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ENZYMESA protein with catalytic properties due to its power of specific activation
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Chemical reactions 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.
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Reaction pathway
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Making reactions go faster 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”
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An enzyme controlled pathway
Enzyme controlled reactions proceed 108 to 1011 times faster than corresponding non-enzymic reactions.
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Enzyme structure Enzymes are
proteins They have a
globular shape A complex 3-D
structure
Human pancreatic amylase
© Dr. Anjuman Begum
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The active site 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
© H.PELLETIER, M.R.SAWAYA ProNuC Database
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Cofactors An additional non-
protein 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 cofactorsJmol from a RCSB PDB file © 2007 Steve Cook
H.SCHINDELIN, C.KISKER, J.L.SCHLESSMAN, J.B.HOWARD, D.C.REESSTRUCTURE OF ADP X ALF4(-)-STABILIZED NITROGENASE COMPLEX AND ITS
IMPLICATIONS FOR SIGNAL TRANSDUCTION; NATURE 387:370 (1997)
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The substrate
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
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The Lock and Key Hypothesis 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
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The Lock and Key Hypothesis
Enzyme may be used again
Enzyme-substrate complex
E
S
P
E
E
P
Reaction coordinate
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The Lock and Key Hypothesis
This explains enzyme specificity This explains the loss of activity
when enzymes denature
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The Induced Fit Hypothesis 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)
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The Induced Fit Hypothesis
This explains the enzymes that can react with a range of substrates of similar types
Hexokinase (a) without (b) with glucose substratehttp://www.biochem.arizona.edu/classes/bioc462/462a/NOTES/ENZYMES/enzyme_mechanism.html
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Understanding Km
The "kinetic activator constant" 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
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Understanding Vmax
The theoretical maximal velocity 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
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The turnover number
A measure of catalytic activity 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
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Factors affecting Enzymes
substrate concentration pH temperature inhibitors
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Substrate concentration: Non-enzymatic reactions
The increase in velocity is proportional to the substrate concentration
Reaction velocity
Substrate concentration
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Substrate concentration: Enzymatic reactions
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.
Reaction velocity
Substrate concentration
Vmax
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Enzymes and [S]
[S]
Init
ial re
act
ion r
ate
/
arb
itra
ry u
nit
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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Enzymes and [S]
[S]
Init
ial r
eact
ion
rate
/ ar
bitr
ary
unit
s
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Enzymes and [S]
[S]
Init
ial r
eact
ion
rate
/ ar
bitr
ary
unit
s
Increasing [S] increases collision rate and increases reaction rate
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Enzymes and [S]
[S]
Init
ial r
eact
ion
rate
/ ar
bitr
ary
unit
sAll active sites are occupied. Enzymes are working at maximum rate.
All active sites are not
occupied
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Enzymes and [Substrate]
[S]
Init
ial r
eact
ion
rate
/ ar
bitr
ary
unit
sMaximum turnover
number or Vmax has been reached
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Enzymes and [enzyme]
[Enzyme]
Init
ial r
eact
ion
rate
/ ar
bitr
ary
unit
s
Can we explain this in terms of the proportions of active sites occupied?
What factor is limiting here?
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Enzymes and temperature: a tale of two effects
Temperature / oC
Collision rate of enzymes and substratesNumber of enzymes remaining undenatured
React
ion r
ate
/ a
rbit
rary
unit
s
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Enzymes and temperature
Temperature / oC
Increasing kinetic energy
increases successful
collision rate
React
ion r
ate
/ a
rbit
rary
unit
s
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Enzymes and temperature
Temperature / oC
Permanent disruption of
tertiary structure leads to loss of
active site shape, loss of binding efficiency and
activity
React
ion r
ate
/ a
rbit
rary
unit
s
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Enzymes and temperature
Temperature / oC
Optimum temperature
React
ion r
ate
/ a
rbit
rary
unit
s
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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…
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Enzymes and pH
pH
Rea
ctio
n ra
te /
arbi
trar
y un
its
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
Optimum pH
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Enzymes and pH
pH
Rea
ctio
n ra
te /
arbi
trar
y un
its
This loss of activity is only truly denaturation at extreme pH since between optimum and these extremes, the loss of activity is reversible
Optimum pH
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Enzymes and pH
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Enzymes and inhibitors Inhibitors are molecules that prevent
enzymes from reaching their maximum turnover numbers
Some inhibitors compete with the substrate for the active site
Some inhibitors affect the active site shape by binding to the enzyme elsewhere on the enzyme
Active site directed inhibition
Non-active site directed inhibition
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Active site directed inhibition (Competitive) Inhibitor resembles the substrate
enough to bind to active site and so prevent the binding of the substrate: Substrat
e
Inhibitor
Enzyme
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Active site directed inhibition (Competitive) Inhibitor resembles the substrate
enough to bind to active site and so prevent the binding of the substrate: Substrat
e
Enzyme/Inhibitor complex
Enzyme activity is
lost
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Enzymes and competitive inhibition
[S]
Init
ial re
act
ion r
ate
/
arb
itra
ry u
nit
sAt low [S], the enzyme is more likely
to bind to the inhibitor and so activity is markedly reduced
Uninhibited
Inhibited
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Enzymes and competitive inhibition
[S]
Init
ial re
act
ion r
ate
/
arb
itra
ry u
nit
sAs [S] rises, the enzyme is
increasingly likely to bind to the substrate and so activity increases
Uninhibited
Inhibited
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Enzymes and competitive inhibition
[S]
Init
ial re
act
ion r
ate
/
arb
itra
ry u
nit
sAt high [S], the enzyme is very unlikely to bind to the inhibitor and so maximum
turnover is achieved
Uninhibited
Inhibited
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Non-active site directed inhibition (Non-competitive) Inhibitor does not resemble the
substrate and binds to the enzyme disrupting the active site
Substrate
Inhibitor
Enzyme
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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 irreversibilit
y
Non-competitive inhibition
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Non-competitive inhibition Inhibitor does not resemble the
substrate and binds to the enzyme disrupting the active site
Substrate
Enzyme
Activity is permanentl
y lost
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Enzymes and non-competitive inhibition
[S]
Init
ial re
act
ion r
ate
/
arb
itra
ry u
nit
s
Can we explain this graph in terms of limiting factors in the parts of
the graph A and B?
A B
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Applications of inhibitors
Negative feedback: end point or end product inhibition
Poisons snake bite, plant alkaloids and nerve gases.
Medicine antibiotics, sulphonamides, sedatives and stimulants