1. Cellular Biochemistry and Metabolism (CLS 331) Dr. Samah Kotb Nasr Eldeen

2. Glycolysis Chapter 9

4. Glycolysis:  Glucose is the major fuel of most organisms; it is rich in energy and can be mobilized from glycogen stores when sudden demands for energy are needed.  Monosaccharides (Glucose + other hexoses) provide upon catabolism 50% of the daily energy needs in humans.

5.  Glycolysis is the catabolic pathway made of 10 reactions responsible for the breakdown of a 6C- glucose molecule into two 3C- pyruvate molecules.  Glycolysis is a central pathway of glucose catabolism that occurs in animal, plant and microbial cells

6. Fate of Pyruvate:-

7. Energetics of Glycolysis

8. Energetics of Glycolysis:- Degradation

9. Energetics of Glycolysis:-  Only 6.9% of the energy present in glucose is released as a result of glycolysis. The remaining 93% remains in the bonds of pyruvate and is only released at later stages of catabolism as pyruvate is completely degraded into CO 2 + H 2 O.

10. Chemistry of Glycolysis

11. Chemistry of Glycolysis:- The 10 reactions of glycolysis all occur in the cytoplasm (cytosol) of cells. Glycolytic reactions can be divided into 2 phases each made of 5 reactions. 1)Preparatory Phase. 2) Secondary phase.

12. 1.Preparatory Phase  The end product of this phase is the phosphorylated triose (Glyceraldehyde – 3 – phosphate).  2 molecules of ATP are consumed during this phase.  Hexoses other than glucose (fructose, galactose & mannose enter into glycolysis for degradation at certain points of this phase.

13. 2. Secondary phase.  The end product of this phase is 2 pyruvate molecules.  4 ATP molecules are produced during this phase. Note: There are 10 enzymes required for catalysis of each of the 10 glycolytic reactions.

14. Chemistry of Glycolysis:-

17. As seen all the intermediates of glycolysis are phosphorylated except for glucose & pyruvate. There are 3 functions of the phosphate group:- 1.The phosphate group is completely ionized at physiological pH and carrier a net negative charge. Cell membranes are impermeable to charged molecules and thus the glycolytic intermediates cannot escape across cell membranes to the outside.

18. Chemistry of Glycolysis:- 2.These phosphate groups will ultimately be cleaved and transferred to ADP to allow ATP synthesis. 3.The phosphate groups act as recognition or binding groups to allow a tighter proper fit of the glycolytic intermediates to the active sites of their respective enzymes.

19. Steps of Glycolysis

20. 1.Glucose is phosphorylated at C6 to yield G-6-P. ATP is required for this reaction which is hydrolyzed to ADP thus providing the phosphate group and the energy required for phosphorylation. Glucokinase is specific for glucose and does not phosphorylate other hexoses.

21. 2.Isomerization of G-6-P into F-6-P.

22. 3.Phosphorylation of F-6-P into F1,6 di P.

23.  Phosphofructokinase (PFK) like hexokinase is a regulatory enzyme.  It is the major regulatory enzyme of muscle glycolysis. Its activity is accelerated whenever the cells ATP supply is depleted, and it is inhibited when the cell has sufficient ATP.  Allosteric inhibitor of PFK is ATP.  Allosteric stimulator of PFK is ADP.

24. 4.Cleavage of Fructose 1,6 diphosphate. The fructose 1,6 diphosphate ring is cleaved down the middle resulting in the formation of two different triose phosphates.

25. These are Dihydroxyacetone phosphate and Glyceraldehyde-3-phosphate. The reaction is catalyzed by the enzyme fructose diphosphate aldolase.

27. 5.Interconversion of Triose Phosphates. Only Glyceraldehyde – 3 – phosphate can be degraded in subsequent reactions of glycolysis. Dihydroxyacetone phosphate is rapidly converted into Glyceraldehyde – 3 – phosphate by isomerization.

29. 6.Oxidation of Glyceraldehyde – 3 – phosphate.

30. In this reaction the aldehyde group of glyceraldehyde – 3 – phosphate is oxidized (by dehydrogenation) not to a free carboxyl group as might be expected but to a carboxylic anhydride carrying a phosphate group.

31. This type of anhydride is called an acyl phosphate and has a very high  G of hydrolysis and is a super-high energy compound named 3-phosphoglyceroyl phosphate. Note that the reaction requires pool inorganic phosphate. 6.Oxidation of Glyceraldehyde – 3 – phosphate.

32. The enzyme responsible for catalysis is glyceraldehyde – 3 – phosphate dehydrogenase.

33. Dehydrogenases Are a group of enzymes responsible for oxidation by dehydrogenation. A hydrogen atom is removed from the substrate which thus gets oxidized.

34. The enzyme requires a coenzyme namely nicotinamide adenine dinucleotide (NAD+) which functions by accepting the removed hydrogen from the substrate and in doing so gets reduced.

35. 6.Oxidation of Glyceraldehyde – 3 – phosphate. There are many examples of dehydrogenation reactions which operate on metabolic pathways.

36. 7.Transfer of the phosphate group for 3- phosphoglyceroyl phosphate to ADP. The enzyme phosphoglycerate kinase transfers the high energy phosphate group from 3-phosphoglyceroyl phosphate (3PGP) to ADP thus forming ATP & leaving behind 3-phosphoglycerate (3PG).

37. 8.Conversion of 3-phosphoglycerate (3PG) to 2-phosphoglycerate (2PG). This reaction involves an intra-molecular rearrangement in which the phosphate group changes position from C 3 TO C 2.

38. 9.Dehydration of 2-phosphoglycerate (2PG) to phosphoenolpyruvate (PEP). Here another super-high energy compound is generated. A molecule of water is removed from 2PG.

39. 10.Formation of Pyruvate. The phosphate group is now hydrolyzed and transferred to ADP to make ATP. Reaction is catalyzed by Pyruvate kinase.  Note: Reaction 6 – 10 should be multiplied by 2 as 2 molecules of glyceraldehyde – 3 – phosphate are formed as a result of reaction 5.

40. Pyruvate Pathways

42. 1. Conversion of Pyruvate to Acetyl CoA Under aerobic conditions, pyruvate from glycolysis to produce acetyl CoA, which enters the citric acid cycle as well as other metabolic pathways- the enzyme involved is pyruvate dehydrogenase and the coenzyme NAD + is also required. The animal tissue under aerobic conditions pyruvate is the product of glycolysis and the NADH formed by reaction 6 is then reoxidized to NAD + by oxygen.

43. Conversion of Pyruvate to Acetyl CoA Under aerobic conditions pyruvate from glycolysis is decarboxylated acetyl CoA enters the citric acid cycle NAD + pyruvate dehydrogenase

44. This pathway provides the most energy from glucose O || CH 3 —C—COO - + HS-CoA + NAD +  pyruvate O || CH 3 —C—S—CoA + CO 2 + NADH + H + acetyl CoA pyruvate dehydrogenase

45. 2. Conversion of Pyruvate to Ethanol Anaerobic microorganisms such as yeast, convert pyruvate to ethanol by fermentation - pyruvate is decarboxylated to acetaldehyde, which is reduced to ethanol- NAD+ is regenerated to continue glycolysis.

46. Anaerobic microorganisms such as yeast, convert pyruvate to ethanol by fermentation. pyruvate is decarboxylated to acetaldehyde, which is reduced to ethanol. NAD+ is regenerated to continue glycolysis

48. Conversion of Pyruvate to Ethanol Anaerobic microorganisms such as yeast pyruvate from glycolysis is decarboxylated acetaldehyde ethanol NAD + pyruvate decarboxylase fermentation reduced to alcohol dehydrogenase CO2

49. 3. Reduction of Pyruvate to Lactate:

50. Reduction of Pyruvate to Lactate:  Under anaerobic conditions in very active skeletal muscle or in lactic acid bacteria, the NADH generated by reaction 6 cannot be reoxidized but it used to reduce pyruvate to lactate which results in the formation of NAD +. The enzyme required for this is Lactate Dehydrogenase.

52.  In summary, a single glucose molecule in glycolysis produces a total of 2 molecules of pyruvic acid, 2 molecules of ATP, 2 molecules of NADH and 2 molecules of water.

53.  Although 2 ATP molecules are used in steps 1-3, 2 ATP molecules are generated in step 7 and 2 more in step 10. This gives a total of 4 ATP molecules produced. If you subtract the 2 ATP molecules used in steps 1-3 from the 4 generated at the end of step 10, you end up with a net total of 2 ATP molecules produced.

54. The net reaction for Glycolysis is : Glucose+2ADP+2Pi+2NAD + --> 2 Pyruvate + 2ATP+2NADH+2H++2H2O