1.  Department of Biochemistry 2011 (E.T.) Basic concept and design of metabolism The glycolytic pathway

3. The metabolic interplay of living organisms in our biosphere T wo large groups of living organisms according to the chemical form of carbon they require from the environment. Autotrophic cells ( " self-feeding " cells) – green leaf cells of plants and photosynthetic bacteria – utilize CO 2 from the atmosphere as the sole source of carbon for construction of all their carbon-containing biomolecules. They absorb energy of the sun light . The synthesis of organic compounds is essentially the reduction (hydrogenation) of CO 2 by means of hydrogen atoms, produced by the photolysis of water (generated dioxygen O 2 is released). Heterotrophic cells – cells of higher animals and most microorganisms – must obtain carbon in the form of relatively complex organic molecules (nutrients such as glucose) formed by other cells. They obtain their energy from the oxidative (mostly aerobic) degradation of organic nutrients made by autotrophs and return CO 2 to the atmosphere. Carbon and oxygen are constantly cycled between the animal and plant worlds, solar energy ultimately providing the driving force for this massive process .

4. CO 2 Small molecules macromolecules proteins, lipids , saccharides energy O 2 The metabolic interplay in human Green plants Absorb energy of the sunlight Autotrophs CO 2 Heterotrophs

5. Energy in chemical reactions Gibbs free energy (  G) The maximal amount of useful energy that can be gained in the reaction (at constant temperature and pressure ) aA+bB  cC+dD The Δ G of a reaction depends on the nature of the reactants (expressed by the Δ G º term) and on their concentrations (expressed by the second term).  G º ´ = – R T ln K  G 0´ pH = 7,0, T=25 o C)

7. Processes exergonic endergonic Endergonic reactions can proceed only in coupling with exergonic reactions Transfer of energy from one proces to another process in enabled by „high-energy“ compounds Mostly ATP is used. Phosphoryl group PO 3 2- is transferred from one to another compound in process of coupling

8. -PO 3 2- is transferred by the enzyme kinase from ATP to glucose. Principles of coupling  G o ´ = +13,8 kJ/mol  G o ´ = -30,5 kJ/mol Example 1: Formation of glucose-6-phosphate glucose + P i  glucose-6-P + H 2 O  G o ´ = - 16,7 kJ/mol glucose + ATP  glucose -6-P + ADP ATP + H 2 O  ADP + P i

9. The term „high-energy compound“ (also „macroergic compound“ or „energy rich compounds“ ) The most important is ATP O O H O H 2 O O C H O P O P O O O ~ P O O O ~ + H + H N N N N N H 2 ATP + P O O O H O 2 O O O H O H N N N N N H 2 2 ADP P O O O C H O P O O O ~

10. ATP provides energy in two reactions: ATP + H 2 O  ADP + P i  G 0 ´ = -30,5 kJ/mol ATP + H 2 O  AMP + PP i  G 0 ´ = -32,0 kJ/mol Reactions are catalyzed by enzymes Similarly GTP, UTP a CTP can provide energy

12. Most important macroergic phosphate compounds These compounds are formed in metabolic processes. Their reaction with ADP can provide ATP = substr ate phosphorylation Compound  G 0 (kJ/mol) typ compound phosphoenolpyruvate -62 enolester carbamoyl phosphate -52 mixed anhydride 1,3-bisphosphoglycerate -50 mixed anhydride phosphocreatine -43 amide

13. Energy- rich compounds may be also thioesters (e.g. acyl group bonded to coenzym A)  G 0 = -31,0 kJ/mol

17. succinyl coenzyme A succinate + CoA Examples of substrate-level phosphorylations In glycolysis 1,3-bisphosphoglycerate 3-phosphoglycerate ADP ATP 3-phosphoglycerate kinase In the citrate cycle GDP + P i GTP thiokinase ADP + H + ATP creatine kinase phosphocreatine creatine In skeletal muscle phosphocreatine serves as a reservoir of high-potential phosphoryl groups that can be readily transferred to ATP: phosphoenolpyruvate pyruvate ADP ATP pyruvate kinase

19. [ATP]/[ADP] ratio ( in most cells 5-200 ) Energ y charge of the cell : The energy charge of most cells ranges from 0.80 to 0.95 Energy status of a cell

21. Metabolism of sacharides 1 Celular metabolism of glucose

22. Transport of Glucose into the cells Glucose transporters Transmembrane proteins facilitating a transport of glucose 2 main types: GLUT (1-14)* and SGLT** * glucose transporter ** sodium-coupled glucose transporter Molecules of glucose are strongly polar, they cannot diffuse freely across the hydrophobic lipid bilayer

23. GLUT 1-GLUT 14, family of transporters with common structural features (isoforms) but a tissue specific pattern of expresion:  500 AA, 12 transmembrane helices Mechanism of transport: facilitated diffusion (follows concentration gradient, do not require energy)

25. Glucose transporters Typ characteristics GLUT 1 Basal glucose uptake (ercs, muscle cells at resting conditions, brain vessels ..) GLUT 2 Liver,  cells of pancreas , kidney GLUT 3 Neurons, placental cells GLUT 4 Muscle, adipocytes – dependent on insulin - GLUT 5 Transport of fructose, small intestine - GLUT 7 Intracelular transport liver

26. Transport of glucose by GLUT Two conformational states of transporters Extracellular space cytosol glucose

27. Extracellular space cytosol

28. cytosol Extracellural space

29. GLUT 4 carriers are regulated by insulin (muscle, adipocytes) insulin receptor "Sleeping“ GLUT4 in the membranes of endosom e s insulin

30. insulin receptor insulin vesicles move to the membrane Binding of insulin to its receptor

31. insulin receptor Vesicles fuse with the plasma membrane Transport of glucose into the cell

32. The presence of insulin leads to a rapid increase in the number of GLUT4 transporters in the plasma membrane. Hence, insulin promotes the uptake of glucose by muscle and adipose tissue. GLUT 4 receptors– conclusion:

34. Cotransport of glucose with Na + Lumen of small intestine enterocyte

35. Transporter changes conformation after the binding glucose and Na + lumen enterocyte

36. Na + and glucose enter the cell (symport) lumen enterocyte

37. Na + /K + -ATPase is located on the capillary side of the cell and pumps sodium outside the cell (active transport) enterocyte Extracelular space

38. glucose is exported to the bloodstream via uniport system GLUT-2 (pasive transport) enterocyte glucose extracellular space

39. Glucose-6-phosphate is formed immediately after it the glucose enters a cell: glucose + ATP glucose-6-P + ADP Glucose metabolism in cells Enzymes hexokinase or glucokinase

40. Glucose phosphorylation phosphorylation glucose glucose ATP ADP glucose-6-P Hexokinase, (glucokinase) The reverse reaction is catalyzed by glucose-6-P phosphatase This occurs only in liver (and in less extent in kidney).

42. GLUCOKINASE X HEXOKINASE liver, pancreas glucose Is not inhibited low by insulin 10 In many tisues broad (hexoses) Glc-6-P high no 0,1 Occurence Specifity Inhibition Afinity to Glc Inducibility K M (mmol/l) Glucokinase Hexokinase Characteristics

43. Glucose concentration, mmol/l Enzyme activity hexokinase glucokinase Glucose concentration and rate of phosphorylation reaction K M - hexokinase K M - glucokinase V max of glucokinase V max of hexokinase 5 10 Concentration of fasting blood glucose

45. Role of glucokinase in pancreas Glucokinase in  - cells of pancreas functions as a blood glucose sensor When blood glucose level is high (after the saccharide rich meal), glucose enters the  -pancreatic cells (by GLUT2) and is phosphorylated by glucokinase Increase of energy status in the cell enhances the release of insulin

46. Conversions of Glc-6P in the cells and their significance Energy gain, synthesis of fatty acids from acetyl-CoA Formation of glucose stores Source of pentoses, source of NADPH Synthesis of glycoproteins, proteoglycans Glycolysis Synthesis of glycogene Pentose phosphate path. Synthesis of derivatives Význam Pathway

48. The glycolysis can be thought of as comprising three stages: Trapping the glucose in the cell and destabilization by phosphorylation. Oxidative stage in which new molecules of ATP are formed by substrate-level phosphorylation of ADP. Cleavage into two three-carbon units.

49. Reactions of glycolysis 1. Formation of Glc-6-P 1 hexokinase, glucokinase Non-reversible glucose glucose-6-P ATP

50. Formation of glucose-6-P from glycogen (no consumption of ATP) phosphorylase glucose-1-P glucose-6-P O phosphoglucomutase

51. 2. Izomerization of glucose-6-P Phosphoglucose isomerase Fructose-6-P reversible

52. 3. Formation fructose-1,6-bisphosphate phosphofructokinase The rate of this reaction determines the rate of the whole glycolysis ATP

55. Regulatory effect of fructose-2,6-biP on glycolysis and gluconeogenesis in liver 2,6-biP is allosteric efector of phosphofructokinase fructose 1,6-bisphosphatase (glycolysis) (gluconeogenesis) activation inhibition Formation of fructose-2,6-biP  Stimulation by fructose-6P  inhibition by glucagone

56. aldolase reversible reversible 4.Formation of triose-phosphates reversible Glyceraldehyde-3-P Dihydroxyaceton-P Triosaphosphate isomerase 5 4 4 5

57. P i Anhydride bond glyceraldehyde-3-P dehydrogenase reversible !! NAD + NADH +H + glyceraldehyde-3-P Ester bond 5. Oxidation and phosphorylation of glyceraldehyde-3-P Note, that reaction enters P i , not ATP !!!!

58. Compare: glyceraldehyde-3-phosphate 3-phosphoglycerate C H 2 C H H O C H O P O O O O - - O P O O O C H 2 C H H O C O O - - -

59. NAD + is consumed in this reaction, NADH is formed Oxidation and phosphorylation of glyceraldehyd-3-P + + Thioester bond anhydride bond 1,3-bisphosphoglycerate Phosphorolytic cleavage

60. Phosphoglycerate kinase 3-phosphoglycerate 2x ATP 1,3-bisphosphoglycerate ADP ATP reversible 6.Formation of 3-phosphoglycerate and ATP Formation of ATP by substrate-level phosphorylation: 1,3 BPG is high-energy compound (mixed anhydride), Energy released during PO 3 2- transfer is utilized for ATP synthesis

61. mutase 2,3-bisphosphoglycerate phosphatase 3-phosphoglycerate + P i 1,3-bisphosphoglycerate reversible Formation of 2,3-bisphosphoglycerate, side reaction in erytrocytes Binding to Hb, significance for release of O 2 This reaction does not provide ATP !!!

62. phosphoglycerate mutase 2-phosphoglycerate 3-phosphoglycerate reversible 7. Formation of 2-phosphoglycerate

63. + H 2 O enolase (inhibition by F - ) enolase reversible 8. Formation of phosphoenolpyruvate 2-phosphoglycerate phosphoenolpyruvate Whem blood samples are taken for mesurement of glucose, it is collected in tubes containing fluoride

64. ATP 2x ATP ADP ATP Pyruvate kinase Non reversible !!! 9. Formation of pyruvate phosphoenolpyruvate Pyruvate kinase Activation by fructose-1,6-bisP Inactivation by glucagon (substrate-level phosphorylation)

65. Conversions of pyruvate lactate acetyl CoA alanine glutamate 2-oxoglutarate ADP,P i ATP,CO 2 oxalacetate ethanol (in microorganisms) NAD + NADH, CO 2 NADH+ H + NAD +

66. Formation of lactate - anaerobic glycolysis LD Significance of this reaction: Regeneration of NAD + consumed in formation 2,3-bisP-glycerate when NAD + is lacking, the glycolysis cannot continue NADH cannot be reoxidized in respiratory chain

67. Lactate dehydrogenase (LD) Enzyme catalyzes the reaction in both directions Isoenzymes LD 1 - LD 5 Subunit H (heart) Subunit M (muscle) LD 1 LD 2 LD 3 LD 4 LD 5

69. Cori cycle – tranport of lactate from tissues into the liver, utilization for gluconeogenesis LIVER glucose MUSCLE glucose pyruvate lactate BLOOD lactate pyruvate LD LD

70. Energetic yield of glycolysis 1. Direct gain of ATP by substrate-level phosphorylation 2 ATP per one glucose Consumption of ATP Gain of ATP  - + This yield is the same for both, anaerobic and aerobic glycolysis. This is the only yield in anaerobic glycolysis

72. Total energy yield of aerobic glycolysis Aerobic glycolysis till pyruvate: * Depending on shuttle type (see lecture Respiratory chain) Further conversions of pyruvate: * (2x NADH to the resp. chain) 2 4-6 * glucose  2 pyruvate (substrate level phosporylation) 2 NADH  2NAD + ATP yield Reaction ATP yield Reakce 36-38 ATP Total maximal energy yield 6* 2x 12 2 pyruvate  2 acetylCoA + 2 NADH 2 acetyl CoA  2 CO 2 + 6 NADH + 2 FADH 2

73. Energy yield of anaerobic glycolysis Anaerobic glycolysis till pyruvate: Formation and consumption of NADH at anaerobic glycolysis 0 2 0 glucose  2 x pyruvate ( substrate-level phosphorylation) 2 NADH  2NAD+ ATP yield Reaction +2 -2 2 glyceraldehyde-3-P  2 1,3-bisP-glycerát 2 pyruvate  2 lactate In sum Yield/loss NADH Reaction