1. Metabolism of carbohydrates

2. Sources of glucose (Glc) ● from food (4 hours after meal) ● from glycogen (from 4 to 24 hours after meal) ● from gluconeogenesis (days after meal, during starvation) Figure was assumed from Devlin, T. M. (editor): Textbook of Biochemistry with Clinical Correlations, 4th ed. Wiley ‑ Liss, Inc., New York, 1997

3. Glycemia glucose concentration in the blood physiological range of fasting glycemia 3,3 – 5,6 mmol/L is regulated by hormones (insulin, glucagon, epinephrine, kortisol, …)

4. Glucose can enter into cells: a) by facilitative diffusion (GLUT 1 – 7) GLUT 1 – blood-brain barrier, erythrocytes GLUT 2 – liver, β-cells in pancreas GLUT 3 – neurons GLUT 4 – skeletal muscles, heart muscle, adipose tissue b) by cotransport with Na + ion (SGLT-1 and 2) Figure was assumed from textbook: Devlin, T. M. (editor): Textbook of Biochemistry with Clinical Correlations, 4th ed. Wiley ‑ Liss, Inc., New York, 1997. small intestine, kidneys

5. An effect of insulin on insulin-sensitive cells Transport of Glc into cells is dependent on insulin effect (GLUT-4) in the following tissues: skeletal and heart muscle and adipose tissue Figure is found on http://www.mfi.ku.dk/ppaulev/chapter27/Chapter%2027.htm

6. Metabolic pathways included in utilization of Glc – glycolysis, pentose cycle, glycogen synthesis Phosphorylation of glucose  after enter into cell Glc is always phosphorylated to form Glc-6-P  enzyme hexokinase catalyzes esterification of Glc  ATP is a donor of phosphate group!  enzyme is inhibited by excess of Glc-6-P  2 isoenzymes of hexokinase exist: hexokinase and glucokinase  hexokinase has a higher affinity to glucose than glucokinase

7. Hexokinase vs. glucokinase Figure is found on http://web.indstate.edu/thcme/mwking/glycolysis.html K M hexokinase = 0,1 mM K M glucokinase = 10 mM

8. Glycolysis substrate: Glc-6-P product: pyruvate function: source of ATP subcellular location: cytosol organ location: all tissues regulatory enzymes: hexokinase/glucokinase, 6-phosphofructokinase-1 (main regulatory enzyme), pyruvatekinase Regulatory enzymes are activated by hormone insulin!

9. Glucose Glucose in blood: Breakdown of polysaccharides Synthesis from noncarbohydrate precursors (gluconeogenesis) Glucose Utilization (Nucleic acid and NAD synthesis) (ATP and intermediates)

10. Glycolysis “Sweet splitting” Catabolism of 1 mol glucose to form 2 mol pyruvate Sequence of 10 enzyme-catalyzed reactions Occurs in almost every living cell Two stages (phases): Hexose/preparatory (stage 1, consumes 2 ATP) Triose/payoff (stage 2, generates 4 ATP and 2NADH) Anaerobic process (no oxygen required) Overall chemical reaction: D-glucose + 2ADP + 2P i + 2NAD + → 2 pyruvate + 2ATP + 2NADH + 2H + + 2H 2 O Provides significant portion of free energy used by most organisms

11. 1 2 3 4 5 6 7 8 9 10 (McKee and McKee, Biochemistry, 3rd ed.) The Glycolytic Pathway

12. Glycolysis Figure is found on http://web.indstate.edu/thcme/mwking/glycolysis.html

13. 1. Synthesis of glucose-6-phosphate (G6P) Glucose is phosphorylated immediately after entering cell Prevent transport out of cell Increase activity of phosphate ester oxygen Enzyme = hexokinase Catalyze phosphorylation of hexoses Remember: kinase = transfer phosphoryl group between ATP and metabolite ATP-Mg 2+ complex is cosubstrate Source of phosphoryl group Metal shields negative charge, making P more accessible and electrophilic Reaction is irreversible Product not accommodated by enzyme active site  G ’ ° = -16.7 kJ/mol

14. Conformational change of Hexokinase Active site engulfed when glucose binds Brings ATP-Mg 2+ closer to glucose C6 Proximity Excludes water from active site Prevents competing phosphoryl group transfer to water Less polar environment increases rate of nucleophilic reaction

15. 2. Conversion of G6P to fructose-6- phosphate (F6P) Aldose converted to Ketose Enzyme = phosphoglucose isomerase (PGI) aka phosphohexose isomerase  G ’ ° = 1.7 kJ/mol Reaction occurs on linear form of G6P Substrate binds to enzyme Ring opening catalyzed by Lys or His residue Proton transfer to/from Glu residue achieves isomerism Ring closes, is released from PGI C1 of F6P now available for phosphorylation

16. Mechanism of the Phosphohexose Isomerase Reaction

17. 3. Phosphorylation of F6P to form fructose-1,6-bisphosphate (FBP) Irreversible reaction  G ’ ° = -14.2 kJ/mol Catalyzed by phosphofructokinase (PFK-1) Regulatory enzyme Inhibited by high levels of ATP and citrate (indicators that citric acid cycle has slowed down) Requires a second mole of ATP- Mg 2+ complex Prevent later products from diffusing out of cell Rate determining step Commit the cell to glycolysis

18. 4. Cleavage of FBP Products: Dihydroxyacetone phosphate (DHAP) from C1-C3 Glyceraldehyde-3-phosphate (GAP) from C4-C6 Enzyme = aldolase Covalent catalysis Acid-base catalysis Electrostatic stabilization of intermediates  G ’ ° = 23.8 kJ/mol (note standard) Reaction = aldol cleavage Reverse of aldol condensation Common C-C bond cleavage reaction Two carbonyl products (aldehyde and ketone), each with 3 carbons Would not be true of G6P

19. Mechanism of the Aldolase Reaction

20. 5. Interconversion of GAP and DHAP GAP is the only substrate for the next reaction in glycolysis DHAP is converted to isomeric GAP to prevent loss of 3 carbon unit Enzyme = triose phosphate isomerase (TPI or TIM) Rate of reaction is diffusion controlled Product formation occurs as quickly as E and S collide Catalytic perfection  G ’ ° = 7.5 kJ/mol

21. Formation of GAP

22. End of Stage 1 1 mol glucose → 2 mol GAP

23. Next…payoff

24. 6. Oxidation/phosphorylation of GAP to 1,3-bisphosphoglycerate (1,3-BPG) Aldehyde is oxidized and phosphorylated Oxidizing agent = NAD + Enzyme = glyceraldehyde-3-phosphate dehydrogenase Binds GAP and NAD +  G ’ ° = 6.3 kJ/mol 1,3-BPG contains high-energy bond Used in next step to generate ATP

25. Mechanism of the Glyceraldehyde-3- phosphate Dehydrogenase Reaction

26. 7. Phosphoryl group transfer to form 3-phosphoglycerate (3PG) Phosphoryl group transfers from 1,3-BPG to ADP Substrate-level phosphorylation ATP is produced (2 mol in overall pathway) Enzyme = phosphoglycerate kinase (PGK) Named for reverse reaction Mg 2+ required Two domains ADP 1,3-BPG Domains swing together to create water-free active site, as with hexokinase  G ’ ° = -18.5 kJ/mol Equilibrium slightly shifted to products

27. 8. Interconversion of 3PG and 2-phosphoglycerate (2PG) 3PG has low phosphoryl-group-transfer potential Isomerization of 3PG to 2PG is first step to forming a molecule with a high energy phosphate bond Enzyme = phosphoglycerate mutase (PGM) An isomerase Mg 2+ ? No, but does need BPG to seed the rxn Phosphoryl group from active site on enzyme transferred to substrate Bisphospho intermediate 2,3-BPG Phosphoryl group from C3 transfer back to His  G ’ ° = 4.4 kJ/mol

28. The Phosphoglycerate Mutase Reaction

29. 9. Dehydration of 2PG to form phosphoenolpyruvate (PEP) Dehydration of primary alcohol Elimination of H 2 O from C2 and C3 PEP has high phosphoryl-group-transfer potential due to enol Phosphoryl group restricts keto-enol tautomerization Enzyme = enolase Requires Mg 2+ for activity (F - inhibits)  G ’ ° = 7.5 kJ/mol

30. Synthesis of pyruvate from PEP Enzyme = pyruvate kinase (PK) Requires both Mg 2+ and K +  G ’ ° = -31.4 kJ/mol Phosphoryl group transfer to ADP Substrate-level phosphorylation Overall, 2 mol ATP produced Reaction is irreversible Large decrease in free energy as keto tautomer formed from enolpyruvate

31. Activity, part 1

32. Activity, part 2

33. Fate of Pyruvate Pyruvate does not accumulate Undergoes one of 3 possible enzyme- catalyzed reactions Reaction depends on type of cell or species or the availability of Oxygen

34. Fate of Pyruvate Anaerobic organisms / conditions: Need to oxidize NADH to regenerate NAD + for glycolysis to continue Pyruvate converted to waste products (ethanol, lactate, acetic acid, etc.) through fermentation Mammals: homolactic or lactate fermentation (lactate dehydrogenase) Yeast/microorganisms: alcoholic fermentation (pyruvate decarboxylase) Aerobic organisms: NADH oxidized by oxidative phosphorylation (using molecular oxygen) Oxidative decarboxylation of pyruvate to form CO 2 and an acetyl group (of Acetyl- CoA) Enzyme = pyruvate dehydrogenase Requires coenzyme A and NAD + Acetyl-CoA becomes fuel for the citric acid cycle (formation of CO 2 and H 2 O) and building block for fatty acid synthesis

35. Regulation of glycolysis Glycolysis operates continuously under steady-state conditions Flux varies to meet the needs of organism Regulation primarily controlled by allosteric enzymes Hexokinase (reaction 1) Inhibited by uncomplexed ATP (no Mg 2+ ) and G6P (product of rxn 1) PFK (reaction 3) Inhibited by ATP and citrate Pyruvate kinase (reaction 10) Inhibited by ATP Activated by FBP (product of rxn 3) Reactions of these enzymes are irreversible

36. Regulation of glycolysis Regulatory enzymes ● Hexokinase – inhibited by Glc-6-P ● Glucokinase – activated by insulin – inhibited by Fru-6-P ● 6-phosphofructokinase-1 (PFK-1) – activated by insulin, ↑ AMP / ATP - inhibited by ↑ ATP /AMP, citrate ● Pyruvatekinase – activated by insulin, Fru-1,6-bisP - inhibited by glucagon, ↑ ATP /AMP, acetyl-CoA

37. Metabolism of other carbohydrates Fructose, galactose, and mannose Convert to glycolytic intermediates Metabolized by glycolytic pathway Pentose phosphate pathway Alternative pathway to glycolysis Glucose degradation Products = ribose-5-phosphate and NADPH Biosynthetic precursors

38. Production of ATP in glycolysis  conversion of 1,3-bisphosphoglycerate to 3-phosphoglycerate  conversion of phosphoenolpyruvate (PEP) to pyruvate These reactions are examples of substrate level phosphorylation!

39. Pentose phosphate pathway substrate: Glc-6-P product: CO 2, NADPH + H + function: gain of NADPH + H +, production of rib-5-P for nucleotide synthesis, mutual conversions of monosacharides subcellular location: cytosol organ location: all tissues regulatory enzyme: glucose 6-phosphate dehydrogenase

40. Pentose phosphate pathway – oxidative stage produces rib-5-P Figure is found on http://web.indstate.edu/thcme/mwking/pentose-phosphate-pathway.htmlhttp://web.indstate.edu/thcme/mwking/pentose-phosphate-pathway.html

41. Pentose phosphate pathway – non-oxidative stage includes interconversions of monosaccharides Figure is found on http://web.indstate.edu/thcme/mwking/pentose-phosphate-pathway.htmlhttp://web.indstate.edu/thcme/mwking/pentose-phosphate-pathway.html

42. Glycogen synthesis (glycogenesis) substrate: Glc-6-P product: glycogen function: glucose storage in the form of glycogen cellular location: cytosol organ location: especially in the liver and skeletal muscles, other tissues have lower glycogen storage regulatory enzyme: glycogen synthase Enzyme glycogen synthase is inhibited by phosphorylation (glucagon in liver and epinephrine in muscles)!

43. Glycogen synthesis Glc-6-P → Glc-1-P Glc-1-P + UTP → UDP-Glc + PP i Glycogen synthase catalyzes the formation of  (1 → 4) glycosidic bonds. Branching (formation of  (1 → 6) glycosidic bonds) is performed by enzyme amylo-(1,4 – 1,6)- transglycosylase („branching enzyme“). Figure is found on http://en.wikipedia.org/wiki/Glycogenhttp://en.wikipedia.org/wiki/Glycogen

44. Metabolic pathways serving to supplementation of Glc into the bloodstream – glycogen degradation and gluconeogenesis Glycogen degradation (glycogenolysis) ● substrate: glycogen product: Glc-6-P function: releasing of Glc from glycogen subcellular location: cytosol organ location: liver, skeletal muscles, but also other tissues regulatory enzyme: glycogen phosphorylase Enzyme glycogen phosphorylase is activated by phosphorylation which is induced by hormones glucagon and epinephrine. Insulin inhibits enzyme phosphorylation.

45. Glycogen degradation Glycogen (n Glc) + P i → Glc-1-P + glycogen (n - 1 Glc) Enzyme glycogen phosphorylase catalyzes the cleavage of  1 → 4 bonds. Enzyme amylo-  1 → 6-glucosidase („debranching enzyme“) cleaves  1 → 6 bonds. Glc-1-P ↔ Glc-6-P phosphoglucomutase Glc-6-P glucose-6-phophatase (liver, kidneys, enterocytes) Glc

46. Gluconeogenesis substrates: lactate, pyruvate, glycerol, amino acids – Ala, Asp, Gln etc. product: glucose function: synthesis of Glc from non-sugar precursors subcellular location: mitochondrial matrix + cytosol organ location: liver + kidneys regulatory enzymes: pyruvate carboxylase and PEP carboxykinase Regulatory enzymes are activated by hormones glucagon and cortisol. Insulin inhibits them.

47. Scheme of gluconeogenesis Figure is found on http://web.indstate.edu/thcme/mwking/gluconeogenesis.htmlhttp://web.indstate.edu/thcme/mwking/gluconeogenesis.html

48. Gluconeogenesis Synthesis of PEP is divided into 2 steps: Pyr → matrix of mitochondria → Pyr is carboxylated to oxaloacetate (OA) by pyruvate carboxylase CH 3 -CO-COO - + CO 2 + ATP → - OOC-CH 2 -CO-COO - + ADP + P i OA is transported to the cytosol and decarboxylated to PEP by PEP carboxykinase - OOC-CH 2 -CO-COO - + GTP → PEP + CO 2 + GDP Synthesis of 1 mol Glc consumes 4 mol ATP and 2 mol GTP!

49. Figure was assumed from http://www.biochem.arizona.edu/classes/bioc462/462b/glycolysis.htmlhttp://www.biochem.arizona.edu/classes/bioc462/462b/glycolysis.html

50. Regulation of gluconeogenesis Hormones: activation: cortisol, glucagon, epinephrine inhibition: insulin Enzyme pyruvate carboxylase activation: acetyl-CoA from β-oxidation of FA → source of ATP Enzyme fructose-1,6-bisphosphatase activation: citrate, starvation inhibition: AMP, Fru-2,6-bisP Enzyme glucose-6-phosphatase (in ER of liver, kidneys and enterocytes !)

51. Cori cycle Figure was assumed from http://web.indstate.edu/thcme/mwking/gluconeogenesis.htmlhttp://web.indstate.edu/thcme/mwking/gluconeogenesis.html

52. Glucose-alanine cycle Figure is found on http://web.indstate.edu/thcme/mwking/gluconeogenesis.htmlhttp://web.indstate.edu/thcme/mwking/gluconeogenesis.html

53. Fructose metabolism Fru is a component of sucrose (Glc + Fru) part of Fru in converted to Glc in enterocytes: Fru-6-P → Glc-6-P → Glc part of Fru is absorbed and it is transferred via blood into liver: Fru + ATP → Fru-1-P + ADP by enzyme fructokinase Fru-1-P is broken down to glyceraldehyde (GA) and dihydroxyacetonephosphate (DHAP) by aldolase DHAP enters glycolysis and GA → glyceraldehyde-3-P → glycolysis

54. Galactose metabolism Gal is a component of lactose (Glc + Gal) Gal is absorbed by the same mechanism in enterocytes like Glc → liver Gal is phosphorylated in liver to form Gal-1-P: Gal + ATP → Gal-1-P + ADP by enzyme galactokinase Gal-1-P is converted to UDP-Gal: Gal-1-P + UTP → UDP-Gal + PP i by uridyltransferase UDP-Gal is used to lactose synthesis in mammary gland during lactation epimerization of UDP-Gal to UDP-Glc → glycogen synthesis / synthesis of glucuronic acid / glycoprotein synthesis