Ans- B Glycolysis (Embden Meyerh of Pathway) Definition: Glycolysis is a process by which glucose molecules are metabolized through a series of enzymatic reactions into 2 molecules of pyruvate. Site Of GlycolysisCytosol Purpose: Aerobic glycolysis produces 7 moles of ATP per mole of glucose and 2 moles of pyruvate (Ref: Harper 28th edition, pg 155) Anaerobic glycolysis produces 2 moles of ATP per mole of glucose and 2 moles of lactate. Tissues Dependent On Glycolysis: Erythrocytes, cornea, lens, kidney medulla. Irreversible Reactions Of Glycolysis are: Hexokinase and Glucokinase Have Different Properties Four different isoenzymes of hexokinase (I, II, III, and IV) are expressed in a tissue - specific manner in the body. The hexokinase isoenzymes found in most tissues (I, II, and III) have a low Km, for glucose (<0.1 mM) relative to its concentration in blood (~ 5 mM) and are strongly inhibited by the product glucose 6-phosphate (G6P). The latter is important as it prevents hexokinase from tying up all of the inorganic phosphate of a cell in the form of phosphorylated hexoses. Although the hexokinase reaction is not at equilibrium because of the inhibition imposed by G6P, the level at which hexokinase is expressed in cells can have a major impact on the rate of glycolysis in cells. Liver parenchymal cells and b cells of pancreas are unique in that they contain hexokinase isoenzyme IV, usually called glucokinase, which has strikingly different kinetic properties. Glucokinase catalyzes ATP-dependent phosphorylation of glucose like other hexokinase, but its S0.5 (Substrate concentration that gives enzyme activity of one half maximum velocity) for glucose is considerably higher than the Km for glucose of the other hexokinases. Furthermore, glucokinase is much less sensitive to product inhibition by G6P, and its glucose saturation curve is sigmoidal, which is indicative of cooperativity. Important Enzymes Of Glycolysis are: Difference between Hexokinase and Glucokinase: Hexokinase Glucokinase 1. Site 2. Substrate 3. Induction 4. Km for glucose 5. Inhibition by glucose-6-phosphate 6. Effect of feeding and insulin All tissues except liver Glucose, fructose or galactose. Non inducible Low Inhibited No change in activity Only in liver Only glucose Inducible High Not inhibited Increased rate. Hexokinase II And Cancer As a rule rapidly growing cancers metabolize glucose faster than normal cells, at least in part because overexpression of hexokinase II gives cancer cells greater enzymatic capacity for glucose phosphorylation. On top of this, tight binding of hexokinase II to the mitochondrial outer membrane provides the enzyme with first dibs for the ATP produced by oxidative phosphorylation. Phosphofructokinase: Major regulatory enzyme of glycolysis Activators of PFK: AMP; F6P; F2,6-BP; Inhibitors of PFK: ATP, citrate Enolase: Enzyme is inhibited by sodium fluoride Embden-Meyerhof pathway Fructose 2,6-Bisphosphate Plays a Unique Role in the Regulation of Glycolysis & Gluconeogenesis in Liver. Comment Energetics of Glycolysis During glycolysis 2 ATP are utilized and 4 ATP are produced at substrate level. 2 reducing equalents NADH+ are produced and reoxidized by electron transport chain, to generate 5 ATP molecules (2.5 ATP per NADH+ molecule). Thus total 9 ATP molecules are produced and 2 are utilized, i.e., There is net gain of 7 ATP molecules in aerobic glycolysis. In anaerobic conditions, the reoxidation of NADH by electron transport chain is prevented and NADH gets reoxidized by conversion of pyruvate to lactate by lactate dehydrogenase. Thus, in anaerobic glycolysis only 4 ATP are produced at substrate level. Therefore, there is net gain of 2 ATP molecules in anaerobic glycolysis. Note: - Previous calculations were made assuming that NADH produces 3 ATPs and FADH2 generates 2 ATPs. This will amount to a net generation of 8ATPs per glucose molecule during glycolysis. Recent experiments show that these old values are overestimates and NADH produces 2.5 ATPs and FADH2 produces 1.5 ATPs. Thus, net generation is only 7 ATPs during glycolysis. Reaction Enzyme No of ATPs gained (new calculation) No of ATPs gained (old calculations) Glucose - Glucose-6-phosphate Hexokinase, glucokinase - 1 - 1 Fructose-6-p -Fructose -1,6-bisphosphate Hexokinase, glucokinase - 1 - 1 Glyceraldehyde-3-P - 1,3 bisphosphoglycerate Glyceraldehyde-3-P, dehydrogenase + 5 (2.5 X 2 NADH) +6 (3.0 X 2 NADH) 1,3 bisphosphoglycerate - 3-phosphoglycerate Phosphoenol pyruvate - Pyruvate Phosphoglycerate mutase +2 +2 +2 +2 o Net generation in aerobic glycolysis Pyruvate kinase 9 - 2 = 7 10 - 2 = 8 Comment Important Points The most potent positive allosteric activator of phosphofructokinase-1 and inhibitor of fructose 1,6-bisphosphatase in liver is fructose 2,6-bisphosphate. It relieves inhibition of phosphofructokinase-1 by ATP and increases the affinity for fructose 6-phosphate. It inhibits fructose 1,6-bisphosphatase by increasing the Km for fructose 1,6-bisphosphate. Its concentration is under both substrate (allosteric) and hormonal control (covalent modification) Fructose 2,6-bisphosphate is formed by phosphorylation of fructose 6-phosphate by phosphofructokinase-2. The same enzyme protein is also responsible for its breakdown, since it has fructose 2,6-bisphosphatase activity. This bifunctional enzyme is under the allosteric control of fructose 6-phosphate, which stimulates the kinase and inhibits the phosphatase. When there is an abundant supply of glucose, the concentration of fructose 2,6-bisphosphate increases, stimulating glycolysis by activating phosphofructokinase-1 and inhibiting fructose 1,6-bisphosphatase. In the fasting state, glucagon stimulates the production of cAMP, activating cAMP-dependent protein kinase, which in turn inactivates phosphofructokinase-2 and activates fructose 2,6-bisphosphatase by phosphorylation. Hence, gluconeogenesis is stimulated by a decrease in the concentration of fructose 2,6-bisphosphate, which inactivates phosphofructokinase-1 and relieves the inhibition of fructose 1,6-bisphosphatase.