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Unit 13 - Assignment 2 - P4
Active site - A region on an enzyme that binds to a protein or other substance during a reaction
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Catalysts lower the activation energy for reactions
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Therefore enzymes speed up reactions by
lowering activation energy
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This is called
induced fit
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Enzymes have active sites
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Enzymes form complexes with their substrates
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Tertiary Structure:

The protein molecule will bend and twist in such a way as to achieve maximum stability or lowest
energy state
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Under physiologic conditions, the hydrophobic side-chains of neutral, non-polar amino acids tend to
be buried on the interior of the protein molecule
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The alkyl groups
often form hydrophobic interactions between one-another, while aromatic groups often stack
together
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The formation of disulphide bridges by oxidation of the sulfhydryl groups on is an important aspect
of the stabilization of protein tertiary structure
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Also, hydrogen bonds may form between different side-chain
groups
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Ionic interactions between positively and
negatively charged sites on amino acid side chains, also help to stabilize the tertiary structure of a
protein
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dreamstime
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The hydrophobic areas are near the centre,
whilst hydrophilic areas are near the edges
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The roles of globular
proteins are usually metabolic
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Alpha-amylase

Reference: https://en
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org/wiki/B-amylase

Reference: http://www
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davidson
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htm

The active site of alpha-amylase contains three acidic groups
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In
amylase, glutamate 233, aspartate 197 and aspartate 300 work together to cleave the connection
between two sugars in a starch chain
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A calcium ion is found nearby where it stabilizes the structure of the enzyme
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Unit 13 - Assignment 2 - P5
Effect of Substrate Concentration on Enzyme Activity – H2O2 and
immobilised yeast (catalase)

Equipment:
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20% yeast suspension
2% Sodium alginate
Hydrogen peroxide solutions – 5%, 4%, 3%, 2%, 1%
Calcium chloride 0
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Stir yeast suspension
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2
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Stir the mixture very gently to mix
thoroughly, but without introducing air bubbles
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Put about 50cm3 of calcium chloride solution into a clean 100cm3 beaker
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This should form beads of immobilised yeast
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Leave in the calcium chloride for 2 minutes, then strain over an empty beaker using small
sieve
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Rinse the beads, in the sieve, with running water (tap water will do)
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6
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7
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Mark a line 10cm from the base of each tube
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Pour in the correct concentration of hydrogen peroxide up to the line in each tube
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9
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The bead will sink, but as the catalase in the yeast releases oxygen – you will see the tiny
bubbles – the bead will rise up again
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Repeat with 2 more beads – no need to remove the bead
already in there
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Repeat with fresh beads for all 5 concentrations
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It goes faster each time
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Enzyme concentration
Method:
In this experiment, I added different concentrations of sucrase to a fixed volume of sucrose
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Results:

Conclusion:
As the enzyme concentration increases, the time it takes for the sucrose to break down decreases
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This increases the rate of reaction
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2% Amylase (buffered at optimum pH4)
Iodine in potassium iodide solution
Spotting tiles
Plastic droppers
Test tubes + rack
Marker pen
Stopwatch
1ml, 10ml syringes
Waterbaths at a range of temperatures (eg 20ºC, 40ºC, 60ºC, 80ºC) with test tube racks

Method
1
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2
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In the test tube labelled amylase, place 1ml of amylase, using a 1ml syringe
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Place the 2 test tubes in the first waterbath
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Repeat with pairs of test tubes for all the waterbaths
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Allow about 10 minutes for the starch and amylase to equilibrate with the temperature of
the waterbath
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Place a drop of iodine in each well of a spotting tile
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8
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Start the stopwatch
and mix the contents of the tube by placing your thumb over the top of the tube and
inverting gently
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At 30 second intervals use a plastic pipette to place a drop of the starch/amylase mixture
into one of the wells in the spotting tile
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e
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(You may need more than one tile) Note the time at
which this occurs
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Repeat for all temperatures
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This photo shows that for every 30 seconds the mixture is in the water bath, the colour gets lighter
and lighter
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By counting how many wells it
takes for the mixture to turn orange, you can estimate how long it takes, as it is 30 seconds for each
well
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You will investigate the
effect of pH on the rate of starch breakdown
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You will investigate the rate of reaction pH
4
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0, 6
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0 and 10
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If starch is present then you will see a blue/black colour and the enzyme still has not managed to
break down starch yet
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Equipment:
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Amylase solution
Starch solution
Buffer solution at pH 4
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0, 6
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0 and 10
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2
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4
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6
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8
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10
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12
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Set up your 600ml beaker as a water bath at 30 degrees Celsius
Label a test tube pH 4
Add 2cm3 of amylase solution to this tube
Add 1cm3 of pH buffer solution to this amylase solution
Label a second test tube starch
Add 2cm3 starch solution to the second tube
Put both tubes in your water bath for 30 seconds
Place a single drop of iodine solution into each well on your spotting tile
After 3 minutes, add the starch solution to the amylase and buffer solution and mix
thoroughly
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You must stay next to the water bath for the next part of the practical
After 20 seconds, dip a glass rod into the mixture and then dab it into the first drop of iodine
solution on the spotting tile
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Wipe
the glass rod using a paper towel ready for the next test
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Repeat step 10 until the black/blue colour fails to appear
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If the
blue/black colour is still present when you have used each well of the spotting tile you will

need to quickly wash it out and reload with iodine
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pH
pH4
pH5
pH6
pH7
pH10

Time taken for glucose to be present (seconds)
480+ seconds
20 seconds
20 seconds
20 seconds
480+ seconds

For pH5, 6, and 7, it the time takes for glucose to be present is 20 seconds
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The reaction is much slower
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Evaluation:
If the water is not at 30 degrees Celsius, then you the reactants may not react as efficiently
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To improve this method, look
at the thermometer at eye-level
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To keep the water at 40 degrees, add more hot water to the beaker
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Parallax error may
also occur in this case
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Also, to decrease the rate at which heat is lost, cover and surround the beaker with a thick
layer of cloth, polystyrene, or anything that can stop the heat escaping
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By improving your
techniques, this will results in an accurate and reliable reading
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This can be due to
parallax error
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Also, make sure that there is you have collected no air in the syringe with the
liquid, as by doing so, you would not collect the right amount of liquid
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After this, collect some more liquid to replace the air
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If there are still many air bubbles, get rid of the whole contents
of the syringe and collect the liquid again
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If you use the same syringe for the pH buffer solution and amylase, then the reactants may react too
early
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This means that you can
ensure that the reactants would not react too early
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If you put the liquids in the wrong or same test tube, then you could get an inaccurate reading
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This means that you will By improving your
techniques, this will results in an accurate and reliable reading
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To improve this method, squeeze the pipette slowly and cautiously to ensure
that you only have one drop of iodine solution in each well of you spotting tile
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By improving your techniques, this will
results in an accurate and reliable reading
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To improve this method, dip the glass rod into the solution, no less
than an inch
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Also, after each
dip, wipe your glass rod to ensure that the liquid on the last dip would not affect the latest results
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Unit 13 - Assignment 2 - M3
Temperature
The activity of an enzyme is affected by the environmental conditions it is in
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An organism can adjust the
conditions of the enzymes to produce an optimum rate of reaction
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If you apply heat to a reaction, increases the kinetic energy that the
molecules possess
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Therefore it will

increase the number of collisions in an amount of time
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Furthermore, increasing the temperature has a significant effect
on the number of collisions with enough energy to react
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As enzymes catalyse reactions by colliding with substrate molecules, this
increases the temperature, which then increases the rate of reaction, forming more product
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This
puts strain on the bonds that hold them together
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Breaking bonds within the enzyme will cause the active site to change shape
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This means that it is
less likely to catalyse the reaction
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As temperature increases, more active sites shapes will be less likely to fit to the shape of
their substrate
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Unit 13 - Assignment 2 - D3
The effect of pH on time taken by Amylase to break down starch
The activity of an enzyme is affected by the environmental conditions it is in
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An organism can adjust the
conditions of the enzymes to produce an optimum rate of reaction
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This means that they interfere with hydrogen and ionic bonds
which hold together an enzyme
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This causes a change in shape of the enzyme and its active site
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At the pH value, bonds within them are influenced by H+ and OH- ions
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At
the Optimum pH, the rate of reaction is at an optimum
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Small changes in pH above or
below the Optimum do not cause a permanent change to the enzyme
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But extreme changes in pH can cause enzymes to Denature and permanently lose their
function

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