Lecture14-chymotrypsin

Lecture14-chymotrypsin - BIOC*2580
Lecture
14:


Info iconThis preview shows page 1. Sign up to view the full content.

View Full Document Right Arrow Icon
This is the end of the preview. Sign up to access the rest of the document.

Unformatted text preview: BIOC*2580
Lecture
14:
 The
Catalytic
mechanism
of
Chymotrypsin
 1
 
 
 
 
 Synopsis:
Chymotrypsin
catalyzes
hydrolysis
of
a
substrate
peptide
at
the
carboxylate
group
of
 phenylalanine,
 tyrosine
 or
 tryptophan,
 unless
 the
 next
 amino
 acid
 is
 proline.
 This
 behaviour
 depends
 on
 the
 coordinated
 action
 of
 several
 groups
 of
 amino
 acids
 in
 chymotrypsin:
 the
 binding
pocket
precisely
positions
the
target
amino
acid;
the
 catalytic
triad
provides
an
active
 nucleophile
 and
 general
 acid/base
 catalysis;
 the
 oxyanion
 hole
 stabilizes
 the
 shape
 of
 the
 transition
state.
 
 Reading:

Lehninger
p.
205‐211
(4th
ed
p.
213‐218).
 
 Read
the
supplementary
material
"What
do
the
curly
arrows
mean
in
reaction
diagrams?"
 
 
 Chymotrypsin
binds
the
substrate
peptide
into
a
groove
on
the
surface
of
the
enzyme.
 
 Weak
binding
occurs
via
hydrogen
bonds
to
 the
 NH
 group
 of
 the
 target
 amino
 acid
 and
 to
 several
 backbone
 CO
 or
 NH
 groups
 preceding,
 so
 the
 enzyme
 selects
 a
 peptide
 chain
as
its
substrate.
 
 Strong
 binding
 of
 the
 target
 amino
 acid
 occurs
 when
 its
 benzene
 ring
 side
 chain
 fits
 in
 a
 hydrophobic
 pocket
 on
 the
 surface
 of
 chymotrypsin.
If
the
substrate
peptide
chain
 contains
 one
 of
 the
 target
 amino
 acids,
 the
 peptide
 bond
 immediately
 following
 the
 target
 is
 positioned
 precisely
 next
 to
 the
 catalytic
components.
 
 Page
1
of
6
 BIOC*2580
Lecture
14:
 The
Catalytic
mechanism
of
Chymotrypsin
 2
 
 Peptide
 hydrolysis
 by
 H2O
 without
 the
 help
 of
 a
 catalyst
 
 H2O
acts
as
a
nucleophile:

 (1)
 a
 lone
 pair
 from
 the
 water
 is
 donated
 to
 C
 of
 C=O,
 due
to
electrophilic
C.
 
 C
can't
accommodate
more
than
8
valence
electrons,
so
 the
 electronegative
 O
 atom
 withdraws
 a
 bonding
 electron
pair
to
itself.
 
 The
 transition
 state
 forms
 a
 semistable
 oxyanion
 –O–,
 and
 the
 attacked
 C
 atom
 goes
 from
 initial
 sp2
 state
 (trigonal
 planar)
 to
 sp3
 (tetrahedral)
 in
 the
 transition
 state
 
 (2)
 If
 the
 transition
 state
 breaks
 up
 allowing
 N
 to
 withdraw
 its
 bonding
 electrons,
 the
 peptide
 bond
 is
 broken.
 The
 products
 stabilize
 by
 exchanging
 protons
 (3)
 to
 become
 neutral;
 additional
 proton
 exchanges
 with
 the
 surrounding
 H2O
 may
 then
 lead
 to
 ‐CO2‐
 and
 + NH3‐
groups,
and
the
reaction
is
complete.
 
 (2B)
 If
 the
 unstable
 transition
 state
 breaks
 up
 by
 withdrawing
 electrons
 back
 to
 the
 original
 H2O,
 the
 reactants
are
restored
without
any
change
having
taken
 place
and
the
reaction
has
no
outcome.
 
 Page
2
of
6
 BIOC*2580
Lecture
14:
 The
Catalytic
mechanism
of
Chymotrypsin
 3
 
 The
problem
with
hydrolysis
by
neutral
H2O:

 
 H2O
 is
 not
 a
‐very
 good
 nucleophile
 because
 O
 is
 too
 electronegative
 to
 share
 its
 lone
 pair
 electrons.
HO 
 would
be
much
better,
but
not
enough
is
available
at
normal
pH.
H2O
 is
also
a
 good
 leaving
 group,
 so
 the
 transition
 state
 may
 not
 lead
 to
 a
 net
 reaction.
 As
 a
 result
 the
 uncatalyzed
reaction
is
excessively
slow,
about
one
reaction
every
10
years.
 
 ‐ At
high
pH,
HO 
will
be
a
better
nucleophile,
and
at
low
pH,
H+
stabilizes
the
transition
state
and
 makes
the
N
into
a
better
leaving
group
by
adding
 H+.

However,
a
biological
catalyst
must
be
 able
to
function
close
to
pH
7
 
 
 Chymotrypsin
makes
a
multi‐pronged
attack
to
give
about
40
reactions
per
 second
(4
 × 
109
 fold
speed
up)
 
 Chymotrypsin
uses
a
better
nucleophile
in
the
form
of
the
catalytic
triad
 
 ‐
Asp
102,
His
57,
Ser
 195
side
chains
 
 The
 transition
 state
 is
 stabilized
 by
 the
 oxyanion
 hole

‐
Gly
193
and
Ser
195
backbone
‐NH‐
groups.
 
 The
 reaction
 is
 taken
 in
 two
 easy
 steps
 instead
 of
 one
difficult
step
 
 
 Step
 1:
 a
 nucleophile
 in
 the
 enzyme
 attacks
 the
 target
 peptide
bond,
splitting
off
the
C‐terminal
half
 of
 the
 substrate
 peptide.
 The
 N‐terminal
 half
 remains
 bonded
 to
 the
 enzyme
 to
 form
 a
 reaction
 intermediate.
 
 Step
2:
The
enzyme
recruits
an
H2O
to
split
off
the
N‐terminal
half
of
the
substrate,
restoring
 the
enzyme
to
its
original
state.
 
 Page
3
of
6
 BIOC*2580
Lecture
14:
 The
Catalytic
mechanism
of
Chymotrypsin
 4
 
 The
catalytic
triad
 
 The
 Catalytic
 triad
 consists
 of
 3
 amino
 acid
 side
 chains,
far
apart
in
the
polypeptide,
but
 brought
 physically
 close
 together
 by
 the
 folding
of
chymotrypsin:
 
 1.
 Aspartate
 102
 is
 a
 negative
 carboxylate,
 isolated
 away
 from
 external
 aqueous
 solution
 at
 chymotrypsin
surface;
it
would
like
to
have
a
+ve
charged
partner.
 2.
Histidine
57,
a
weak
base,
could
be
positive
but
is
only
weakly
protonated
since
its
side
chain
 has
pKa
=
6.5.
 3.
 Serine
195,
side
chain
CH2‐OH,
is
not
a
good
nucleophile
unless
it
could
get
rid
of
the
proton,
 but
by
itself
it's
not
acidic.
 
 The
combined
action
of
the
catalytic
triad
is
to
make
Ser
195
into
a
better
nucleophile.
 
 Step
1
of
the
catalytic
reaction
(See
also
Fig
6‐21
Lehninger,
p208‐9)
 
 The
 substrate
 binds
 with
 its
 target
 C=O
 group
 next
 to
 Ser
 195.
 Substrate
 is
 shown
 in
 blue.
 The
 catalytic
 triad,
 which
 is
 part
 of
 chymotrypsin,
 is
 in
 black.
 Electron
 exchanges
 are
 in
 red.
 Cooperative
 action
 among
 the
 catalytic
 triad
 helps
form
the
first
transition
state.
 
 His
 57
 acts
 as
 a
 general
 base
 by
 removing
a
proton
from
Ser‐OH,
and
 this
 helps
 the
 Ser
 O:
 make
 its
 nucleophilic
 attack
 on
 the
 substrate
 C=O,
 while
 negative
 Asp
 102
 promotes
formation
of
HisH+.
 
 The
 oxyanion
 hole
 is
 also
 part
 of
 chymotrypsin,
 and
 consists
 of
 the
 backbone
 ‐NH‐
 groups
 of
 Gly
 193
 and
Ser
195
(shown
in
pink).
The
 N‐ H
 groups
 are
 positioned
 in
 such
 a
 way
 that
 they
 will
 donate
 strong
 H‐ bonds
to
the
substrate
C=O,
if
the
 C
 atom
is
tetrahedral,
as
found
in
the
 transition
 state.
 This
 strains
 the
 bonds
 of
 the
 trigonal
 planar
 C=O
 of
 the
 original
 substrate,
 helping
the
reaction
to
proceed
to
the
transition
state.
 
 Page
4
of
6
 BIOC*2580
Lecture
14:
 The
Catalytic
mechanism
of
Chymotrypsin
 5
 
 Breakdown
 of
 the
 first
 transition
 state
 and
 formation
 of
the
intermediate:
 
 The
transition
state
is
broken
up
by
 loss
 of
 the
 peptide
 bond
 after
 the
 target
amino
acid.

 
 Histidine
 now
 acts
 as
 a
 general
 acid,
 donating
 its
 proton
 to
 the
 N
 atom
 so
 that
 it
 is
 a
 better
 leaving
 group.
 
 After
 gaining
a
 proton
 from
His
57,
 a
 neutral
 NH2‐
 group
 is
 formed,
 so
 the
C‐terminal
half
of
the
substrate
 is
no
longed
bonded
to
the
enzyme
 and
is
now
free
to
leave,

 
 The
 remainder
 of
 the
 substrate
 remains
bonded
via
its
 carboxylate
 group
in
an
ester
 bond
to
Ser
195.
 This
 is
 called
 the
 acyl‐enzyme
 intermediate.
 
 Step
2
 With
 half
 of
 the
 substrate
 out
 of
 the
 way,
 there's
 now
 room
 for
 an
 H2O
 to
 get
 into
 range
 of
 the
 catalytic
triad.
 
 His
 57
 acts
 as
 a
 general
 base
 to
 remove
 a
 proton
 from
 H2O,
 enhancing
 the
 nucleophilic
 power
 of
 the
 O
 in
 H2O
 (much
 like
 the
 earlier
step
with
Ser
195).
 
 H2O
can
now
attack
the
substrate
 
 Transition
 state
 2
 breaks
 up
 by
 HisH+
57
acting
as
a
general
acid
to
 donate
 the
 proton
 back
 to
 Ser
 195,
 in
 turn
 breaking
 the
 Ser‐O
 to
 substrate
C‐‐O
ester
bond.
 
 
 Page
5
of
6
 BIOC*2580
Lecture
14:
 The
Catalytic
mechanism
of
Chymotrypsin
 6
 
 Final
result:

 
 The
 N‐terminal
 half
 of
 the
 original
 peptide
 substrate
 now
 carries
 the
 target
 amino
 acid
 at
 its
 newly
 formed
 C‐terminus.
This
is
now
free
 to
 leave
 in
 the
 form
 of
 a
 carboxylic
 acid,
since
 the
C=O
is
trigonal
planar
 and
pops
out
of
the
oxyanion
hole.
 
 
 Some
points
to
note
 
 At
 the
 end
 of
 the
 reaction,
 the
 catalytic
 triad
 is
 back
 to
 the
 original
 state.
 A
 single
 enzyme
 molecule
can
go
through
this
cycle
of
reactions
with
new
substrate
millions
of
times
over.
 
 The
ambivalent
behaviour
of
His
57
is
critical
to
the
process.
Because
the
pKa
of
His
is
about
6.5,
 His
 is
 able
 to
 protonate
 and
 deprotonate
 almost
 equally
 well
 and
 won't
 commit
 to
 staying
 protonated.

 Deprotonated
His
57
acts
as
a
general
base,
removing
the
proton
from
Ser
 195
to
 form
transition
state
1,
and
again
removing
a
proton
from
H2O
to
form
transition
state
2.
 
 Protonated
HisH+
57
acts
as
a
general
acid
donating
a
proton
back
to
the
departing
amino
group,
 and
then
finally
to
replace
the
proton
on
Ser
195.
 
 This
 is
 just
 an
 example
 of
 an
 enzyme
 mechanism.
 Trypsin
 and
 other
 members
 of
 the
 serine
 protease
 family
 of
 related
 enzymes
 share
 this
 mechanism,
 differing
 only
 in
 the
 identity
 of
 the
 targeted
 amino
 acid.
 Other
 enzymes
 use
 a
 variety
 of
 chemical
 processes
 to
 bring
 about
 the
 appropriate
catalytic
process.
 
 Site
 directed
 mutagenesis
 is
 an
 important
 experimental
 tool
 for
 testing
 theories
 of
 enzyme
 mechanism.
A
genetically
engineered
version
of
the
enzyme
is
produced
with
a
chosen
amino
 acid
in
the
sequence
modified.
This
allows
one
to
test
the
contribution
of
each
amino
acid
to
the
 whole
 process
 As
 one
 might
 predict,
 when
 Asp
 102,
 His
 57
 or
 Ser
 195
 are
 changed
 into
 other
 amino
 acids,
 this
 disrupts
 the
 catalytic
 triad
 and
 grossly
 reduces
 the
 catalytic
 efficiency
 of
 chymotrypsin.
 
 • Changing
Asp
to
Ala
reduces
catalytic
rate
by
a
factor
of
10.
 • Changing
His
to
Lys
reduces
catalytic
rate
by
a
factor
of
1000.
 • Changing
Ser
to
Ala
reduces
catalytic
rate
by
106
fold.
 
 Page
6
of
6
 ...
View Full Document

Ask a homework question - tutors are online