Introduction to Biophysics, WS 2017/18 Exercise 7: Enzyme Kinetics

Problem 1: Competitive Inhibition II

A system has a Michaelis-Menten constant KM = 2.8105 M and a maximal reaction velocity
of vmax = 53 M/min.

1a) Using the expression derived in the previous exercise, calculate and compare the
product formation rates v if the substrate concentration is [S] = 3.7 104 M and
the inhibitor concentration is [I] = 4.8 104 M, for the cases

i) no inhibition

ii) competitive inhibition

The equilibrium constant of the inhibitor is given by KI = 1.7 105 M. (Note that KI is the
equilibrium constant of the dissociation reaction, as it is commonly defined!)

Besides competitive inhibition, where the inhibitor competes with the substrate for binding
sites on the enzyme, there are other similar mechanisms that modulate the product formation
rate. One is called uncompetitive inhibition, where the inhibitor I binds to the enzyme-
substrate complex ES to form ESI, which blocks the reaction. In this case

[S] [ES][I]
vUI = vmax 0
with 0 = 1 + [I]/KI0 ,where KI0 = (1)
KM + [S] [ESI]

Furthermore, it is possible that both inhibition mechanisms take place at the same time,
which is then called mixed inhibition. This modifies the product formation rate to

[S]
vMI = vmax (2)
KM + 0 [S]

with = 1 + [I]/KI as derived in the previous exercise and 0 as stated above.

1b) What are the qualitative effects of changes in and 0 on the Lineweaver-Burk
representations of equations 1 and 2?

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Introduction to Biophysics, WS 2017/18 Exercise 7: Enzyme Kinetics

Problem 2: Inhibition Character from Measurements

The enzyme invertase hydrolyses sucrose (table sugar). The reaction is reversibly inhibited
by the addition of urea, a small molecule. The initial rate v of this reaction, for a certain
concentration cinvertase of enzyme, is measured in terms of the initial sucrose concentration,
both with and without urea. The following table shows the reaction rates depending on
sucrose concentration.

csucrose [M] v (no urea) [M/s] v (with urea) [M/s]

0.0292 0.182 0.083
0.0584 0.265 0.119
0.0876 0.311 0.154
0.117 0.33 0.167
0.175 0.372 0.192
0.234 0.371 0.188

Q) Use the Lineweaver-Burk representation of the data in the table and your results
from the first question to deduce if the inhibition described here is competitive,
uncompetitive, or mixed.

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Introduction to Biophysics, WS 2017/18 Exercise 7: Enzyme Kinetics

Problem 3: Probability of capture

Particles diffusing randomly in an infinite space are absorbed on the surface of a sphere of
radius a. A source maintains the concentration of particles at c0 on a spherical shell at radial
distance r0 from the centre of the sphere.

3a) Show that the steady-state concentration profile varies as

c0  a
c(r) = 1 , a r r0 (3)
1 a/r0 r
r0
= c0 , r r0 . (4)
r
Hint: Remember the solution of the diffusion equation in exercise 4.

3b) By considering the inward and outward radial flux at r = r0 , show that the proba-
bility that a particle initially at r = r0 being absorbed is
a
p(r0 ) = .
r0

3c) A small enzyme is initially a distance r0 from its substrate, a globular protein of
radius a. Using the above result, show that the average number of times that the
enzyme visits the substrate and returns to r = r0 , before it wanders away for good,
is
a
hni = .
r0 a

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Introduction to Biophysics, WS 2017/18 Exercise 7: Enzyme Kinetics

Problem 4: Chemotaxis of E.Coli

In chemotaxis experiments a source of nutrient molecules is typically introduced into the
medium containing bacteria via a micropipette. The outward diffusion of the nutrient
molecules creates a position-dependent concentration gradient and the chemotactic response
of the bacteria can be observed under a microscope.

4a) Estimate the nutrient gradient in steady state as a function of the distance from the
micropipette r by assuming that it keeps the concentration fixed at c0 for distances
r < r0 . Make a plot of the concentration gradient as a function of r for typical
values c0 = 1 mM and r0 = 1 m. Hint: Remember the solution of the diffusion
equation in exercise 4.

4b) Assuming that a bacterium of 2 m length makes two measurements of the concen-
tration using one array of receptor proteins at one of its ends and another array at
the other, estimate the maximum distance from the nutrient source for which the
bacterium is still able to detect a gradient. Assume that the receptor array counts
the number of molecules present in a cubic volume with side a = 100 nm.
Hint: The counting error for N molecules is roughly N and in order to detect the
difference in concentration between the two ends of the bacterium, the measure-
ment error should be less than the difference itself.
Also note that the calculations become rather tricky so solve this graphically or by
trying out a few numbers.

4c) Now assume a different strategy, where one receptor is employed but the bacterium
compares the concentration at two different positions along a run, separated by a
distance of 10 m. Compute the maximum distance from the nutrient source at
which the bacterium will be able to detect the gradient in this case.