Enzyme Activity: Skills (DP IB Biology)

Effects of Temperature, pH & Substrate Concentration

NOS: Describing patterns and trends in graphs

• You are required to describe the relationship between variables shown in graphs

• Generalised sketches of these relationships are examples of models in Biology

• Enzyme experiments can be conducted to investigate the effects of the following factors on the rate of enzyme activity:

  • Temperature

  • pH

  • Substrate concentration

• Sketch graphs can be drawn and evaluated using the results from these experiments

Designing experiments to test the effect of temperature, pH and substrate concentration on the activity of enzymes

• Three different independent variables can be tested

  • Temperature

  • pH

  • Substrate concentration

• You should plan how the dependent variable is going to be measured

  • With appropriate units

• Also, what intervals of the independent variable are going to be chosen

• These factors dictate the choice of apparatus and other equipment required for the experiment

• The control variables need to be identified and monitored e.g. temperature when measuring the effect of pH

Investigating the effects of temperature or pH on catalase activity

• The progress of enzyme-catalysed reactions can be investigated by:

  • Measuring the rate of formation of a product

  • Measuring the rate of disappearance of a substrate

• In this investigation, the rate of product formation is used to measure the rate of an enzyme-controlled reaction:

  • Hydrogen peroxide is a common but toxic by-product of metabolism

  • This means it must be broken down quickly

  • Catalase is an enzyme found in the cells of most organisms that breaks down hydrogen peroxide into water and oxygen

  • Hydrogen peroxide and catalase are combined and the volume of oxygen generated is measured in a set time

  • The rate of reaction can then be calculated

Investigating catalase diagram

Experimental set-up for investigating the rate of formation of a product using catalase

• If measuring the effect of temperature on enzyme activity, the conical flask containing potato pieces can be held in a water bath at the required temperature

  • The water level in the water bath must be higher than the level of H₂O₂ in the conical flask, to ensure even heating

  • The conical flask can also be swirled gently to mix the contents and maintain an even temperature

Investigating the effect of substrate concentration on amylase activity

• In this investigation, the rate of substrate disappearance is used to compare rates of reaction under different conditions

• Amylase is a digestive enzyme that hydrolyses starch into maltose and glucose

• Amylase functions best at pH 7 and 37^oC (all enzymes operate best under specific conditions)

• Amylase and starch are combined and this reaction mixture is then tested for starch at regular time intervals

• This can be done by taking samples from the reaction mixture at each time interval and adding each sample to some iodine in potassium iodide solution

  • Starch forms a blue-black colour with this solution

  • If no starch is present, the iodine solution remains yellow-brown

• In this way, the time taken for starch to be broken down can be measured

• The investigation can be repeated under different starch concentrations and the reaction rates can then be compared

  • This experiment also can be adapted to measure the effects of altering pH, temperature or enzyme concentration

Investigating amylase diagram

Experimental set-up for investigating the rate of disappearance of a substrate using amylase

Investigating the effect of starch concentration on amylase using colorimetry

• A colorimeter is able to measure light absorbance (how much light is absorbed) or light transmission (how much light passes through) a substance

• Colorimetry can be used in any enzyme-catalysed reaction that involves a colour change

• As the colour breaks down the transmission increases or light absorption decreases and this can be used to measure the rate of the reaction

• For example, a colorimeter can be used to follow the progress of a starch-amylase catalysed reaction as the amylase breaks the starch down into maltose

• This can be carried out as follows:

  • Colorimeter calibration: this is an important step in a colorimetric investigation and in this case, a weak iodine solution can be used to calibrate the colorimeter as the endpoint (or 100% transmission)

  • Preparation of a starch solution of known concentration (stock solution), from which a range of concentrations are made using serial dilutions (method outlined in diagram below)

  • Following calibration and switching on the red filter (to maximise the percentage transmission or absorbance), the colorimeter is used to measure the percentage absorbance or percentage transmission values

  • Sometimes a reagent or indicator is used to produce the colours detected by the colorimeter and sometimes the solutions themselves absorb light waves

  • A calibration graph is then plotted of starch concentration (x-axis) vs percentage absorbance or percentage transmission (y-axis)

Serial starch dilutions diagram

Serial dilution of starch to make a range of concentrations

Interpreting graphs on the effects of temperature, pH and substrate concentration on the rate of enzyme activity

• Temperature, pH and substrate concentration affect the rate of activity of enzymes

• Enzymes have a specific optimum temperature – the temperature at which they catalyse a reaction at the maximum rate

• Lower temperatures either prevent reactions from proceeding or slow them down:

  • Molecules move relatively slowly due to having less kinetic energy

  • Lower frequency of successful collisions between a substrate molecule and the active site of enzyme

  • Less frequent enzyme-substrate complex formation

  • Substrate and enzyme collide with less energy, making it less likely for bonds to be formed or broken (stopping the reaction from occurring)

• Higher temperatures speed up reactions:

  • Molecules move more quickly due to having more kinetic energy

  • Higher frequency of successful collisions between a substrate molecule and the active site of enzyme

  • More frequent enzyme-substrate complex formation

  • Substrate and enzyme collide with more energy, making it more likely for bonds to be formed or broken (allowing the reaction to occur)

• However, as temperatures continue to increase, the rate at which an enzyme catalyses a reaction drops sharply, as the enzyme begins to denature

The effect of temperature on enzyme action diagram

The effect of temperature on the rate of an enzyme-catalysed reaction

Changes in pH

• pH is a result of the hydrogen ion concentration in a solution

• A low pH is acid and has a high hydrogen ion concentration

• A high pH is alkaline and has a low hydrogen ion concentration

• A 10× increase in hydrogen ion concentration lowers the pH by 1 unit

  • pH is therefore measured on a logarithmic scale of hydrogen ion concentration, not a linear scale

• Water has a pH of 7, regarded as neutral

• Extremes of pH can also alter hydrogen bonding within an enzyme's structure and cause irreversible denaturation

• Each enzyme has an optimum pH

• Not all enzymes have an optimum pH near to neutral. For example

  • The stomach enzyme pepsin is adapted to work best at pH 2

  • Certain bacterial enzymes work at pH 9-10, in line with the pH of the bacteria's main habitat

The effect of pH on enzyme action diagram

The effect of pH on three enzymes' rates of reaction

Changes in substrate concentration

• The more substrate molecules are present in a solution, this increases the frequency of collisions with the enzyme's active site

• Active sites are occupied or 'blocked' by substrates whilst the reaction is taking place

• The more active sites are occupied, the fewer are available to catalyse other substrate molecules

• As substrate concentration rises, the slower the rise in the rate of the enzyme-catalysed reaction

• The active sites have become saturated

• At the point of active site saturation, increasing the substrate concentration will cause no further increase in the rate of reaction

• At the point of active site saturation, a method of increasing the rate of reaction would be to make more active sites available by increasing the enzyme concentration

The effect of substrate concentration on enzyme action diagram

The effect of substrate concentration on enzyme activity