Effect Of An Inhibitor Such As Copper Sulphate On The Activity Of Catalase

Biological Principal

Enzymes are biological catalysts that make possible chemical changes under the condition in cells. They are globular proteins and are coiled into a precise, three-dimensional shape, with hydrophilic R groups on the side of the molecules enabling them to be lobule in water.

Enzymes work by binding to their substrate molecules at their active site, as the enzyme (E) and substrate (S) form an enzyme substrate complex (ES). The substrate is either broken down on mended to form product (Pr).

E + S ---------> ES  -----------> Pr + E

                                            <---------         <----------

The activities of all enzymes are affected by: temperature, pH level, substrate concentration, enzyme concentration and inhibitors. There are two types of inhibitors – competitive and non-competitive inhibitors. The competitive inhibition, that is said to be reversible, because it can be reversed by increasing the substrate concentration. The non-competitive inhibition tend to bind permanently within the enzyme active site and it is known as non-competitive irreversible inhibition, as it is not affected by the concentration of the substrate. They cause a permanent block to the active site of an enzyme. Thus no substrate can compete with them to bind with the active site. On the other hand, the competitive inhibitors slow down the activity of an enzyme. But as the substrate concentration is increased, the slow down caused by the inhibition is overcome and the inhibitor molecules progressively displaced from the active site.

Research that I carried out on the internet, I found out that the Fe ion is found in the active site of yeast (Catalase). This means that Cu  ions can displace the Fe ions (in enzyme) thus inhibit Catalase, and consequently disruption can be caused to the configuration of hydrogen bonds and the hydrophobic interaction which is holding the enzyme molecule.

Method

Part A: Apparatus

• A conical flask to place the mixture of the enzyme, substrate and the inhibitor
• A 5ml syringe to inject 2ml of yeast (catalase) enzyme into the mixture
• A gas syringe to collect and measure the volume of Oxygen gas is produced within specific time interval
• A clamp to hold the gas syringe
• 13 test tubes 6 for different concentration of inhibitor and 6 for the substrate and 1 for water
• 3 100ml measuring cylinders to measure out the volume of each variables
• A stop clock to measure rate of production of Oxygen gas
• A test tubes rack will be needed to hold the test tubes
• A pipettes to insert the reactant from their bottles to the measuring cylinders

Safety Precautions

• Through out the experiment must wear goggles, so spillage of H2O2 can’t get into your eyes
• Wear gloves whilst handling H2O2 as it is irritant and could burn your skin if comes into contact
• Wear a lab coat to prevent spoiling you clothes
• Handle glass wear equipment cautiously to prevent breaking them
• Get rid of used reactants appropriately

TABLES FOR THE VARIABLES

• There are six test tubes labelled A-F, which A contain the lowest concentration of CuSO4 and the test tube F containing the highest concentration.
• The substrate concentration and volume will be kept constant (same) for all carried out reaction (12cm3) in each separate test tube.
• The six test tubes labelled A to F will have different concentration on each as shown on above table.
• To make the volume same for the substrate and the inhibitor I will add water to the inhibitor. The volume of water I will add is shown on the table of variables
• Place the both substrate and inhibitor in the conical flask form each of the test tubes and then inject 2ml (1cm3) of yeast into the mixture in flask and simultaneously start the stop clock. And see how much oxygen gas is collect within every five seconds of the reaction time
• I will carry out each reaction for 60 seconds
• Record the amount gas produced in my results table by reading of the gas syringe
• I will repeat each experiment twice. This will be done to give an average volume of gas produce within the given time

MY RESULT TABLE