Enzymes

Enzymes:
- Are proteins
- Are biological catalysts (Catalyst speeds up chemical reactions)
- Are specific to one particular ‘Substrate’ (The molecule that an enzyme
acts on is called substrate)
- Are affected by temperature and pH
- Are not used up in the reaction they catalyze
Mode of action of enzyme-
Each enzyme has its own active site. The substrate attaches to the active site like lock and key to
form enzyme-substrate complex to produce products by lowering the energy needed for the
reaction to start. It is the reason why enzymes are specific, i.e. an enzyme will only catalyse one
reaction.

Enzymes also catalyse reactions where large molecules are built up from smaller ones. In this
case, several substrate molecules attach to the active site, the reaction takes place and the larger
product molecules are formed. The product then leaves the active site and the enzyme is free to
act on more substrate molecules.

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Enzymes may work either extracellularly or intracellularly. Extracellular enzymes catalyse
reaction outside the cell. For example, trypsin, amylase and lipase. Intracellular enzymes catalyse
the reactions inside the cells. For example, DNA helicase, DNA polymerase, ligase etc.
Factors affecting the rate of enzymatic actions:
• Temperature
• pH
• Substrate concentration
Temperature-
With the increase in temperature the rate of enzymatic action increases. Every 10 degrees
increase in temperature the rate increases twice. This continues upto optimum temperature. After
optimum temperature, the rate of enzymatic action does not increase although temperature
increases. At very high temperature after optimim, the enzymatic action stops.

Explanation- Higher temperatures give the molecules of the enzyme and the substrate more
kinetic energy, so they collide more often. More collisions mean that the reaction will take place
more freequently to form more enzyme-substrate complex. At very high temperature after
optimum, the enzymes get denatured and their active sites are altered. Thus, the substrates no
longer fit with the active site. As a result, very few or no enzyme-substrate complex is formed.

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pH-
Initially, increasing the pH increases the rate of reaction. However, after the optimum pH is
reached the enzyme begins to change shape and the active site stops being able to bind to the
substrate.
The enzyme becomes denatured and stops working (the rate of reaction is zero at this point).

Substrate concentration-
When there is a low concentration of substrate, the active sites of some enzyme molecules will
be empty, so the rate of reaction will be low. If the concentration of substrate is increased, there
will be more collisions between enzyme and substrate molecules and more active sites will be
filled. As a result, the rate of reaction will increase.
This increase in rate wiil be continued for a limited period. Then the enzyme become saturated-
all of the active sites are occupied by substrate molecules- and a further increase in substrate
concentration will not increase the rate of reaction further. At this point, only an increase in
enzyme concentration will increase the rate of reaction.

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Describe an Investigation to see the effect of temperature on the activity of amylase.
To carry out the investigation, I need the following steps to follow:
• I will select five different temperature such as 10, 20, 30, 40 and 500C using either
thermostically controlled water bath or thermostatically controlled room or incubators.
• I will prepare amylase enzyme solution of a particular concentration (e.g. 1%).
• I will also prepare 1% starch suspension.
• A graduated syringe is used to place 10 cm3 of 1% amylase in a boiling tube.
• Another syringe is used to place 10 cm3 of 1% starch suspension in another test tube.
• These two test tubes are then placed in a thermostatically controlled room set at 300C and
allow them to stay there for five minutes for eqilibration.
• Then, I will mix contents of two test tubes and time how long it takes for the amylase to
digest the starch.
• To tell when the starch has been digested, I will remove a sample of the mixture at 30
seconds intervals and add one drop of iodine solution to it. The iodine solution will start
off blue-black when the starch is present, but change colour to yellow when the starch has
been digested, so I will time how long it takes for this to happen.
• I can then calculate the rate of digestion using the following formula:
Volume of starch solution in cm3
𝑅𝑅𝑅𝑅𝑅𝑅𝑅𝑅 =
Time needed to digest in mins
• To ensure reliability, I will repeat the experiment three times and calculate an average.
• I will repeat the whole experiment at other temperature keeping all other factors such as
volume and concentration of enzymes and starch suspension, source of enzymes, length
of time of experiment etc. same.
• I will then plot the values in the graph, temperature at ‘X’ axis and rate at ‘Y’ axis for
easy comparison.

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An investigation into the effect of pH on the activity of catalase.

Buffer solutions resist changes in pH. Different buffer solution can be prepared to maintain
different values of pH and are useful for finding the effect of pH on enzyme activity. Potato cells
contain a high concentration of enzyme catalase. To carry out this investigation, I need to follow
the following steps:
• I need to prepare a range of pH such as pH-6, pH-7 and pH-8 using buffer
• I need to obtain potato extract as a source of catalase
• A graduated syringe is used to place 5 cm3 of potato extract in a boiling tube
• Another syringe is used to add 5 cm3 of pH-7 buffer solution to the same boiling tube
• The tube is shaken gently to mix the buffer with the potato extract
• The mixture is left at room temperature for five minutes for eqilibration
• Then 5 cm3 of 5% hydrogen peroxide solution kept at room temperature is added to the
tube from a third syringe
• A bung and delivery tube are quickly inserted into the boiling tube and the end of the
delivery tube is placed in a beaker of water as follows:

• The bubbles of oxygen gas produced in the first minute after the hydrogen peroxide is
added are counted
• The number of bubbles per minute is a measure of the initial reaction rate
• The experiment is repeated, using different buffers for pH-6 and pH-8 and their initial
reaction rate is calculated.
• During repeat, I will keep all other factors such as temperature, volume of potato extract
and volume and concentration of hydrogen peroxide etc. same.
• I will polt a bar chart of the results. Hence, pH is placed at ‘X’ axis and number of
bubbles per minute or initial reaction rate at ‘Y’ axis for easy comparison.

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