Enzyme
(E)
and
substrate
(S)
rst
reversibly
combine
to
give
an
enzyme
substrate
complex
(ES).
Chemical
processes
then
occur
in
a
second
step
with
a
rate
constant
called
kcat,
or
the
turnover
number,
which
is
the
maximum
number
of
substrate
molecules
converted
to
product
per
ac6ve
site
of
the
enzyme
per
unit
of
6me.
The
kcat
is
a
rate
constant
that
refers
to
the
proper6es
and
reac6ons
of
the
ES
complex.
For
simple
reac6ons,
kcat
is
the
rate
constant
for
the
chemical
conversion
of
the
ES
complex
to
free
enzyme
and
products.
Important:
These
deni6ons
are
valid
only
when
the
concentra6on
of
the
enzyme
is
very
small
compared
with
that
of
the
substrate.
They
apply
only
to
the
ini6al
rate
of
forma6on
of
products:
the
rate
of
forma6on
of
the
rst
few
percent
of
the
product,
before
the
substrate
has
been
depleted
and
products
that
can
interfere
with
the
cataly6c
reac6on
have
accumulated.
For example, the cytoplasm inside a cell behaves more like a gel than a
freely flowable or watery liquid, due to the very high concentration of
protein (up to ~400 mg/mL) and other solutes, which can severely limit
molecular movements (e.g., diffusion or collision). This causes
macromolecular crowding, which can alter reaction rates and
dissociation constants.
The
Michaelis
Menten
scheme
explains
why
a
maximum
rate
of
reac'on,
Vmax,
is
always
observed
when
the
substrate
concentra6on
is
much
higher
than
the
enzyme
concentra6on.
This
is
the
fastest
a
reac,on
can
go.
Vmax
is
obtained
when
the
enzyme
is
saturated
with
substrate.
There
are
then
no
free
enzyme
molecules
available
to
turn
over
addi6onal
substrate.
Hence,
the
rate
is
constant,
Vmax,
and
is
independent
of
further
increase
in
the
substrate
concentra,on.
The
substrate
concentra6on
when
the
half
maximal
rate,
Vmax/2,
is
achieved
is
called
the
Km.
For
many
simple
reac6ons,
it
can
easily
be
shown
that
the
Km
is
equal
to
the
dissocia6on
constant,
Kd,
of
the
ES
complex.
The
Km
describes
the
anity
of
the
enzyme
for
the
substrate.
For
more
complex
reac6ons,
Km
may
be
regarded
as
the
overall
dissocia6on
constant
of
all
enzyme
bound
species.
A
plot
of
the
reac6on
rate
as
a
func6on
of
the
substrate
concentra6on
for
an
enzyme
catalyzed
reac6on.
Vmax
is
the
maximal
velocity.
The
Michaelis
constant,
Km,
is
the
substrate
concentra6on
at
half
Vmax.
The
rate
is
related
to
the
substrate
concentra6on,
[S],
by
the
Michaelis
Menten
equa6on:
Determination of constants - LineweaverBurk plot
To determine the maximum rate of an enzyme-mediated reaction, a series of experiments is carried
out with varying substrate concentrations ([S]) and the initial rate of product formation is measured.
'Initial' here is taken to mean that the reaction rate is measured after a relatively short time period,
during which complex builds up but the substrate concentration remains approximately constant and
the quasi-steady-state assumption will hold. The measurements can then be plotted in a
LineweaverBurk plot, plotting the inverse of substrate concentration against the inverse of
the initial velocity
1 = Km + [S] = Km x 1 + 1
V0 Vmax [S] Vmax [S] Vmax
The
ul6mate
limit
to
this
value
is
set
by
the
rate
constant
for
the
ini6al
forma6on
of
the
ES
complex.
This
rate
cannot
be
faster
than
the
diusion-
controlled
encounter
of
an
enzyme
and
its
substrate,
which
is
between
108
to
109
per
mole
per
second.
The
quan6ty
is
some6mes
called
the
specicity
constant
because
it
describes
the
specicity
of
an
enzyme
for
compe6ng
substrates.
It
is
a
useful
quan6ty
for
kine'c
comparison
of
mutant
proteins.
Measure
these
numbers
to
assess
how
a
muta6on
aects
a
par6cular
process.
Its
therefore
a
way
to
see
how
important
that
part
of
your
protein
is.
The advantages of the induced fit mechanism arise due to the stabilizing effect of strong enzyme
binding. There are two different mechanisms of substrate binding: uniform binding, which has strong
substrate binding, and differential binding, which has strong transition state binding.
The stabilizing effect of uniform binding increases both substrate and transition state binding
affinity, while differential binding increases only transition state binding affinity. Most proteins
seem to use the differential binding mechanism to reduce the Ea, so most proteins have high affinity
of the enzyme to the transition state. The substrate first binds weakly, then the enzyme changes
conformation increasing the affinity to the transition state and stabilizing it, so reducing the
activation energy to reach it.
Mechanisms of transition state stabilization
These conformational changes also bring catalytic residues in the active site close to the chemical
bonds in the substrate that will be altered in the reaction. After binding takes place, one or more
mechanisms of catalysis lowers the energy of the reaction's transition state, by providing an
alternative chemical pathway for the reaction. There are six possible mechanisms of "over the
barrier" catalysis as well as a "through the barrier" mechanism:
1: Catalysis by bond strain - this is the principal effect of induced fit binding, where the affinity of the
enzyme to the transition state is greater than to the substrate itself. This induces structural
rearrangements which strain substrate bonds into a position closer to the conformation of
the transition state, so lowering the energy difference between the substrate and transition state
and helping catalyze the reaction.
2: Catalysis by proximity and orientation - this increases the rate of the reaction as enzyme-
substrate interactions by aligning reactive chemical groups and holding them close together. This
reduces the entropy of the reactants and thus makes reactions such as ligations or addition
reactions more favorable, there is a reduction in the overall loss of entropy when two
reactants become a single product.
3: Catalysis involving proton donors or acceptors (Acid/Base Catalysis) - proton donors and
acceptors, i.e. acids and bases, may donate and accept protons in order to stabilize developing
charges in the transition state. This typically has the effect of activating nucleophile and
electrophile groups, or stabilizing leaving groups. Histidine is often the residue involved in these
acid/base reactions, since it has a pKa close to neutral pH and can therefore both accept
and donate protons.
Mechanisms of transition state stabilization
4: Electrostatic catalysis - stabilization of charged transition states can also be by residues in the
active site forming ionic bonds (or partial ionic charge interactions) with the intermediate.
These bonds can either come from acidic or basic side chains found on amino acids such as lysine,
arginine, aspartic acid or glutamic acid or come from metal cofactors such as zinc. Metal ions are
particularly effective and can reduce the pKa of water enough to make it an effective nucleophile.
5: Covalent catalysis - involves the substrate forming a transient covalent bond with residues in the
active site or with a cofactor. This adds an additional covalent intermediate to the reaction, and
helps to reduce the energy of later transition states of the reaction. The covalent bond must,
at a later stage in the reaction, be broken to regenerate the enzyme. This mechanism is found in
enzymes such as proteases like chymotrypsin and trypsin, where an acyl-enzyme intermediate is
formed.
6: Quantum tunneling the "over the barrier" mechanisms above have been challenged in some
cases by models and observations of "through the barrier" mechanisms (quantum tunneling). Some
enzymes operate with kinetics which are faster than what would be predicted by the classical G.
In "through the barrier" models, a proton or an electron can tunnel through activation barriers.
Quantum tunneling for protons has been observed in tryptamine oxidation by aromatic amine
dehydrogenase. Not super convincing.
General:
Enzymes
decrease
the
ac'va'on
energy
of
chemical
reac'ons
The
Michaelis
complex,
ES,
undergoes
rearrangement
to
one
or
several
transi'on
states
before
the
product
is
formed.
Energy
is
required
for
these
rearrangements.
The
input
energy
required
to
bring
free
enzyme
and
substrate
to
the
highest
transi6on
state
of
the
ES
complex
is
called
the
ac'va'on
energy
of
the
reac6on.
In
the
absence
of
enzyme,
spontaneous
conversion
of
substrate
to
product
also
proceeds
through
transi'on
states
that
require
ac'va'on
energy.
The
rate
of
chemical
reac6on
is
strictly
dependent
on
its
ac6va6on
energy,
and
the
more
than
1
million-fold
enhancement
of
rate
achieved
by
enzyme
catalysis
results
from
the
ability
of
the
enzyme
to
decrease
the
ac6va6on
energy
of
the
reac6on.
This
decrease
in
ac6va6on
energy
is
achieved
by
enzymes
in
several
dierent
ways
like
weve
seen.
Generally
-
the
higher
the
anity
of
the
enzyme
for
the
transi'on
state
makes
the
transi'on
energe'cally
favorable
and
thus
decreases
the
ac'va'on
energy.
If,
on
the
other
hand,
the
enzyme
were
to
bind
the
unaltered
substrate
more
strongly
than
the
transi6on
state,
the
decrease
in
binding
energy
on
the
forma6on
of
the
transi6on
state
would
increase
the
ac6va6on
energy
and
catalysis
would
not
be
achieved.
The enzyme likes substrates transi'on state more than its ground state
This
means
that
the
ac6va6on
energy
required
for
forming
the
ES
transi6on
complex
drops
Catalysis occurs