بسم الله الرحمن الرحيم

1. بسم الله الرحمن الرحيم

2. Major Metabolic Pathways of Glucose By Reem M. Sallam, MD, PhD. Clinical Chemistry Unit, Pathology Dept. College of Medicine, KSU

3. Metabolic Pathway • A pathway is made out of multistep sequences of reaction. The product of one reaction is the substrate of the subsequent reaction. • Metabolism is the sum of all the chemical changes occurring in a cell, a tissue or the body. • Pathways can be either catabolic (degradative) or anabolic (synthetic) • The pathways of metabolism must be regulated. Regulation occurs at certain steps called rate-limiting reactions.

4. Metabolic Pathway • Regulatory mechanisms are either rapid & short term or slow and long term. • Rapid regulation can be by covalent modification (e.g. phosphorylation/dephosphorylation) or allosteric. • Slow regulation occurs at the gene level by induction or repression.

5. Metabolic Pathways of Glucose:Production and Utilization Glycogenolysis Hexose interconversion Gluconeogenesis Production Glucose Utilization Glycolysis HMP/PPP Hexose interconversion Glycogenesis Krebs cycle

6. Metabolic Pathways of Glucose:Catabolic and Anabolic Catabolic cycles Glycolysis (Mainly) Krebs (Mainly) Glycogenolysis HMP Anabolic cycles Gluconeogenesis Glycogenesis

7. Glycogenesis and Glycogenolysis Glycogenesis: Synthesis of glycogen from glucose Mainly liver and muscle, Cytosol Glycogenolysis Degradation of glycogen into glucose Mainly liver and muscle, Cytosol

8. Hexose Monophosphate Pathway (HMP) orPentose Phosphate Pathway (PPP) 1- Important source for NADPH Which is used in reductive syntheses 2- Source for metabolically active ribose Which is used for production of nucleotides: For nucleic acids For co-enzymes

9. Glucose Transport Na+-Monosaccharide Cotransporter: Against concentration gradient Energy dependent Carrier-mediated Coupled to Na+ transport Small intestine, renal tubules Na+-Independent Facilitated Diffusion: With concentration gradient Energy Independent Glucose Transporters (GLUT 1-14)

10. Glucose Transport: Facilitated Diffusion

11. Glucose Transporters • Tissue-specific expression pattern • GLUT-1 RBCs and brain • GLUT-2 Liver, kidney & pancreas • GLUT-3 Neurons • GLUT-4 Adipose tissue & skeletal muscle • GLUT-5 Small intestine & testes • GLUT-7 Liver (ER-membrane) • Functions: • GLUT-1, 3 & 4 Glucose uptake from blood • GLUT-2 Blood & cells (either direction) • GLUT-5 Fructose transport

12. Glycolysis: Objectives • Major oxidative pathway of glucose • The main reactions of glycolytic pathway • The rate-limiting enzymes/Regulation • ATP production (aerobic/anaerobic) • Pyruvatekinase deficiency hemolytic anemia

13. Glycolysis: An Overview • Glycolysis, the major pathway for glucose oxidation, occurs in the cytosol of all cells. • It is unique, in that it can function either aerobically or anaerobically, depending on the availability of oxygen and intact mitochondria. • It allows tissues to survive in presence or absence of oxygen, e.g., skeletal muscle. • RBCs, which lack mitochondria, are completely reliant on glucose as their metabolic fuel, and metabolizes it by anaerobic glycolysis.

14. Reactions of Aerobic Glycolysis • The conversion of glucose to pyruvate occurs in 2 stages. • The first 5 reactions is the energy investment phase (ATP is used to synthesize phosphorylated intermediates). • The second phase is the energy generation phase in which a net of 2 molecules of ATP is made by the substrate-level phosphorylation/glucose molecule, and 2 molecules of NADH are formed when pyruvate is produced (aerobic glycolysis), whereas NADH is reconverted to NAD+ when lactate is the end product (anaerobic glycolysis).

15. Reactions of Glycolysis • Phosphorylation of glucose into glucose 6-P by hexokinase (in most tissues) or glucokinase (in liver). This is irreversible reaction. It is one of the regulatory enzyme. The enzyme’s gene in induced by insulin and repressed by glucagon • Isomerization of glucose 6-P to fructose 6-P by phosphoglucose isomerase. Reversible reaction, so not regulated step.

16. Reactions of Glycolysis • Phosphorylation of Fructose 6-P to fructose 1,6 bisphosphate by phosphofructokinase1 (PFK-1). Irreversible reaction. It is the most important regulatory step and the rate limiting step of glycolysis. It is regulated: • Allosteric inhibition by elevated levels of ATP, and of citrate. • Allosteric activation by high level of AMP, and by fructose 2,6 bisphosphate (the most potent activator of PFK-1) • Cleavage of fructose 1,6 bisphosphate by aldolase into dihydroxyacetone phosphate (DHAP) and glyceraldehyde 3-P. This is reversible reaction and not regulated.

17. Reactions of Glycolysis • Isomerization of dihydroxyacetone phosphate (DHAP) and glyceraldehyde 3-P by triose-P isomerase. This is reversible reaction and not regulated. Because DHAP has to be isomerized to glyceraldehyde 3-P for further metabolism by glycolysis, there is a net production of 2 molecules of glyceraldehyde 3-P . • Oxidation of glyceraldehyde 3-P to 1,3-bisphosphoglycerate (1,3-BPG) by glyceraldehyde 3-P dehydrogenase. 1,3-BPG contains high-energy P group.

18. Reactions of Glycolysis • Synthesis of 3-phosphoglycerate from 1,3-BPG by phosphoglycerate kinase. There is formation of 1 ATP molecule. This is a substrate-level phosphorylation. Because 2 molecules of 1,3-BPG are formed from each glucose molecule, there is formation of 2 ATP molecule in this step. • Shift of P group from carbon 3 to carbon 2 by phosphoglycerate mutase. Reversible reaction. • Dehydration of 2-P glycerate by enolase resulting in the formation of phosphoenolpyruvate (PEP) that has high energy P- bond. It is reversible reaction.

19. Reactions of Glycolysis • Formation of pyruvate from PEP by pyruvate kinase (PK). It is irreversible step, so it is regulated. It results in the formation of ATP by substrate level phosphorylation. It is activated by fructose 1,6 bisphosphate (feed-forward regulation). The enzyme is covalently regulated by phosphorylation/dephosphorylation: hypoglycemia  glucagon release from  cells of pancreas  increase intracellular level of cAMP  phosphorylation and inactivation of PK. Dephosphorylation of PK by phosphoprotein phosphatase  reactivation of the enzyme.

20. Glycolysis in RBC: formation of 2,3-BPG • In RBCs, some of the 1,3-BPG is converted to 2,3-BPG by the enzyme bisphosphoglycerate mutase. • 2,3-BPG is an important regulator of the binding of oxygen to hemoglobin, because the presence of 2,3-BPG reduces the affinity of hemoglobin for oxygen enabling hemoglobin to release oxygen efficiently to the tissues. • 2,3-BPG is then hydrolyzed by a phosphatase to 3-phosphoglycerate, which can continue glycolysis.

21. Pyruvate Kinase DeficiencyHemolytic Anemia

22. Pyruvate Kinase DeficiencyHemolytic Anemia • Normal mature RBC lacks mitochondria and is completely dependent on glycolysis for production of ATP. • If there is genetic deficiency in glycolysis enzymes, the rate of glycolysis will decrease in RBCs decreased ATP production  alteration in RBCs membranes and cell shape  premature death & lysis of RBCs  hemolytic anemia. • Pyruvate kinase deficiency is the most common genetic defects of glycolytic enzymes. • The severity of the hemolytic anemia depends on the degree of enzyme deficiency and the degree of RBC compensation by increasing the level of synthesis of 2,3-BPG to facilitate the release of Oxygen from hemoglobin to tissues.

23. Substrate-level phosphorylation Vs. Oxidative phosphorylation • Phosphorylation is the metabolic reaction of introducing a phosphate group into an organic molecule. • Oxidative phosphorylation: The formation of high-energy phosphate bonds by phosphorylation of ADP to ATP coupled to the transfer of electrons from reduced coenzymes to molecular oxygen via the electron transport chain (ETC); it occurs in the mitochondria. • Substrate-level phosphorylation: The formation of high-energy phosphate bonds by phosphorylation of ADP to ATP (or GDP to GTP) coupled to cleavage of a high-energy metabolic intermediate (substrate). It may occur in cytosol or mitochondria

24. Summary: Regulation of Glycolysis Regulatory Enzymes (Irreversible reactions): Glucokinase/hexokinase PFK-1 Pyruvate kinase Regulatory Mechanisms: Rapid, short-term: Allosteric Covalent modifications Slow, long-term: Induction/repression Apply the above mechanisms for each enzyme where applicable

25. Aerobic Glycolysis: ATP Production ATP Consumed: 2 ATP ATP Produced: Substrate-level 2 X 2 = 4 ATP Oxidative-level 2 X 3 = 6 ATP Total 10 ATP Net:10 – 2=8 ATP

26. Take Home Message • Glycolysis is the major oxidative pathway for • glucose • Glycolysis is employed by all tissues • Glycolysis is a tightly-regulated pathway • PFK-1 is the rate-limiting regulatory enzyme

27. Take Home Message • Glycolysis is mainly a catabolic pathway for ATP production, But it has some anabolic features (amphibolic) • Pyruvate kinase deficiency in RBCs results in hemolytic anemia

28. THANK YOU