Lecture18-Inhibition

Lecture18-Inhibition -...

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Unformatted text preview: BIOC*2580
Lecture
18:

Enzyme
Inhibition
 
1 Synopsis:
 Inhibitors
 control
 enzyme
 activity
 by
 reversibly
 decreasing
 the
 enzyme
 activity.
 Different
mechanisms
of
inhibition
depend
on
the
relationship
between
inhibitor
and
substrate,
 and
can
be
distinguished
by
observing
how
inhibitor
affects
KM
or
Vmax
of
the
enzyme.
 Competitive
inhibition
increases
KM
with
no
effect
on
Vmax.
 Non‐competitive
inhibition
decreases
Vmax
with
no
effect
on
KM.

 
 Reading:
Lehninger,
p.
194‐205
(4th
ed
p.209‐212)
 
 
 Enzymes
are
subject
to
various
substances
that
act
to
reduce
their
activity.
 
 Inactivation
 results
 from
 a
 reactive
 molecule
 that
 may
 form
 covalent
 bonds
 with
 key
 amino
 acids,
 preventing
 the
 enzyme
 from
 completing
 its
 reaction
 cycle.
 Inactivation
 tends
 to
 be
 irreversible.
Essentially,
an
inactivator
reduces
the
quantity
of
available
enzyme
irreversibly
and
 in
a
stoichiometric
manner:
 
 3
µmol
enzyme
+
2
µmol
inactivator
leaves
1
µmol
enzyme
to
continue
working.
 
 e.g.
 Disopropylfluorophosphate
 reacts
 with
 acetylcholinesterase
 irreversibly,
 blocking
 transmission
of
nerve
impulses.
Many
so‐called
nerve
gases
act
this
way
and
many
are
 halogen‐ phosphorus
compounds.

 
 Reversible
Inhibition
results
from
a
substance
 that
binds
to
an
enzyme
and
limits
its
capacity
 to
 catalyze
reaction.
The
binding
is
non‐covalent
and
reversible,
and
if
inhibitor
is
removed,
normal
 activity
is
restored.
The
concentration
of
inhibitor,
like
substrate,
is
typically
much
higher
than
 enzyme
concentration.
 
 Enzymes
 need
 to
 be
 regulated
 in
 the
 course
 of
 normal
 metabolism,
 i.e.
 an
 enzyme
 that
 is
 temporarily
 not
 needed
 is
 turned
 off.
 Reversible
 inhibition
 can
 contribute
 to
 regulation,
 since
 activity
can
be
restored
by
removing
the
inhibitor
without
having
to
make
new
enzyme.
Enzyme
 regulation
and
its
consequences
are
major
themes
of
BIOC*3560.
 
 Many
 of
 the
 substances
 that
 we
 use
 as
 drugs
 act
 by
 inhibiting
 a
 key
 enzyme
 in
 the
 body.
 For
 example,
 acetylsalicylic
 acid
 (ASA
 or
 aspirin)
 inhibits
 an
 enzyme
 called
 cyclooxygenase,
 responsible
 for
 making
 prostaglandins
 that
 stimulate
 the
 inflammatory
 response.
 When
 ASA
 inhibits
 cyclooxygenase,
 less
 prostaglandin
 is
 made
 and
 inflammation
 is
 kept
 under
 control.
 Finding
and
analyzing
properties
of
enzyme
inhibitors
is
an
important
aspect
of
pharmaceutical
 research.
 
 Page
1
of
5
 BIOC*2580
Lecture
18:

Enzyme
Inhibition
 
2 There
are
several
reversible
inhibition
mechanisms,
distinguished
by
the
relationship
between
 inhibitor
and
the
substrate
of
the
enzyme.

 
 1. Competitive
 inhibition:
 the
 enzyme
 either
 binds
 substrate
 or
 binds
 inhibitor,
 but
 not
 both.
In
 other
words,
the
substrate
and
inhibitor
compete
for
occupation
of
the
enzyme
 molecule.

 2. Non‐competitive
 inhibition:
 inhibitor
 can
 bind
 to
 enzyme
 whether
 substrate
 is
 also
 bound
or
not,
i.e.
substrate
binding
has
no
effect
on
inhibition.

 3. Uncompetitive
inhibition:
opposite
to
competitive,
the
inhibitor
can
 only
bind
to
the
ES
 complex,
 and
 substrate
 must
 bind
 first.
 
 This
 mode
 may
 occur
 with
 two‐substrate
 enzymes.
We
won’t
be
discussing
this
mode
other
than
to
mention
it
here.
 4. Mixed
 inhibition:
 is
 some
 combination
 of
 non‐competitive
 with
 either
 of
 the
 other
 mechanisms
 True
non‐competitive
inhibition
is
rare,
and
most
cases
are
actually
mixed
 inhibition
that
closely
approximates
the
non‐competitive
case.
 
 1.
Competitive
inhibition:
 
 A
 competitive
 inhibitor
 I
 can
 only
 bind
 to
 the
 unoccupied
enzyme
E,
not
to
the
ES
complex.
The
 quantity
 of
 complex
 EI
 that
 forms
 is
 governed
 by
 the
 equilibrium
 constant
 Ki,
 known
 as
 the
 inhibition
 constant.
If
more
EI
forms,
less
enzyme
 is
available
to
form
productive
ES
complex.
 
 However,
since
inhibitor
I
can't
bind
to
ES,
very
high
substrate
concentrations
can
overcome
the
 inhibitor
by
forcing
the
substrate
binding
equilibrium
in
the
direction
of
ES,
and
this
brings
the
 enzyme
up
to
its
normal
Vmax.
Hence
the
term
competitive
to
describe
this
inhibition.
 
 Michaelis
 Menten
 hyperbolic
 plot:
 shows
 the
 rate
 vo
 of
 the
 enyme
 reaction
 with
 a
 constant
 concentration
[I]
of
inhibitor
as
[S]
is
varied.
 
 The
 rate
 rises
 more
 gradually
 when
 inhibitor
 is
 present,
 but
 eventually
 reaches
 normal
 Vmax
 when
[S]
is
very
high.
 
 If
 inhibitor
 concentration
 [I]
 is
 set
equal
to
Ki,
this
causes
the
KM'
 observed
 to
 be
 doubled
 relative
 to
uninhibited
enzyme.
 Page
2
of
5
 BIOC*2580
Lecture
18:

Enzyme
Inhibition
 
3 Characteristics
of
a
competitive
inhibitor:


 Vmax
is
unchanged,
observed
KM’
increases.
 
 [I] In
presence
of
inhibitor
at
concentration
[I],

KM'
=
KM

 1 + ) ( 
 

 K i Remember
that
higher
KM
implies
that
the
enzyme
binds
its
substrate
with
less
affinity.
 i.e.

a
higher
concentration
of
[S]
is
needed
before
the
enzyme
can
reach
50%
of
Vmax.
 
 € Ki
is
equal
to
the
concentration
of
inhibitor
that
doubles
the
observed
KM
of
the
enzyme.
 
 [I] If
we
set
the
inhibitor
concentration
[I]
=
Ki,
then
KM'
=
KM













=
KM
(1
+
1)
 (1 + ) 
 

 K i [I] (1 








 The
term





+ ) is
the
inhibition
factor,
and
appears
in
all
inhibition
equations.
 

 K i 
 The
 change
 in
 KM
 is
 detected
 by
 plotting
 the
 data
 in
 one
 of
 the
 linear
 forms,
 e.g.
 in
 the
 € Lineweaver
Burk
Plot
 
 € The
 dashed
 line
 represents
 the
 activity
 of
 the
 normal
 uninhibited
 enzyme,
 and
 then
 the
 experiment
 is
 repeated
 with
 a
 series
 of
 different
concentrations
of
I.
The
other
two
 lines
show
the
effect
increasing
[I].


 
 Each
 line
 shows
 the
 behaviour
 of
 the
 enzyme
 for
 a
 given
 value
 of
 [I].
 The
 higher
 concentration
 of
 inhibitor
 gives
 a
 steeper
 slope
to
the
line.

The
series
of
lines
pivot
on
 the
y
intercept,
 since
Vmax
is
not
changed
for
 competitive
 inhibition.
 The
 X‐intercept
 becomes
 smaller
 as
 [I]
 increases,
 since
 KM
 increases
for
competitive
inhibition.
 
 To
measure
Ki,
one
finds
the
inhibitor
concentration
[I]
that
just
doubles
the
 observed
KM’.
 This
 is
represented
by
the
middle
line
in
the
figure
(x‐intercept
halved
means
KM’
was
doubled.).
 
 Page
3
of
5
 BIOC*2580
Lecture
18:

Enzyme
Inhibition
 
4 2.
Non‐Competitive
inhibition:
 
 A
 non‐competitive
 inhibitor
 I
 can
 bind
 both
 to
 unoccupied
 enzyme
 E,
 and
 to
 ES
 complex.
 The
 EI
 complex
can
bind
S,
but
EIS
is
unable
to
proceed
to
 give
products.
 
 The
quantity
of
complexes
EI
and
EIS
that
form
 are
 governed
by
the
equilibrium
constant
Ki.
If
more
EI
or
EIS
forms,
less
enzyme
is
available
to
form
 productive
 ES
 complex.
 Since
 I
 can
 also
 bind
 to
 ES,
 high
 substrate
 concentrations
 do
 not
 overcome
 the
 inhibitor.
 Hence
 the
 term
 noncompetitive
 to
describe
this
inhibition.
True
 non‐ competitive
inhibition
requires
Ki
to
be
the
same
at
both
stages.
If
the
Ki
for
I
binding
to
empty
E
 is
not
the
same
as
for
I
binding
to
occupied
ES,
mixed
inhibition
will
be
observed.


 
 Michaelis
 Menten
 hyperbolic
 plot:
 shows
 the
 rate
 vo
 of
 the
 enzyme
 reaction
 with
a
 constant
concentration
 [I]
 of
inhibitor
as
[S]
is
 varied.
The
rate
 rises
 more
 gradually
 when
 inhibitor
 is
 present,
and
levels
off
at
a
lower
V'max.
 
 If
 inhibitor
 concentration
 [I]
 is
 set
 equal
 to
 Ki,
 this
 causes
 the
 V'max
 observed
 to
 be
 halved
 relative
 to
 uninhibited
enzyme.
 
 Characteristics
of
a
non‐competitive
inhibitor:

V’max
decreases,
KM
is
unchanged.
 
 V Vmax’
=

 max (1 [I] 
 

 + K i ) Lineweaver‐Burk
Plot:

 
 The
 dashed
 line
 represents
 the
 activity
 € of
 the
 uninhibited
 enzyme.
 
 The
 other
 two
 lines
 show
 the
 effect
 of
 added
 inhibitor
 I.
 The
 series
 of
 lines
 pivot
 on
 the
 ‐ve
 x‐intercept,
 since
 KM
 is
 unchanged
 for
 non‐competitive
 inhibition.
 Y‐intercept
 and
 slope
 increase
 due
 to
 the
 reciprocal
 dependence
 on
 Vmax,
 which
 decreases.
 To
 measure
 Ki,
 one
 finds
 the
 inhibitor
 concentration
 [I]
 that
 just
 halves
 the
 observed
V’max.
 
 Page
4
of
5
 BIOC*2580
Lecture
18:

Enzyme
Inhibition
 
5 4.
Mix
Inhibition
 
 Mixed
inhibition
is
indicated
by
decrease
in
Vmax
coupled
to
increase
in
KM.

If
the
change
in
 Vmax
 is
large
and
the
change
in
KM
is
small,
then
it
is
a
reasonable
approximation
(and
mathematically
 much
simpler)
to
treat
the
case
as
non‐competitive.
 
 Page
5
of
5
 ...
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This note was uploaded on 09/21/2011 for the course BIOOC 2580 taught by Professor Douger during the Fall '10 term at University of Guelph.

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