CPAC 12: The Effect of Temperature on an Enzyme-Catalysed Reaction

Aim:
To investigate the effect of temperature on the initial rate of an enzyme-catalysed reaction.

Independent Variable:
Temperature of each enzyme solution (water baths at 10C, 20C, 30C, 40C and 50C will be used)

Dependent Variable:
Volume of oxygen produced in cm3

Control Variables:
Volume of hydrogen peroxide used should remain constant or the volume of oxygen produced will
increase or decrease based on the change in the volume of hydrogen peroxide. Also, the mass of liver
should remain the same or the volume of oxygen produced at each temperature may vary. At each
temperature, you should allow the liver sample to acclimatise to the temperature of the water bath
therefore allow the sample to remain in the solution for a constant time. Furthermore, the collection
syringe should be held horizontally in place utilising a clamp stand and ensure the volume of oxygen
at each interval is read by the same person to reduce human error in the practical.

Apparatus
Boiling tubes Clamp stand with boss and clamp
100 and 250 cm3 beakers Basic laboratory equipment
Graduated pipettes Water bath
Syringes
Measuring cylinders

Methodology
1) Set up the apparatus as shown. The collecting syringe should not be over the end of the
delivery tube when the boiling tube is first attached to the bung.
2) Measure a known mass of liver (5g).
3) Then place this into the boiling tube and add 5 cm 3 of hydrogen peroxide to the pea solution
4) Immediately reconnect the boiling tube to the apparatus making sure the bung has a gas
tight fit
5) After, place the syringe over the end of the delivery tube, horizontally by using a clamp stand
6) At regular intervals of 10 seconds measure the volume of oxygen produced.

Mohammed Kibriah Core Practical 12

 7) Repeat the procedure at a range of different temperatures, and at each temperature repeat
the procedure 3 times to find any anomalous results.

Results & Calculations

A graph can be made where initial rate (1/T) can be plotted on the y-axis and temperature on the x-
axis. You should see a graph looking similar to this:

A Q10 value can also be calculated for this reaction using the data below the optimum temperatures.
The equation for Q10 is as follows:
(Rate of reaction at temperature + 10C) (Rate of reaction at temperature T)

A Q10 value of 2 means that for every increase in 10C, the initial rate doubles. Likewise, a value of 3
means that every increase in 10C triples the initial rate and so on. This deduction only works for
temperatures up to the optimum temperature. The Q 10 value we obtained was 2.04 this suggests for
every increase of 10oC the initial rate of reaction doubled.

Conclusion:
The shape of the graph is largely down to kinetics and protein structure. A low temperature provides
less kinetic energy to the enzymes so fewer enzyme-substrate complexes are formed in the same
amount of time. Therefore, fewer product molecules are produced. This is the reason why increasing
temperature initially increases the rate of reaction.
The peak of the graph indicates the optimum temperature. This is when enzymes have the greatest
amount of kinetic energy they can have whilst maintaining their protein structure. At this point, the
enzymes are working most efficiently and effectively.

Beyond this optimum temperature the protein structure starts to change. Large amounts of kinetic
energy overcome the hydrogen bonds in the tertiary and secondary structures of the proteins. This
causes the enzyme to change shape and so the shape of the active site is also altered. For this
reason, fewer substrates can bind to form enzyme-substrate complexes. Therefore, increasing the
temperature beyond a certain point slows down the rate of reaction.

Evaluation points
Inconsistency when calculating the volume of oxygen at each regular interval (random error) this is
because it is impossible to estimate the volume of oxygen at each interval precisely because humans
make errors. Therefore, the measurements at each regular interval are qualitative in nature, which
essentially leads to unreliable results.

Mohammed Kibriah Core Practical 12

Mohammed Kibriah Core Practical 12