Factors Affecting
Enzyme Activity
Enzymes are large globular
proteins…
• They have a precise 3-D
shape
• Some have quaternary
structure
• The ‘active site’ (blue)
represents a tiny part of
the molecule
RuBisCo
Amino Acid
A reminder about
protein structure
Amylase
• Protein
structure
is
achieved by the precise
folding of secondary
structures to form a
tertiary structure held
together by a range of
bond types between Rgroups (or ‘side-chains’)
Some reaction kinetics…
Some reaction kinetics…
The ‘Lock and Key’ analogy
The ‘Lock and Key’ analogy
Induced fit
Reaction rate / arbitrary units
Enzymes and temperature: a tale of
two effects
Collision rate of
enzymes and
substrates
Number of
enzymes remaining
undenatured
Temperature / oC
Reaction rate / arbitrary units
Enzymes and temperature
Increasing kinetic
energy increases
successful
collision rate
Temperature / oC
Reaction rate / arbitrary units
Enzymes and temperature
Permanent disruption
of tertiary structure
leads to loss of active
site shape, loss of
binding efficiency and
activity
Temperature / oC
Enzymes and temperature
Reaction rate / arbitrary units
Optimum temperature
Temperature / oC
Enzymes and pH
• The precise shape of an enzyme (and hence
its active site) depends on the tertiary
structure of the protein
• Tertiary structure is held together by weak
bonds (including hydrogen bonds) between
R-groups (or ‘side-chains’)
• Changing pH can cause these side chains to
ionise resulting in the loss of H-bonding…
Enzymes and pH
Reaction rate / arbitrary units
Optimum pH
Either side of the optimum
pH, the gradual ionising of
the side-chains (R-groups)
results in loss of Hbonding, 3o structure,
active site shape loss of
binding efficiency and
eventually enzyme activity
pH
Enzymes and pH
Reaction rate / arbitrary units
Optimum pH
This loss of activity is only
truly
denaturation
at
extreme pH since between
optimum
and
these
extremes, the loss of
activity is reversible
pH
Enzymes and pH
Initial reaction rate /
arbitrary units
Enzymes and [S]
As soon as a reaction begins,
[S] begins to fall and so it is
important that initial
reaction rates are measured
[S]
Initial reaction rate /
arbitrary units
Enzymes and [S]
[S]
Initial reaction rate /
arbitrary units
Enzymes and [S]
Increasing
[S]
increases collision
rate and increases
reaction rate
[S]
Initial reaction rate /
arbitrary units
Enzymes and [S]
All active sites are
occupied. Enzymes
are
working
at
maximum rate.
All active sites
are not occupied
[S]
Initial reaction rate /
arbitrary units
Enzymes and [S]
Maximum
turnover number
or Vmax has been
reached
[S]
Initial reaction rate /
arbitrary units
Enzymes and [enzyme]
Can we explain this in terms of
the proportions of active sites
occupied?
What factor is limiting
here?
[Enzyme]
Enzymes and inhibitors
• Inhibitors are molecules that prevent
enzymes reaching their maximum turnover
numbers
• Some inhibitors compete with the substrate
Active site directed inhibition
for the active site
• Some inhibitors affect the active site shape
Non-active
siteenzyme
directed inhibition
by binding
to the
elsewhere on the
enzyme
Active site directed inhibition
• Inhibitor resembles the substrate enough to
bind to active site and so prevent the
binding of the substrate:
Substrate
Inhibitor
Enzyme
Active site directed inhibition
• Inhibitor resembles the substrate enough to
bind to active site and so prevent the
binding of the substrate:
Substrate
Enzyme
activity is lost
Enzyme/Inhibitor
complex
Enzymes and active site directed inhibition
Initial reaction rate /
arbitrary units
At low [S], the enzyme is more
likely to bind to the inhibitor and
so activity is markedly reduced
Uninhibited
Inhibited
[S]
Enzymes and active site directed inhibition
Initial reaction rate /
arbitrary units
As [S] rises, the enzyme is
increasingly likely to bind to the
substrate and so activity increases
Uninhibited
Inhibited
[S]
Enzymes and active site directed inhibition
Initial reaction rate /
arbitrary units
At high [S], the enzyme is very
unlikely to bind to the inhibitor and so
maximum turnover is achieved
Uninhibited
Inhibited
[S]
Non-active site directed inhibition
• Inhibitor does not resemble the substrate
and binds to the enzyme disrupting the
active site
Substrate
Inhibitor
Enzyme
Non-active site directed inhibition
• Inhibitor does not resemble the substrate
and binds to the enzyme disrupting the
active site
Substrate
Enzyme
Active site is
changed
irreversibility
Non-active site directed inhibition
• Inhibitor does not resemble the substrate
and binds to the enzyme disrupting the
active site
Substrate
Enzyme
Activity is
permanently
lost
Enzymes and non-active site directed
inhibition
Initial reaction rate /
arbitrary units
Can we explain this graph in
terms of limiting factors in the
parts of the graph A and B?
A
B
[S]