Glycolysis Dr.S.Chakravarty MD
Learning objectives: • Analyze the importance of Glycolytic pathway that it can produce ATPs in both aerobic and anaerobic environment • Differentiate between substrate level and oxidative phosphorylation • List the GLUT transporters and classify them based on insulin dependency • List the rate limiting and irreversible steps of Glycolysis and their regulation • Explain the Importance of Embden Meyerhof pathway • Describe the clinical features of pyruvate kinase deficiency • Calculate the Energy generated during aerobic and anaerobic Glycolysis
Metabolism: • Defined as sum of all chemical changes that occur in the body • Divided into two groups : • Anabolism : synthesis of complex molecules from simple molecules like glucose to glycogen. • Catabolism : breakdown of complex molecules like proteins, carbohydrates and lipids to simple molecules such as CO2, H2O and NH2
Three stages of catabolism : Lipids Proteins Carbohydrates Fatty acids Glycerol Monosaccharides Aminoacids Acetyl Co-A CO2 + H2O + ATP TCA cycle
Glucose uptake by cells:Major Glucose transporters (GLUT): • Km is inversely proportional to affinity:
Salient features of Glycolysis: • Occurs in the cytoplasm of all the cells in the body • Immediate /basal source of energy (ATP) is provided by this pathway. • It provides intermediates for other pathways like Pyruvate, glucose-6-PO4, and Dihydroxyacetone phosphate etc. • Hub of carbohydrate metabolism – all carbs are finally converted to glucose or intermediates of Glycolysis before being metabolized.
ALL CELLS CARRY OUT GLYCOLYSIS • Glycolysis is the ONLY source of ATPs in: • Cornea and lens of the eye • Renal medulla • RBCs • Skin • Cancerous cells.
Two types of Glycolysis: • Aerobic Glycolysis : formation of Pyruvate as end product with production of ATP and NADH when oxygen is available • Anaerobic Glycolysis : formation of lactate as end product with production of only ATP in the absence of oxygen . • Allows continuous production of ATPs in cells without mitochondria or cells deprived of oxygen
Glycolysis Glucose Energy consuming phase ATP Glucokinase /Hexokinase Irreversible step -1 • Glycolsis, • Gluconeogenesis, • The HMP shunt , • Glycogenesis • Glycogenolysis ADP Glucose -6-PO4 Reversible but driven forward because of a low concentration of F6P, which is constantly consumed during the next step of glycolysis. Phosphohexoseisomerase Rate limiting step Fructose -6-PO4 ATP Irreversible step -2 Phosphofructokinase-1 ADP Fructose -1,6-bisphosphate
Glycolysis Splitting phase – into molecules of 3 carbons each Fructose -1,6-bisphosphate 6C Aldolase A Fatty acid synthesis Glycerol -3-po4 Glycerol -3-po4 dehydrogenase Dihydroxyacetone phosphate Glyceraldehyde-3-PO4 Isomerase 3C 3C
Glyceraldehyde-3-PO4 NAD Glyceraldehyde-3-PO4 dehydrogenase Energy yielding phase NADH 1,3 bisphosphoglycerate Pathway repeats twice because of 2 molecules of Glyceraldehye 3-PO4 formed ADP Phosphoglyceratekinase ATP 3-phosphoglycerate Phosphoglyceratemutase Substrate level phosphorylation 2-phosphoglycerate Enolase (-) Fluoride Phosphoenolpyruvate ADP Irreversible step -3 Pyruvate Kinase ATP Pyruvate
Regeneration of NAD+ • Very little NAD in the cytosol. • NADH NAD+ + 2 electrons • In Aerobic tissues: by transferring the electrons to mitochondria to produce ATP by shuttle mechanisms. • In Anaerobic tissues or aerobic tissues devoid of oxygen: by producing lactic acid.
Anerobic glycolysis: Pyruvate NADH NADH Lactate Dehydrogenase NAD NAD Lactate • Net energy gain during anaerobic Glycolysis is only 2 ATPs • NADH produced during anaerobic Glycolysis is utilized during lactate dehydrogenase step
Glycolysis in Erythrocytes: 1,3 Bisphosphoglycerate Mutase ADP 2,3 Bisphosphoglycerate (2,3BPG) Phosphoglycerate kinase ATP Phosphatase 3-phosphoglycerate • Net ATP production during production of 2,3 BPG in RBCs = 0 ATPs • Increase in 2,3 BPG shifts the oxygen dissociation curve to the right
Regulation of Glycolysis: • Regulation at the level of Glucokinase/Hexokinase • Regulation at Phosphofructokinase • Regulation of Pyruvate kinase Hormonal regulation (mainly liver): Insulin favors Glycolysis and Glucagon inhibits Glycolysis
Diabetes Mellitus : • Insulin dependent Diabetes Mellitus (IDDM) – def of insulin due to autoantibodies against Beta cells • Non insulin dependent Diabetes mellitus (NIDDM) – insulin receptor resistance • Maturity onset diabetes of the young – (MODY) – mutation in the Glucokinase gene.
Allosteric Regulation of PFK-1: • Situation of high energy levels in the cells indicated by: • High ATP: • High citrate levels : • Situation of low energy in the cells indicated by: • High ADP /AMP level • High fructose 2,6 bisphosphate Allosteric inhibition of PFK-1 Allosteric activation
Insulin Fructose -6-po4 PFK-2 Glucagon PFK-1 Fructose -2,6- Bisphosphate Fructose -1,6- Bisphosphate Regulation of PFK -1 :
Covalent modification of Pyruvate kinase : cAMP Glucagon (+) Inhibition of Glycolysis in liver and increase blood glucose Protein kinase A (+) po4 ATP ADP Pyruvate Kinase Pyruvate Kinase Active Inactive (+) Protein phosphatase (+) Insulin
Pyruvate kinase def : in RBCs • Second most common cause for enzyme deficiency related hemolytic anemia. • Def causes decreased ATP production in RBCs • Decreased energy to fuel the pumps required to maintain the biconcave, flexible shape of RBCs. • Red cell damage and phagocytosis – premature death and lysis – hemolytic anemia (chronic hemolysis) • Absense of Heinz bodies ( to differentiate G6PD def)
Under conditions of anaerobic glycolysis, the NAD+ required by glyceraldehyde-3-phosphate dehydrogenase is supplied by a reaction catalyzed by which of the following enzymes? • Glycerol-3-phosphate dehydrogenase • Alpha-ketoglutarate dehydrogenase • Lactate dehydrogenase • Malate dehydrogenase • PDH
After consumption of a carbohydrate-rich meal, the liver continues to convert glucose to glucose-6-phosphate. The liver’s ability to continue this processing of high levels of glucose is important in minimizing increases in blood glucose after eating. What is the best explanation for the liver’s ability to continue this conversion after eating a carbohydrate-rich meal? • The Hepatocyte cell membrane’s permeability for glucose-6-phosphate • The high maximum reaction rate (high Vmax) of Glucokinase • The inhibition of Glucokinase by high glucose-6-phosphate • The lack of Glucokinase level regulation by insulin • The low Michaelis-Menten (Km) constant of Glucokinase
1. Conversion to Acetyl co-A • Pyruvate can enter mitochondria – Symport with H+ ions. • Converted by Pyruvate Dehydrogenase to acetyl Co-A, which can enter the TCA cycle. • Irreversible reaction.
2. Formation of 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 in anaerobic metabolism/ RBCs. • 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 – 5 types. • Lactate is released by tissues (e.g., RBCs or exercising muscle) and is used by the liver for Gluconeogenesis or by tissues such as the heart and kidney
3. Conversion to Oxaloacetate • Pyruvate can be converted to OAA by Pyruvate carboxylase. • Replenish intermediates of the TCA cycle as well as substrates for Gluconeogenesis. • Requires biotin as co-factor. • The enzyme is activated by acetyl CoA.
4. Conversion to Alanine • Pyruvate can be transaminated to form the amino acid alanine. • The enzyme involved is alaninetransaminase, which requires pyridoxal phosphate (B6) as a cofactor.
Transamination: Amino acid 1 Keto acid 1 PLP NH2 NH2 Keto acid 2 Amino acid 2
Which of the following is required for cholesterol synthesis in hepatocytes? • A. Citrate shuttle • B. Glycerphosphate shuttle • C. Malate-Aspartate shuttle • D. Carnitine shuttle • E. Adenine nucleotide shuttle
A 55 year old alcoholic was brought to the emergency department by his friends. During their usual nightly gathering at the local bar, he had passed out and they had been unable to revive him. • The physician ordered an injection of thiamine followed by overnight parental glucose. The next morning the patient was alert and serum thiamine was normal and blood glucose was 73mg/dl. • The IV line was removed and he was taken home. At the time of discharge from hospital which of the following proteins would have no significant physiological activity in this patient? • Malatedehydrogenase • Glucokinase • GLUT 1 transporter • PFK-1 • Glucose 6 PO4 dehydrogenase