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Required Practical 1 enzymes

Laura Armstrong

Teacher

Laura Armstrong

Contents

Explainer Video

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Controlling and Varying Variables

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Measuring the Rate of Reaction Experimentally

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Plotting Results and Finding the Initial Rate of Reaction

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Recall Questions

This topic requires prior knowledge of enzymes and factors that affect their rate of reaction. You can test your knowledge on these below.

What is an enzyme and how does it function in biological reactions?

  • Enzymes are biological catalysts that speed up reactions by lowering the activation energy.
  • They have an active site that binds to a specific substrate to form an enzyme-substrate complex.

What are the factors that affect enzyme activity?

  • Temperature, pH, substrate concentration, enzyme concentration

Explain how temperature affects the rate of an enzyme controlled reaction.

  • As temperature increases the kinetic energy of the substrate and enzymes increases, increasing the rate of formation of enzyme substrate complexes

  • At the optimum temperature the rate of enzyme activity is at its maximum

  • Above the optimum temperature the bonds within the tertiary structure of the enzyme start to break. The enzyme denatures

  • When the enzyme has denatured the active site is no longer complementary to the substrate and an enzyme substrate complex cannot form

Explainer Video

For any experiment there are 3 types of variables; the independent, dependent and control variables.

Core practical 1 is about measuring the rate of an enzyme controlled reaction and investigating how different variables affect the rate. You need to know how to vary each of the different factors and how to control each of the other factors. Remember only 1 variable should be changed in an experiment all the rest should be controlled.

Controlling and Varying Variables

To collect enough data to plot a graph and observe a trend, you need at least 5 different values for your independent variable.

When investigating the effect of temperature it is important to make sure that the enzyme and substrate solutions are at the same temperature. To do this place them into the water bath for sufficient time before mixing them to allow them to equilibrate.

Measuring the Rate of Reaction Experimentally

Method 1: Investigating Amylase Activity

Example: Measuring the rate of starch hydrolysis by amylase using iodine solution

  1. Add iodine solution to a spotting tile.

  2. Prepare a mixture of starch and amylase in a test tube and place it in a water bath at a controlled temperature.

  3. At regular time intervals, remove a small sample and add it to the iodine on the spotting tile.

  1. Observe the colour change:

    • Blue-black = Starch present

    • Orange-brown = Starch absent (reaction complete)

  1. Record the time taken for the starch to be completely broken down. This will be when the iodine solution remains yellow/brown. It will no longer turn blue/black when all the starch has been hydrolysed to glucose.

  2. Repeat with different temperatures or pH values to investigate their effect.

Alternative Method: Using a Colorimeter

  1. Prepare test tubes containing starch and amylase and place them in a water bath at a controlled temperature.

  2. At regular time intervals, take a small sample and add it to a cuvette.

  3. Place the cuvette into a colorimeter set to measure absorbance. 

  4. Record the absorbance readings at each time interval.

  5. As starch is broken down, the absorbance decreases, indicating the reaction progress.

  6. Plot a graph of absorbance against time and use it to determine the rate of reaction.

To check for repeatability, repeat the experiment at each value of the independent variable at least three times and calculate a mean after discarding anomalies. 

To improve accuracy use smaller time intervals between taking samples.

 

Method 2: Investigating Catalase Activity

Example: Measuring the rate of hydrogen peroxide breakdown by catalase

Catalase is an enzyme that breaks down hydrogen peroxide into water and oxygen.

  1. Place a set volume of hydrogen peroxide solution into a test tube.

  2. Add a set mass or volume of catalase enzyme solution (e.g., from potato extract).

  3. Collect the oxygen gas produced over time using a gas syringe or an inverted measuring cylinder in a water bath.

  4. Record the volume of oxygen produced at regular intervals (e.g., every 10 seconds).

  5. Repeat the experiment at different temperatures or pH values to determine their effect on the rate of reaction.

To check for repeatability, repeat the experiment at each value of the independent variable at least three times and calculate a mean after discarding anomalies.

To improve accuracy use a measuring cylinder with greater resolution (smaller intervals).

Plotting Results and Finding the Initial Rate of Reaction

1. How to Plot the Graph

  • X-axis: Time (seconds)

  • Y-axis: Volume of oxygen produced (cm³)

  • Plot the data points and draw a smooth curve (not a straight line).

2. How to Determine the Initial Rate of Reaction

  • The rate of reaction will decrease over time as the substrate is used up so we need to find the initial rate of reaction to be able to compare.

  • The initial rate of reaction is the rate at the very start of the reaction when substrate concentration is highest.

  • To find this:

    1. Draw a tangent at t = 0 on the curve.

    2. Calculate the gradient of the tangent: Rate=change in y ÷ change in x

E.g. Rate = 50.0 ÷ 18.0 = 2.778 cm³s⁻¹

The steeper (greater the gradient) the greater the initial rate of reaction. 

 

Key Terms

  • Active site: The region on the enzyme where the substrate binds.
  • Denaturation: Loss of enzyme structure due to high temperature or extreme pH.
  • Substrate concentration: The amount of substrate available for the enzyme to act upon.
  • Enzyme concentration: The number of enzyme molecules available for catalysis.
  • Tangent: A straight line touching the curve at one point, used to estimate initial rate.
No answer provided.

Exam Tips

Always identify the key variables when planning and experiment.

Independent variable - what is it and how will you change it?

Dependent variable - what is it and how will you measure it?

Controlled variables - what are they and how will you control them?

Remember to include repeats to enable you to calculate a reliable mean.

Remember to include a control experiment to compare to the results of your actual experiment. In the case of an enzyme experiment, you could use a boiled and denatured enzyme in your control experiment to prove it is the enzyme causing the rate of reaction to change.

When reading about an experiment consider whether there are any control variables that have not been mentioned.

No answer provided.

The enzyme catalase is found in many living organisms and catalyses the breakdown of hydrogen peroxide (H₂O₂) into water and oxygen. A student investigates how the concentration of hydrogen peroxide affects the rate of reaction. The student sets up an experiment using yeast as a source of catalase.

Describe a method the student could use to measure the rate of reaction. (2 marks)

  • Collect the oxygen gas produced using an inverted measuring cylinder filled with water or a gas syringe.

  • Measure the volume of oxygen produced in a given time (e.g. 5 minutes).

Practice Question

Try to answer the practice question from the TikTok on your own, then watch the video to see how well you did!

Factors that affect the rate of an enzyme controlled reaction Enzyme inhibition