Introduction to Biophysics, WS 2017/18 Exercise 6: Signaling

Problem 1: Kinetic Proofreading

An enzyme E catalyses the conversion of substrate S to product P, but also catalyses the
conversion of substrate S to product P. The only difference in the kinetics is that the
0
complex ES dissociates more readily than the complex ES, i.e. k1 > k1 in the following
reaction scheme:
1 k
k2
E+S

)*
ES * E + P
k1
k1 k

E + S )*

2
ES * E + P
0
k1

1a) Assumg the substrate concentrations in both processes are the same, show that, at
steady state, the relative rate of production of P and P is given by
0
v k1
(1)
v0 k1

1b) Hence show that the maximal specificity with which the enzyme creates products is
 
v GS GS 0
= exp (2)
v 0 max kB T

where GS = kT ln(k1 /k1 ) is the free energy change when E binds to S and
G0S is the free energy change when E binds to S.

Suppose that an additional step is inserted into the reaction scheme:

k1 k k

E+S)*

0 2
ES * ES* * E + P
k1
k
1 k0 k2
E + S

)*
ES * ES* * E + P
0
k1

where ES* is a modified state of the complex which has higher energy than ES.
There is also an additional direct dissociation and association process involved between
the modified complexes and the initial substrate, that might happen before the reaction is
complete:
k3

E+S)*
ES*
k3
k3
E + S

)*
ES*
0
k3

The modification reaction ES ES* is made almost irreversible by coupling it to a reaction,

such as ATP hydrolysis, which has a large, negative free energy change.

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Introduction to Biophysics, WS 2017/18 Exercise 6: Signaling

1c) Assuming that the rate k3 of direct formation of ES* is negligible, show that
 0  0 
v k1 + k0 k3 + k2
= (3)
v0 k1 + k0 k3 + k2

1d) Assuming that the dissociation of the primed modified complex happens faster
0
then for the unprimed one, i.e. k3 > k3 , show that the maximal specificity of the
enzyme is now  
v GS GS 0
= exp 2 (4)
v 0 max kB T

1e) The specificity increases as k2 decreases, but what are the disadvantages of having
k2 too small? Could additional modification reactions be used to enhance the
specificity even more?

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Introduction to Biophysics, WS 2017/18 Exercise 6: Signaling

Problem 2: Allosteric Transitions

2a) Explain what is meant by allostery.

Enzymes that undergo allosteric transitions are typically composed of a large number of
identical subunits. A complex is made of N identical subunits, each of which can bind to a
ligand molecule. The ligand occupancy of the ith subunit is given by i for vacant (i = 0)
and occupied (i = 1) subunits. In the all-or-none MWC (Monod, Wyman, and Changeux)
model, the activity s of the entire complex is either active (s = 1) or inactive (s = 0). For
the MWC model, the energy of the complex depends on s and i according to:
X X
H = (E +  i )s + i .

E is the energy difference between active and inactive state in the absence of the ligand.
Each occupied subunit enhances the activity by changing the energy of the active state
by  < 0; is the free energy for ligand binding in either state, depends on the ligand
concentration and a dissociation constant, K0 , for the inactive state. All energies are in units
of the thermal energy kT .
The parameters used in the orignal MWC model are the equilibrium constant L for the
transition from the inactive to the active state in the absence of the ligand, and the ratio of
the dissociation constants for the inactive and active states, C = K0 /K1 .

2b*) Show that the partition function is

Z = [1 + exp()]N + exp(E)[1 + exp( )]N ,

P
Hint: If n = i is the number of bound ligands, what is the number states for a
given n? Use the binomic formula to simplify the expressions for Zs=0 and Zs=1 .

2c*) Show that the parameters L and C are related to the above energy parameters by:

exp(E) = L , exp() = C , exp() = [L]/K0 ,

where [L] is the concentration of the ligand.

2d*) Finally, show that the average activity of the complex is

L(1 + C[L]/K0 )N
hsi = .
(1 + [L]/K0 )N + L(1 + C[L]/K0 )N

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Introduction to Biophysics, WS 2017/18 Exercise 6: Signaling

Problem 3: Bacterial Flagellar Rotor*

In an experiment, the bacterial flagellar motor is found to rotate through 10 revolutions in a
mean time hT10 i = 11.9 s with standard deviation (T10 ) = 0.19 s. According to one model,
the rotor turns by making a large number of small steps.

3a) Assume the relative error of the time measurements corresponds to the relative
error of counting a large number of stochastic and independent steps. How many
steps does the motor take to finish one revolution?

3b) How does this correlate with the observation that approximately 800 protons tra-
verse the membrane for each revolution of the motor.

3c) The membrane potential of E. coli is 150 mV. The observed value of torque required
to stop its motor turning is Gstall = 3 1018 Nm. Assuming the energy stems from
the protons, calculate the efficiency of the machine.