Practical: Enzyme Action (SQA National 5 Biology): Revision Note
Exam code: X807 75
Investigating enzyme specificity: the action of potato phosphorylase
Enzymes are highly specific, meaning that they act only on substrates that are complementary to their active site
Phosphorylase enzyme catalyses the formation of starch from glucose-1-phosphate
glucose-1-phosphate (+ potato phosphorylase) → starch
It is possible to demonstrate that starch is only produced when both the enzyme and its correct substrate are present, giving evidence of enzyme specificity
Examiner Tips and Tricks
Note that this practical is a 'suggested practical' in the specification, rather than content that all students are expected to learn. Some schools may choose to complete alternative practicals, or may miss out practical work that is not realistic, e.g. due to equipment or time constraints.
Apparatus
Fresh potato tissue (source of phosphorylase enzyme)
Glucose-1-phosphate solution
Alternative sugar, e.g. glucose-6-phosphate, glucose, maltose
Distilled water
Test tubes and rack
Measuring syringes
Stopwatch
Iodine solution (for starch test)
Beaker of warm water (optional, to maintain room temperature)
Filter paper or muslin (to strain enzyme extract)
Labels or marker pen
Method
Prepare the enzyme extract:
Chop the potato into small pieces and grind with a little distilled water using a pestle and mortar
Filter the mixture through muslin or filter paper to collect the enzyme extract
Label four test tubes:
A: enzyme + glucose-1-phosphate
B: enzyme + alternative sugar
C: water + glucose-1-phosphate
Measure out solutions:
Use a measuring syringe or cylinder to measure out equal volumes of each of the solutions listed in step 2, e.g.:
tube A = 2 cm3 enzyme + 2cm3 glucose-1-phosphate
tube B = 2 cm3 enzyme + 2 cm3 alternative sugar
tube C = 2 cm3 water + 2 cm3 glucose-1-phosphate
Mix gently and leave the tubes to stand for 15–20 minutes at room temperature
Test for starch:
Add a few drops of iodine solution to each tube
Record any colour change
Expected results
Tube | Contents | Iodine test | Explanation | Purpose of tube |
|---|---|---|---|---|
A | Phosphatase Glucose-1-phosphate | Blue-black | Phosphorylase has converted glucose-1-phosphate into starch | Shows that the enzyme and substrate produce starch when combined |
B | Phosphatase Alternative sugar | No colour change | Phosphorylase has not converted the alternative sugar into starch | Shows that the enzyme can only catalyse the reaction using the correct substrate |
C | Water Glucose-1-phosphate | No colour change | No reaction can occur in the absence of the enzyme | Experimental control: shows that any starch produced is due to the specific action of the enzyme on its correct substrate, and not another factor |
Starch is only produced when both the phosphorylase and its specific substrate are present
This provides clear evidence for enzyme specificity — similar molecules cannot bind effectively to the enzyme’s active site
Limitations
Limitation | Possible solution |
|---|---|
Judging the iodine colour by eye can be subjective | Compare to a colour chart Use a colorimeter to measure colour intensity |
Potato extract strength may vary between preparations | Prepare a stock enzyme extract rather than using potatoes |
Incomplete mixing can cause uneven starch formation | Use a consistent mixing technique, e.g. inverting the tube or stirring a specific number of times |
Room temperature fluctuations | Place tubes in a water bath at a set temperature throughout the reaction |
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