The Effect of substrate concentration on the enzyme catalase.

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The Effect of substrate concentration on the enzyme catalase

Hypothesis - Hydrogen peroxide will breakdown to oxygen and water in the presence of Catalase. The reaction will increase with increasing enzyme concentration when molecules of hydrogen peroxide are freely available. However, when molecules of the substrate are in short supply, the increase in rate of reaction is limited and will have little effect.

Variables - In this investigation, the variables that affect the activity of the enzyme, Catalase, were considered and controlled so that they would not disrupt the success of the experiment.

i) Temperature - As temperature increases, molecules move faster (kinetic theory). In an enzyme catalysed reaction, such as the decomposition of hydrogen peroxide, this increases the rate at which the enzyme and substrate molecules meet and therefore the rate at which the products are formed. As the temperature continues to rise, however, the hydrogen and ionic bonds, which hold the enzyme molecules in shape, are broken. If the molecular structure is disrupted, the enzyme ceases to function as the active site no longer accommodates the substrate. The enzyme is denatured.

To control this variable, the temperature was maintained at a fairly constant level that allowed the enzyme to work effectively 40oC. This will be achieved by using a test tube rack and tongs to handle the apparatus so that the heat from my hands did not affect the Catalase.

ii) pH - Any change in pH affects the ionic and hydrogen bonding in an enzyme and so alters it shape. Each enzyme has an optimum pH at which its active site best fits the substrate. Variation either side of pH results in denaturation of the enzyme and a slower rate of reaction.

In this experiment, the pH was kept constant using a pH 7 buffer, selected to maintain a pH level suited to the enzyme by being equal to the natural environment of the enzyme (potato tissue).

iii) Enzyme Concentration - When there is an excess of substrate molecules, an increase in the enzyme concentration, produces a corresponding increase in the rate of reaction. If there are sufficient substrate molecules to occupy all of the enzymes´ active sites, the rate of reaction is unaffected by further increases in substrate concentration as the enzymes are unable to break down the greater quantity of substrate.

To control the enzyme concentration, identical quantities of the substrate were used for each reading. To ensure that this was measured precisely, 5ml syringes were used to accurately gauge to exact quantities.

iv) Inhibition - Inhibitors compete with the substrate for the active sites of the enzyme (competitive inhibitors) or attach themselves to the enzyme, altering the shape of the active site so that the substrate is unable to occupy it and the enzyme cannot function (non-competitive inhibitors). Inhibitors therefore slow the rate of reaction. They should not have affected this investigation, however, as none were added.

v) Enzyme cofactors - cofactors are none protein substances which influence the functioning of enzymes. They include activators that are essential for the activation of some enzymes. Coenzymes also influence the functioning of enzymes although are not bonded to the enzyme.

Unless enzyme cofactors were present in the potato tissue containing the Catalase, they were not included in this investigation and therefore would not have affected the rate of reaction and the results of this experiment.
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vi) Substrate Concentration - Provided there is an excess enzyme, an increase in enzyme concentration will lead to a corresponding increase in rate of reaction. Where the enzyme is in short supply (i.e. it is limiting) an increase in substrate concentration has no effect.

I varied the substrate concentration by altering the concentration of the hydrogen peroxide in the reaction.

Apparatus -

i) A manometer

ii) 30ml hydrogen peroxide

iii) Manometer fluid

iv) 6 boiling tubes

v) Tongs

vi) A test tube rack

vii) A potato
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