Microbial Metabolism Chapter 4

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Microbial Metabolism(Chapter 4)

• Louise S. Thai, M.D.

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Objectives

• Understand major metabolic pathways
• Know the differences between aerobic and
  anaerobic respirations
• Know the diagnostic application of commonly known
  metabolic patterns

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Definitions

• Catabolism
• Anabolism
• Metabolism
• ATP

• Enzyme
• Coenzyme
• Ribozyme
• Substrate

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• Catabolism
• The process of substrate breakdown
• generates energy (ATP)
• Anabolism
• The process of synthesis of cellular elements
• Requires energy (ATP)
• Metabolism Catabolism Anabolism

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ATP (Adenosine Triphosphate)

• Structure adenine ribose 3 P
• Link between catabolic and anabolic pathways
• Stores the energy captured from catabolic
  breakdown reactions in P-bonds
• Is broken down to provide energy in anabolic
  biosynthetic reactions

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Enzymes

• A special category of proteins
• Found in all living organisms
• Are substrate specific
• Are catalysts
• Speed up reaction rate a million times
• Remain unchanged while doing that
• Named by the name of the substrate suffix ase
  (e.g., protease, phosphatase, lipase etc.)
• Ribozyme
• An RNA molecule that catalyzes RNA reactions

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Coenzymes and Cofactors

• Coenzymes
• Organic by nature
• A non-protein portion of the enzyme molecule
• Many are synthesized from vitamins
• NAD is made from niacin

• Cofactors
• Inorganic by nature
• Improve the fit of the enzyme with its substrate
  
• Ions of
• Mg
• Zn
• Mn

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Enzyme Inhibition

• Regulates the enzymatic activity
• Similar in structure to substrate
• Competitively inhibits the reaction
• Competes with the substrate for the active site
• Examples
• Sulfa drugs compete with PABA for the enzyme that
  converts it to folic acid
• Penicillins compete with the substrate for
  transpeptidation enzyme during bacterial cell
  wall synthesis

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Noncompetitive Inhibition

• Noncompetitive inhibitors bind to a site other
  than the active site
• Reversible
• Irreversible
• Distort the tertiary protein structure
• Alter the shape of the active site
• Prevent the substrate binding
• Examples Pb, Hg, and other heavy metals

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Factors that affect the enzymatic reactions

• Temperature
• pH
• Concentrations of substrate, product , and
  enzyme
• Example A B ? AB or AB ? A B

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Feedback Inhibition

• The end product of an enzymatic reaction binds to
  and inactivates the enzyme
• Reversible
• Noncompetitive

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CATABOLISM
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Carbohydrate Catabolism

• Sugars are broken down to generate energy
• Some ATP is used, but more produced
• Glucose breakdown
• Glycolysis or Embden-Meyerhof Pathway
• Pentose phosphate pathway - parallel with
  glycolysis
• Entner-Doudoroff pathway - alternative to
  glycolysis and pentose phosphate pathway

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Glycolysis (Embden-Meyerhof Pathway)

• The most common pathway
• Does not require oxygen
• Used by both aerobes and anaerobes
• Key events
• Phosphorylation of glucose
• Breaking of a 6-C-molecule of glucose into two
  3-C-molecules
• Electron transfer to coenzyme NAD
• Capture of energy in the form of ATP

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Pentose Phosphate Pathway (Hexose Monophosphate
Shunt)

• Runs concurrently with glycolysis
• Provides precursors for biosynthesis (e.g.,
  ribose for nucleic acids, erythrose for amino
  acid synthesis)
• Provides reducing power in the form of NADPH for
  use in biosynthesis
• Generates 1 ATP
• Examples
• E. coli
• B. subtilis

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Entner-Doudoroff Pathway

• Alternative to glycolytic and pentose phosphate
  pathways
• Glucose undergoes a short series of reactions
  until glyceraldehyde 3-P
• Glyceraldehyde 3-P follows the last 5 steps of
  glycolysis
• 1 ATP is generated
• Example
• Pseudomonas aeruginosa

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What happens after glycolysis?

• Respiration
• The final electron acceptor is an inorganic
  molecule (e.g., oxidation of pyruvic acid to CO2
  and H2O)
• Fermentation
• The final electron acceptor is an organic
  molecule (e.g., conversion of pyruvic acid to
  ethanol, lactic acid etc.)

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In the presence of O2

• Pyruvate enters the TCA Krebs cycle
• Oxidative decarboxylation ? Acetyl-CoA
• Acetyl-CoA Oxaloacetate Citrate
• Series of oxidative reactions
• Back to oxaloacetate
• Yield 2 moles of CO2, 3 moles of NADH, 1 FADH2,
  1 GTP

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Aerobic Respiration

• NADH and FADH2 enter the electron transport chain
  (respiratory chain)
• Electrons are passed from carrier to carrier
• Each carrier is reduced as it picks up electrons
  and is oxidized as it passes it on to the next
  carrier in the chain
• Energy released is captured in ATP molecules
• The final electron acceptor is O2 ? H2O

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In the absence of O2

• Anaerobic Respiration
• Electron acceptor other than O2
• NO3-
• SO4-
• CO2
• Less efficient than aerobic in ATP generation

• Fermentation
• Electron acceptor is an organic compound
• Lactic acid
• Propionic acid
• Butyric acid
• Mixed acid
• No ATP produced

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Aerobic and Anaerobic Growth

• For most organisms
• Oxygen enhances metabolism and growth
• Oxygen acts as the hydrogen acceptor during
  energy (ATP) production
• Use of oxygen generates toxic H2O2 O2-
• Superoxide dismutase and catalase are essential

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Enzyme Action

• Superoxide dismutase
• 2O2- 2H ? H2O2 O2
• Catalase
• 2H2O2 ? 2H2O O2

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Response to O2

• Obligate aerobes have the enzymes
• Neisseria meningitidis
• Facultative anaerobes have the enzymes
• Escherichia coli
• Aerobic respiration in the presence of O2
• Fermentation in the absence of O2
• Obligate anaerobes lack the enzymes
• Clostridium tetani

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Fermentation Tests in Clinical Laboratory

• Sugar containing medium is inoculated with
  bacteria
• If fermentation occurs ? pyruvate and lactate are
  produced
• The medium turns acidic
• Acid lowers the pH
• pH indicator changes color
• Phenol red ? yellow

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Examples of Sugar Fermentation Tests

• Shigella sonnei
• Glucose
• E. coli
• Glucose
• Lactose
• S. aureus
• Mannitol

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Clinical Relevance

• Clostridium perfringens
• Ferments glucose
• Butyric acid is produced
• Butyric acid causes tissue damage

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Oxidation of Sugars

• Neisseria meningitidis utilizes
• Glucose
• Maltose
• Neisseria gonorrhoeae utilizes
• Glucose

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ANABOLISM
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Carbohydrate Anabolism

• Carbohydrates
• Catabolic intermediates are used for biosynthesis
• Energy is supplied by ATP

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Protein Catabolism

• Hydrolysis by proteolytic digestion
• Production of amino acids
• Deamination
• Glutamic acid ? ?-ketoglutarate
• Aspartic acid ? oxaloacetate
• Oxidation in Krebs cycle

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Protein Anabolism

• Some bacteria can synthesize all the necessary
  amino acids from the intermediates of glucose
  metabolism
• Fastidious bacteria require external amino acids
  for their growth
• Proteins are synthesized from amino acids
• Genes (DNA) regulate the synthesis
• The information is transcribed onto mRNA
• Then translated into tRNA
• tRNA-aa is read on ribosomes

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Lipid (Fat) Metabolism

• Fats are hydrolyzed to glycerol and 3 fatty acids
• Glycerol is used in glycolytic pathway
• Fatty acids undergo beta-oxidation ?Acetyl-CoA
• Oxidized in Krebs Cycle
• Precursor for lipid synthesis