Objectives:

Investigate the kinetics of urea decomposition by urease

Use the urease enzyme extracted from soybeans.
Study the reaction mechanism for biocatalytic reactions
Obtain the kinetic parameters: Michaelis constant (Km) and maximum rate
(rmax) through the Lineweaver-Burk equation.
Monitor the progress of the reaction by conductivity measurements
Investigate the effect of temperature on the reaction speed in catalysis.
enzymatic.
Obtain the maximum activity temperature of a biocatalyst (urease)
Determine the activation energy of a reaction.
Calculate internal energy, enthalpy, entropy, and activation Gibbs energy (ΔU≠,

ΔH≠, ΔS≠ and ΔG≠ of the biocatalytic hydrolysis of urea.

Introduction
In general, all metabolic reactions are regulated by enzymes.
globular proteins that act as catalysts, increasing the rate of those
reactions that are energetically possible. Enzymes allow reactions in the
temperature, pressure, and pH conditions specific to the intracellular medium, reducing the
activation energy needed for the reaction to occur. Enzymes do not
they undergo structural changes at the end of the chemical process they catalyze.

Enzymes act on specific substances known as substrates, whose

transformation makes it possible. In many cases, the substrate is a very complex substance.
which must be transformed into one or more simpler ones; the enzyme binds to the substrate through
numerous weak interactions such as: hydrogen bonds, electrostatic,
hydrophobics, etc., in a specific place, the active site or catalytic center. This center is a
small portion of the enzyme made up of a series of amino acids that interact
specifically with the substrate weakening the bonds that hold the atoms together
that forms it and making its transformation easier. Normally the active site of the
an enzyme is like a groove, which can be modified by binding with the substrate.

Urease, whose systematic name is urea amidohydrolase, has a molecular weight of

483,000 Dalton. Urease consists of 6 identical structural subunits. It is an enzyme.
very specific, that exists in various types of vegetables, for example, in soybeans.
urease is used extensively as a catalytic agent for the quantitative measurement of urea. The
the ammonium hydroxide that forms can be titrated y can be related,
stoichiometrically, with the amount of urea present in a sample. Although urease
It is not very common in nature, this enzyme has had more importance in development.
of modern enzymology than any other. Research on urease has
it is allowed to deduce the important principle in enzymatic reactions: the function of the
SH (sulfhydryl) groups in catalysis. Urease has 3 or 4 active groups, it is a
representative of a large number of enzymes whose activity depends on the existence of
SH groups, intact, derived from cysteines that are part of the polypeptide chain
of the enzyme.
Materials, equipment, and reagents
Experimental procedure
For the extraction of urease, 1 g of soy was ground in a mortar, subsequently
they added 20 ml of a previously prepared solution of 30% ethanol/water and finally
It was placed for 15 minutes at 2500 rpm in the centrifuge.

For the determination of maximum urease activity; 20 ml of a solution were placed

of 0.02 M urea previously prepared in a test tube and placed in a water bath.
at 30°C. After a few minutes when the temperature in the bath and in the solution was
uniformly, with the help of a syringe, 0.5 ml of urease extract was added to the tube of
the stopwatch was started, taking measurements of
conductivity every 10 seconds for 2 minutes. The procedure was repeated for
temperaturas de 40,50 y 60 °C .

Finally, in another test tube, 10 ml of a 0.004M urea solution was added,

In the same way, it was placed in a water bath at the temperature of maximum activity, fixing the
tube to the universal support. At the moment when the bath reached the ideal temperature,
0.5 ml of urease extract was added to the test tube, starting the stopwatch and
introducing the cell into the tube immediately. The measurements were taken.
conductivity every 10 seconds for 2 minutes and the previous steps were repeated
for concentrations of 0.008, 0.012, 0.016, and 0.02M

Results
Table 1. Conductivity results over time.
 Time (s) Temperatura (°C)

Environment 40 50 60

10 100.0 124.3 130.6 162.0

20 100.9 118.8 125.5 155.1

30 101.7 118.2 125.7 155.5

40 103.4 118.5 125.7 159.0

50 103.7 119.7 126.0 161.7

60 104.5 120.1 127.5 164.6

70 105.5 122.0 130.1 199.9

80 106.3 121.9 130.4 199.9

90 107.0 124.0 131.9 199.9

100 107.9 124.5 133.3 199.9

110 108.5 125.5 134.5 191.0

120 109.2 126.9 136.3 192.0

Tabla 2. Resultados de conductividad a 40°C

Time (s) Conductivity

0.004M 0.008M 0.012M 0.016M 0.02M

10 326 377 349 231 124.3

20 317 373 352 241 118.8

30 318 376 356 250 118.2

40 323 381 363 260 118.5

50 327 387 370 268 119.7

60 330 393 379 276 120.1

70 336 398 386 282 122.0

80 343 408 393 292 121.9

90 345 415 402 300 124.0

100 353 422 409 317 124.5

110 359 427 4117 320 125.5

 120 364 434 426 323 126.9

Analysis of results

A comparative graph of conductivity vs time was created at the temperatures of

work, linear regression was applied to each dataset to determine 0for each
temperature, obtaining graph 1 and table 3

Graph 1. Conductivity VS time at different temperatures

Table 3. Values of 0 for each operating temperature

Temperature (Unit
(°C) s)

28 0.0838

40 0.0591

50 0.0852

60 0.4609

From table 3, graph 2 was created to determine the maximum temperature.

catalytic activity, obtaining an optimal temperature of 40°C, which was the temperature of
I work in experimental activity.

Graph 3. 0VS Temperature

To determine 0 At different concentrations, it was necessary to create a graph with the
data obtained from conductivity at each reaction time of the solutions at different
concentrations, resulting in graph 4 and table 4

Graph 4. Conductivity VS time at different concentrations.

Table 4. Values of 0 for each work concentration

Concentration (M) (united
des)

0.004 0.4213
 0.008 0.585

0.012 0.7085

0.016 0.8636

0.02 0.0591

Graph 6. 0VS Concentration

4. Profile the graph of (ro) vs [S]o, based on what has been obtained, in which region are they located
its results (first-order kinetics, fractional or zero).
Profiling the graph of ro vs concentration leads to the conclusion that the results are
within the region of zero order.

5. Using the Lineweaver-Burck equation, determine maximum rate (rmax) and the
KM
The Lineweaver-Burk equation is:

Concentration r0 1/(M) 1/ro

(M)
 0.004 0.4213 250 2.37360551

0.008 0.585 125 1.70940171

0.012 0.7085 83.3333333 1.4114326

0.016 0.8636 62.5 1.15794349

0.02 0.0591 50 16.9204738

According to the linear equation:

1 1
= 0.8577 =0.8577 = 1.1659
= 0.0062 = 0.0062(1.1659) =0.00722

6. Determine the rate constant at each experimental temperature. Do not consider

the results after the maximum catalytic activity temperature.

It is known that the initial velocity equation has the form:

[ ] 0 [ ]0
0=
+ [ ]0

And because it has been previously shown that the reaction kinetics follow a
zero-order equation, therefore will be despicable in the face of[ ]0 therefore the equation
the initial speed will remain:
 [ ] 0 [ ]0
0= = [ ] 0= á =
[ ]0

So the speed constant will be constant at all temperatures and its value will be
equal to the maximum speed.
á = = 0.8577

7. Using the Arrhenius equation, calculate the activation energy of the reaction.
and the frequency factor.
The linearized form of the Arrhenius equation is:

T (K) 1/T K InK

301.15 0.00332 0.8577 -0.1535 If it is supposed to be the same in

all the temperatures?
313.15 0.00319 0.8577 -0.1535 If it is of order zero, yes; but still
I have a doubt about this
323.15 0.00309 0.8577 -0.1535
Aside from that, Ea is negative :/
333.15 0.003 0.8577 -0.1535 If it is supposed to be the same in
all the temperatures?
If it is of zero order, yes; but still
I have a question about this

Aside from that, Ea gives a negative :/

Commented [7]:Si se supone que sea la misma en

all the temperatures?
If it is zero order, yes; but still
I have a doubt about this.

Aside from that, Ea is negative :/

= 3.83 ×10-12 Eₕ = 3.83 ×10-12 (8.314 J/mol = 3.18 10-11

= . = . = .

8. Determine the activation parameters: ΔH≠, ΔS≠, and ΔG≠. (Use the Eyring equation), at
each experimental temperature.

The Eyring equation has the form:

Where: = í ó
ℎ=
=
Clearing we obtain:
⋅ℎ
≠
=−

Obtaining the for each temperature:

Temperatura [°C] ≠
[J/mlo]

25 1305378.852

40 829994.68

50 669363.642

60 561457.946

To obtain ≠ the equation is used:

≠= −

Knowing that m=n and that the reaction is of order 0:

≠=

Therefore: ≠= 3.18 ×10-11

So for the calculation of ≠ the equation is used:

By clearing, the equation is obtained:

≠ ⋅ℎ
= − /
So for each T:

Temperatura [°C] ≠
[J/mol]

25 -225.5743

40 -229.4819

50 -231.3371

60 -232.8529
Conclusions
The temperature of maximum activity of urease was determined.
Enzyme-catalyzed reactions have a greater effect in favor of kinetics and for
both in the thermodynamic properties of it, such as the decrease
of the activation energy.
The procedure for enzyme extraction was applied and learned.
The effect of temperature on the reaction speed in enzymatic catalysis was observed.

References
-Vargas, M. & Obaya A. (2005). Calculation of rate parameters in Chemical Kinetics and
enzymatic. (1st Ed.) National Autonomous University of Mexico, Mexico
-Chang, R. (2000) Physical Chemistry for the Chemical and Biological Sciences, Mc Graw Hill. 3rd Ed.
Mexico.