• Understand techniques used for measuring enzyme
activity
• Explain the principles of using of starch - agar plates
for assaying carbohydrase activity
•Use precise experimental techniques in assaying
enzyme actiivity
•To interpret enzyme activity graphs
•To complete serial dilutions using precise
measurements
•To set up starch agar plates with amylase
Here are some answers to enzyme related
questions
Can you write a question for each?
Answer
- Lock and key, induced fit
Answer
- Ionic, disulphide (bridges) and hydrogen bonds
Answer - Increased (kinetic) energy enzyme/substrate
moves faster, including collisions
Answer
- Denatured
Answer
- Amylase
Enzyme Kinetics - measuring the rate of enzyme reactions.
To measure an enzyme catalysed reaction you need a
signal to measure that shows the progress of the reaction.
The signal should change with either substrate or product
concentration, and it should preferably be something that
can be measured continuously.
Can you think of possible signals?
Typical signals include colour changes, pH changes, mass
changes, gas production, volume changes or turbidity
changes.
If the reaction has none of these properties, it can
sometimes be linked to a second reaction, which does
generate one of these changes.
One useful method is to dissolve the substrate in
agar in an agar plate. If a source of enzyme is placed
in the agar plate, the enzyme will diffuse out through
the agar, turning the substrate into product as it goes.
Well
containing
enzyme
There must be a way to distinguish the substrate from
the product, and the reaction will then show up as a ring
around the enzyme source.
What signal could be used to show the digestion of
starch?
The higher the concentration of enzyme, the
higher the diffusion gradient, so the faster the
enzyme diffuses through the agar, so the larger
the ring in a given time.
The diameter of the ring is therefore proportional to the
enzyme concentration. This can be done for many
enzymes, e.g. a protein agar plate can be used for a
protease enzyme, or a starch agar plate can be used for
the enzyme amylase.
Starch Agar Plate
You are now going to set up an agar plate to assay the
activity of amylase
(a) Name the part of the enzyme labelled D.
Active site; 1
........................................................................................................
..............................................
(1 mark)
(b) Explain how substrate C is broken down by the enzyme.
Substrate
enters active site; Complimentary shapes / Lock and
........................................................................................................
Key;
(Binding) to form enzyme-substrate complex; Lowering of
........................................................................................................
activation
energy; Conformational / shape change;
........................................................................................................
Breaking of bonds in substrate; Products no longer fit active
site and so are released;
(c)
Molecules A and B inhibit the enzyme in different ways.
Explain how each molecule inhibits the enzyme.
Molecule A
Molecule A binds at site away from active site / allosteric site;
........................................................................................................
Causes enzyme / active site to change shape;
........................................................................................................
.......................................................................................................
.......................................................................................................
Molecule B
Prevents substrate from entering / no
.......................................................................................................
enzyme-substrate
.......................................................................................................
complex formed / active site blocked;
.......................................................................................................
........................................................................................................
(4 marks)
(i) Describe how a saprophytic fungus obtains its food.
Secretes enzymes (for extra-cellular digestion);
......................................................................................................
Absorbs products;
......................................
......................................................................................................
(2 marks)
(ii) What conclusion can be drawn from the results?
ii)
Optimum pH is 7 / neutral / between 6 and 8 / between 7
.................................................................................................
and
8;
................................................................................................
(1 mark)
(iii) Use an appropriate method to estimate the maximum
rate of reaction that was observed in this investigation.
Show your working.
Maximum rate of reaction
Max rate = Distance
11 × 60
.................................................................................................
.................................................................................................
Time
4
.................................................................................................
2.75 mm / hour, 0.046mm/min, 4.6 x 10-3 mm/min
.................................................................................................
1 mm/ 21.8 mins, 23.76mm2/hour]