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
 A reminder about
protein structure
• 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 R-
groups (or ‘side-chains’)
Amylase
Some reaction kinetics…
Some reaction kinetics…
The ‘Lock and Key’
analogy
The ‘Lock and Key’
analogy
Induced fit
 Enzymes and
temperature: a tale of two
effects
Collision rate of
enzymes and
substrates

Number of
enzymes remaining
undenatured
oit cae R

Temperature / oC
 Enzymes and temperature
Increasing kinetic
energy increases
successful
collision rate
oit cae R

Temperature / oC
 Enzymes and temperature
Permanent disruption
of tertiary structure
leads to loss of active
site shape, loss of
binding efficiency and
activity
oit cae R

Temperature / oC
 Enzymes and temperature
Optimum temperature
oit cae R

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

Optimum pH Either side of the optimum

pH, the gradual ionising of
the side-chains (R-groups)
results in loss of H-
bonding, 3o structure,
active site shape loss of
binding efficiency and
eventually enzyme activity
oit cae R

pH
 Enzymes and pH

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
oit cae R

pH
Enzymes and pH
 Enzymes and [S]

As soon as a reaction begins,

[S] begins to fall and so it is
important that initial
reaction rates are measured
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[S]
oit caer l aiti nI
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[S]
Enzymes and [S]
 Enzymes and [S]
Increasing [S]
increases collision
rate and increases
reaction rate
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[S]
 Enzymes and [S]
All active sites are
occupied. Enzymes
are working at
maximum rate.

All active sites

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are not occupied

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[S]
 Enzymes and [S]
Maximum
turnover number
or Vmax has been
reached
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[S]
Enzymes and [enzyme]
Can we explain this in terms of
the proportions of active sites
occupied?

What factor is
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limiting here?
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[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
by binding to the
Non-active siteenzyme elsewhere on the
directed inhibition
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
At low [S], the enzyme is more
likely to bind to the inhibitor and
so activity is markedly reduced

Uninhibited
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Inhibited
[S]
Enzymes and active site directed
inhibition
As [S] rises, the enzyme is
increasingly likely to bind to the
substrate and so activity increases

Uninhibited
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Inhibited
[S]
Enzymes and active site directed
inhibition
At high [S], the enzyme is very
unlikely to bind to the inhibitor and so
maximum turnover is achieved

Uninhibited
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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

Active site is
changed
Enzyme
irreversibility
 Non-active site directed
inhibition
• Inhibitor does not resemble the substrate
and binds to the enzyme disrupting the
active site
Substrate

Activity is
permanently
Enzyme
lost
Enzymes and non-active site
directed inhibition
Can we explain this graph in
terms of limiting factors in the
parts of the graph A and B?
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[S]
A B