Temperature affects the reaction rate of enzymes, as do pH, substrate concentration and enzyme concentration. At low temperatures, enzymes have low activity. As the temperature rises the rate of reaction increases, usually 2-fold for every 10 degree Celsius rise.
The activity peaks at a specific temperature unique to the enzyme. This is known as the optimum temperature - the temperature at which an enzyme is maximally active. Beyond the optimum temperature the activity of the enzyme decreases. At extreme temperatures, the enzymes are denatured and activity ceases.
investigate the effect of temperature on the Activity of catalase
In the following experiment the activity of catalase is measured and graphedover a range of temperatures. The substrate for catalase is hydrogen peroxide and the products of its decomposition are water and oxygen. Hydrogen peroxide is a reactive oxygen species and by-product of several biochemical reactions. Since hydrogen peroxide becomes toxic if it accumulates, catalase protects cells from oxidative damage.
The rate of reaction is monitored by the rate of appearance of the product oxygen.
Equation for Catalase Activity
Equipment/Materials
Fresh catalase solution ( potato tuber or celery homogenate buffered at pH7 or pH 9 respectively ). Stored on ice until needed.
20% Hydrogen peroxide solution
Water baths*
10 mL graduated cylinder
teat dropper
8 identical boiling tubes
Stop clock
* Water baths maintained at 0oC, 10oC, 20oC, 30o C, 40oC, 50oC, 60oC and 70oC. The number of temperatures over which enzyme activity is measure can be reduced depending on the capacity of the biology lab.
Procedure
Label test tubes - 8 for catalase and with each temperature and the other 8 for hydrogen peroxide and with each temperature. Place the boling tubes in the test tube rack
Add 1 drop of catalase enzyme into a the catalase tube labeled 0oC
Add 1mL of Hydrogen peroxide to the hydrogen peroxide tube labeled 0oC.
Place the two boiling tubes - one catalase and one hydrogen peroxide into the 0oC water bath for 10 minutes to allow for the equilibration of temperature.*
Add the hydrogen peroxide to the catalase solution and start the stop clock.
Measure the bubble column after 30 seconds and record in a table.
Repeat steps 2-6 for the other temperatures.
Plot a graph with the height of the bubble column in 30s against the temperature for each temperature. Determine the optimum temperature.
*It is recommended that this step be staggered with tubes placed in the water baths 5 minutes apart, or students can work in groups, dividing the water baths amongst themselves.
Discussion/ANALYSIS Questions & Answers
Q. Define enzyme. A. An enzyme is a biological catalyst.
Q. Define optimum temperature. A. Optimum temperature is the temperature at which the enzyme has it maximum activity (highest rate of reaction)
Q. From your graph of activity against temperature, determine the optimum temperature. A.(Read off your graph the temperature on the x axis which corresponds to the maximum activity) NB, that for catalase - and as for other enzymes, the optimum temperature varies with the species. Q. Predict the the optimum temperature of a digestive enzyme such as pancreatic amylase. A. Since pancreatic amylase is a digestive enzyme its optimum temperature would be boy temperature ie.37oC. Q. Account for the increase in slope/ the rate of reaction before the optimal temperature is attained. A. Temperature is a measure of the kinetic energy of the molecules in a system. As the temperature increases, the kinetic energy and thus the number of random collisions of enzyme with substrate increases per unit time. With the increase in frequency in the number of collisions, the probability of the enzyme and substrate colliding in the correct orientation (ie. the substrate fits into the active site) also increases. Hence rate of reaction increases with temperature increases, up until the optimum temperature is attained.
Q. Account for the decline in the slope after the optimal temperature is attained. A. A further increase in the temperature beyond the optimal temperature leads to disruption of the weak bonds of the enzyme - enzymes are proteins. The unfolding of the protein due to the disruption of bonds is called denaturing. During denturation the precise configuration of the active site is lost. As the active sites are lost, the catalytic activity and hence rate of reaction decrease.
Q. Calculate the temperature coefficient (Q10) for your enzyme controlled reaction. A. Use the formula: Temperature coefficient (Q10) = rate of reaction at (x +10oC) / rate of reaction at xoC (Use only the value of x and x + 10 that are between 0 and 45o C; Q10 is unit-less as the units cancel out)
Q. The temperature coefficient (Q10) for an enzyme controlled reaction between 0 and 45oC is 2. What does this mean? Account for any disparity between this figure and your calculated value. A. A temperature coefficient of 2 means that the rate of the an enzyme controlled reaction doubles with every 10oC rise in temperature.
Q. Apart from temperature, name the other factors which affect the rate of reaction of enzymes. A. Other factors which affect rate of enzyme action are 1. enzyme concentration, 2. substrate concentration, 3. pH, and 4. inhibition (competitive, non-competitive reversible and non-competitive irreversible).