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Medical Biochemistry

Medical Biochemistry. Glycolysis/TCA/ETS Robert F. Waters, PhD. Glycolysis(Introduction). Glucose + 2 ADP + 2 NAD + + 2 P i -----> 2 Pyruvate + 2 ATP + 2 NADH + 2 H + Cofactors needed Mg++ for ATP Ca++, Zn++, Cd++ for absorption of glucose. Glycolysis. Anaerobic (Hypoxia)

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Medical Biochemistry

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  1. Medical Biochemistry Glycolysis/TCA/ETS Robert F. Waters, PhD

  2. Glycolysis(Introduction) Glucose + 2 ADP + 2 NAD+ + 2 Pi -----> 2 Pyruvate + 2 ATP + 2 NADH + 2 H+ • Cofactors needed • Mg++ for ATP • Ca++, Zn++, Cd++ for absorption of glucose

  3. Glycolysis • Anaerobic (Hypoxia) • Aerobic (Inhibits Glycolysis) • Tries to make pyruvate • Cytosol • Glucose Absorption and Transport

  4. Glucose General Information • Glucose turnover of 70kg (154 lb) person • ~2mg/kg/min or 200g/24hrs. • Hypoglycemic substances • Insulin • Binds on cells to IRS-1 (Insulin receptor substrate) • Stimulates TG synthesis • Blocks Lipolysis (Increases LDLs) • Hyperglycemic substances • Glucagon, epinephrine, HGH, cortisol

  5. Stimulatory Substances for Insulin Production • GIP-glucose dependent insulinotropic peptide • CCK-cholecystokinin (pancreozymin) • GLP-1 glucagon like peptide • VIP-vasoactive intestinal peptide NOTE: This is the reason the body has a better insulin response orally rather than IV.

  6. Digestion Cont:

  7. Glucose Absorption • Glucose Transporters (GLUTs) • Glut-1 = erythrocytes • Glut-2 = liver and pancreas • Glut-3 = brain • Glut-4 = skeletal muscle and adipose tissue • Glut-5 = small intestine (Fructose Transport) • Cotransported with Na+ • Na+ dependent glucose transport • Active Transport • Most monosaccharides can cross brush border but extremely slow (diffusion) • Fructose is absorbed by Na+ independent facilitated transport (ATP consumed as well)

  8. Glycolysis • Glucose to Glucose-6-phosphate • Hexokinase • glucose-6-phosphate (G6P)is the first reaction of glycolysis, and is catalyzed by tissue-specific isoenzymes known as hexokinases • Glucokinase • Four mammalian isozymes of hexokinase are known (Types I - IV), with the Type IV isozyme often referred to as glucokinase. Glucokinase is the form of the enzyme found in hepatocytes. The high Km of glucokinase for glucose means that this enzyme is saturated only at very high concentrations of substrate.

  9. The Km for hexokinase is significantly lower (0.1mM) than that of glucokinase (10mM). Glycolysis

  10. Glycolysis • G-6-P inhibits hexokinase • Glucose stimulates hexokinase production • Concept of Km? • Definition of a Kinase • Mg++ or Mn++ • Inhibited by Fluorine

  11. Glycolysis • Glucose-6-Phosphate to Fructose-6-Phosphate • Isomerase b-D-Fructose-6-phosphate

  12. Glycolysis • Fructose-6-phosphate to Fructose-1,6-bisphosphate • PFK-1 • PFK-2 • Insulin to Glucagon ratio • Citrate Inhibits • ATP inhibits • Mn++ or Mg++ • Inhibited by fluorine b-D-Fructose-1,6-bisphosphate

  13. Glycolysis • Split of F-1,6-bisphosphate into dihydroxyacetone phosphate and phosphoglyceraldehyde (DHAP and PGAL) • Triose phosphate isomerase • Aldolase A (Isoenzyme) • isomerase

  14. Glycolysis • PGAL to 1,3 bisphosphoglycerate • Substrate level Phosphorylation • Inhibited by Arsenate • Add Pi

  15. Glycolysis • Erythrocytes • 1,3-bisphosphoglycerate to 2,3-bisphosphoglycerate to 3-phosphoglycerate • Mutase • phosphatase • All cells metabolizing • 1,3-bisphosphoglycerate to 3-phosphoglycerate • phosphoglycerokinase

  16. Glycolysis • 3-phosphoglycerate to 2-phosphoglycerate • Phosphoglyceromutase

  17. Glycolysis • 2-phosphoglycerate to phosphoenolpyruvate • Enolase • Inhibited by fluorine (halogens)

  18. Glycolysis • PEP to Pyruvate • Pyruvate kinase

  19. Glycolysis • Anaerobic conditions • Pyruvate to lactate • Lactate dehydrogenase • Aerobic conditions • Pyruvate to mitochondrial TCA cycle • Pyruvate-keto acid form of alanine

  20. Glycolytic Control Mechanisms • Hexokinase • Insulin Stimulates • Glucose Stimulates • Inhibited by G-6-P • G-6-P does not inhibit glucokinase • Inhibited by Glucagon

  21. Glycolytic Control—PFK-1 • Stimulated by AMP • Stimulated by Fructose 2,6-bisphosphate • Inhibited by ATP • Inhibited by Citrate • Inhibited by Glucagon

  22. Glycolysis Control Mechanisms PFK-1 and PFK-2 Insulin to Glucagon Ratio

  23. Glycolytic Control-Pyruvate Kinase • Stimulated by Fructose 1,6-bisphosphate • Inhibited by Glucagon

  24. Glycolytic Metabolic Lesions • Hexokinase Deficiency • Hemolytic Anemia • Lactic Acidosis • Normal Blood Levels-1.2 mM • High Levels 5 mM or more • May be due to high lactate production or utilization • Hypoxia • Reduces Mitochondrial ATP Production • Activates PFK-1 • Stimulates Glycolysis • Increased Lactate Production • May be caused [hypoxia] by reduced blood flow in tissue (shock), respiratory disorders, etc.

  25. Pyruvate Kinase Deficiency • Pyruvate Kinase exists as isoenzymes • One or more subunits may be affected • Ionic imbalance causing erythrocytes to swell • Hemolytic anemia-excessive RBC destruction

  26. Pyruvate Kinase Deficiency-Cont. • Presentation • Newborn anemic and jaunticed • Hematology • Variability in cell morphology • Above normal reticulocytes/total RBC ratio • Pyruvate Kinase activity is ~20% of normal • Treatment • None for the most part • Splenectomy under high anemic conditions • Probably splenomegaly and hepatomegaly

  27. The Mitochondria • mtDNA • mtRNA • Inorganic Phosphate Carrier Molecule • ATP Transport Molecules

  28. Pyruvate Dehydrogenase Complex • Decarboxylation • Vitamins and pseudo-vitamins • Niacin, Riboflavin, -lipoic acid, Thiamine, CoA (Pantothenate) • Enzyme Complex (E1,E2,E3) • Pyruvate Decarboxylase (CO2) 3C to 2C, dihydrolipoyltransacetylase, dihydrolipoyldehydrogenase

  29. PDH Regulation • Inhibited by; • Acetyl CoA • NADH • Stimulated by; • CoASH • NAD+ • Pyruvate

  30. PDH Stimulation Through Inhibition • For example NAD+ inhibits Protein Kinase which does NOT phosphorylate PDH enzymes and keeps them active. • ATP and Mg++ are necessary for Protein Kinase • Phosphoprotein Phosphatase removes phosphates from phosphorylated enzymes. • This enzyme is stimulated by Ca++ • NOTE: [Ca] concentration and [ATP] concentration in mitochondria are inversely related.

  31. Further PDH Regulation • Insulin Stimulates PDH in Adipose Tissue • Catecholamines in cardiac muscle stimulates PDH • Dopamine • Norepinephrine • epinephrine

  32. Example of Cortisol Activating PDH in Cardiac Muscle • Adrenal Cortex Secretes Cortisol • A glucocorticoid (Also anti-inflammatory) • Cortisol moves into adrenal medulla • Stimulates Phenylethanolamine N-methyltransferase • Converts norepinephrine to epinephrine • Epinephrine secreted into blood and activates PDH complex • Genetic inhibition by epinephrine

  33. Epinephrine Production

  34. Defects in PDH Complex • Severe cases are fatal • Symptoms • Lactic Acidosis • Neurological Disorders • High Serum [Pyruvate] • High Serum [Alanine]

  35. PDH Defect Treatments • Large doses of thiamin • Helps with E1 defect • Large doses of lipoic acid • Helps with E2 defect • Ketogenic Diet rather than Glucogenic • Ketogenic Amino Acids • Isoleucine, leucine, tryptophan, lysine, phenylalanine, tyrosine

  36. TCA Cycle • Tricarboxylic Acid Cycle • Krebs Cycle • Citric Acid Cycle • Mitochondrial Matrix

  37. TCA Cycle • Oxaloacetate • Keto Acid form of Aspartate • Regenerating Substrate (4-carbons) • Acetyl-CoA • Stoichiometric Substrate (2-carbons) • Citrate Synthase (Irreversible) • Produce Citrate (6-carbons)

  38. TCA Cycle • Citrate to Isocitrate • Citrate less water  cis-Aconitate • Cis-Aconitate plus water  isocitrate • Prochiral carbon • Carbon with three different groups therefore distinguish between which COO- and stereospecificity with enzyme • Cis-aconitase • Bidirectional (isoergonic) • Reactants to products favored (exergonic) • Products to reactants favored (endergonic)

  39. TCA Cycle • Isocitrate to -ketoglutarate • Isocitrate dehydrogenase • Regulatory enzyme • NADH • CO2 • Oxidative decarboxylation • Coupled with reduced NAD and oxidative phosphorylation

  40. TCA Cycle • -ketoglutarate to Succinyl-CoA • -ketoglutarate dehydrogenase • Niacin, Riboflavin, Thiamine, -lipoic acid • Multi-subunit enzyme structure • CO2 • Keto acid form of Glutamate

  41. TCA Cycle • Succinyl CoA to Succinate • Succinate thiokinase (kinase) • Coupled Reaction • GDP + Pi  GTP • ADP  ATP • Substrate level Phosphorylation NOT Oxidative Phosphorylation in the production of ATP

  42. TCA Cycle • Succinate to Fumarate • Succinate Dehydrogenase • Oxidation of succinate to fumarate • Reduction of FAD+ to FADH

  43. TCA Cycle • Fumarate to Malate • Fumarase • Hydration of fumarate to malate • isoergonic

  44. TCA Cycle • Malate to Oxaloacetate • Malate dehydrogenase • Reduced NAD (NADH) • Isoergonic • Slighty endergonic (Slightly favors malate formation)

  45. ATP Production • 8 ATP - Glycolysis • 30 ATP - PDH and TCA Cycle • Theoretical Number of ATP (38) • Actual ~36 ATP per mole of glucose

  46. TCA Cycle Control • Citrate Synthase (Synthetase) • Condensing Enzyme • Inhibited By: • ATP • NADH • Succinyl CoA

  47. TCA Cycle Control-Cont: • Isocitrate Dehydrogenase • Activated By: • ADP • Inhibited By: • ATP • NADH

  48. TCA Cycle Control-Cont: • -Ketoglutarate Dehydrogenase • Inhibited by: • Succinyl CoA • NADH • ATP • Contains tightly bound Tpp, lipoamide, FAD • Similar to PDH complex • E3 subunit the same

  49. TCA Cycle Control-Cont: • Succinyl CoA Synthetase • Coupled reaction with GTP • Enzyme that catalyses coupled reaction is called Nucleotidediphosphate Kinase

  50. TCA Cycle Control-Cont: • Succinate Dehydrogenase • Has Iron-Sulfur Centers • Covalently Bound with FAD

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