Learning Intention: Students will better understand the use of mass spectroscopy in the diagnosis of a medical condition and explore the implications of enzyme deficiencies in protein metabolism. They will alsounderstand the effect of pH on enzyme activity and apply their knowledge of scientific method to writing the procedure for a practical experiment.
Success criteria: Students will complete the practical activity “Absent enzymes – phenyketonuria and albinism”.
Liver contains an enzyme that catalyses the reaction to breakdown hydrogen peroxide (H2O2)
Enzyme activity in this case is indicated by the production of oxygen – a splint re-ignites in the presence of oxygen.
High temperatures denature proteins and prevent enzyme activity.
Diastase in an enzyme in plants that converts starch into glucose at an optimum temperature of 20 degrees Celcius.
The presence of starch in solution is indicated by iodine turning blue-black.
Your task is to devise an experiment that demonstrates the effect of pH on enzyme activity. Remember that pH is a logarithmic scale – a pH of 4 is ten times more H+ ions than a pH of 5. pH and Enzymes Effect of pH on enzyme activity
Proteins are polypeptides or chains of peptides (amino acids) joined together by peptide bonds. These large organic molecules have four levels of structure –
Primary – order of amino acids in the chain
Secondary – alpha-helices, beta-pleats and random coils
Tertiary – the folding of the chains due to the presence of disulphide bonds
Quarternary – when two or more polypeptide chains are folded together in a complex molecule
Enzymes are a specific type of protein that play a critically important role in living organisms. The molecules in cells are constantly interacting – being broken down, built up or exchanged. These chemical reactions constitute an organism’s metabolism. An organism is regulated and the rate of it’s chemical activity is maintained by these special proteins, known as biological catalysts. Like all proteins, enzymes are made in the ribosomes by linking together specific amino acids in the cytoplasm, according to the DNA code. Each cell contains and needs a very large number of different enzymes, but not all cells produce all enzymes – it depends on the structure and function of the cell as to which genes are ‘switched on’.
Enzymes are proteins and are therefore made up of amino acids (containing carbon, hydrogen, oxygen and nitrogen)
Enzymes are ‘biological catalysts’ because they speed up the rate of a chemical reaction
Enzymes remain unchanged at the end of the reaction (not used up)
Enzymes are only required in small amounts
Enzymes are highly specific (one enzyme catalyses one type of reaction)
Enzymes work best under optimum conditions of temperature and acidity
Enzymes are ‘denatured’ (destroyed) by heat and sensitive to pH
Enzymes work like a key fits into a lock – their shape complements the shape of the substrate materials.
The ‘active site’ of a particular enzyme has a specific shape into which only one kind of substrate will fit
Enzymes may need ‘co-enzymes’ (specific vitamins) or ‘co-factors’ (minerals) to help functioning
The Gene Technology Access Centre have some excellent resources for VCE Biology, including this slideshow and activity sheets “exploring protein structure“. The image above is one view of a representation of the enzyme amylase, which breaks starch down into sugars. You can see the green alpha-helices, yellow beta-sheets and blue random coils in the secondary structure of this protein. You may also be able to see the ‘co-factors’ or molecules which assist at the active site of this enzyme. Amylase relies on the co-factors calcium and chloride to function efficiently. What are the dietary sources of calcium and chloride?
Learning Intention: Students will understand that enzymes are proteins and biological catalysts that speed up chemical reactions in living organisms. They will also understand two of the factors that affect the action of enzymes, temperature and pH.
Success Criteria: Students will be able to design, perform, describe and report on an experimental procedure demonstrating the effect of temperature and pH on enzyme activity.
Students used potato, alfalfa sprouts and liver extract (most successful) to demonstrate how catalase (enzyme) breaks down hydrogen peroxide (H2O2) into water and oxygen. This process is essential to maintain a safe and healthy internal environment. When hydrogen peroxide was added to the liver extract and different concentrations of HCl (distilled water, 0.01M, 0.05M, 0.1M, 0.5M and 1.0M hydrochloric acid), only the distilled water and 0.01M HCl tubes released significant quantities of oxygen. At higher acid concentrations (lower pH) no enzyme activity was apparent, because the acid destroys the protein or denatures the enzyme.
Diastase is an enzyme that catalyses the conversion of starch (polysaccharide) into sugars (di- and mono-saccharides). Iodine is an indicator that turns from yellow to blue-black in the presence of starch. Students used two sets of five test tubes with 10 ml of starch solution in each. In the control set, distilled water was added to to each test tube. In the second set, the enzyme diastase was added. One tube from each set was then placed into water baths at different temperatures (room temp, 40C, 60C, 80C and 100C). Iodine was used to indicate which tubes contained active enzyme. If the tube contained starch, the colour was blue-black, indicating that there was little or no enzyme activity (control tubes). The lighter the colour, the greater the conversion, therefor the more enzyme activity. The tubes at room temperature, 40C and 60C showed the most enzyme activity.
At the Gene Technology Access Centre on Monday we spent the day learning about the structure and function of enzymes. As well as a lecture and practical experiment, we had the opportunity to use a computer program for protein modelling. GTAC has some great online resources for teaching and learning, including this slideshow about Enzyme Action.
Enzymes have specific characteristics:
– enzymes are proteins, made up of amino acids
– enzymes have specific primary, secondary and tertiary structures
– enzymes are specific to substrates
– enzymes are biological catalysts (they speed up a reaction)
– enzymes have optimum temperature and pH ranges
– enzymes are not changed or used up in a reaction
– enzymes have an active site, which is where the substrate is broken down or the products are made
– enzymes can contain co-factors (ions, such as chlorine or calcium) that assist to attract the reactants to the active site
We worked with amylase, an enzyme that breaks starch down into disaccharides. We used iodine to indicate the presence of starch and a photo spectrometer to measure the degree of staining of the medium. The higher the photo spectrometer reading, the more starch, which meant the less enzyme action. We stopped the reaction using an acid, which denatures the enzyme and prevents the break down of starch. Our results showed that the optimum temperature of amylase activity was about 40 degrees and the optimum pH was 6. This is what you might expect from human amylase, which would be working at normal body temperature (37 degrees) and neutral (or slightly acidic) pH in the mouth.
These pitcher plants and other ‘carnivorous’ plants produce digestive enzymes that can break down the flesh of small invertebrates, such as flies, spiders and ants. Often they grow in soil that is deficient in specific inorganic nutrients, such as nitrates and phosphoros, and can get these essential elements from the dead animals that are attracted by sweet and sticky liquids.
If you choose to study microbiology at University, you may be involved with experiments such as this, working in an anaerobic chamber. When you exclude oxygen from the environment, respiration takes place anaerobically – without oxygen. Read more here. Anaerobic respiration is used both in the brewing and baking industries, as alcohol and carbon dioxide are produced when specific organisms respire without oxygen. This is called fermentation. If you have access to YouTube, you can see a quick Food Science video about fermentation here.
This site, from Thomas M. Terry of the University of Connecticut, has some excellent, very detailed animations of cellular respiration. Chapter three also deals with enzymes – proteins that accelerate biological reactions. It is important to remember that enzymes are not reactants or products of a reaction – they are not ‘used up’ during the process. Enzymes facilitate, or speed up, a specific reaction. For a good tutorial check out: “What is an Enzyme?”.