Download Enzymes Enzymes are characterized by

Enzymes
Enzymes are characterized by:
12 greater than
Catalytic
Catalytic Power
Power -- rates
rates are
are 10
1066-10
-1012
greater than
corresponding
corresponding uncatalyzed
uncatalyzed reactions
reactions
Specificity
Specificity -- highly
highly specific
specific for
for substrates
substrates
Regulation
Regulation -- acheived
acheived in
in many
many ways
ways including
including
changes
changes in
in amount
amount of
of enzyme
enzyme synthesized;
synthesized;
covalent
covalent modification;
modification; interaction
interaction with
with
metabolic
metabolic inhibitors
inhibitors and
and activators;
activators;
comparmentation;
comparmentation; etc.
etc.
Terminology
Commonly named by adding the suffix -ase
to the name of the substrate or a phrase
describing the catalytic reaction.
Urease - catalyzes the hyrolysis of urea
Alcohol dehydrogenase - catalyzes the
oxidation of alcohols to aldehydes
Enzymes catalyze reactions in association
with other molecules called cofactors:
2+ or Zn2+
2+
Metal ions - Ca2+
Coenzyme - not covalently bound to the
enzyme; most derived from vitamins
(NAD+)
Prosthetic group - permanently associated
with their protein; heme group of
hemoglobin
HOLOENZYME - Active enzyme-cofactor
complex
APOENZYME - Enzymatically inactive
protein from which the cofactor has
been removed
How do enzymes act as catalysts?
Rate
Rate of
of aa reaction
reaction depends
depends on
on how
how effectively
effectively
reactants
reactants collide
collide to
to form
form aa transition
transition state.
state. The
The
colliding
colliding substances
substances must
must be
be in
in the
the correct
correct
orientation
orientation and
and must
must possess
possess sufficient
sufficient energy
energy
to
to approach
approach the
the physical
physical configuration
configuration of
of the
the
atoms
atoms and
and bonds
bonds of
of the
the product.
product.
The
The energy
energy required
required to
to reach
reach the
the transition
transition
state
energy of
of
state from
from the
the ground
ground state
state is
is the
the energy
activation.
activation.
How do enzymes act as catalysts?
R
R == reactants
reactants
P
P == products
products
Activation
Activation energy
energy
R
R
E
E
P
P
Energy
Energy difference
difference between
between
reactant
reactant and
and products
products
Progress
Progress of
of reaction
reaction
Uncatalyzed Reaction
Enzymes
Enzymes work
work by
by lowering
lowering the
the overall
overall
activation
activation energy:
energy:
R
R
Activation
Activation energy
energy is
is
lowered
lowered
E
E
P
P
Progress
Progress of
of reaction
reaction
Catalyzed Reaction
Enzymes
Enzymes lower
lower activation
activation energy
energy by
by
orienting
orienting the
the reacting
reacting molecules
molecules so
so that
that the
the
most
most favorable
favorable collisions
collisions can
can occur.
occur.
Enzymes
Enzymes cause
cause substrates
substrates to
to come
come together
together
in
in favorable
favorable orientations
orientations in
in an
an
enzyme-substrate
enzyme-substrate [ES]
[ES] complex
complex where
where
substrates
substrates are
are bound
bound to
to aa specific
specific region
region of
of
the
active site.
site.
the enzyme
enzyme called
called the
the active
Active Site:
Three-dimensional
Three-dimensional entity
entity formed
formed by
by groups
groups that
that
come
come from
from different
different parts
parts of
of the
the linear
linear sequence
sequence
of
of amino
amino acids.
acids.
A
A cleft
cleft or
or crevice
crevice in
in the
the tertiary
tertiary structure.
structure.
Water
Water is
is excluded
excluded unless
unless aa reactant.
reactant.
Non-polar
Non-polar nature
nature enhances
enhances the
the binding
binding of
of
substrates.
substrates.
Lock
Lock and
and Key
Key Theory
Theory -- Emil
Emil Fisher
Fisher (1894)
(1894)
proposed
proposed that
that the
the active
active site
site of
of an
an enzyme
enzyme
alone
alone is
is complementary
complementary in
in shape
shape to
to that
that of
of
the
the substrate.
substrate. The
The enzyme
enzyme is
is the
the lock
lock and
and
the
the substrate
substrate the
the key
key that
that fits
fits it.
it.
Induced
Induced Fit
Fit Model
Model -- Daniel
Daniel Koshland
Koshland (1958).
(1958).
Enzymes
Enzymes change
change their
their conformation
conformation after
after
binding
binding the
the substrate.
substrate. The
The active
active site
site has
has aa
shape
shape complementary
complementary to
to the
the substrate
substrate only
only
after
after the
the substrate
substrate is
is bound.
bound.
Kinetic Properties of Enzymes:
For many enzymes, the rate of catalysis (v)
varies with the substrate concentration [S]
as follows:
Vmax (evidence for
formation of an ES
complex)
v
Km [S]
Km = Substrate
concentation at which
the velocity is 1/2
Vmax.
v
[S]
v
[S]
v
[S]
When all active sites are filled, the rate is
at a maximum (Vmax).
v
[S]
Leonor Michaelis and Maud Menten (1913)
proposed a model to account for the
behavior shown in the V vs. [s] curve:
E+S
k11
ES
k33
E+P
k22
One assumption is that P cannot revert
back to S (there is no k44).
Michaelis and Menten derived an
equation that relates the rate of catalysis
(v) to the concentration of enzyme and
substrate and the rates of the individual
steps in the kinetic pathway:
Starting point is that the rate at which
product is formed is: V = k3[ES].
Expressing [ES] in terms of known
quantities:
rate of formation of [ES] = k11 [E] [S]
rate of breakdown of [ES] = (k22 + k33) [ES]
E+S
k11
k22
ES
k33
E+P
Michaelis-Menten kinetics apply to
catalytic rates occuring under
steady-state conditions where the
concentration of ES stays the same
while the concentrations of reactants
and products changes. This happens
when the rate of formation of ES is equal
to the rate of breakdown of ES:
k11 [E] [S] = (k22 + k33) [ES]
Rearrange equation:
[ES] = k11 [E] [S]
=
[E] [S]
(k22 + k33)
(k22 + k33)
k11
(k22 + k33)
By definition
is a constant
k11
termed the Michaelis constant, Km.
[ES] = k11 [E] [S]
(k22 + k33)
=
[E] [S]
(k22 + k33)
k11
[ES] = k11 [E] [S]
(k22 + k33)
=
[E] [S]
Km
[ES] =
[E] [S]
Km
[ES] = [E] [S] Km
Another
Another assumption
assumption -- [S]
[S] is
is in
in vast
vast
excess
excess (so
(so that
that the
the rate
rate is
is proportional
proportional
to
to [ES])
[ES]) and
and [E]
[E] is
is very
very small
small compared
compared
to
to [S];
[S]; thus
thus
[ES] = [E] [S] / Km
[E] = [Etotal
] - [ES]; substitute:
total
[ES] = ([Ett] - [ES]) [S]
Km
Solve for ES:
[ES] = [Et][S] - [ES][S] = [Et][S] - [ES][S]
Km
Km
Add [ES][S] to both sides:
Km
[ES]
[ES] ++ [ES][S]
[ES][S] == [Et][S]
[Et][S]
Km
11
Km
Km
Factor out [ES]:
[ES] 1+ [S]
Km
=
[Et][S]
Km
Divide by 1 + [S]
Km
substitute
substitute Km
Km for
for 11
Km
Km
[ES] = [Et][S] / Km
1 + [S]
Km
Rearrange to:
[ES] = [Et] [S]
[S] + Km
Substitute this for [ES] in V = k33[ES]
V = k33[Et] [S]
[S] + Km
V = k33[Et] [S]
[S] + Km
Vmax
Vmax is
is attained
attained when
when all
all enzyme
enzyme sites
sites are
are
saturated
saturated with
with substrate
substrate (S
(S >>>>>
>>>>> Km)
Km) so
so
that
[S]
that
so that
that Vmax
Vmax == K
K33[Et]
[Et]
[S] == 1;
1; so
[S]
[S] ++ Km
Km
V = Vmax [S]
Km + [S]
The Michaelis-Menten
Equation!!!
The M-M equation accounts for the
kinetic data in the v vs. S curve:
Vmax
Vmax
v
[S]
[S]
Km
Km
V = Vmax [S]
Km + [S]
When
When [S]
[S] is
is low
low (<<<
(<<< Km)
Km) then
then
V
V == [S]
[S] Vmax
Vmax
Km
Km
When
When [S]
[S] is
is >>>
>>> Km
Km then
then V
V == Vmax
Vmax
V = Vmax [S]
Km + [S]
The
The meaning
meaning of
of Km
Km is
is evident
evident from
from the
the M-M
M-M
equation
equation when
when [S]
[S] == Km;
Km; then
then V=Vmax
V=Vmax (Km)
(Km)
2Km
2Km
or
or V
V == Vmax
Vmax
22
and
and Km
Km is
is thus
thus equal
equal to
to
the
the substrate
substrate
concentration
concentration at
at which
which the
the
velocity
velocity is
is 1/2
1/2 maximal.
maximal.
For
For convenience
convenience sake
sake the
the M-M
M-M equation
equation
can
can be
be converted
converted into
into aa form
form that
that gives
gives
aa straight
straight line:
line:
11
vv
yy
==
11 ++
Vmax
Vmax
==
b
b
++
Km
Km xx
Vmax
Vmax
m
m
11
[S]
[S]
X
X
A plot of 1/v vs. 1/[S] yields a
Lineweaver-Burke plot:
slope
slope == Km/Vmax
Km/Vmax
1/v
1/Vmax
1/Vmax
0
-- 1/Km
1/Km
1/[S]
Significance of Km and Vmax:
Km
Km has
has two
two meanings:
meanings: 1)
1) concentration
concentration of
of
substrate
substrate at
at which
which 1/2
1/2 of
of active
active sites
sites are
are
filled;
filled; 2)
2) is
is aa ratio
ratio of
of rate
rate constants
constants k2
k2 ++ k3
k3
k1
k1
If
If k2
k2 >>>
>>> k3;
k3; then
then Km=
Km= k2/k1
k2/k1 and
and is
is equal
equal
to
to the
the dissociation
dissociation constant
constant for
for the
the ES
ES
complex.
complex.
Turnover number:
The
The number
number of
of substrate
substrate molecules
molecules converted
converted to
to
product
product per
per unit
unit time
time when
when the
the enzyme
enzyme is
is fully
fully
saturated
saturated with
with substrate.
substrate. Equal
Equal to
to k3
k3 and
and is
is
termed
termed kcat.
kcat. Vmax
Vmax reveals
reveals the
the turnover
turnover number
number
ifif the
the conentration
conentration of
of active
active sites
sites (Et)
(Et) is
is known
known
because
because Vmax
Vmax == k3[Et]
k3[Et] (or,
(or, Vmax
Vmax == kcat
kcat [Et]
[Et] ).).
When
When all
all of
of the
the enzyme
enzyme sites
sites are
are occupied
occupied by
by S,
S,
then
then ES
ES == Et.
Et.
Enzyme Inhibition
Small
Small molecules
molecules or
or ions
ions can
can inhibit
inhibit
Irreversible
Irreversible inhibitors
inhibitors dissociate
dissociate very
very
slowly,
slowly, if
if at
at all
all because
because they
they are
are
tighly
tighly bound.
bound.
Reversible
Reversible inhibition
inhibition is
is characterized
characterized
by
by rapid
rapid dissociation
dissociation of
of the
the enzyme
enzyme
inhibior
inhibior (EI)
(EI) complex.
complex.
E+S
k11
ES
k33
E+P
k22
+I
kii
EI
The
The binding
binding affinity
affinity for
for II
is
is described
described by
by Ki,
Ki, the
the
dissociation
dissociation constant
constant of
of
the
the enzyme-inhibitor
enzyme-inhibitor
complex.
complex.
Three types of reversible inhibition:
1. Competitive Inhibition
The
The enzyme binds either substrate or
inhibitor, but NOT both.
Rate
Rate is diminished by reducing the
proportion
proportion of
of enzyme molecules bound
to
to substrate.
substrate.
Can
Can be
be overcome
overcome by high substrate
concentrations,
concentrations, and thus Vmax is
unaffected.
unaffected.
Competitive Inhibition
+I
-I
1/v
Vmax
Vmax stays
stays
the
the same
same
Km
Km changes
changes
0
1/[S]
2. Non-competitive Inhibition
The
The substrate
substrate and
and inhibitor
inhibitor can
can bind
bind
simultaneously
simultaneously to
to the
the enzyme
enzyme (i.e.,
(i.e., their
their
binding
binding sites
sites do
do not
not overlap).
overlap).
Non-competitive
Non-competitive inhibitors
inhibitors decrease
decrease the
the
turnover
turnover number
number of
of an
an enzyme.
enzyme.
Since
Since the
the turnover
turnover number
number is
is dependent
dependent
upon
upon Vmax,
Vmax, non-competitive
non-competitive inhibitors
inhibitors
affect
affect Vmax.
Vmax.
Non-competitive Inhibition
+I
-I
1/v
Vmax
Vmax
changes
changes
Km
Km stays
stays the
the
same
same
0
1/[S]
Allosteric Enzymes
Most are multisubunit enzymes.
Vmax
Vmax
Show
Show sigmoidal
sigmoidal plots
plots of
of vv
V
V vs. S.
K
K0.5
0.5
[S]
[S]
The binding of substrates or modulators
to one active site affects properties of the
other active sites.