Biochemical Kinetics

k
on
k
off
+
Binding
• The most common interaction in biological system
• Non-covalent interaction between two molecules:
• (usually one is a macromolecule)
• Protein-Protein (Very common)
• Protein-Small molecule (Very common)
• Protein-lipid
• Protein-Sugar
• Binding is reversible
Binding in Cellular Signaling
Ligand: A hormone (small peptide), a secreted protein, or small molecule
Receptor: Protein
Membrane bound Ligand: Usually Protein
Binding in Cellular Signaling
R
e
c
e
p
t
o
r
Ligand
R
e
c
e
p
t
o
r
Ligand
P
R
e
c
e
p
t
o
r
Ligand
P
A
R
e
c
e
p
t
o
r
Ligand
P
A
B
R
e
c
e
p
t
o
r
Ligand
P
A
B
P
(binding)
(binding)
(binding)
GENE
B
P
(binding)
Protein
Nucleus
Application of Molecular Binding
• Most of the time it is simple 1-to-1 associations of the form
A + B ↔AB
• The most common variation where one partner (e.g. B) has
additional binding sites.
A + B ↔AB
AB + A ↔A
2
B or
AB + C ↔ABC
• If these binding sites are independent then one simply treats
each interaction as a 1-to-1 association and adds them together.
• If the binding sites are not independent then one has positive or
negative cooperativity
Binding
Binding Affinity
A + B AB
Rate of forward reaction, .[ ].[ ]
Rate of backward reaction, .[ ]
At equilibrium,

.[ ].[ ] .[ ]
[ ]
[ ][ ]
In this case, is called Affinity Const
F on
B off
F B
on off
on
eq
off
eq
R k A B
R k AB
R R
k A B k AB
k AB
K
k A B
K
=
=
=
=
= =

ant ,
1 [ ][ ]
Dissociation Constant,
[ ]
A
off
D
A on
K
k
A B
K
K k AB
= = =
Low K
D
means stronger binding
Binding Affinity
x y
Generalized Formulation:
xA + yB A B
1 [ ] [ ]
Dissociation Constant,
[ ]
x y
off
D
A on x y
k
A B
K
K k A B
= = =

Energetics of Binding
∆G = ∆G
o
+ RT. ln([AB]/[A][B])
At equlibrium, ∆G = 0 :
∆G
o
= -RT. ln(K
A
) = RTln(K
D
)
For binding to happen, ∆G <0
∆G = ∆H –T.∆S
Contribution of Enthalpy and Entropy?
Change in enthalpy (∆H)
– Release of heat (∆H <0) favours binding
– This happens when bonds are formed
• e.g. hydrogen bonds, salt bridges, van der Waals
contacts
– However bonds are also broken upon binding
• displacement of water and ions (always)
• conformational change (sometimes)
Energetics of Binding
Change in entropy (T∆S)
- Increase in entropy favours binding
- The protein/protein interaction leads to decrease in
entropy
• Stabilise conformation at the binding
interface.
• Decreased rotation/translation of proteins
- Displacement of water from the binding interface leads to
an increase in entropy
Energetics of Binding
Measuring Binding Affinity
Equilibrium Technique
1. Take a fixed amount of B.
2. Allow to bind with different amount of A.
3. Let the binding reach equilibrium (incubation for long period of time)
4. Measure free A, and AB complex formed
A + B AB
1 [ ] [ ]
Dissociation Constant,
[ ]
So,
[ ]([ ] [ ]) [ ][ ]
[ ] [ ]
.[ ] [ ].[ ] [ ].[ ]
[ ].[ ]
[ ]
[ ]
off
D
A on
Total
D
D Total
Total
D
k
A B
K
K k AB
A B AB A B
K
AB AB
K AB A AB A B
A B
AB
K A
= = =

= =
+ =
=
+

B
o
u
n
d

(
[
A
B
]
)
[A]
• Data are circles
• Line is non-linear fit of the equation performed
• If the fit is good it indicates that binding follows the simple 1:1 model
• But many a time non-linear fitting is not good
[ ].[ ]
[ ]
[ ]
Total
D
A B
AB
K A
=
+
Measuring Binding Affinity
Measuring Binding Affinity
D
[ ].[ ]
[ ]
[ ]
[ ]. [ ].[ ] [ ].[ ]
Devide both side by [A].K and rearrange
[ ] [ ] [ ]
[ ]
Total
D
D Total
Total
D D
A B
AB
K A
AB K AB A A B
B AB AB
A K K
=
+
+ =
= − +
B
o
u
n
d
/
[
A
]
[
A
B
]
/
[
A
]
[AB]
Scatchard Plot
Equilibrium Dialysis to Measure K
D
SPR to Study Binding Kinetics
SPR Sensogram

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