Microbial Metabolism

1
Microbial Metabolism

• Enzymes

2
What is metabolism?

• The sum of chemical reactions within a living
  organism

C6H12O6 6O2
H2O
6CO2 6H2O energy
NAD
e-
ATP
H
O2
NADH
3
Why do we need to know about microbial metabolism?

• Metabolism is the basis of all life, not just
  microbes
• Metabolism forms the basis of all forms of
  microbiology including taxonomy to medical
  microbiology
• If your interest is in human health, knowledge of
  metabolism forms the basis of antibiotic therapy.
  Some antibiotics interfere with metabolic
  reactions

4
Metabolism Catabolism and Anabolism

• Catabolic Reactions
• The breakdown of complex organic molecules into
  simpler molecules
• Generally hydrolytic
• exergonic (produce energy)-energy stored in
  chemical bonds is released
• Anabolic Reactions
• The synthesis of complex organic molecules from
  simpler molecules
• Generally dehydration synthesis reactions
  (release water)
• Endergonic (consume energy)

5
Role of ATP in metabolism

• ATP (adenosine triphosphate) stores the energy
  generated by catabolic reactions and makes it
  available for anabolic reactions

6
Enzymes-the driving force of metabolic reactions

• Catalyze the chemical reactions of life
• Enzymes an example of catalysts, chemicals that
  increase the rate of a chemical reaction without
  becoming part of the products or being consumed
  in the reaction
• Specific for a particular substrate and reaction
• The unique three-dimensional shape of an enzyme
  allows it to recognize its substrate

7
How do enzymes work?

• By decreasing the activation energy, the energy
  required to initiate a chemical reaction
• Enzymes have an active site at which only
  specific reactants or substrates are positioned
  for various interactions

8
Enzyme-substrate interaction
Active site
9
Turnover number

• Enzymes participate in chemical reactions but are
  not consumed by them (can function over and over
  again)
• An enzymes speed or turnover number is the
  maximum number of substrate molecules an enzyme
  molecule can convert to product each second
• Enzyme speeds can range over several orders of
  magnitude but are characteristic of a particular
  enzyme
• Examples
• DNA polymerase (DNA synthesis) 15
• Catalase (breakdown of H2O2)
  20,000

10
Enzyme components

• Simple enzymes-entirely protein
• Conjugated enzymes consist of
• Apoenzyme-the protein component
• Cofactor-non protein component e.g., Mg2 or Ca2
  (metal) ions. If cofactor is an organic molecule
  it is called a coenzyme
• Apoenzyme cofactor Holoenzyme
• In the absence of the cofactor, the apoenzyme is
  inactive

11
Coenzymes

• Can act in catalysis by accepting a chemical
  group from one substrate and transferring it to
  another substrate
• Some act as electron carriers
• Many are derived from vitamins. Examples
• vitamin B6-coenzyme in amino acid metabolism,
• Folic acid-coenzyme in the synthesis of
  nucleotides

12
Important coenzymes in cellular metabolism

• Nicotinamide adenine dinucleotide (NAD)
• NAD is involved in catabolic reactions
• Nicotinamide adenine dinucleotide phosphate
  (NADP)
• NADP is involved in anabolic reactions
• Both NAD and NADPH are derivatives of vitamin B1
  (niacin) and they both function as electron
  carriers

13
Other key coenzymes

• The flavin coenzymes
• Flavin mononucleotide (FMN) and flavin adenine
  dinucleotide (FAD)
• Derivatives of vitamin B2 (riboflavin)
• Also act as electron carriers
• Coenzyme A
• Derivative of vitamin B5 (pantothenic acid)
• Important roles in fat metabolism and the TCA
  cycle

14
Naming enzymes
15
Factors affecting enzymatic activity
-rate of chemical reactions increases with
temperature -elevation above a certain
temperature reduces enzymatic activity due to
denaturation of the enzyme
-most enzymes have a pH optimum -changes in pH
can cause result in alterations in the
3D-structure of the enzyme leading to denaturation
-high substrate concentration leads to maximal
enzyme activity, the enzyme is said to be
saturated -under normal conditions enzymes are
not saturated
16
Exo- and Endoenzymes

• Exoenzymes
• Active outside the cell
• Breakdown of nutrients that are too large to
  enter the cell.
• Some play a role in disease e.g., Streptokinase

• Endoenzymes
• Most metabolic enzymes are endoenzymes

17
Control of metabolic pathways

• Metabolic pathways are controlled at the level of
  their enzymes
• Control of enzymes
• Synthesis
• Activity

18
Production of enzymes in the cell
Their production can be regulated in response to
substrate or product concentrations.
Enzymes can be produced at constant levels in the
cell OR
19
Enzyme Inhibitors

• An effective way to control the growth of
  bacteria is to control their enzymes
• Certain poisons such as cyanide, arsenic and
  mercury combine with enzymes and inhibit their
  activity
• Enzyme inhibitors can be classed as
• Competitive inhibitors
• Noncompetitive inhibitors

20
Competitive inhibitors

• Fill the active site and compete with substrate
• Similar in shape and chemical structure to the
  substrate

e.g., Inhibition of folic acid synthesis by
sulfanilamide
21
Noncompetitive inhibitors

• Interact with a site other than the active site
• Binding of the inhibitor causes a change in the
  shape of the active site, making it nonfunctional

22
Enzyme Repression

• The end-product of the reaction signals back to
  the DNA to turn off expression of the gene
• Prevents the cell from wasting energy

23
The Pursuit and Utilization of Energy

• Energy in Cells
• Exergonic reaction a reaction that releases
  energy as it goes forward
• Endergonic reaction a reaction that is driven
  forward with the addition of energy

24
The Cells Energy Machine
25
Basic Chemical Reactions Underlying Metabolism

• Catabolism and Anabolism
• Oxidation and Reduction Reactions
• ATP Production and Energy Storage

26
Energy production

• Nutrient molecules have energy associated with
  the electrons that form bonds between atoms
• Catabolic reactions oxidize nutrients by removing
  electrons and concentrate their energy into the
  bonds of ATP
• ATP has high energy or unstable bonds which
  allows the energy to be released quickly and
  easily.

27
ATP
28
ATP generation

• Cells use oxidation-reduction (redox) reactions
  in catabolism to extract energy from nutrient
  molecules
• This energy is trapped by the generation of ATP
  by phosphorylation of ADP

29
Oxidation-reduction reactions

• Oxidation is the removal of electrons from a
  molecule
• Reduction is the gaining of electrons by a
  molecule
• Oxidation and reduction reactions are always
  coupled (redox reaction)

Acronym for Redox reactions O.I.L R.I.G.
(Oxidation
Involves Loss Reduction Involves Gain)
30
Many catabolic oxidation-reduction reactions are
also dehydrogenation reactions

• The removal of electrons also means the removal
  of hydrogen atoms i.e., not just an electron but
  also a proton (H)
• These are transferred to an electron carrier

31
Electron carriers
2H 2e-

• In catabolic reactions, energy is extracted from
  molecules in the form of electrons, which are
  transferred, along with H ions, to electron
  carriers like NAD.

NAD
NADH H
32
Mechanisms of ATP generation

• Substrate-level phosphorylation
• Oxidative phosphorylation
• Photophosphorylation(only in photosynthetic cells)

33
Substrate-level phosphoryation

• ATP is generated when a high-energy phosphate is
  transferred directly to ADP from a phosphorylated
  substrate

34
Oxidative phosphorylation

• Electrons are transferred from organic compounds
  through a series of electron carriers to O2 or
  other oxidized inorganic or organic molecules
• The sequence of electron carriers is called the
  electron transport chain

• The transfer of electrons from one carrier to the
  next generates energy which is used to make ATP
  from ADP by chemiosmosis

35
How do chemoheterotrophs generate energy?

• Sources of energy carbohydrate, fat, protein,
  minerals.
• Most microorganisms oxidize carbohydrates as the
  major source of cellular energy
• Energy can also be derived from the oxidation of
  fats, proteins, and minerals.

36
Carbohydrate catabolism

• Microbes use three general processes to generate
  energy from glucose
• Aerobic respiration
• Anaerobic respiration
• Fermentation
• All 3 start with glycolysis
• ( Emden Meyerhoff pathway)

37
Figure 8.15
38
Aerobic Respiration

• Glycolysis
• Glucose is oxidized to pyruvic acid
• Pyruvic acid is oxidized to acetyl CoA
• TCA cycle (Krebs cycle)
• Acetyl CoA is oxidized to CO2
• Electron transport chain
• Reduced NADH and FADH2 from the above are
  oxidized through a series of redox reactions
  through an electron transport chain.

39
Glycolysis

• Starting point for cellular respiration and
  fermentation.
• 10 step catabolic pathway
• Two stages
• Preparatory stage
• Energy conserving stage

40
Glycolysis preparatory stage
Glucose
Hexokinase

• 2 ATPs are used
• Glucose is split to form 2 molecules of
  Glyceraldehyde-3-phosphate

ADP
Glucose 6-phosphate
Phosphoglucoisomerase
Fructose 6-phosphate
Phosphofructokinase
ADP
Fructose 1,6-diphosphate
aldolase
Dihydroxyacetone phosphate
Triose phosphate isomerase
Glyceraldehyde 3-phosphate
41
Glycolysis energy conserving stage
Glyceraldehyde 3-phosphate
NAD
NAD
Triose phosphate dehydrogenase
NADH

• For each initial glucose molecule
• 2 Glyceraldehyde-3-phosphate oxidized to 2
  Pyruvic acid
• 4 ATP produced
• 2 NADH produced

Diphosphoglyceric acid
ADP
ADP
Phosphoglycerokinase
3-phosphoglyceric acid
Phosphoglyceromutase
2-phosphoglyceric acid
H2O
H2O
Enolase
Phosphoenolpyruvic acid
ADP
ADP
Pyruvate kinase
Pyruvic acid
42
Summary of glycolysis

• Glucose (C6H12O6) is split and oxidized through a
  ten step pathway to two molecules of pyruvic acid
  (C3H4O3)
• Net gain of 2 ATP molecules, 4 from energy
  conserving phase (by substrate level
  phosphorylation) minus 2 from preparatory phase
• 2 NADH molecules produced
• Pyruvic acid can now undergo either fermentation
  or respiration

43
Alternatives to glycolysis

• Many bacteria have an alternative pathway to
  glycolysis for the oxidation of glucose
• Entner-doudoroff reaction
• Phosphogluconate pathway
• Some bacteria oxidize inorganic compounds instead
  of glucose to get energy. (the Lithotrophs)

44
Use of Inorganic ions as electron SOURCES
(Lithotrophs)
45
Bacteria, by Energy sources

• Phototrophs
• Chemotrophs
• Oxidize organic compounds for Energy
• Chemoorganotrophs
• Oxidize inorganic compounds for Energy
• Chemolithotrophs

46
Microbe of the Week

• Streptococcus pyogenes
• - Spherical
• - Gram-Positive
• - Grows in long chains
• - Cause of Group A streptococcal infections
• (e.g. pharyngitis Strep Throat and impetigo)
• - Infrequent, but usually pathogenic, part of
  the
• skin flora

47
Enzymes as Virulence Factors

• Streptococcus pyogenes
• Virulence factors include several extracellular
  enzymes e.g. Streptokinase, Hyaluronidase, C5
  peptidase.
• Help invade and
• Colonize host tissues.

Streptokinase - an enzyme that dissolves blood
clots Hyaluronidase - breaks down connective
tissue, allowing spread of the bacteria in skin
infections
pharyngitis
48
Impetigo
Group A streptococcal infection