ENZYMES
A protein with catalytic properties due to its
power of specific activation
Chemical reactions
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Chemical reactions need an initial input of
energy = THE ACTIVATION ENERGY
During this part of the reaction the molecules
are said to be in a transition state.
Reaction pathway
Making reactions go faster
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Increasing the temperature make molecules
move faster
Biological systems are very sensitive to
temperature changes.
Enzymes can increase the rate of reactions
without increasing the temperature.
They do this by lowering the activation energy.
They create a new reaction pathway “a
short cut”
An enzyme controlled
pathway
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Enzyme controlled reactions proceed 108 to 1011 times
faster than corresponding non-enzymic reactions.
Enzyme structure
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Enzymes are
proteins
They have a
globular shape
A complex 3-D
structure
Human pancreatic
amylase
© Dr. Anjuman Begum
The active site
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© H.PELLETIER, M.R.SAWAYA
ProNuC Database
One part of an
enzyme, the active
site, is particularly
important
The shape and the
chemical
environment inside
the active site
permits a chemical
reaction to proceed
more easily
Cofactors
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An additional nonprotein molecule that
is needed by some
enzymes to help the
reaction
Tightly bound
cofactors are called
prosthetic groups
Cofactors that are
bound and released
easily are called
coenzymes
Many vitamins are
coenzymes
Nitrogenase enzyme with Fe, Mo and ADP cofactors
Jmol from a RCSB PDB file © 2007 Steve Cook
H.SCHINDELIN, C.KISKER, J.L.SCHLESSMAN, J.B.HOWARD, D.C.REES
STRUCTURE OF ADP X ALF4(-)-STABILIZED NITROGENASE COMPLEX AND ITS
IMPLICATIONS FOR SIGNAL TRANSDUCTION; NATURE 387:370 (1997)
The substrate
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The substrate of an enzyme are the
reactants that are activated by the
enzyme
Enzymes are specific to their substrates
The specificity is determined by the
active site
The Lock and Key
Hypothesis
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Fit between the substrate and the active site of the
enzyme is exact
Like a key fits into a lock very precisely
The key is analogous to the enzyme and the substrate
analogous to the lock.
Temporary structure called the enzyme-substrate
complex formed
Products have a different shape from the substrate
Once formed, they are released from the active site
Leaving it free to become attached to another substrate
The Lock and Key
Hypothesis
S
E
E
E
Enzymesubstrate
complex
Enzyme may
be used again
P
P
Reaction coordinate
The Lock and Key
Hypothesis
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This explains enzyme specificity
This explains the loss of activity when
enzymes denature
The Induced Fit Hypothesis
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Some proteins can change their shape
(conformation)
When a substrate combines with an enzyme, it
induces a change in the enzyme’s conformation
The active site is then moulded into a precise
conformation
Making the chemical environment suitable for
the reaction
The bonds of the substrate are stretched to
make the reaction easier (lowers activation
energy)
The Induced Fit Hypothesis
Hexokinase (a) without (b) with glucose substrate
http://www.biochem.arizona.edu/classes/bioc462/462a/NOTES/ENZYMES/enzyme_mechanism.html
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This explains the enzymes that can react with a
range of substrates of similar types
Factors affecting Enzymes
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substrate concentration
pH
temperature
inhibitors
Substrate concentration: Nonenzymatic reactions
Reaction
velocity
Substrate concentration
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The increase in velocity is proportional to the
substrate concentration
Substrate concentration: Enzymatic
reactions
Vmax
Reaction
velocity
Substrate concentration
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Faster reaction but it reaches a saturation point when
all the enzyme molecules are occupied.
If you alter the concentration of the enzyme then Vmax
will change too.
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 [Substrate]
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]
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
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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 Hbonding…
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
Enzymes and inhibitors
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Inhibitors are molecules that prevent
enzymes reaching their maximum
turnover numbers
Some inhibitors compete with the
substrate for the active site
Active site directed
inhibition
 Some inhibitors affect
the active site
shape by binding to the enzyme
elsewhere on the enzyme
Non-active site directed
Active site directed inhibition
(Competitive)
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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
(Competitive)

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 competitive
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 competitive
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 competitive
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 (Non-competitive)
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Inhibitor does not resemble the
substrate and binds to the enzyme
disrupting the active site
Substrate
Inhibitor
Enzyme
Non-competitive inhibition
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Inhibitor does not resemble the
substrate and binds to the enzyme
disrupting the active site
Substrate
Enzyme
Active site is
changed
irreversibility
Non-competitive 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-competitive
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]
Applications of inhibitors
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Negative feedback: end point or end
product inhibition
Poisons snake bite, plant alkaloids and
nerve gases.
Medicine antibiotics, sulphonamides,
sedatives and stimulants
Understanding Km
The "kinetic activator constant"
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Km is a constant
Km is a constant derived from rate constants
Km is an approximation of the dissociation
constant of E from S
Small Km means tight binding; high Km
means weak binding
Understanding Vmax
The theoretical maximal velocity
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Vmax is a constant
Vmax is the theoretical maximal rate of the
reaction - but it is NEVER achieved in reality
To reach Vmax would require that ALL enzyme
molecules are tightly bound with substrate
Vmax is asymptotically approached as
substrate is increased
The turnover number
A measure of catalytic activity
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kcat, the turnover number, is the number
of substrate molecules converted to
product per enzyme molecule per unit of
time, when E is saturated with substrate.
Values of kcat range from less than 1/sec
to many millions per sec
Michaelis-Menten equation
Lineweaver-Burke plot
Hanes-Woolf plot