Due Nov.

10

Bi/Ch110 Problem Set 3 Solutions

Problem 1: Enzymes and Inhibitors (30 points)

a) (15 points) The following kinetic data were obtained for an enzyme with and without the
inhibitor salicylate (I). Generate a Lineweaver-Burk plot with the data and calculate Vmax and KM
for this enzyme in the presence and absence of inhibitor.
Rate with Rate with
[Substrate] no inhibitor inhibitor
mM (mM/min) (mM/min)
1.42 0.200 0.180
0.80 0.180 0.150
0.45 0.160 0.120
0.20 0.120 0.080
0.14 0.100 0.060
0.08 0.070 0.040

The following kinetic data were obtained for an enzyme with and without the inhibitor salicylate
(I). Generate a Lineweaver-Burk plot with the data and calculate Vmax and KM for this enzyme in
the presence and absence of inhibitor (25 points).

30

25

20
y = 1.6452x + 4.5515

15

10
y = 0.7797x + 4.4925
5

0
0 2 4 6 8 10 12 14

Using the equation and relations for the Lineweaver-burke plot, we have slope = Km/Vmax, the
y-intercept 1/Vmax and the x-intercept -1/Km.

Thus, for the case with no inhibitor, we have Vmax = 0.220. With inhibitor, we have Vmax =
0.222 (units of mM/min). (These values are approximately the same)

Km is therefore determined using this data and the slope. With no inhibitor, Km = 0.171 With
inhibitor, Km = 0.361 (units of concentration).
 Due Nov. 10

b) (10 points) What type of inhibitor is I?

The y-intercept of both plots are approximately equal. In a Lineweaver-burke plot, this
represents 1/Vmax. Hence, if the substrate concentration is high enough the enzyme will reach
the same Vmax as without the inhibitor. However, it will require a higher concentration of
substrate to achieve this and so the Km of the enzyme will also be higher. As a result, this best
fits competitive inhibition.

c) (10 points) From the following choices (24 mM, 48 mM, 96 mM), which is closest to the
value of Ki ?

For competitive inhibition:

Plugging in the appropriate numbers where [I] = 40 mM, we get a value of 36 mM.

Problem 2: Michaelis-Menten Kinetics (30 points)

In Michaelis-Menten kinetics, enzyme (E) interacts with substrate (S) witha forward rate
constant of k1 and a reverse rate constant of k-1 to form complex (ES) where substrate is then
converted into product (P).

a) (10 points) In the chart below, label the steady state portions of the graph. What will happen to
this graph if it is taken out to a time long enough to deplete the substrate to 1/2 of its original
level?
 Due Nov. 10

a)

After a long time, ES is depleted (substrate is all transformed into product and enzyme is free).

b) (5 points) What is the Steady-State assumption?

d[ES]/dt=0 There is no change in the concentration of the enzyme-substrate complex over time at
steady state.

c) (10 points) What assumption(s) are made in a Michaelis-Menten treatment of enzyme

kinetics? Using these assumption(s), derive a rate equation for product formation.

kcat << k-1

c) Assumption: Complex formation in fast and chemistry is slow.
 Due Nov. 10

d) (5 points) Using the assumption for Michaelis-Menten treatment, label the rate-limiting (rate-
determining) step under each condition for the free energy diagrams at below.
 Due Nov. 10

d)

Problem 3: Glycolysis and ATP (20 points)

a) (10 points) Which steps of glycolysis are made more exergonic by high levels of ATP ?

b) (10 points) What feature of one of these enzymes allow the cell to attenuate the rate of
glycolysis when ATP levels are sufficiently high?

a) Glucose -> G6P and F6P -> FBP because both involve ATP -> ADP + Pi
b) Phosphofructokinase is inhibited by ATP. It is the committed step of
glycolysis, so we don’t want it to go faster when [ATP] increases.
 Due Nov. 10

Problem 4: Metabolism and Disease (20 points)

The website www.climb.org.uk is a charitable organization in the UK that helps families with
children who have been diagnosed with metabolic diseases. Their website lists a number of
diseases, many of which, sadly, still result in a short life expectancy for children diagnosed with
these diseases around the world.

(a) (10 points) From this website, find a metabolic disease that is associated with a component of
glycolysis and describe it briefly.

Glycolytic Diseases: TIM, PK, G6P Isomerase, GLUT1, PGKinase deficiencies

(b) (10 points) Does the disease you found cause red blood cell issues and anemia? Explain how
mutations in glycolytic enzymes would relate to this question.

Since red blood cells have no mitochondria and rely entirely on glycolysis for their energy,
hemolytic phenotypes generally accompany glycolytic diseases.