The effect of pH on enzyme activity
Table of Contents
The aim of this experiment is to find
the optimum pH for the potato extract catalase.
globular proteins that act as catalysts, they alter the rate of a chemical
reaction without changing themselves. They can be reused, therefore effective
in small amounts. (Tool and Toole 2014: 23). Speeding reactions up by factors of 1010. (Lister and Renshaw 2015: 208).
Amino acids combine
in condensation reactions to make a polymer called a polypeptide. Polypeptides combine
to form a protein. This is the primary structure of a protein this determines the
ultimate shape, hence its function. Secondary structure is where the polypeptide chain can
coil into a a-helix as
the amine and carbonyl group form hydrogen bonds. a-helices can be twisted and folded even
more to give tertiary structure. Structure includes; disulfide bridges, ionic and
hydrogen bonds. This gives the specific 3-D structure of the protein making
each protein distinctive. (Tool and Toole 2014: 20). An enzymes active site can be selective of the shape of the
substrate that several enzymes only catalyse of one of the pair of enantiomers.
These are called stereoisomers, so enzyme is said to be stereospecific. (Lister and Renshaw 2015: 208)
Figure 1 – shows how amino acids
form active site in ESC (Tool and Toole 2014:
Enzymes have an
active site. Made up of relatively small amino acids (figure 1). The molecule which
the enzyme acts on is called the substrate. This fits into the active site of
the enzyme to form an enzyme-substrate complex (ESC). This molecule is held
within the active site by bonds that temporarily form. The induced fit model suggests
that the active site forms as the enzyme and the substrate interacts. Proximity
of the substrate leads to a change in the enzyme that creates the functional
active site. Strain on the substrate molecule, distorts particular bonds in the
substrate and lowers the activation energy (Ea). For reactions to
occur naturally molecules must collide with enough energy. Reactions require an
initial amount of energy to start. The minimum amount is called the activation
energy. Enzymes work by lowering the Ea level. Enzymes allow
reactions to occur at lower temperatures than normal (figure 2). (Tool and Toole 2014:
Figure 2 – How enzymes lower activation
and Toole 2014: 23)
A rise in the temperature increases the
kinetic energy of molecules. As a result, more frequent collisions so more ESC are
formed so an increase in the rate of the reaction. As the temperature rises a certain point
bonds begin to break in the enzyme molecule. The active site of the enzyme changes
shape so substrates don’t fit lowering the rate of reaction. The enzyme stops
working and is said to be denatured. The pH of a solution is the measure of its
hydrogen ion concentration. Each enzyme has its own optimum pH. This can be
pH = -log10H+.
A change in pH from
the optimum affects the rate of enzyme action. If the change of pH is more
extreme, beyond a certain pH, enzymes becomes denatured. Arrangement of active
site is partly determined by the hydrogen and ionic bonds between -NH2 and
-COOH groups that make up the enzyme. Change in H+ ions affect the
bonding, causing the active site to change shape. (Tool and Toole 2014: 28)
If there is an
excess of substrate, an increase in quantity of enzyme leads to proportionate
increase in the rate of a reaction. If concentration of enzyme is fixed at a persistent
level and substrate concentration is increased, the rate of reaction increases
in proportion to the increase in substrate concentration. (Toole and Toole 2013:
inhibitors are substances that directly or indirectly interfere with the
functioning of the active site of an enzyme and so reduce the activity. There
are two types; competitive – bind to active site of the enzyme and compete with
the substrate molecule, and non-competitive – which bind to enzyme at a
position other than the active site changing the shape of the active site, so
no substrate molecules can fit (figure 3 & 4). (Tool and Toole 2014: 32)
Figure 3 & 4 – competitive inhibition and
non-competitive inhibition (Toole and Toole 2013: 32)
In the human
digestive system, chemical digestion occurs. Large insoluble molecules are hydrolysed
into smaller soluble ones. It is carried out by enzymes. it is necessary for
these enzymes to remain in their optimum pH’s so that they can work
and Toole 2014: 152)
Table 1 – shows the different enzymes and their
optimum pH’s (Tool
and Toole 2014: 152)
coenzymes are molecules
that some enzymes require in order to function. Coenzymes play major role in
photosynthesis and respiration. They carry hydrogen atoms from one molecule to
and Toole 2015: 23). Cofactor is an ion or molecule that must bind to
the enzyme before substrates can also bind (Mason et al., 2014:117)
Enzymes play an
important role in industry as they help in the polymerase chain reaction (PCR)
as DNA polymerase Is used as it joins nucleotides together. valuable if only
minute amount of DNA is available. Usually used during forensic analysis on
crime scenes. (Tool
and Toole 2015: 276) Also enzymes also have important role in in vivo gene cloning as restriction
endonucleases are used to cut DNA. This is useful as able to introduce new gene
into another organism. Also, it produces transformed bacteria that can produce
proteins for commercial or medical use such as hormones one being insulin. (Tool and Toole 2015:
The enzyme activity will be at its peak at
around neutral pH.
coat, gloves and safety goggles must be worn throughout the experiment. Long
hair must be tied back. Take care when handling hydrogen peroxide as it is
corrosive to the skin.
25ml of hydrogen peroxide into beaker and measure the depth. Produce 50/50 mix
of potato extract and enzyme solution (pH 5). Using forceps immerse filter
paper into mixture and drain on paper towel for 10 seconds. Place filter paper
at the bottom of the hydrogen peroxide in the beaker and measure time taken to
reach to the top. Repeat steps again for pH 5 to get an average time and
complete same procedure for all pH’s.
As you can see
from the results as the pH increased the enzyme activity had increased too,
till it got to its optimum point which is pH 7 and started to decrease. At this
point the enzymes are starting to denature, as there is a change in the H+
ions which alter the hydrogen and ionic bonds between -NH2 and -COOH
groups. Causing the active site to change shape therefore no substrates can sit
onto the enzyme. So, less ESC formed, meaning a reduce in the enzyme activity. This
means the hypothesis can be accepted as it had stated that ‘the enzyme activity
will be at its peak at around neutral pH’ and it was correct.
One error in
the experiment is that when drying the filter paper for 10 minutes, it was not
the same time for each solution to dry. The practical could have been more
accurate if one person was measuring out the hydrogen peroxide and measuring
the depth as it was different at some points meaning the distance travelled by
filter paper was more/less than others affecting the enzyme activity.
In conclusion, the
experiment carried out had went well as obtained the results needed, also it
was good as it was clear and simple as it was one set objective. One way to
improve the experiment is to have a wider range or pH’s so we can find the
exact point at which an enzyme really denatures and to have other different
enzymes and substrates to show how each enzyme had a different optimum pH.
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