Medical Biochemistry

1
Medical Biochemistry

• Glycolysis/TCA/ETS

2
Glycolysis(Introduction)

• Glucose 2 ADP 2 NAD 2 Pi -----gt 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

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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

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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.

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Digestion Cont

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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)

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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.

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Glycolysis

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Glycolysis

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

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Glycolysis

• Glucose-6-Phosphate to Fructose-6-Phosphate
• Isomerase

b-D-Fructose-6-phosphate
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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
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Glycolysis

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

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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

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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

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Glycolysis

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

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Glycolytic Control Mechanisms

• Hexokinase
• Insulin Stimulates
• Glucose Stimulates
• Inhibited by G-6-P
• G-6-P does not inhibit glucokinase
• Inhibited by Glucagon

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Glycolytic ControlPFK-1

• Stimulated by AMP
• Stimulated by Fructose 2,6-bisphosphate
• Inhibited by ATP
• Inhibited by Citrate
• Inhibited by Glucagon

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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

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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.

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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

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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

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The Mitochondria

• mtDNA
• mtRNA
• Inorganic Phosphate Carrier Molecule
• ATP Transport Molecules

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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, dihydrolipoyldehydrog
  enase

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PDH Regulation

• Inhibited by
• Acetyl CoA
• NADH
• Stimulated by
• CoASH
• NAD
• Pyruvate

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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.

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Further PDH Regulation

• Insulin Stimulates PDH in Adipose Tissue
• Catecholamines in cardiac muscle stimulates PDH
• Dopamine
• Norepinephrine
• epinephrine

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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

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Epinephrine Production

34
Defects in PDH Complex

• Severe cases are fatal
• Symptoms
• Lactic Acidosis
• Neurological Disorders
• High Serum Pyruvate
• High Serum Alanine

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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

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TCA Cycle

• Tricarboxylic Acid Cycle
• Krebs Cycle
• Citric Acid Cycle
• Mitochondrial Matrix

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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)

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ATP Production

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

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TCA Cycle Control

• Citrate Synthase (Synthetase)
• Condensing Enzyme
• Inhibited By
• ATP
• NADH
• Succinyl CoA

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TCA Cycle Control-Cont

• Isocitrate Dehydrogenase
• Activated By
• ADP
• Inhibited By
• ATP
• NADH

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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

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TCA Cycle Control-Cont

• Succinyl CoA Synthetase
• Coupled reaction with GTP
• Enzyme that catalyses coupled reaction is called
  Nucleotidediphosphate Kinase

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TCA Cycle Control-Cont

• Succinate Dehydrogenase
• Has Iron-Sulfur Centers
• Covalently Bound with FAD

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General TCA Information

• Amphibolic
• Involved in catabolic and anabolic processes
• Anaplerotic Reactions
• Increase concentrations of TCA cycle
  intermediates
• Example Amino Acid Metabolism
• Aminotransferase Glutamate and ?-ketoglutarate
• Aminotransferase Aspartate and Oxaloacetate
• Pyruvate Carboxylase Gluconeogenesis
• Pyruvate CO2 ? Oxaloacetate
• The aminotransferase associated with alanine and
  pyruvate is not anaplerotic because pyruvate is
  not TCA intermediate.

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Specifics of Pyruvate Dehydrogenase Complex

• E1
• Pyruvate Decarboxylase
• Liberates CO2
• Thiamine (TPP)
• E2
• Dihydrolipoyltransacetylase
• Produces Acetyl-CoA
• ?-lipoic Acid
• CoA-Pantothenic Acid
• E3
• Dihydrolipoyldehydrogenase
• Produces FADH to NADH
• Riboflavin and Niacin

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Malate Aspartate Shuttle

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Glycerophosphate Shuttle

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Cytochromes

Cytochrome c oxidase contains two haem a groups,
one binuclear copper site (CuA), one mononuclear
copper site (CuB) and one bound Mg2 per monomer
plus FeII and FeIII iron associated with sulfur
(S).
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Cytochrome c Oxidase (COX)

• Do NOT get confused with cyclooxygenase also
  called COX!
• Cytochrome c Oxidase Cycle

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Co-enzyme Qx

• Impaired coenzyme Q10 synthesis due to
  nutritional deficiencies. (Ubiquinone)
• Genetic or acquired defect in coenzyme Q10
  synthesis.
• Increased tissue needs resulting from a
  particular medical condition.

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Co-enzyme Q10

• Interconversion
• Isoprenoid Units

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Electron Transport System

• Oxidative Phosphorylation

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Electron Transport Continued

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Electron Transport Continued

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Electron Transport Continued

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Electron Transport Continued

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Electron Transport Continued

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Electron Transport Continued

Chemistry of Complex V
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ETS-Cont

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Electron Transport Inhibitors

• Complex I
• Many insecticides
• Barbiturates
• Amobarbital
• Secobarbital
• Some antibiotics

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Electron Transport Inhibitors-Cont

• Complex III
• Some antibiotics
• Complex IV
• H2S
• Cyanide
• Azide
• CO

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Electron Transport Inhibitors-Cont

• Cyanide Poisoning (Example)
• Almond smell of gaseous CN (breath)
• Severe Acidosis
• A cancer treatment in Mexico is called Amygdalin
  (extracted from almonds) may be harmful due to CN
  is metabolite.
• Treatment
• Infusion of thiosulfate (binds CN)
• Ventilation with 100 O2
• Administer Sodium Bicarbonate