1. Dr.S.Chakravarty MD

2. A year and a half old Amish girl from Pennsylvania girl is being seen by the hematologist after her pediatrician found her to be severely anemic with splenomegaly and jaundice. Her mother gives a possible history of a “blood problem” in her family but doesn’t know for sure. Her hemoglobin electrophoresis was normal, and the complete blood count (CBC) revealed a normocytic anemia. The platelet and white blood cell counts are normal. On the peripheral smear, there are many bizarre erythrocytes including spiculated cells. Heinz bodies are absent. Questions:- What can be the diagnosis? What is the biochemical basis of the clinical features?

3. 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

4. Metabolism: Defined as sum of all chemical changes that occur in the body Divided into two groups : 1. Anabolism : synthesis of complex molecules from simple molecules like glucose to glycogen. 2. Catabolism : breakdown of complex molecules like proteins, carbohydrates and lipids to simple molecules such as CO 2, H 2 O and NH 2

5. Three stages of catabolism : Carbohydrates LipidsProteins Monosaccharides Fatty acids Glycerol Aminoacids Acetyl Co-A TCA cycle CO 2 + H2O + ATP

6. Glucose uptake by cells : Major Glucose transporters (GLUT): ReceptorTissuesKmFunction Facilitative bidirectional transporters GLUT – 1Most tissues (Brain, RBCs, Colon,Placenta) 1 mM LOW Km-High affinity Basal uptake GLUT – 2Liver Pancreas Small intestine Kidney 15 mM HIGH Km (Low affinity transporter) Uptake and release of glucose by liver( AFTER A MEAL ) Glucose sensor GLUT-3Brain Kidney Placenta 1mM Low Km-High AFFINITY Basal uptake GLUT – 4Skeletal muscle Adipose tissue Heart 5 mM Insulin stimulated glucose uptake GLUT -5Small Intestine Absorption Sodium dependent unidirectional transporter SGLT1Small Intestine and Kidney Active uptake of glucose against a concentration gradient NORMAL BLOOD GLUCOSE CONCENTRATION 4-6 mM (70-110 mg/dl) Glut 1 and Glut 3 are at Vmax at Normal glucose concentration RECALL :Km is inversely proportional to affinity

7. 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.

8. 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.

9. Two types of Glycolysis: A. Aerobic Glycolysis : formation of Pyruvate as end product with production of ATP and NADH when oxygen is available B. 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

10. Glycolysis Glucose Glucose -6-PO4 Fructose -6-PO4 Fructose -1,6-bisphosphate Glucokinase /Hexokinase Phosphofructokinase-1 ATP ADP ATP ADP Energy consuming phase Irreversible step -1 Irreversible step -2 Rate limiting step Phosphohexose isomerase Glycolysis, Gluconeogenesis, The HMP shunt, Glycogenesis Glycogenolysis Reversible but driven forward because of a low concentration of F6P, which is constantly consumed during the next step of glycolysis.

11. Glycolysis Splitting phase – into molecules of 3 carbons each Fructose -1,6-bisphosphate Glyceraldehyde-3-PO4 Dihydroxyacetone phosphate Aldolase A 6C 3C Isomerase Glycerol -3-po4 Glycerol -3-po4 dehydrogenase Fatty acid synthesis

12. Energy yielding phase Glyceraldehyde-3-PO4 1,3 bis phosphoglycerate NAD NADH Glyceraldehyde-3- PO4 dehydrogenase 3-phosphoglycerate 2-phosphoglycerate Phosphoenolpyruvate Pyruvate ADP ATP Pyruvate Kinase Irreversible step -3 Pathway repeats twice because of 2 molecules of Glyceraldehye 3-PO4 formed ADP ATP Phosphoglycerate kinase Enolase (-) Fluoride Substrate level phosphorylation Phosphoglycerate mutase

13. Energy yield from one molecule of glucose ATPs consumed during Glycolysis 1 – Glucokinase 1 – Phosphofructokinase ATPs produced during Glycolysis 2 – Phosphoglycerate kinase 2 – Pyruvate kinase NADH produced Glycolysis (Aerobic pathway / or cells with mitochondria) 2 – Glyceraldehyde-3-PO4 dehydrogenase (NADH = 2.5 ATPs) Net gain in ATPs during Aerobic glycolysis = (4 + 5 – 2 = 7 ATPs)

14. 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.

15. Anerobic glycolysis: Pyruvate Lactate Lactate Dehydrogenase NADH NAD Net energy gain during anaerobic Glycolysis is only 2 ATPs NADH produced during anaerobic Glycolysis is utilized during lactate dehydrogenase step

17. Glycolysis in Erythrocytes: 1,3 Bis phosphoglycerate 3-phosphoglycerate 2,3 Bis phosphoglycerate (2,3BPG) 2,3 Bis phosphoglycerate (2,3BPG) Mutase Phosphatase Phosphoglycerate kinase ADP ATP 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

18. 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

19. Difference between Hexokinase and Glucokinase HexokinaseGlucokinase Substrate specificity All hexosesMainly Glucose Km Low (high affinity) Works at normal glucose concentration High (low affinity) works only when glucose levels are elevated LocationUniversal Mainly liver and Beta cells of pancreas Vmax (rate of reaction)LowHigh Glucose-6-PO4 (Allosteric inhibition)Inhibits the enzymeNo inhibition InsulinNo regulationPositive regulation

20. 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.

21. Allosteric Regulation of PFK-1: Situation of high energy levels in the cells indicated by: 1. High ATP: 2. High citrate levels : Situation of low energy in the cells indicated by: 1. High ADP /AMP level 2. High fructose 2,6 bisphosphate Allosteric inhibition of PFK-1 Allosteric activation

22. Fructose -6-po4 Fructose -1,6- Bisphosphate Fructose -2,6- Bisphosphate PFK-1 PFK-2 Insulin Glucagon Regulation of PFK -1 :

23. Covalent modification of Pyruvate kinase : Pyruvate Kinase po4 ATP ADP Protein kinase A Glucagon cAMP (+) InactiveActive (+) Protein phosphatase Insulin (+) Inhibition of Glycolysis in liver and increase blood glucose

24. 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) Absence of Heinz bodies ( to differentiate G6PD def)

25. 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

26. 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

28. Various fates of Pyruvate:

29. 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.

30. 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

31. 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.

32. 4. Conversion to Alanine Pyruvate can be transaminated to form the amino acid alanine. The enzyme involved is alanine transaminase, which requires pyridoxal phosphate (B6) as a cofactor.

33. Transamination: Amino acid 1 Amino acid 2 Keto acid 1 Keto acid 2 PLP NH2

34. How many ATPs are produced from oxidation of 2 molecules of Glucose ? A. 32 B. 38 C. 64 D. 48 E. 0

35. Which of the following best describes the effect of ATP on PFK 1 ?

36. 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

37. 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? Malate dehydrogenase Glucokinase GLUT 1 transporter PFK-1 Glucose 6 PO4 dehydrogenase