Lecture 16
– Inhibition
– Quiz on Friday, Oct. 14 (MM derivation (assume
no E+P -> ES) and 6 enzyme classes-what
types of reactions do they catalyze?)
Enzymes
•
The 6 enzyme classes can be illustrated by the general reactions catalyzed
1. Oxidoreductases:
A- + B  A + B-
2. Transferases:
A-B + C  A + B-C
3. Hydrolase:
A-B + H2O  A-H + B-OH
XY
4. Lyases:
A-B  A=B + X-Y
XY
YX
5. Isomerases:
A-B  A-B
6. Ligases (synthases)
A + B  A-B
Enzyme reactions can be slowed by the presence
of inhibitors
• Other inhibitors bind noncovalently and reversibly to their
target enzymes.
• These are usually divided into three broad classes,
competitive, noncompetitive, and uncompetitive,
depending on their manner of binding.
• Kinetic analysis can distinguish among these inhibitors if
the reaction rate is measured against substrate
concentration at different inhibitor concentrations.
Figure U2-4.1 Competitive, noncompetitive
and uncompetitive inhibition
Figure U2-4.1a Competitive
inhibition
(a) A competitive inhibitor (I) binds to
the same site as does the substrate
(S; top).
The inhibitor changes the apparent Km
for the reaction, but not the Vmax
because enough substrate can keep
any inhibitor from binding.
A Lineweaver-Burk plot (bottom) for
the reaction at various concentrations
of the inhibitor reflects this behavior.
E
ES
EI
Competitive Inhibition
S+E
ES
P
v0 =
+I
Vmax[S]
KM + [S] +[I] (KM/KI)
EI
KI = [E][I]
[EI]
v (µmol/min)
70
Vmax
60
50
w/o I
w/ I
40
30
20
10
0
1.00E-05
1.00E-04
1.00E-03
1.00E-02
[S]
1.00E-01
1.00E+00
Competitive Inhibition
ES
S+E
v0 =
P
+I
Vmax[S]
KM + [S] +[I] (KM/KI)
1/V0 = KM 1
Vmax [S]
EI
1/V0 = KM 1
Vmax [S]
(1+ [I] ) + V1
KI
() + V1
max
I
w/o I
1/v0
-1/KM
-1
KM(1+ [I] )
KI
1/Vmax
-1
KM
1/[S]
-1/KM’ or apparent KM
max
Competitive inhibition
•  = 1 +[I]/KI
• As [I] increases, v decreases (1/v increases)
• As [I] increases, KM decreases (1/KM increases)
• Vmax is the same and the inhibition can be overcome by
high [S]
Figure U2-4.1b Noncompetitive inhibition
(b) A noncompetitive inhibitor (I) (green)
does not bind to the substrate binding
site and can bind to both the free enzyme
or the ES complex.
Usually a noncompetitive inhibitor
resembles one substrate (S2) in a twosubstrate reaction, as shown here, where
both substrates are present on the
enzyme at the same time.
In the simplest cases, noncompetitive
inhibitors don't change the Km for the first
substrate (S), because they don't affect
its binding.
But providing the concentration of S2 is
not high enough to out-compete all the
inhibitor, the inhibitor does reduce the
Vmax for the reaction.
E
ES
EI
Noncompetitive Inhibition (Mixed Inhibition)
ES
S+E
KI
+I
KI’
P
v0 =
+I
KM + ’[S]
 = 1 +[I]/KI
’ = 1 +[I]/KI’
EIS
EI + S
KI’ = [ES][I]
[ESI]
KI = [E][I]
[EI]
v (µmol/min)
70
Vmax[S]
Vmax
60
50
w/o I
40
w/ I
30
20
10
0
1.00E-05
1.00E-04
1.00E-03
1.00E-02
[S]
1.00E-01
1.00E+00
Noncompetitive Inhibition (Mixed Inhibition)
ES
S+E
KI
+I
EI + S
KI’
P
v0 =
Vmax[S]
+I
KM + ’[S]
EIS
1/V0 = KM 1
Vmax [S]
() + V’
max
I
w/o I
1/v0
-1/KM
1/Vmax
1/[S]
Noncompetitive inhibition
•  = 1 +[I]/KI, ’ = 1 +[I]/KI’
• As [I] increases, v decreases (1/v increases)
• As [I] increases, KM decreases (1/KM increases), but in
the simplest case does not change (very slight).
• Cannot be overcome by increasing [S]
• May intersect above, blow or even on the line (x or y axis)
• x-axis - no affect on KM (KM = KM’)
• y-axis - no affect on Vmax
Figure U2-4.1c
Uncompetitive inhibition
(c) An uncompetitive inhibitor (blue)
binds only to the enzyme-substrate
(ES) complex and slows down the
reaction probably by inducing a
conformational change in the
enzyme.
Both the apparent Km and Vmax are
affected proportionally by such an
inhibitor, leading to parallel
Lineweaver-Burk plots for different
inhibitor concentrations.
Uncompetitive Inhibition
ES
P
S+E
KI’
+I
KI’ = [ES][I]
[ESI]
EIS
v0 =
Vmax[S]
KM + ’[S]
1/V0 = KM 1
Vmax [S]
’ = 1 +[I]/KI’
+
I
w/o I
1/v0
1/Vmax
1/[S]
’
Vmax
Uncompetitive inhibition
• ’ = 1 +[I]/KI’
• Reacts only with ES complex
• As [I] increases, v decreases (1/v increases)
• As [I] increases, apparent KM increases (apparent 1/KM
decreases), but there is no effect on binding of E to S.
• Cannot be overcome by increasing [S]
• Relatively rare in single substrate reactions but can be
more common in complex cases.
Enzyme reactions can be slowed by the presence
of inhibitors
• A key parameter that can be obtained from such an
analysis is the affinity of the inhibitor for the enzyme, the
inhibition constant Ki.
• By convention, Ki is given as the dissociation constant for
the enzyme-inhibitor equilibrium:
Ki = [E][I]
[EI]
Ki’ = [ES][I]
[ESI]
• The lower the value of Ki the tighter the inhibitor binds. In
pharmacology, the value of Ki is often used as a measure of the
effectiveness of a drug.
• A compound with a very low Ki, say 10-9 M (nanomolar) or less, can
be given at very low doses and will still be able to bind its target.
Enzyme Inhibitor Classification
1. Competitive inhibitors:
•
Most common class of reversible inhibitor consists of
compounds that resemble the substrate.
•
Such molecules can fit into the substrate binding site,
thereby blocking access from substrate molecules.
These inhibitors compete with the substrate for the
active site.
•
Example: Many HIV protease inhibitors that have proven
to be effective in treatment of AIDS are competitive
inhibitors that were designed to resemble the peptide
substrate of the HIV protease.
Enzyme Inhibitor Classification
•
Not all inhibitors compete with the substrate for the active site of the
enzyme; other inhibitors bind to a separate site.
2. Noncompetitive inhibitors bind to both the free enzyme
and the enzyme-substrate complex.
•
If an enzyme has two substrates that must bind simultaneously for
the reaction to occur, an inhibitor might compete with one substrate
but not the other.
Enzyme Inhibitor Classification
•
Not all inhibitors compete with the substrate for the active site of the
enzyme; other inhibitors bind to a separate site.
3. Uncompetitive inhibitors bind only to the enzymesubstrate complex.
•
In effect, the inhibitor reduces the amount of ES that can
go on to form product.
•
Lineweaver-Burk plots characteristically show a series of
parallel lines (Fig. U2-4.1c).
•
Uncompetitive inhibitors often stabilize an alternative
conformation of the protein, and in this case they are
called allosteric inhibitors.
•
There are also allosteric activators: molecules that
activate enzymes by stabilizing a conformation of the
enzyme that is more active than the conformation that
exists in their absence.
Figure 3-10 Ligand-induced conformational change activates
aspartate transcarbamoylase
Binding of the allosteric activator ATP to its
intersubunit binding sites on the regulatory
subunits (that between R1, outlined in purple,
and R6 is arrowed) of the T state of ATCase
(top) causes a massive conformational
change of the enzyme to the R state
(bottom).
In this state the structure of the enzyme is
opened up, making the active sites on the
catalytic subunits (C) accessible to substrate.
Al and Zn in the lower diagram indicate the
allosteric regions and the zinc-binding region,
respectively; cp and asp indicate the binding
sites for the substrates carbamoyl phosphate
and aspartate, respectively.
The red and yellow regions are the
intersubunit interfaces that are disrupted by
this allosteric transition.
Allostery
• Does not follow Michalis-Menton kinetics!
• EIS goes to products at the same rate as ES but with
lower affinity for [S]
• Doesn’t affect Vmax.
• Does affect KM
• Can operate in both directions and involves a second
binding site.
• Positive-activator, cooperative
• Negative-inhibitor, antagonistic
• Enzyme controlled by binding at second site
homotrophic (modifier is related to the substrate)
heterotrophic (related to substrate).
Enzyme-catalyzed reactions can have multiple
steps with several intermediates
• Multi-step reactions can have very complicated kinetics,
e.g., “double-displacement” or “ping-pong” enzymes.
• These are enzymes that use two or more substrates but
catalyze reactions that are strictly ordered in the sequence
in which substrates bind and products are released (Figure
U2-3.3a).
• Lineweaver-Burk plots of the velocity against one substrate
concentration at a series of fixed concentrations of other
substrate give a family of parallel lines (Fig. U2-3.3b).
Insert: double reciprocal plot of
observed initial
velocities versus CO2 concentration for
CA at different concentrations of
TAPS buffer: 5 mM, 10 mM, 20 mM, 50
mM.
Biochemistry 1999, 38, 13119-13128
Enzyme-catalyzed reactions can have multiple
steps with several intermediates
Example of Ping-Pong Enzyme: Aspartate aminotransferase: Catalyzes the conversion
of aspartate to glutamate with production of oxaloacetate and consumption of alphaketoglutarate.
The reaction sequence starts with the binding of aspartate to the enzyme followed by its
conversion to oxaloacetate, in the process of which the aspartate leaves behind its amino
group bound to a cofactor in the active site.
After oxaloacetate departs (the so-called ping step), alpha-ketoglutarate reacts with the
amino group and is converted to glutamate (the so-called pong step), bringing the enzyme
back to its original state.
The two substrates, aspartate and alpha-ketoglutarate, never encounter each other on the
enzyme. The kinetics are characteristically simple for this kind of reaction:
Lineweaver-Burk plots of the velocity against aspartate concentration at a series of fixed
concentrations of alpha-ketoglutarate, give a family of parallel lines (Fig. U2-3.3b).
Figure U2-3.3 Ping-pong or doubledisplacement kinetic behavior
•
•
(a) In ping-pong reactions, two substrates bind sequentially to an
enzyme. In this example, a chemical group (green) is transferred from
substrate A (red) to substrate B (blue).
(b) A Lineweaver Burk plot of 1/v against 1/[substrate A] at various
fixed concentrations of substrate B shows a set of parallel lines which
are diagnostic for the ping-pong reaction mechanism.
Enzyme reactions can be slowed by the presence
of inhibitors
• The rate of an enzyme-catalyzed reaction can be affected by
molecules that do not themselves participate in the chemical
reaction.
• Activators increase the reaction rate and inhibitors decrease the rate.
• Many drugs, including aspirin, penicillin, statins and Viagra are
enzyme inhibitors:
• they achieve their pharmacological effects by reducing the rate of a
key enzyme-catalyzed reaction.
• In some cases the effect is achieved by forming a dead-end covalent
complex between the inhibitor and enzyme.
• Penicillin and aspirin work this way: they form stable chemical bonds
with residues in the active sites of the enzymes they inhibit.
• Such “suicide inhibitors” permanently inactivate their target
enzyme molecules, and cells can only overcome their effects by
synthesizing fresh enzyme.
Figure U2-3.4 Effect of temperature on
reaction rate
Reaction rates depend on
collisions between reacting
species, which in turn depend on
concentrations and temperature.
The temperature dependence of a
reaction thus relates a
thermodynamic quantity (the free
energy of the transition state) to
kinetics (the rate of the reaction).
The equation that expresses this
relationship is called the Arrhenius
Equation.
E 
kcat  Aexp  a 
RT 
Predicts a two-fold increase in rate
for every 10 °C rise in temperature
As the temperature increases, the
rate of an enzyme-catalyzed reaction
increases until the protein unfolds
and the rate then rapidly drops.
How do kinetics relate to biochemistry?
•
Classic collision theory - to increase the rate of P
–
–
–
•
Increase the concentration of A or B
Increase the temp.
Decrease the concentration of the products
Catalysis is a process that increases the rate at which
a reaction approaches equilibrium.
Proximity and Orientation
• By simply positioning
2 molecules in such
a way that they are
close together, the
probability of reaction
is increased.
• Example is anhydride
formation with
different degrees of
rotational freedom.
• By limiting motions,
intramolecular
reactions are greatly
increased
Proximity and Orientation
• Probably the least important by itself
• Uses binding energy to position correctly.
• Koshland figures only 2-3-fold increase, but can see
increases up to 107.
Strain or Distortion
• Binding of substrate or conformational change in enzyme
may induce strain in bond.
• This lowers the required energy to reach EA.
• Stabilize or force bonds closer to the transition state.
Ex. Proline racemase
sp2 planar
D-pro
CO2-
+
-H
H+
L-pro
H
N
H
N
N
CO2H
H
H
If we look at the binding of inhibitors to active site:
N
H
CO2-
N
+
>>
CO2-
N
H
CO2-
CO2H
Strain or Distortion
Vitamin B1
• Model reaction - Vitamin B1
(thiamine)
• Part of thiamine pyrophosphate
(TPP)
• Mechanism for pyruvate
decarboxylase
TPP
pyruvate
-O
O
TPP
R +
CH3
O
N
S
R’
CH
3
R +
O HO N
-O
C
C
CH3
S
R’
Strain or Distortion
• Compound I is stable
in H2O, so
decarboxylation is
slow.
• Compound II is
stable in DMSO, so
decarboxylation is
fast
• Pyruvate
decarboxylase puts
TPP in very
hydrophobic region
of enzyme.
R +
O HO N
C
C
-O
I
S
R’
S
R’
CH3
R
O HO
N
C
O
C
CH3
R
HO
N
O
C
O
+
C
CH3
II
S
R’
General Acid-Base Catalysis
• General acid catalysis- a process in which partial proton
transfer from a Brønstead acid (a species that can donate
protons) lowers the free energy of a reaction’s transition
state.
• General base catalysis - process in which partial proton
abstraction by a Brønstead base (a species that can
combine with a proton) lowers the free energy of a
reaction’s transition state.
• General acid-base catalysis-a combination of both.
Page 497
Figure 15-1a Mechanisms of keto–enol tautomerization.
(a) Uncatalyzed.
Page 497
Figure 15-1bMechanisms of keto–enol tautomerization.
(b) General acid catalyzed.
Page 497
Figure 15-1c Mechanisms of keto–enol
tautomerization.
(c) General base catalyzed.
General Acid Base Catalysis
• Ex. Ester hydrolysis
O
C
d
H
H
d
O
+ H+
OR
C
H2O
d
OR
C OR
O+
H
H
H+
O
C
O
O
- H+
OH
C
+ ROH
O
H
OR
H+
General Acid-Base Catalysis
• Large number of possible amino acids
• Requires that they can accept and donate a proton
• Glu, Asp
• Lys, His, Arg
• Cys, Ser, Thr
• Also can include metal cofactors
• Example can be observed in carboxypeptidase A (both acid
and base catalysis)
General Acid-Base Catalysis
• Ex. Carboxypeptidase A
Zn plays role of acid (4th ligand
is normally H2O, but it is
displaced by peptide binding)
Glu72
His196
His69
O
Zn++
H O


d
d
H
Glu270 C-OGlu acts as base catalyst
to polarize water and
form nucleophile
R
H-C
N
 d
dO
C
+ Arg145
CO2H
Key aas that holds
molecule in place
HO-Tyr248
H-C-R
NH
C
Tyr also plays role
as 2nd acid catalyst
O
+ Arg