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Factors Affecting Enzyme Action

Factors Affecting Enzyme Action

1. Temperature

In humans, enzymes function best at 37°C (see figure below). This is the optimum temperature. At very high temperatures proteins denature; this means that the hydrogen, hydrophobic and electrostatic interactions that result in the protein’s three-dimensional shape break down, unravelling the protein into its primary structure, a long chain of amino acids.

When an enzyme is denatured, the shape of its active site, as well as the rest of the protein shape is altered. The substrate can no longer fit in the active site of the enzyme and chemical reactions cannot take place. Low temperatures can slow down or even inactivate enzymes, as low temperature means less available kinetic energy, so that even the lower energy of activation that the enzyme allows is not available. The first graph shows the effect of temperature on enzyme activity.

2. pH

Enzyme activity is sensitive to pH. Enzymes have an optimum pH as shown on the graph, but they can function effectively within a pH range. The effectiveness of the enzyme falls sharply when the pH is outside its optimum range. An enzyme can become denatured when exposed to a pH outside its pH range, as pH affects the charge on some amino acids, and therefore affects the electrostatic interactions holding the tertiary structure together. The second graph shows the effect of pH on enzyme activity.


The optimal pH and temperature for an enzyme will be determined by the kind of living thing it is found in. The enzymes in the human body have an optimum temperature of 37 °C. Bacteria that live in compost heaps have enzymes with an optimal range in the 40’s, and bacteria called hyperthermophiles (lovers of very high temperatures) that live in hot springs have enzymes with optimum temperatures above 80 °C.

Image credit: Siyavula

In the investigation that follows, the effect of temperature on catalase enzyme activity will be investigated. Hydrogen peroxide is potentially toxic and so living tissues contain an enzyme named catalase to break it down into non-toxic compounds, namely water and oxygen. You will study the effect of the enzyme catalase on the breakdown of hydrogen peroxide. You will further examine the effect of pH and temperature on enzyme activity.

Investigation: Investigating the Effect of Catalase from Chicken Liver on Hydrogen Peroxide


To demonstrate the effect of catalase on hydrogen peroxide


  • 10 ml measuring cylinders
  • pipette
  • 3% Hydrogen peroxide solution
  • scalpel
  • forceps
  • balance
  • chicken liver at room temperature
  • boiled chicken liver
  • frozen chicken liver
  • stirring rod


Follow the instructions below:

  • Cut two square pieces weighing 0.1 g from the fresh liver sample and place each in a separate 10 ml measuring cylinder.
  • Use a clean measuring cylinder to measure 3 ml water. Pour into one of the fresh liver-containing cylinders. This is your negative control.
  • Use a clean measuring cylinder to measure 3 ml hydrogen peroxide. Pour into the remaining fresh liver-containing cylinder. This is your positive control.
  • Wait for four minutes and then measure and record the height of the resulting oxygen bubbles in each cylinder.


  1. Name the three variables that must remain stable throughout these experiments and explain why they must be kept stable.
  2. What kind of reaction is taking place?
  3. How could you make this experiment more accurate?
  4. In addition to temperature, what other factors influence the rate of reaction?

Part B:


To demonstrate the effect of temperature on catalase activity


  • Add 3 ml of hydrogen peroxide to two separate 10 ml graduated measuring cylinders. Mark one cylinder “frozen chicken liver” and the other “boiled chicken liver”.
  • Cut a 0.1 g square from each of the frozen and boiled chicken livers. Add the liver pieces to the correspondingly labelled measuring cylinder with hydrogen peroxide in it.
  • Leave both pieces of liver for four minutes and measure the height of bubbles produced.


  1. Give reasons for the differences observed across the three measuring cylinders.
  2. Name the dependent and independent variables in this experiment.
  3. How could you make this experiment more accurate?
  4. What would you conclude from your observations?

Video: Enzymes Summary

The Amoeba Sisters video below explains enzymes and how they interact with their substrates.

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