IPN-UPIIG Ingeniera Biotecnolgica Ingeniera Enzimtica

Problem 1.-An assay was performed with the penicillinase enzyme. The amount of hydrolyzed penicillin per
minute was measured as a function of the penicillin concentration (see table). Calculate the value of K M and
Vmax.
Data Solution
Penicillin Hydrolyzed
M (nmoles/min)
1 0.11
3 0.25
5 0.34
10 0.45
30 0.58
50 0.61

Problem 2.-The kinetics of an enzyme is measured as a function of the substrates concentration ([S]) in the
presence of an inhibitor at a concentration of 2mM.
Calculate K M and V max in the absence and presence of an inhibitor
Mention what type of inhibition is involved
What is the value of K I ?
Data Solution
[S], Velocity (mol/min)
mM Sin I Con I
3 10.4 4.1
5 14.5 6.4
10 22.5 11.3
30 33.8 22.6
90 40.5 33.8

Problem 3.- The kinetics of an enzyme is measured as a function of the substrate concentration ([S]) with
another type of inhibitor at a concentration of 100M.
Calculate K M and Vmax in the absence and presence of an inhibitor.
Mention what type of inhibition is involved
What is the value of K I ?

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IPN-UPIIG Ingeniera Biotecnolgica Ingeniera Enzimtica

Data Solution
[S], Velocity (mol/min)
mM Sin I Con I
3 10.4 2.1
5 14.5 2.9
10 22.5 4.5
30 33.8 6.8
90 40.5 8.1

Problem 4- A yeast disaccharide can hydrolyze sucrose and maltose, according to the table below. It is
understood that sucrose and maltose are the substrate, and the glucose the released product expressed as the
reaction rate. Investigate which are the reactions in each case and calculate the K M and V max for each substrate,
and identify which is the natural substrate of the enzyme.
Saccharose Vo (mol Glucose /10 Maltose Vo (mol Glucose
(mM) min) (mM) /10 min)
5 40 5 48
10 60 10 80
20 80 20 120
50 100 50 172
100 109 100 200

Problem 5.- An experiment was conducted where the rate of reaction of an enzyme with different inhibitor
concentrations was measured, the results are below.
a) Using the representation L-B determine the type of inhibition
b) Calculate the kinetic parameters K M , V max and K I for each concentration of inhibitor.
Remember that the parameter K M and V max that varies more in percentage in relation to the kinetics without
inhibitor would indicate which type of inhibition is involved.
Data
Vo, [mol/min]
[S] mM [I]= 0.00 [I]= 1.393 [I]= 2.790 [I]= 4.180
0.05 2.62 1.77 1.40 1.29
0.1 4.54 3.00 2.32 2.10
0.15 5.72 3.84 3.00 2.66
0.2 6.67 4.77 3.50 3.11
0.25 7.43 5.00 4.06 3.55
0.3 7.90 5.42 4.48 3.74
0.4 9.04 6.07 4.80 4.17

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IPN-UPIIG Ingeniera Biotecnolgica Ingeniera Enzimtica
Problem 6.- From a series of flasks with a constant concentration of enzyme the following initial velocities were
taken, they were obtained as a function of the concentration of the substrate.
a) Calculate the K M and V max kinetic parameters of the three forms (Lineweaver-Burk, Eadie-Hofstee, Dixon).
b) Analyze which are the atypical data that cause a low correlation, which can be eliminated and explain your
answer.
Data Solution
[Velocidad
[S] reaccin]
mmol/L mmol/L min
1 0.2
2 0.22
3 0.3
5 0.45
7 0.41
10 0.5
15 0.4
20 0.33

Problem 7.- An enzyme whose K M is 10-4 M in the presence of a 10-2M substrate concentration, is capable of
transforming 20% of the S into 10 min. Calculate the approximate percentage of S transformed in 20 min.

Problem 8.- The following table shows fractional saturation data for hemoglobin (S) and the partial oxygen
pressure in plasma (pO 2 ). Determine the value of the Hill coefficient and p 0.5 for this enzyme.
pO 2 , mmHg 10 20 30 40 50 60 70 80 90 100 110 120
S 0.18 0.40 0.65 0.80 0.87 0.92 0.94 0.95 0.95 0.96 0.96 0.97

Problem 9.- Calculate the denaturing constant (k d ) and the reaction rate at 55 minutes of an enzyme having a
thermal denaturation at 50 C following a first order kinetics; this enzyme has a reaction rate at the initial time
of [r] 0 = 0.11 mmol / min
Time (min) [] mmol/min
10 0.0976
20 0.0867
30 0.0770
40 0.0680
50 0.0606
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IPN-UPIIG Ingeniera Biotecnolgica Ingeniera Enzimtica
60 0.0538
70 0.0478
80 0.0424
90 0.0377

Problem 10.- The amyloglucosidase enzyme is immobilized on polyacrylamide gels. The activities of the
immobilized enzyme and the soluble enzyme are compared at 80 C. The speed data is shown in the following
table. Determine for both systems the values of k d as well as half-life.
Time Soluble, Immobilized,
(min) [=]mol/ mL*min [=]mol/ mL*min

0 0.86 0.45
3 0.79 0.44
6 0.70 0.43
9 0.65 0.43
15 0.58 0.41
20 0.469 0.40
25 0.41 0.39
30 0.38
40 0.37

Problem 11.- The following graphs show 3 straight lines associated with a kinetics without inhibitor (S), with
inhibitor (I), and double concentration of inhibitor (2I). Explain for each set of lines what type of inhibition they
present and which straight line corresponds to the one with the highest concentration of inhibitor. Justify your
answers.

a b c
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IPN-UPIIG Ingeniera Biotecnolgica Ingeniera Enzimtica

a b c
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a b c
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Bibliography.
1) Chang Raymond. 2009. Fisicoqumica Editorial McGraw Hill Interamericana. 3ra edicin / Rstica / Castellano / Libro
ISBN13:9789701066522
2) Doran M. P. Principios de Ingeniera de los Bioprocesos. Editorial Acribia S. A. ISBN: 84-200-0853-2
3) Illanes Andres. Enzyme biocatalysis, principles and applications. Editorial Springer Science. ISBN 978-1-4020-8360-
0.ZMG49
4) Levenspiel Octave. 2009. Ingeniera de las Reacciones Qumicas. Editorial Limusa Wiley. 3ra edicin.
5) Fogler H. S.