFK-2)

The only example of a citric acid cycle intermediate regulating a glycolytic enzyme Citrate and ATP Phosphofructokinase- 1(PFK-1)Inhibitors- Citrate and ATP

Pyruvate Kinase

Glucagon - Pyruvate Kinase-P (Inactive) -PFK-2-P (Inactive) Pyruvate Kinase

(Active) Pyruvate Kinase (Inactive) GLYCOLYSIS IS INHIBITED Pyruvate Kinase Glucagon/Epinephrine -Pyruvate Kinase-P (Inactive) -PFK-2-P (Inactive)

F26BPase inactive PFK-2 Inactive F26BPase active PFK-2 Active P P Active Protein kinase A High Glucagon (cAMP) as in hypoglycemia Decreased GLYCOLYSIS ATPADP F 2,6BP F 6BP

High Glucagon (cAMP) –PFK-2 Inactive ………  Fructose-2,6-bisphosphate thus decreases in liver cells in response to a glucagon-activated cAMP signal cascade. Downstream effects include:  Glycolysis slows because of the decreased concentration of fructose-2,6-bisphosphate  Gluconeogenesis Increases because of the decreased concentration of fructose-2,6- bisphosphate

 Well fed state/Carbohydrate rich diet /High Insulin- INCREASE in the :  Uptake of Glucose by GLUT 4 (Skeletal muscle and adipose tissue)  Utilization of glucose by oxidation  Anabolic pathways  Hypoglycemia/Fasting /Starvation/ High Glucagon/Epinephrine- DECREASE in the :  Utilization of glucose by oxidation by Inactivation/Repression of the key Glycolytic enzymes Hormonal Control over Glycolysis

Clinical Significance   Enzyme deficiencies -Pyruvate kinase Deficiency   Lactic Acidosis   Cancer cells   2,3,Bisphosphoglycerate   Fluoride and blood glucose estimations

Pyruvate kinase deficiency  Genetic defect associated with mutant enzyme-A severe decrease in Pyruvate kinase activity and rate of glycolysis  Affects mainly RBCs that depend solely on anaerobic glycolysis for energy and maintaining structural integrity  Associated with the rise in 2,3-BPG levels-Shift of ODC to the right  Clinical presentation: # Hemolytic Anemia due to lysis of RBCs; usually associated with precipitating factors (oxidant stress, associated with precipitating factors (oxidant stress, infection) infection) #Diagnosis: Levels of pyruvate kinase activity in RBCs #Diagnosis: Levels of pyruvate kinase activity in RBCs #Treatment: Usually no therapy required

Lactic Acidosis Accumulation of lactic acid in blood Accumulation of lactic acid in blood As a result of failure of delivery of oxygen to tissues – Dependent on anerobic glycolysis As a result of failure of delivery of oxygen to tissues – Dependent on anerobic glycolysis May be seen associated with: May be seen associated with:  Severe myocardial infarction Cardiac muscles-Adapted for aerobic Glycolysis Cardiac muscles-Adapted for aerobic Glycolysis Poor survival under ischemic conditions Poor survival under ischemic conditions  Uncontrolled hemorrhage

Cancer cells  Very rapid glucose uptake (shown in 1928 by the German Biochemist, Otto Warburg)  Commonly experience hypoxia and depend on anaerobic glycolysis for most of their ATP  Have a very HIGH glycolytic rate (lesser mitochondria)

Mature Erythrocytes - Mature Erythrocytes -  Dependent on Glucose as fuel  Glucose oxidized by Anaerobic Glycolysis(Lack Mitochondria) for Energy,maintain ion channel and structural integrity  Enzyme Bisphosphoglycerate mutase Synthesize 2,3- Bisphosphoglycerate is synthesized from an intermediate of glycolysis, 1,3 Bisphosphoglycerate The 2,3-Bisphosphoglycerate Pathway in Erythrocytes

2,3-BPG in the RBC’s 2,3-BPG in the RBC’s Stabilises the Taut form/ “T” /deoxy form of Hb Stabilises the Taut form/ “T” /deoxy form of Hb Favours unloading of Oxygen to the tissues Favours unloading of Oxygen to the tissues High BPG content shifts Oxygen dissociation curve to the right High BPG content shifts Oxygen dissociation curve to the right High Altitude –Increased 2,3-BPG High Altitude –Increased 2,3-BPG Fetus-Inability of 2,3-BPG to bind to fetal Hb (  chains) Fetus-Inability of 2,3-BPG to bind to fetal Hb (  chains) RECALL…………………..

Fluoride and Blood Glucose estimations Blood glucose concentration is used for the diagnosis of Diabetes Blood glucose concentration is used for the diagnosis of Diabetes Fluoride bulb is used for blood glucose estimation - Mixture of anticoagulant with Sodium Fluoride (Enzyme inhibitor) Fluoride bulb is used for blood glucose estimation - Mixture of anticoagulant with Sodium Fluoride (Enzyme inhibitor) Fluoride (used as sodium fluoride) is a strong competitive inhibitor of Enolase, blocking glycolysis. Fluoride (used as sodium fluoride) is a strong competitive inhibitor of Enolase, blocking glycolysis. Addition of Enzyme inhibitor to the blood inhibits Glucose utilisation in vitro. Addition of Enzyme inhibitor to the blood inhibits Glucose utilisation in vitro. Prevents false low values for blood glucose Prevents false low values for blood glucose

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