G0G71A: Physical Chemistry of
Biological Systems
Binding Kinetics
Prof. Dr. Hideaki Mizuno
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faster, less aftertaste
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aftertaste
1. Collisional encounter of molecules
1.1 Association of E and L
Association of A and B leading to the formation of
an active complex occurs in (at least) 2 steps:
k1 k2
E+L EL EL*
k-1 k-2
collisional functional
complex complex
1.2 Magnitude of k1 E+L
k1
EL
k2
EL*
k-1 k-2
From the theory of Von Smoluchowski
k1 = 4πN(DA + DB)⋅rAB N: Avogadro number
with DA = kT/fA the diffusion coefficient and fA the
friction coefficient for a sphere with radius rA.
k : Boltzmann constant R=kN
fA = 6πηrA (Law of Stokes-Einstein) with
η the viscosity.
rAB = rA + rB is the radius of encounter.
f(r) = (1/rA+1/rB)(rA + rB)
1.3 Numerical values of k1 E+L
k1
EL
k2
EL*
k-1 k-2
R = 8.31 JK-1mol-1 (molar gas constant)
η in Poise (CGS unit):
for water at 25°C,
η = 0.01 Poise = 0.001 kg⋅m-1⋅s-1
(1 Poise = 1 g⋅cm--1⋅s-1 = 0.1 kg⋅m--1⋅s-1)
1.3 Numerical values of k1 E+L
k1
EL
k2
EL*
k-1 k-2
f(r) = (1/rA+1/rB)(rA + rB)
1) for rA = rB , f(r) = (1/r + 1/r)⋅(r+r) = 4
M-1⋅s-1
2) for rA = 10⋅rB e.g. a protein with rA =4 nm and
a ligand with rB = 0.4 nm,
M-1⋅s-1
2. Formal kinetic analysis:
Reversible association of molecules
2.1 single step reversible association
k1
E+L EL
k-1
Simplification for pseudo first order conditions:
L0 >> E0 such that [L] ≅L0
k’1
E EL
k-1
remove EL via mass balance !" = ! + !%
!"
= −&' ′ " + &*' "+ − "
!#
2.1 single step reversible association
k1 !"
E+L EL = −&' ′ " + &*' "+ − "
k-1 !#
= − #$% + #'$ ( + #'$ ()
Integration leads to the following equation for
the time dependent [E]:
& ( )+* ,)-* .
$% ' + $(%
! = !# &
$% + $(%
!"
!" = −&' ′ " + &*' "+ − "
= −&" + ( !#
!#
!" = − #$% + #'$ ( + #'$ ()
= !#
−&" + ( 𝑎 𝑏
!" #$% &'
) = ) !# ! = − +, - . #(% )#$% /
−&" + ( #(% )#$%
1 −&!"
− ) = ) !# $ 4(% 54$% 6
12 3 )#$%
& −&" + ( ! =! 0 #(% )#$%
1
− ln −&" + ( = # + .1
&
ln −&" + ( = −&# + .2 at t=0, ! = !#
.3 1 −&# = −&" + ( $% & " + ()*
&" = ( − .3 1 −&# !" = !" + !5 = $%&
(* + ()*
" = (2& − .4 1 −&#
& ( )+* ,)-* .
$% ' + $(%
! = !# &
Cn : constants of integration $% + $(%
2.1 single step reversible association
Graphical representation
1) Time course of the reaction k’1
( )+* ,)-* .
E EL
$%& ' + $(% k-1
! = !#
$%& + $(%
establishment of the equilibrium
[EL]
[EL] or [E]
[E]
2.1 single step reversible association
In the case of a reversible process, the observed rate
constant is the sum of forward and backward rate constants.
L0
Whatever you observe the disappearance of E or the
appearance of EL, the rate constant is the same.
k1
E+L EL
k-1
2.1 single step reversible association
Graphical representation
2) Variation of the observed rate constant with increasing L0
L0
How do you get the
individual rate constants?
kobs
slope: k1
y intercept: k-1
L0
2.2 Consecutive reversible steps
k1 k2
E+L EL EL*
k-1 k-2
collisional functional
complex complex
This leads to an experimental curve that can be described
by a sum of two exponentials. Depending on the relative
rate of the two processes these can be separated or not.
Different possibilities are worked out as follows.
2.2 Consecutive reversible steps
k1 k2
E+L EL EL*
k-1 k-2
collisional functional
complex complex
2.2.1 Fast binding followed by a slower
conformational change
2.2.2 Fast second step
2.2.3 First and second step equally fast;
Coupled sequential reversible reactions
2.2 Consecutive reversible steps
k1 k2
E+L EL EL*
k-1 k-2
collisional functional
complex complex
2.2.1 Fast binding followed by a slower
conformational change
2.2.2 Fast second step
2.2.3 First and second step equally fast;
Coupled sequential reversible reactions
2.2.1 Fast binding followed by a slower
conformational change k1 k2
E+L EL EL*
k-1 k-2
Two reactions are uncoupled
k1, k-1 >> k2, k-2
therefor the two processes can be separated in time.
A: fast time scale
kobs = k1L0 + k−1
Gives the same concentration dependence as
“single step reversible association”
2.2.1 Fast binding followed by a slower
conformational change k1 k2
E+L EL EL*
k-1 k-2
B: slow time scale k1, k-1 >> k2, k-2
On the slow time scale, the first process can always
be assumed to be in equilibrium. The equilibrium is
slowly displaced to the right, due to the second step.
Remark: in the second line of derivation (in the next
slide), the equilibrium K1 is introduced, as well as the
mass balance for [E] + [EL]:
K1 k2
E+L EL EL*
k-2
2.2.1 Fast binding followed by a slower
conformational change K1 k2
E+L EL EL*
k-2
K1 =
[ EL ]
K1 =
[ EL ]
([ 0 ] [ ] " $) L0
E − EL − ! EL*#
[ E ] L0
[ ] [ 0] [ ] # %
E = E − EL − " EL*$ 1
=
( E − ! EL*#
[ 0 ] " $ L0 ) − L0
K1 [ EL ]
K1L0
[ ]
EL =
1+ K1L0
E − !(
[ 0] " $EL*#
)
! *#
d " EL $ K1L0
dt
= k2
1+ K1L0
( ! *#
) !
[ E0 ] − "EL $ − k−2 "EL $ *#
! *#
d " EL $ " % * [
K1L0 ! # K 1 L0 E 0]
= − $ k2 + k−2 '" EL $ + k2
dt # 1+ K1L0 & 1+ K1L0
2.2.1 Fast binding followed by a slower
conformational change K1 k2
E+L EL EL*
k-2
d !" EL* #$ " K1L0 % * K L [ E 0]
= − $ k2 ! #
+ k−2 '" EL $ + k2 1 0
dt # 1+ K1L0 & 1+ K1L0
with kobs the coefficient of EL*
2.2.1 Fast binding followed by a slower
conformational change K1 k2
E+L EL EL*
k-2
Graphical representation:
K1L0
kobs = k2 + k−2
1+ K1L0
1
kobs = k2 + k−2
(1 / K1L0 ) +1
y intercept: k-2
kobs
L0
2.2 Consecutive reversible steps
k1 k2
E+L EL EL*
k-1 k-2
collisional functional
complex complex
2.2.1 Fast binding followed by a slower
conformational change
2.2.2 Fast second step
2.2.3 First and second step equally fast;
Coupled sequential reversible reactions
2.2.3 Fast second step
Second step is much faster than first, while the first is
diffusion limited. The whole process is therefore a diffusion
controlled association.
k1 k2
E+L EL EL*
k-1 k-2
encounter
complex
k1 k2
2.2.3 Fast second step E+L EL EL*
k-1 k-2
encounter
complex
Since the second step is much faster, a steady state
for EL can be assumes.
steady
state
EL << E0
(diffusion limited first step)
2.2.3 Fast second step k1 k2
E+L EL EL*
k-1 k-2
encounter
complex
2.2.3 Fast second step k1 k2
E+L EL EL*
k-1 k-2
encounter
complex
Remark that kobs is the sum of two net rate constants.
net rate constant
What is the net rate constants?
the net rate constants
A net rate constant occurs when we have the situation:
k1 k2
A B C
k-1
The net rate constant from A→C (k12) is as follows.
k12 = k1 ×(fraction in forward direction)
appearance disappearance
fraction in
forward direction
reverse direction
2.2.3 Fast second step k1 k2
E+L EL EL*
k-1 k-2
encounter
complex
net rate constant
- if k2 >> k-1, k12 = k1’ and k21 = k-1 k-2 /k2
i.e. formation of EL is diffusion-controlled
- if k2 << k-1, k12= K1k2 and k21= k-2
i.e. formation of EL is reaction-controlled
There is no uncoupling: only one step is observed.
2.2 Consecutive reversible steps
k1 k2
E+L EL EL*
k-1 k-2
collisional functional
complex complex
2.2.1 Fast binding followed by a slower
conformational change
2.2.2 Fast second step
2.2.3 First and second step equally fast;
Coupled sequential reversible reactions
2.2.4 First and second step equally fast;
Coupled sequential reversible reactions
k1 k2
E+L EL EL*
k-1 k-2
This situation arises when all rate constants are of comparable
magnitude, such that we get a set of simultaneous differential
equations:
— Eq.a
— Eq.b
— Eq.c
!"
from Eq.b = &' ′ ! − &*' + &, !" + &*, !"∗
#$
mass balance !0 = ! + !% + !%∗ !"∗ = !0 − ! − !"
— Eq.d
from Eq.a
— Eq.e
— Eq.f
from Eq.d and f
1 %& ' %'
&
+"$ ′ = "$, − "#& ' + "#& '. − "#$ + "& + "#& '/
"#$ %( %(
put Eq.e
1 d [E]
⎛ 2
d [ E ]⎞ 1 ⎛ d[E] ⎞
⎜ + k1 ' ⎟ = ( k1 '− k−2 )[ E ] + k−2 E0 − ( k−1 + k2 + k−2 ) ⎜ + k1 '[ E ]⎟
k−1 ⎝ dt 2
dt ⎠ k−1 ⎝ dt ⎠
d2 [E] d[E] ⎛ d[E] ⎞
+ k1 ' = ( k−1k1 '− k−1k−2 )[ E ] + k−2 k−1E0 − ( k−1 + k2 + k−2 ) ⎜ + k1 '[ E ]⎟
dt 2
dt ⎝ dt ⎠
d2 [E] d[E]
2
+ ( k1 '+ k−1 + k2 + k−2 ) + ( k−1k−2 + k1 ' k2 + k1 ' k−2 )[ E ] − k−2 k−1E0 = 0
dt dt
set ! " = ! − %&' %&( !)⁄ %&( %&' + %( ′%' + %( ′%&'
!" # ′ )
!# ′
"
+ '( + '*( + '" + '*" + '*( '*" + '(′'" + '( ′'*" # ′ = 0
!% !%
!" # ′ )
!# ′
"
+ '( + '*( + '" + '*" + '*( '*" + '(′'" + '( ′'*" # ′ = 0
!% !%
S P
set ! = #$% + #'$ + #( + #'( " $% $% $%
! e + '!e +(e = 0
) = #'$ #'( + #$ ′#( + #$ ′#'(
"
! #′"
!# ′ ! + '! + ( = 0
"
+' +( # ′=0
!% !%
set ! ′ = e%& (λ: observed rate constant)
$/% $0 %
!" ′ ()
! "′ ,
, ()
+ ′ = α. e +α" e
= &e , ,
= & e
!$ !$
The solution of this set of differential
equation is a sum of two exponentials:
2.2.4 First and second step equally fast;
Coupled sequential reversible reactions
k1 k2
E+L EL EL*
k-1 k-2
! ′ = α% e'() +α+ e',)
(double exponential)
E EL* −' ± ' 2 − 4*
!1, !2 =
2
Concentration
EL
S = − ( λ1 + λ2 )
P = λ1 × λ2
Time ! = #$% + #'$ + #( + #'(
) = #'$ #'( + #$ ′#( + #$ ′#'(
2.2.4 First and second step equally fast;
Coupled sequential reversible reactions
S S = − ( λ1 + λ2 ) P P = λ1 × λ2
k1 k1(k2 + k-2)
k-1 + k2 + k-2 k-1k-2
L0 L0
! = #$ ′ + #'$ + #( + #'( = #$ )* + #'$ + #( + #'(
! = #$% #$& + #% ′#& + #%′#$& = #& + #$& #%)* + #$% #$&
2.3 Parallel reactions: competition
2.3.1 Example: the ligand S is of interest to us, but does not
give a signal. The ligand I competes for the same site and
gives a signal. We use ligand I as an indicator!
The experiment is done in conditions of high S and high I
concentration. The reaction is induced by mixing E with (S+I).
First fast phase of binding with k'S >> k-S and k'I >> k-I
Model:
kS
E+S ES
kI
E+I EI
with I = indicator that binds at the same place as S
d[E] that binds at the same place as S:
with I = indicator =-(
k S S 0competition
2.3 Parallel reactions: kII0 + )[E] kS
dt E+S ES
d[E]at the same place as S:
= indicator that binds
= - ( k S S 0 + k I Id[E] 0 )[E] kI
dt = - ( k S + k I )[E] E+I EI
-( k S S 0 + k I I 0 )t S 0 I 0
d[E] [E] = E0 e dt
= - ( k S S 0 + k I I 0 )[E]
dt
[E] = E0 e -( k S S 0 + k I I 0 )t
-( k S S 0 + k I I 0 )t
d[EI] [E] = E0 e
= k I I-(0k[E] S S 0 + k I I 0 )t
[E]
dt = E 0e
d[EI]
= k I I 0[E] d[EI]
dt = k I I 0[E]
d[EI] -( k S S 0 + dt
[EI] = k I I 0=Ek0eI [E] k I I 0 )t
I 0
dt -( + )t
=
Integration kbetween
I I 0 E 0e
k S S0 k I I 0
0 and t gives-( k S S0 + k I I 0 )t
ion between 0 and t gives = k I I 0 E 0e
-( k S S 0 + k I I 0 )t
= k I I 0 E 0e
kI I0
EI = E 0 ( 1 e - ( k S S 0 + k I I 0 )t )
k S S
Binding Kinetics 0 + kI I0 86
Binding Kinetics 86
0 and t gives
*** Exercice: estimate rate constants from the following curves.
2.3 Parallel reactions: competition E+S
kS
ES
kI I0 - ( k kinetics
S S 0 + k I I 0 )t
EI = E 0 (1 e
Competition ) kI
kS S0+ kI I0 E+I EI
100
80
ate rate constants from the following curves.
60
[EI]
Competition kinetics
40
20
0
0.0 0.2 0.4 0.6 0.8 1.0 1.2
time (sec)
Conclusion:
- rate constant increases with increasing [S];
- amplitude
Conclusion: decreases;
- what
- rate determines
constant increases the plateau?
with increasing [S];
2.3 Parallel reactions: competition E+S
kS
ES
kI
E+I EI
2.3.2. Second slower phase:
The initial plateau is determined by the ratio of the on-rate
constants. But the final level should be determined by the
equilibrium constants. Therefore the fast phase will be followed
by a reshuffling towards the equilibrium distribution. This
phase is described by displacement kinetics.
3. DISPLACEMENT KINETICS
3.1 One-Step binding
In the experiment the protein E is in the presence of A,
and is mixed with an excess of B:
k-A
EA E+A
k+A
k+B
E+B EB
k-B
Conditions of derivation of rate equation:
- pseudo first order association reaction, i.e. [A] and [B] >> E0
- steady state for [E], i.e. [EA], [EB] >> [E].
], i.e.: [EA],
reaction, i.e.: [EB]
[A] >> [B]
and [E]. >> E0
’ ’ k-A
EB] - ( k +A[E].k +B )[E] (= 0)KINETICS
3. DISPLACEMENT
[EB] >> + SS EA E+A
] ’ ’ + ’ ’ )[E] k+A
=
= k -Ak[EA] -A [EA] +
+ k -Bk[EB] -B [EB] - (
- ( k +Ak+ +Ak +Bk )[E]
+B (=(=
0)SS0)SS
d[E] k+B
= k -A[EA] + k -B [EB] - ( k’+A + k’+B )[E] (= 0)SS E+B EB
A [EA] dt + k -B [EB] ’ ’
k-B
A [EA] ’ + k ’-B [EB] - ( k +A + k +B )[E] (= 0)SS
k +A + k +B k -Ak[EA] -A [EA] + k+-Bk[EB] -B [EB] k’+A=k+A[A]
[E] [E] SS = SS = ’ ’ ’ ’ k’+B=k+B[B]
+ +
k +Ak +Ak k-A+B[EA]
k +B + k -B [EB]
’ [EA]
[E] ’ +SS =
] putting k+A-A + k +B =kS-B [EB] k
’
+A + k +B
’
[E] SS = ’ ’
[EA][EA] = = - [EB]
E 0E 0 - [EB] k + k
putting
+A putting+B k +A
’ ’ ’ ’
k +Ak++Bk=
+ S
+B = S
’ ’
’
[EA] = E 0 - [EB] putting k +A + k +B = S
k]+B=[E] -- [EB] k -B [EB] putting ’ ’
E 0d[EB]d[EB] ’ ’ k +A k +B = S
+
= k=+Bk[E] [E] - k -
-B k[EB]
-B [EB]
dt dt d[EB] +B
’
= k +B [E] - k -B [EB]
k -Ad[EB]
’
[EB] k +B ’ k -Bdt [EB]
’ + = k +B ’ [E] - k -B- [EB]k EB
’
k
S dt
k
’ E k k’
S [EB] -B
k ’ -B [EB]
k
=] = k +B k -A E-0 - k +B k -A[EB]
+B -A 0 +B -A
+ + k +B k -B [EB]
+B
- k --BEBEB
S ’ S ’ S ’ k -B
d[EB]S k +B k -A E 0 S k +B k -A[EB] S k +B k -B [EB]
=’ - ’ + - k -BEB
B k -A dtE 0 k +B k -AS[EB] k +BSk -B [EB] S
- + - k -BEB
k-A
3. DISPLACEMENT KINETICS EA E+A
k+A
k+B
E+B EB
k-B
Rate constant kobs = coefficient of [EB]:
If B0>>>A0 ,then kobs =k-A .
Performing experiments at increasing concentrations of B
should evolve to a plateau value.