Effect of environment on enzyme activity
Substrate concentration
pH
Temperature
Substrate concentration
Enzyme activity increases
with increasing substrate
concentrations
At a certain concentration
the enzyme will be saturated
and operate at maximum
velocity = Vmax
Substrate concentration
Plot of velocity (activity) vs.
substrate concentration
results in a hyperbola
Michaelis constant (Km)
Used to measure the affinity
of an enzyme for its substrate
Km = substrate concentration
required to achieve half
maximal velocity
Effect of pH and temperature on enzyme activity
Enzymes are most active at optimum pH and temperatures
Deviations from the optima can slow activity and damage the
enzyme
Effect of pH and temperature on enzyme activity
Loss of activity due to extreme pH, temperature or other factors
is known to as denaturation
Temperature and pH optima of microorganism’s enzymes usually
reflect the microorganism’s environment
Enzyme inhibition
Many poisons and antimicrobial agents are enzyme inhibitors
Can be accomplished by competitive or noncompetitive inhibitors
Competitive inhibitors - compete with substrate for the active site
Noncompetitive inhibitors - bind at another location
Competitive inhibitors
Usually resemble the
substrate but cannot be
converted to products
Noncompetitive inhibitors
Bind to the enzyme at some location other than the active site
Do not compete with substrate for the active site
Binding alters enzyme shape and slows or inactivates the enzyme
Heavy metals often act as noncompetitive inhibitors
(e.g. Mercury)
Metabolic regulation
Important to conserve energy and resources
Cell must be able to respond to changes in the environment
Changes in available nutrients will result in changes in
metabolic pathways
Metabolic regulation
Metabolic channeling
Stimulation or inhibition of enzyme activity
Transcriptional regulation of enzyme production
Allosteric enzymes
Activity of enzymes are
altered by small molecules
known as effectors or
modulators
Effectors bind reversibly and
noncovalently to the
regulatory site
Binding alters the
conformation of the enzyme
Allosteric enzymes
Positive effectors increase
activity
Negative effectors decrease
activity
ACTase regulation
Regulation of aspartate carbamyltransferase is a well studied
example of allosteric regulation
CTP inhibits activity and ATP stimulates activity
ACTase regulation
Binding of effectors cause conformational changes that result in
more or less active forms of the enzyme
ACTase regulation
Binding of substrate also
increases enzyme activity
(more than one active site)
Velocity vs. substrate curve
is sigmoid
Covalent modification of enzymes
Attachment of group to
enzyme can result in
stimulation or inhibition of
activity
Attachment is covalent and
reversible
Covalent modification of enzymes
Attachment of phosphate
groups often used to regulate
enzyme activity
Other groups can also be
used to regulate enzyme
activity
Feedback inhibition
Metabolic pathways contain
at least one pacemaker
enzyme
Usually catalyzes the first
reaction in the pathway
Activity of the enzyme
determines the activity of the
entire pathway
Feedback inhibition
Feedback inhibition occurs
when the end product
interacts with the pacemaker
enzyme to inhibit its activity
Branching pathways
regulate enzymes at branch
points
Overview of metabolism
Metabolism = the total of all chemical reactions occurring within
the cell
Catabolism = the breaking down of complex molecules into
simple molecules with the release of energy
Anabolism = the synthesis of complex molecules from simple
molecules with the use of energy
Sources of energy
Microorganisms use one of
three sources of energy
Phototrophs - radiant
energy of the sun
Chemoorganotrophs oxidation of organic
molecules
Chemolithotrophs oxidation of inorganic
molecules
Electron acceptors
Chemotrophs vary regarding their final electron acceptors
Fermentation - no exogenous electron acceptor is required
Aerobic respiration - oxygen is the final electron acceptor
Anaerobic respiration - another inorganic molecule is acceptor
Electron acceptors
Chemolithotrophs can use oxygen or another inorganic molecule
as the final electron acceptor
The three stages of catabolism
Catabolism can be broken
down into three stages
Stage 1
Larger molecules (proteins,
polysaccharides, lipids) are
broken down into their
constituents
Little or no energy is
generated
The three stages of catabolism
Stage 2
Amino acids,
monosaccharides, fatty
acids, glycerol and other
products degraded to a few
simpler products
Can operate aerobically or
anaerobically
Generates some ATP and
NADH or FADH
The three stages of catabolism
Stage 3
Nutrient carbon is fed into
the tricarboxylic pathway
and oxidized to CO2
ATP, NADH and FADH
produced
ATP generated from
oxidation of NADH and
FADH in electron transport
chain
Amphibolic pathways
Pathways that can function
both catabolically and
anabolically
Glycolysis and the
tricarboxylic acid cycle are
two of the most important
amphibolic pathways
Most reactions are reversible
The glycolytic pathway/glycolysis
Also known as the EmbdenMeyerhof pathway
Most common pathway of
degradation of glucose to
pyruvate
Found in all major groups of
microorganisms
Can function aerobically or
anaerobically
The glycolytic pathway/glycolysis
Occurs in 2 stages
The six-carbon stage
Glucose is phosphorylated
2x and converted to
fructose-1,6-bisphosphate
Other sugars converted to
glucose-6-phosphate or
fructose-6-phosphate and
fed into pathway
The glycolytic pathway/glycolysis
The six-carbon stage
Does not yield energy
Uses 2 ATPs
Serves to “prime the pump”
The glycolytic pathway/glycolysis
The three-carbon stage
Fructose-1,6-bisphosphate
split in half by fructose-1,6bisphosphate aldolase
Yields glyceraldehyde-3phosphate and
dihydroxyacetone phosphate
The glycolytic pathway/glycolysis
The three-carbon stage
Dihydroxyacetone phosphate
readily converts to glyceraldhyde3-phosphate
Fructose-1,6-bisphosphate
 2 glyceraldehyde-3-phosphate
The glycolytic pathway/glycolysis
The three-carbon stage
Glyceraldehyde-3-phosphate
converted into pyruvate in 5 steps
Oxidized by NAD+ and a
phosphate is added
 1,3-bisphosphoglycerate
Phosphate on carbon 1 donated to
ADP to form ATP
Substrate-level phosphorylation
The glycolytic pathway/glycolysis
The three-carbon stage
3-phosphoglycerate shifted to
carbon 2  2-phosphoglycerate
Dehydration results in high energy
phosphate bond in
phosphoenolpyruvate
Phosphate transferred to ADP to
form ATP (substrate-level
phosphorylation)
The glycolytic pathway/glycolysis
Glucose  2 pyruvates + ATP
+NADH
2 ATP used in six-carbon stage
4 ATP + 2 NADH formed in threecarbon stage
The glycolytic pathway/glycolysis
Glucose + 2ADP + Pi + 2NAD+  2 pyruvate + 2ATP + 2NADH