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Chapter 5
Microbial Metabolism
Part 2
Factors Influencing Enzyme Activity
• Cellular controls of enzymes
– enzyme synthesis
– enzyme activity
•
•
•
•
temperature
pH
substrate concentration
presence or absence of inhibitors
Factors Influencing Enzyme Activity
• Temperature
– Increase the rate of most chemical reactions
– Elevation of temperature beyond optimal
temperature denatures enzymes
– Denaturation: loss of its characteristic threedimensional (3D) structure (tertiary
configuration)
can no longer function
• involves breakage of H-bonds and other noncovalent
bonds
• in some cases can be partially or fully reversible
Factors Influencing Enzyme Activity
• Temperature
Figure 5.5a
Factors Influencing Enzyme Activity
• Enzymes can be denatured by temperature, pH
(concentrated acids & bases), heavy-metal ions,
alcohol, and UV radiation
Figure 5.6
Factors Influencing Enzyme Activity
• pH most enzymes have an optimum pH
• Extreme changes in pH cause denaturation
Figure 5.5b
Factors Influencing Enzyme Activity
• Substrate concentration
– Within limits, enzymatic activity increases as
substrate concentration increases
• Saturation: condition in which the active
site on an enzyme is occupied by the
substrate or product all the time
Factors Influencing Enzyme Activity
• Substrate concentration
Figure 5.5c
Factors Influencing Enzyme Activity
• Competitive Inhibitors: compete with the
normal substrate for the active site of the
enzyme
– Binds to the active site but does not form
products
– Irreversible inhibitor: binds to the active site
and prevent any further interaction with the
substrate
– Reversible inhibitor: slow down the enzyme’s
interaction with the substrate by alternately
occupying and leaving the active site
Factors Influencing Enzyme Activity
• Competitive inhibitor has similar shape and
chemical structure to those of the normal
substrate
Figure 5.7a, b
Factors Influencing Enzyme Activity
(substrate)
Factors Influencing Enzyme Activity
• Noncompetitive inhibitors: cause allosteric
(“other space”) inhibition by interacting
with another part of the enzyme (allosteric
site), or act on cofactor
– Causes the active site to change its shape &
makes it nonfunctional
– Effect can be reversible or irreversible
– Sometimes can activate an enzyme
Factors Influencing Enzyme Activity
• Noncompetitive inhibitor alters active site
or bind/tie up cofactors
Figure 5.7a, c
Factors Influencing Enzyme Activity
• Feedback inhibition (end-product inhibition):
accumulation of the end-product in a
particular pathway inhibits the first enzyme’s
activity in the pathway
– Regulate cell’s production of amino acids,
vitamins, purines, and pyrimidines
– Mechanism stops the cell from wasting chemical
resources
– Allosteric inhibitors play a role
Factors Influencing Enzyme Activity
• Feedback
inhibition
Figure 5.8
Ribozymes
• Function as catalysts
– have active sites for substrate binding & not
used up in a chemical reaction
• More restricted than protein enzymes
• RNA with enzymatic activity
– specifically act on strands of RNA to remove
sections and splice together the remaining pieces
Energy Production
• Energy is associated with the electrons that
form bonds between their atoms
– Various reactions in catabolic pathways
concentrate the energy into the bonds of ATP.
– “High-energy” unstable bonds of ATP provide
the cell with readily available energy for
anabolic reactions
• Two general aspects of energy production:
– Oxidation-reduction
– Mechanism of ATP generation
Oxidation-Reduction Reactions
• Oxidation: removal of electrons from an
atom or molecule. (produce energy)
– In many cellular oxidations, electrons and
protons (H+) are removed at the same time ;
equivalent to the removal of H atoms.
• Reduction: the gain of electrons.
• Oxidation and reduction reactions are
always coupled.
Oxidation-Reduction
• Redox reaction is an oxidation reaction paired
with a reduction reaction.
Figure 5.9
Oxidation-Reduction
• In biological systems, the electrons are often
associated with hydrogen atoms. Biological
oxidations are often dehydrogenations (loss of H
atoms).
Figure 5.10
Oxidation-Reduction
• Cells use redox reaction to extract energy
from nutrient molecules (some serve as
energy source)
– cells degrade highly reduced compounds (with
many H atoms) to highly oxidized compounds
e.g. glucose (highly reduced compounds)
• energy is removed in stepwise manner and trapped
by ATP
The Generation of ATP
• Much of the energy released during redox
reactions is trapped within the cell by the
formation of ATP.
• ATP is generated by the phosphorylation of
ADP.
– Phosphorylation: addition of a phosphate
group to a chemical compound
The Generation of ATP
• Three mechanisms of phosphorylation to
generate ATP from ADP
– Substrate-level phosphorylation, oxidative
phosphorylation, & photophosphorylation
The Generation of ATP
• Substrate-level phosphorylation: synthesis
of ATP by direct transfer of a high-energy
PO4- to ADP.
– From a phosphorylated intermediate metabolic
compound (usually in catabolism) to ADP
The Generation of ATP
• Oxidative phosphorylation: synthesis of ATP
coupled with electron transport
– electrons are transferred from organic compounds
to one group of electron carriers (NAD+ and FAD)
pass through electron transport chain (via
redox reaction)
to O2 or another inorganic
compound
– generate ATP from ADP through chemiosmosis
– occurs in the plasma membrane of prokaryotes
and in the inner mitochondrial membrane of
eukaryotes
The Generation of ATP
• Photophosphorylation: production of ATP
in a series of redox reactions; reactions is
initiated by electrons from chlorophyll
– energy from light is trapped by chlorophyll and
causes chlorophyll to give up electrons
electrons pass through an electron transport
chain
energy released from the transfer of
electrons (oxidation) via an electron transport
chain is used to generate ATP
– occurs only in photosynthetic cells (contains
chlorophylls)
Metabolic Pathways of
Energy Production
• Energy is released and stored from organic
molecules by a series of controlled reactions
• Energy is extracted from organic
compounds and stored in chemical form by
passing electrons from one compound to
another through a series of redox
(oxidation-reduction) reactions
Metabolic Pathways
Reduction
(Reactant)
(By-products)
Carbohydrate Catabolism
• Breakdown of carbohydrate molecules to
produce energy
• Carbohydrates are oxidized and used as
primary source of cellular energy by most
microorganisms.
– Glucose most common carbohydrate energy
source
• Energy is produced through cellular respiration and
fermentation
Figure 5.14
Carbohydrate Catabolism
• Cellular respiration (respiration) of glucose
– Glycolysis
– Krebs cycle
– Electron transport chain
• Respiration involves a long series of redox
reactions
– Flow of electrons from the energy-rich glucose
to the relatively energy-poor CO2 and H2O
– Production of ATP is coupled to the flow of
electrons
Glycolysis
• The oxidation of glucose to pyruvic acid,
produces ATP and NADH. (O not required)
– Most microorganisms use this pathway
Also known as
Embden-Meyerhof
pathway
Two basic stages:
a preparatory stage
and an energyconserving stage
Preparatory Stage
Preparatory
Stage
Glucose
• 2 ATPs are used
• Glucose is split
to form 2
Glucose-3phosphate
(DHAP or GP)
1
Glucose
6-phosphate
2
Fructose
6-phosphate
3
4
Fructose
1,6-diphosphate
5
Dihydroxyacetone
phosphate (DHAP)
Glyceraldehyde
3-phosphate
(GP)
Figure 5.12.1
Energy-Conserving Stage
6
• 2 Glucose-3phosphate
oxidized to 2
Pyruvic acid
• 4 ATP produced
(substrate-level
phosphorylation)
• 2 NADH
produced
1,3-diphosphoglyceric acid
7
3-phosphoglyceric acid
8
2-phosphoglyceric acid
9
Phosphoenolpyruvic acid
(PEP)
10
Pyruvic acid
Figure 5.12.2
Glycolysis
• Glucose + 2 ATP + 2 ADP + 2 PO4– + 2 NAD+
 2 pyruvic acid + 4 ATP + 2 NADH + 2H+
• Net gain of 2 ATP from each molecule of
glucose oxidized
Alternatives to Glycolysis
• Glucose can be oxidized through another
pathway in many bacteria.
– Most common alternatives are pentose phosphate
pathway & Entner-Doudoroff pathway
• Pentose phosphate pathway (hexose
monophosphate shunt:
– produce pentoses (5-C sugars) and NADPH
• pentoses used in synthesis of nucleic acids, glucose from
CO2 in photosynthesis, and certain amino acids
– intermediates produced can enter glycolysis
Alternatives to Glycolysis
– Operates simultaneously with glycolysis
– net gain of 1 ATP
– Bacillus subtilis, E. coli, Leuconostoc
mesenterioides, and Enterococcus faecalis
• Entner-Doudoroff pathway:
– Produces 2 NADPH and 1 ATP (net gain) to be
used in cellular biosynthesis
– Does not involve glycolysis
– Pseudomonas, Rhizobium, Agrobacterium
(gram-negative)
Cellular Respiration (Respiration)
• ATP-generating process in which molecules
are oxidized and the final electron acceptor is
(almost always) an inorganic molecule
– Pyruvic acid produced from glycolysis can feed
into either cellular respiration or fermentation
• Respiration can be aerobic or anaerobic
• Oxidation of molecules liberates electrons for
an electron transport chain
• ATP generated by oxidative phosphorylation
Respiration
• Aerobic respiration: the final electron acceptor
in the electron transport chain is molecular
oxygen (O2).
• Anaerobic respiration: the final electron acceptor
in the electron transport chain is not O2. Yields
less energy than aerobic respiration because only
part of the Krebs cycles operates under anaerobic
conditions.
Intermediate Step
• Pyruvic acid (from
glycolysis) is
oxidized and
decarboxylated
– pyruvic acid cannot
enter the Krebs
cycle directly
– Decarboxylation:
removal of CO2
from an amino acid
Figure 5.13.1
Aerobic Respiration (Krebs Cycle)
• Krebs Cycle, also known as tricarboxylic
acid (TCA) cycle or citric acid cycle
– uses decarboxylation and redox reactions
• Oxidation of acetyl CoA (pyruvic acid
derivatives) produces NADH and FADH2
– Large amount of potential chemical energy
stored in acetyl CoA is released step by step
– Potential energy is transferred in the form of
electrons to coenzymes (electron carrier)
Krebs Cycle
•2 acetyl CoA molecules
•4 CO2 (decarboxylation)
•6 NADH + 2 FADH2
(redox reaction)
contain most of the energy
originally stored in
glucose
•2 ATP (substrate-level
phosphorylation)
Figure 5.13.2
Aerobic Respiration
(The Electron Transport Chain, ETC)
• A series of carrier molecules that are, in
turn, oxidized and reduced as electrons are
passed down the chain.
– NADH and FADH2 are oxidized to contribute
electrons to ETC
– Three classes or carrier molecules in ETC are
flavoproteins, cytochromes, and ubiquinones
(coenzyme Q)
• Energy released can be used to produce
ATP by chemiosmosis.
Chemiosmosis
A mechanism that uses a proton
gradient across a cytoplasmic
membrane to generate ATP.
Figure 5.15
Chemiosmosis
Figure 5.16.2
Anaerobic respiration
• Anaerobic respiration by bacteria using
nitrate and sulfate as final acceptors is
essential for the nitrogen and sulfur cycles in
nature
Electron acceptor
Products
NO3– (Bacillus & Pseudomonas) NO2–, N2 + H2O
SO4– (Desulfovibrio)
H2S + H2O
CO32 –
CH4 + H2O
Anaerobic Respiration
• Amount of ATP produced varies with the
organism and the pathway (ATP yield is
never as high as in aerobic respiration).
– Only part of the Krebs cycle operates under
anaerobic conditions; not all the electron carriers
in the ETC participate in anaerobic respiration
• Anaerobes tend to grow more slowly due to
less ATP generated.
Pathway
Eukaryote
Prokaryote
Glycolysis
Cytoplasm
Cytoplasm
Intermediate step
Cytoplasm
Cytoplasm
Krebs cycle
Mitochondrial
Cytoplasm
matrix
Mitochondrial inner Plasma
membrane
membrane
ETC
• Energy produced from complete oxidation of 1
glucose using aerobic respiration
Pathway
NADH
FADH2
ATP produced produced
produced
Glycolysis
2
2
0
Intermediate
step
0
2
Krebs cycle
2
6
2
Total
4
10
2
• ATP produced from complete oxidation of 1
glucose using aerobic respiration
Pathway
By substratelevel
phosphorylation
Glycolysis
Intermediate
step
Krebs cycle
2
Total
By oxidative
phosphorylation
From
From
NADH FADH
6
0
0
6
2
18
4
4
30
4
• 36 ATPs are produced in eukaryotes.