Post on 14-Jul-2016
description
2/5/2016
1
1
� It is a substance which binds with the
enzyme and brings about decrease in the
catalytic activity of the enzyme.
�Enzyme inhibitors are molecules that
interact in some way with the enzyme to
prevent it from working in the normal
manner.
�Organic or inorganic
�Reversible or irreversible
2
2/5/2016
2
�Reversible
• Competitive
• Non- competitive
• uncompetitive
� Irreversible
�Allosteric
3
4
2/5/2016
3
5
6
2/5/2016
4
7
8
2/5/2016
5
9
Enzyme Inhibition (Mechanism)
I
I
S
S
S I
I
I II
S
Competitive Non-competitive Uncompetitive
E
E
Different siteCompete for
active siteInhibitor
Substrate
Ca
rto
on
Gu
ide
Equ
atio
n an
d D
escr
iptio
n
[II] binds to free [E] only,
and competes with [S];increasing [S] overcomesInhibition by [II].
[II] binds to free [E] or [ES]
complex; Increasing [S] can
not overcome [II] inhibition.
[II] binds to [ES] complex
only, increasing [S] favors
the inhibition by [II].
E + S→ES→E + P
+II
↓
EII
←
↑
E + S→ES→E + P
+ +II II
↓ ↓
EII+ S→EIIS
←
↑ ↑
E + S→ES→E + P
+II
↓
EIIS
←
↑
E
I
S
10
2/5/2016
6
Km
Enzyme Inhibition (Plots)
I II Competitive Non-competitive Uncompetitive
Dir
ect P
lots
Do
ub
le R
ecip
roca
l
Vmax Vmax
Km Km’ [S], mM
vo
[S], mM
vo
II II
Km [S], mM
Vmax
II
Km’
Vmax’Vmax’
Vmax unchangedKm increased
Vmax decreasedKm unchanged Both Vmax & Km decreased
II
1/[S]1/Km
1/vo
1/Vmax
II
Two parallellines
II
Intersect at X axis
1/vo
1/Vmax
1/[S]1/Km 1/[S]1/Km
1/Vmax
1/vo
Intersect at Y axis
= Km’
11
Reaction of the irreversible inhibitor
diisopropylfluorophosphate (DFP) with a
serine protease
12
2/5/2016
7
Iodoacetate is an irreversible inhibitor of all cysteine
peptidases
13
� Allosteric protein:-two or moretopological distinct bindingsites which interact functionallywith each other.
� Cooperatability:-modification ofthe binding constant of theprotein for a small molecule bythe prior binding of anothersmall molecule.• +ve:-binding ability or affinity
increases
• -ve:- binding ability or affinitydecreases
� Allosteric effectors(inhibitors &activators):- for speed up & tospeed down
when 2,3-BPG binds to an allosteric site
on hemoglobin, the affinity for oxygen of
all subunits decreases.
14
2/5/2016
8
Where n is cooperativity and n>1
,Indicates positive cooperativity.
15
High substrate concentrations
may
cause inhibition in some
enzymatic
reactions, known as substrate
inhibition
16
2/5/2016
9
� Assumption :-A second substrate molecule
can bind to the enzyme when S binds the ES
complex, an unreactive intermediate results.
17
� Unit of enzyme activity:
Used to measure total units of activity in a given
volume of solution.
� Specific activity:
Used to follow the increasing purity of an enzyme
through several procedural steps.
� Molecular activity:
Used to compare activities of different enzymes.
Also called the turn-over number (TON = kcat)
18
2/5/2016
10
� Unit of enzyme activity:
µµµµmol substrate transformed/min = unit
� Specific activity:
µµµµmol substrate/min-mg E = unit/mg E
� Molecular activity:
µµµµmol substrate/min- µµµµmol E = units/µµµµmol E
19
�Temperature
�pH
�Concentration
�Activators
�Product concentration
�Time
�Radiations
20
2/5/2016
11
Temperature / oC
Collision rate of
enzymes and
substrates
Number of
enzymes
remaining
undenatured
Reaction rate
/ a
rbitra
ry u
nits
21
Temperature / oC
Increasing kinetic
energy increases
successful collision
rate
Reaction rate
/ a
rbitra
ry u
nits
22
2/5/2016
12
Temperature / oC
Permanent disruption
of tertiary structure
leads to loss of active
site shape, loss of
binding efficiency and
activity
Reaction rate
/ a
rbitra
ry u
nits
23
Temperature / oC
Optimum temperature
Reaction rate
/ a
rbitra
ry u
nits
24
2/5/2016
13
�The precise shape of an enzyme (andhence its active site) depends on thetertiary structure of the protein
�Tertiary structure is held together byweak bonds (including hydrogen bonds)between R-groups (or ‘side-chains’)
�Changing pH can cause these side chainsto ionise resulting in the loss of H-bonding…
25
� Temperature: enzymes work best at an optimum temperature.
Below this, an increase in temperature provides more kinetic energy to the molecules involved. The numbers of collisions between enzyme and substrate will increase so the rate will too.
Above the optimum temperature, and the enzymes are denatured. Bonds holding the structure together will be broken and the active site loses its shape and will no longer work
26
2/5/2016
14
Even if temperature lowered – enzyme
can’t regain its correct shape
27
pH
Reaction rate
/ a
rbitra
ry u
nits
Either side of the
optimum pH, the
gradual ionising of the
side-chains (R-groups)
results in loss of H-
bonding, 3D structure,
active site shape loss of
binding efficiency and
eventually enzyme
activity
Optimum pH
28
2/5/2016
15
pH
Reaction rate
/ a
rbitra
ry u
nits
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
29
�pH: as with temperature, enzymes have an optimum pH. If the pH changes much from the optimum, the chemical nature of the amino acids can change.
This may result in a change in the bonds and so the tertiary structure may break down. The active site will be disrupted and the enzyme will be denatured.
30
2/5/2016
16
31
32