O-Level Biology · Secondary 3 and 4
Biological Molecules and Enzymes
Living things are built from a small set of biological molecules: carbohydrates, fats and proteins, together with water. This topic explains what each molecule is made of, how to test for it in the laboratory, and how enzymes, the protein catalysts of the cell, speed up the reactions that build and break these molecules. You will use these ideas directly in nutrition, digestion, photosynthesis and respiration, so the food tests and the effect of temperature and pH on enzymes are worth learning well early on.
What the syllabus expects
- State the roles of water in living organisms
- Describe the building blocks of carbohydrates, fats and proteins
- Describe and carry out the tests for starch, reducing sugars, proteins and fats
- Explain enzyme action using the lock and key and induced fit hypotheses
- Explain the effects of temperature and pH on the rate of enzyme activity
- Calculate a rate of reaction from the time taken for an enzyme reaction
Carbohydrates, fats and proteins
Carbohydrates contain carbon, hydrogen and oxygen. Simple sugars such as glucose are monosaccharides. Two monosaccharides join to make a disaccharide, such as maltose or sucrose. Many glucose units join to form polysaccharides: starch is the storage form in plants, glycogen is the storage form in animals, and cellulose makes plant cell walls. Fats are made from glycerol and fatty acids. They store energy, insulate the body and form part of cell membranes. Proteins are long chains of amino acids, and the order of amino acids decides the shape of each protein. Proteins are needed for growth and repair and make enzymes, antibodies and some hormones. Large molecules are broken into their building blocks during digestion, and built up again inside cells. Water is also needed, as a solvent for reactions, as the medium of transport in blood and in xylem, and in temperature control by sweating.
The food tests
For starch, add a few drops of iodine solution. A blue-black colour shows starch is present, while the solution stays brown if there is none. For reducing sugars such as glucose and maltose, add Benedict's solution and heat in a boiling water bath for about two to three minutes. The blue solution forms a green, yellow, orange or brick-red precipitate as the amount of reducing sugar increases. Sucrose is not a reducing sugar, so it gives a negative result. For protein, use the biuret test: add sodium hydroxide followed by a few drops of copper(II) sulfate, or add biuret solution directly. A violet colour shows protein. For fats, use the ethanol emulsion test: shake the sample with ethanol, then pour the ethanol into water. A cloudy white emulsion shows fat. Always describe the colour at the start and at the end.
How enzymes work
Enzymes are proteins that act as biological catalysts. They speed up chemical reactions without being chemically changed at the end, so they can be used again. Each enzyme has an active site with a specific shape. In the lock and key hypothesis, only a substrate with a complementary shape fits into the active site, forming an enzyme-substrate complex. The reaction takes place and the products leave, freeing the active site. The induced fit hypothesis adds that the active site changes shape slightly as the substrate binds, so that it fits more closely around it and the reaction happens more easily. This explains why enzymes are specific: amylase breaks down starch but not protein. Enzymes that break large molecules into smaller ones are used in digestion, and other enzymes join small molecules into larger ones inside cells.
Temperature and pH
As temperature rises from a low value, the enzyme and substrate molecules gain kinetic energy and collide more often, so more enzyme-substrate complexes form and the rate increases. The rate is highest at the optimum temperature, which is about 37 °C to 40 °C for most human enzymes. Above the optimum, the bonds holding the enzyme's shape break, the active site changes shape and the substrate no longer fits. The enzyme is denatured, and this cannot be reversed. At low temperatures the enzyme is inactive but not denatured, so it works again when warmed. Each enzyme also has an optimum pH. Pepsin in the stomach works best at about pH 2, while amylase in saliva works best at about pH 7. Away from the optimum pH the active site changes shape and the rate falls, and extreme pH denatures the enzyme. Rate can be calculated as 1 ÷ time taken.
Worked example
A student mixed starch solution with amylase at three temperatures and tested a drop of the mixture with iodine every 30 seconds. The time for the blue-black colour to stop appearing was 8 minutes at 20 °C and 2 minutes at 40 °C. At 60 °C the mixture still gave a blue-black colour after 20 minutes. (a) Calculate the rate of reaction at 20 °C and at 40 °C. (b) Explain the results at all three temperatures.
- Use rate = 1 ÷ time taken. At 20 °C: 1 ÷ 8 = 0.125 per minute.
- At 40 °C: 1 ÷ 2 = 0.5 per minute, which is four times the rate at 20 °C.
- Explain 20 °C: the molecules have less kinetic energy, so there are fewer collisions between amylase and starch and fewer enzyme-substrate complexes form per minute.
- Explain 40 °C: this is close to the optimum temperature for amylase, so the frequency of effective collisions is highest and starch is broken down to maltose fastest.
- Explain 60 °C: the amylase is denatured. Its active site has changed shape, starch can no longer bind, so starch remains and iodine stays blue-black.
Answer: (a) 0.125 per minute at 20 °C and 0.5 per minute at 40 °C. (b) The rate rises from 20 °C to 40 °C because of more frequent collisions, peaks near the optimum, and is zero at 60 °C because the enzyme is denatured.
Common mistakes
- Saying enzymes are 'killed' at high temperatures. Enzymes are not alive. Say they are denatured, meaning the active site has lost its shape.
- Saying low temperature denatures enzymes. At low temperature they are only inactive, with fewer collisions, and they work again when warmed.
- Forgetting to heat in the Benedict's test. Without heating in a water bath there is no colour change even if reducing sugar is present.
- Calling sucrose a reducing sugar. It gives a negative Benedict's result unless it is first broken down.
- Writing that the substrate has the 'same shape' as the active site. The shapes are complementary, which is what makes the fit specific.
Exam tips
- In any temperature or pH explanation, use the chain: shape of active site, fit of substrate, number of enzyme-substrate complexes, rate. Missing a link often costs a mark.
- For food tests, state the reagent, any heating, the starting colour and the final colour. 'Turns brick-red' alone is not a full answer.
- When a graph shows the rate falling above the optimum, use the word 'denatured' and mention the active site, not just 'the enzyme stops working'.
Practice questions
1. Easy: Describe how you would test a sample of milk for protein, and state the positive result.
Show answer
Add an equal volume of sodium hydroxide solution, then a few drops of dilute copper(II) sulfate and shake (or add biuret solution). A positive result is a colour change from blue to violet.
2. Medium: Lipase breaks down fats. Name the products and explain why lipase cannot break down starch.
Show answer
The products are fatty acids and glycerol. Enzymes are specific: the active site of lipase has a shape complementary only to fat molecules, so starch cannot fit into it and no enzyme-substrate complex forms.
3. Harder: Pepsin works in the stomach at about pH 2. Predict and explain what happens to pepsin activity when it passes into the small intestine, where the pH is about 8.
Show answer
Pepsin activity falls sharply and stops. At pH 8, far from its optimum, the bonds holding its shape are disrupted, the active site changes shape, protein molecules no longer fit and pepsin is denatured.
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