Glycolysis and Gluconeogenesis Dr M. D. Lloyd 5W 2.13;

Steps in Glycolysis

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);

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

A Summary of Glycolysis

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.

Reactions and Enzymes involved in glycolysis

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);

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);

Gluconeogenesis

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.

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);

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’ = kJ/mol for two steps compared to +31 kJ/mol for direct conversion.

Control of Glycolysis and Gluconeogenesis

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.

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

Hormonal Control of Glycolysis

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.

Control of glycolysis and gluconeogenesis

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.