Universidad Autónoma del Estado de México

Facultad de Química
Químico Farmacéutico Biólogo
Bioquímica Básica

Problemario 3

1. You are a research scientist studying a novel enzyme X, and you want to characterize this
new enzyme. You measure the velocity of the reaction with different substrate concentrations and
get the following data:

[substrate] (mM) Initial Velocity (mmol/min)

0.3 10.4
5.0 14.5
10.0 22.5
30.0 33.8
90.0 40.5

a) Graph the above data. From the graph, estimate Km.

b) Calculate Vmax. Show any equations and calculations.

c) Is X an allosteric enzyme? Explain.

d) You decide to do this experiment again, but this time with only one third of the enzyme X
concentration used in the first experiment. Draw a new graph on the same graph that you did the
first graph on. Estimate Km and Vmax from the new graph.

e) You wish to find the amino acid sequence of the enzyme X. What methods might you use to
determine this? Name at least three.

2. Given the following kinetic data, estimate Km and Vmax for the enzyme:

Sustrate Initial reaction

[μM] velocity [μmol/min]
2 7
5 17
10 29
20 44
50 67
100 80
200 89
500 95
1000 98
 Universidad Autónoma del Estado de México
Facultad de Química
Químico Farmacéutico Biólogo
Bioquímica Básica

The graphs below show two ways to estimate Km and Vmax from the above data:

b) What would be the effect on the initial reaction velocities if [enzyme] was reduced to 10% of the
amount used above?

c) How would this change in [E] effect the observed KM and Vmax?

3. Lactose is a disaccharide found in milk. Many adults throughout the world get sick from
drinking milk because they cannot digest lactose. Lactose intolerance varies markedly among
various human populations. (For example, only about 3% of people of Danish descent are lactose
intolerant, compared with 97% of people of Thai descent.) When someone who is lactose intolerant
ingests milk, the lactose accumulates in the lumen of the small intestine because there is no
mechanism for uptake of the disaccharide. This causes abdominal distension, cramping, and watery
diarrhea.

a) Why can't lactose diffuse across the membranes of the intestinal epithelial cells in the absence of
a carrier-mediated uptake system?

b) Why does the accumulation of sugar (or any solute) in the intestinal lumen cause an influx of
water that leads to watery diarrhea?

c) Adults who can drink milk can do so because of the enzyme lactase which is located on the outer
surface of epithelial cells lining the small intestine. Lactase hydrolyzes lactose into its two
component monosaccharides, glucose and galactose. Both glucose and galactose can cross the
epithelial cells, and therefore do not cause illness.

Based on your knowledge of transport across cell membranes, propose a mechanism by which
galactose is transported into the intestinal epithelial cells. Include a diagram of your mechanism.
(There are several possible solutions- you only need to propose one.)
 Universidad Autónoma del Estado de México
Facultad de Química
Químico Farmacéutico Biólogo
Bioquímica Básica

d) You decide to study lactase further, and see whether it can also cleave other common
disaccharides, such as maltose. (Maltose = glucose + glucose.) You find that maltose is NOT
cleaved by lactase, and furthermore, maltose appears to have some kind of inhibitory effect on
lactase's ability to cleave lactose.

Is maltose a more likely candidate for competitive or noncompetitive inhibition of lactase? Explain.

In order to confirm your hypothesis in part (d), you quantitatively study the kinetics of lactase with
lactose alone, and in the presence of both lactose and maltose. You measure the initial velocity of
the reaction (rate at which lactose is cleaved) at varying concentrations of substrate. The data is
given below.

[Lactose] moles/liter Velocity (moles/min)

lactose only with maltose
0.3 x 10-5 10.4 4.1
0.5 x 10-5 14.5 6.4
1.0 x 10-5 22.5 11.3
3.0 x 10-5 33.8 22.6
9.0 x 10-5 40.5 33
e) Make a Lineweaver-Burke plot for lactase both with and without maltose. Does your graph
confirm or contradict your prediction in part (d)? Why?

4. A mutation that changes an alanine residue in the interior of a protein to a valine residue is
found to lead to a loss of activity. However, activity is restored when a second mutation at another
position changes an isoleucine residue to a glycine residue. How might this second mutation lead to
a restoration of activity?

5. The following data were obtained from an enzyme kinetics experiment. Graph the data using
a Lineweaver-Burk plot and determine, by inspection of the graph, the values for K m and Vmax.

[S] (µM) V (nmol/min)

0.20 1.43
0..26 1.67
0.33 2.08
1.0 3.33

Use the Michaelis-Menton Equation to calculate the missing values of [S] given below if V max = 5
mmol/min. Plot [S] versus V (NOT the reciprocals!). Draw line parallel to the x-axis at Vmax and
extend your plotted line to show its approach to Vmax.

[S] (mM) V (mmol/min)

0 1.2
[S]1 1.7
[S]2 2.1
[S]3 2.2
[S]4 2.5
 Universidad Autónoma del Estado de México
Facultad de Química
Químico Farmacéutico Biólogo
Bioquímica Básica

The effect of an inhibitor on an enzyme was tested and the experiment gave the results below. Plot
the data and determine, by inspection of the graph, what type of inhibition is involved.

[S] µM V (µmol/min) with V (µmol/min) with V (µmol/min) with

0.0 nM Inhibitor 25 nM Inhibitor 50 nM Inhibitor
0.4 0.22 0.21 0.20
0.67 0.29 0.26 0.24
1.00 0.32 0.30 0.28
2.00 0.40 0.36 0.32

6. Explain mathematically how a value for Km can be obtained from the Vo vs So graph when
Vo = 1/2 Vmax.

7. How does the Michaelis-Menten equation explain why the rate of an enzyme-catalyzed
reaction is proportional to the amount of enzyme?

8. How does the Michaelis-Menten equation explain why the rate of an enzyme-catalyzed
reaction reaches a maximum value at high substrate?

9. You measure the kinetic of an enzyme E as a function of substrate concentration, first

without inhibitor. The enzyme concentration is maintained constant at a level of 1 μM.

S [μM] Vo [μM/min]
2 2.9
3 3.8
4 4.4
6 5.0
7 5.4
8 5.8
9 6.2
10 6.7

a. Plot the data using the double reciprocal Lineweaver-Burk (1/[S] vs 1/Vo).
b. From these data, determine Vmax, KM, kcat, and the turnover number for the enzyme.

10. For the reaction A <---> B catalyzed by enzyme samples e1, e2 or e3, I obtained the
following data for maximum rates for the forward reaction for the initial concentrations listed:

Do all versions of this enzyme catalyze this particular reaction? How do you know?
a. What is the Km for each enzyme sample?
b. Which sample has the greatest apparent [E]?
c. What is the difference between samples 1 and 2? Between samples 1 and 3? Samples 2 and 3?
d. Which sample will catalyze a reaction at the greatest rate if substrate is not limiting?
e. What two factors determine reaction rate for enzyme catalyzed reactions when the [A] (or any
substrate) is below saturation?
Be able to explain the significance of affinity – we will continue to encounter this concept.
 Universidad Autónoma del Estado de México
Facultad de Química
Químico Farmacéutico Biólogo
Bioquímica Básica

Enzyme 1 Enzyme 2 Enzyme 3

11. Given the reaction

k1 kp
E + S  ES  E + P
k-1
where k1 = 1 x 107 M-1 sec-1 k-1 = 1 x 102 sec-1, and kp = 3 x 102 sec-1

a. Calculate Ks
b. Calculate Km

12. An enzyme was assayed at an initial substrate concentration of 2 x 10-5 M. In 6 minutes half
of the substrate had been consumed. Km for this enzyme’s substrate is 5 x 10 -3 M.
a. Calculate k
b. Calculate Vmax
c. Calculate the concentration of product produced in 15 minutes.

13. An enzyme catalyzes the reaction S  P. The following data has been obtained. Plot the data
to determine Km and V max using both a Lineweaver-Burke plot and also an Eadie-Hofstee plot.

S [M] V [lit-1 min-1]

8.33E-06 1.38E-08
1.00E-05 1.60E-08
1.25E-05 1.90E-08
1.67E-05 2.36E-08
2.00E-05 2.67E-08
2.50E-05 3.08E-08
3.33E-05 3.63E-08
4.00E-05 4.00E-08
5.00E-05 4.44E-08
6.00E-05 4.80E-08
8.00E-05 5.34E-08
1.00E-04 5.71E-08
2.00E-04 6.67E-08
 Universidad Autónoma del Estado de México
Facultad de Química
Químico Farmacéutico Biólogo
Bioquímica Básica

14. Given a crude extract from a cell that contains 20 mg of protein per milliliter of solution. You
took ten microliters (10 l) of this extract and added it to get a total reaction volume of 0.5 ml. The
reaction resulted in the formation of 60 nmoles of product in a time of 1 minute under optimal
experimental conditions.

a) Express ν as each of the following terms:

nmoles/assay
nmoles ml-1 min-1
nmoles liter-1 min-1
moles liter-1 min-1
M min-1
M s-1

b) What would ν be in the same 10 l of the extract were assayed in a 1.0 ml total reaction
volume?
c) What is the enzyme concentration in the assay mixture and on the original extract ( expressed
as units/ml)?
d) What is the specific activity of the preparation?

15. Given the reaction of an enzyme that follows Michaelis-Menten kinetics:

k1 kp
E + S  ES  E + P
k-1

If Km = 30 mM and Vmax = 60 uM min-1

a) What is the initial reaction velocity at a substrate concentration of 0.1 mM?
b) What is the initial reaction velocity at a substrate concentration of 30 mM?
c) What is the initial reaction velocity at a substrate concentration of 1000 mM?