1. Glycolysis and Gluconeogenesis Dr M. D. Lloyd 5W 2.13; M.D.Lloyd@bath.ac.uk

2. Steps in Glycolysis

3. Glycolysis of glucose is a central metabolic pathway and takes place in the cytosol; Energy (as 2 x ATP) has to be put in at the beginning; Most intermediates are phosphorylated (helps compartmentalisation) The products are 2 x pyruvate, 2 x NADH and 4 x ATP (energy); Net energy gain is 2 x NADH and 2 x ATP; Pyruvate is converted to lactate (anaerobic respiration) or completely oxidised to CO 2 and H 2 O (Lectures 28 & 29) (aerobic respiration);

5. Aldolase splits a C6 phosphorylated sugar into two C3 phosphorylated sugars; DHAP and G-3-P can be intercoverted by Triose Phosphate Isomerase; Glyceraldehyde-3-phosphate (G-3-P) is further processed by glycolysis Structure of Triose Phosphate Isomerase

7. A Summary of Glycolysis

8. Anaerobic Metabolism of Pyruvate In the absence of O 2, pyruvate is converted to lactate in humans. e.g. in muscle tissue; In other organisms (e.g. yeast) pyruvate is converted into ethanol or other products.

9. Reactions and Enzymes involved in glycolysis

10. Hexakinase is an ‘induced fit’ enzyme. Binding of the substrate brings about a gross conformational change in the protein; Hexakinase ‘traps’ glucose in the cell as glucose-6-phosphate (G-6-P); G-6-P is a key starting material for several pathways (therefore hexakinase is a secondary control point in glycolysis);

11. Most enzymes perform ‘normal’ chemical reactions; Example from glycolysis: hexakinase. This reaction involves transfer of a phosphate group from a donor (ATP) to an acceptor (Glucose); Energy is put into the system (activation); Charged intermediates are produced (allows compartmentalisation);

12. Gluconeogenesis

13. Glucose is required for brain tissue and erythrocytes. Most humans require around 160 g of glucose per day; Can be synthesised from pyruvate, oxaloacetate or glycerol in the liver (also kidneys). These are derived from amino acids and fats; Gluconeogenesis is not a direct reversal of glycolysis. This is because the hexakinase, phosphofructokinase and pyruvate kinase reactions are effectively irreversible; Energy & reducing power needs to be put into the system.

14. Pyruvate carboxylase reaction Metabolic blocks are overcome by carboxylation of pyruvate followed by decarboxylation to phosphoenol-pyruvate; Carboxylation requires ATP (to synthesise carbamoyl phosphate) and thiamine (to capture CO 2 ); Reaction is mitochondrial – oxaloacetate exported to cytosol as malate (Lecture 31);

15. Phosphoenolpyruvate carboxykinase Reaction is cytosolic (as for rest of gluconeogenesis); Loss of CO 2 from oxaloacetate drives formation of high-energy mixed anhydride bond (phosphoenol pyruvate formation); Overall  Gº’ = +0.83 kJ/mol for two steps compared to +31 kJ/mol for direct conversion.

16. Control of Glycolysis and Gluconeogenesis

17. The key regulatory site in glycolysis is phosphofructokinase (concommittent step); High levels of ATP (high energy) inhibit activity by decreasing the affinity for substrate; Citrate (intermediate in the TCA cycle, Lecture 28) signals high energy and increases effect of ATP (decreases activity); High levels of AMP (low energy) reduces the effect of ATP; Low pH inhibits activity (prevents lactic acidosis); Secondary control enzymes are hexakinase and pyruvate kinase.

18. Phosphofructokinase is allosterically regulated by Fructose-2,6- bisphosphate. F-2,6-BP is synthesised and degraded by phosphofructokinase 2 (PFK2); F-2,6-BP changes behaviour of phosphofructokinase from Sigmoidal to hyperbolic; The enzyme is bifunctional and has kinase (adds PO 4 3- ) and phosphatase (removes PO 4 3- ) activity; Activity of PF2 is hormonally regulated

19. Hormonal Control of Glycolysis

20. Control of gluconeogenesis Gluconeogenesis produces glucose-6-phosphate – prevents diffusion from cell and allows other metabolic uses; Hydrolysis of Glucose-6-phosphate by a phosphatase occurs in liver (and kidneys). Location is lumen of endoplasmic reticulum (ER); Glucose is produced from glucose-6-phosphate in response to low blood glucose levels (glucose homeostasis). Phosphatase requires calcium-binding stabilising protein and transporters for glucose and phosphate.

21. Control of glycolysis and gluconeogenesis

22. Summary Glycolysis is a central pathway in the metabolism of sugars. Reactions take place in the cytosol; Intermediates are phosphorylated (prevents leakage of compounds into other compartments); Energy (2 x ATP) is put into the system. Energy (as pyruvate, 4 x ATP and 2 x NADH) come out of the pathway; Key control points are phosphofructokinase (allosteric and hormonal control), hexakinase and pyruvate kinase. Low energy in the cell increases flux and vice versa. Gluconeogenesis is not a complete reversal of the glycolytic pathway; Glycolysis and gluconeogenesis are reciprocally controlled.