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ICA
PRACT S
SKILL
L

Factors
Enzyme-Controlled
Affecting Enzyme
Reactions
Activity Enzyme-Controlled
DNA and RNA Reactions PRACT
ICA
SKILL L
S

Science
Now youisn’t
knowall about
what enzymes
words andaretheory,
and howit’s they
also about
work, getting
let’s take
your
a look
pipette
at what
dirtymakes
and making
them tick.
bad smells
Humans(inneed
the
things of
name like
discovery
money andof course)
the newest
. These
mobile
pages phone,
showbutyouenzymes
how to measure
are quitethe
content
rate ofwith
an enzyme-controlled
the right temperature
reaction.
and pH. Nucleotides Join
You Need to be Together
Able to Interpret Graphs
to Form Polynucleotides
of Enzyme-Controlled Reactions
Part of a single
The results of enzyme-controlled reactions are usually shown in line graphs. You mightpolynucleotide interpret the
be asked tostrand
Temperature
You can Measure a Big
hasthe Rate
Influence
of an Enzyme-Controlled
on Enzyme Activity Reaction 1 ) A polynucleotide is a polymer of nucleotides .
graph of an enzyme-controlled reaction in the exam. The graph below shows the release of a product over time:
Both DNA and RNA nucleotides form polynucleotides.
Like any
Here are chemical
two ways reaction, the rate
of measuring the of anofenzyme-controlled
rate an enzyme-controlled reaction increases when the temperature’s increased .
reaction:
21) FirstThe nucleotides
look at the start j oin of up via a condensation reaction 2 Now look at what else the graphs are
More heat means more kinetic energy, so molecules move faster. This makes the enzymes more likely to collide Volume of productofreleased
How one by an enzyme-controlled
1 ) the
Yousubstrate
Can Measure TheFast theofProduct of thealso
Reaction is, Made the graph and comparephosphate group
(see p. 2 ) between the showing you and make comparisons
with molecules. energy these collisions increases which means each collision is more reaction at different temperatures
nucleotide
the rates of and reaction the sugar of another. between the different Ester temperatures.
bond
likely to result in a reaction. But, if the temperature gets too high, the reaction stops.

Volume of product released (cm3 )

50 65 °C 37 °C 25 °C Phosphodiester
Catalase catalyses the breakdown of hydrogen peroxide into water and oxygen. It’s easy to measure the 3) here. This forms E.g. the a phosphodiester
rate of bond (consisting of 37 ˚C the graph has plateaued
Atbond
1 ) volume
The riseof in temperature
oxygen produced makes
and the
to work out how fast it’s given off. The diagram below shows the apparatus the phosphate
reaction is fastest group at and two40 ester bonds). (flattened out) because all the
enzyme’s
you’ll need. molecules
The oxygen vibrate more
released .
displaces the water from the measuring cylinder. (A stand and clamp would 4) 65 TheoC. chain Useofwhat sugars you and phosphates is known substrate has been used up.
30 Sugar-phosphate
2 ) also be pretty
If the useful goes
temperature to holdabove a certainupside down, as would a stopwatch and a water bath.)
the cylinder know
as the about factors
sugar-phosphate backbone. At 65 ˚C the graph has plateaued
backbone
Here’s
level,howthis to carry out
vibration the experiment:
breaks some of the | | | | | | cylinder
upside down measuring | | | | | | | |
| | | | | | | |
affecting enzyme 20 earlier than at 37 ˚C, because the
| |

|
Every enzy me has an | | activity to explain why

| | | | | | |
1 ) bonds
Set up that hold the
boiling tubesenzyme in shape.
containing the same volume high temperature caused the enzyme

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volume
optimum temperature of
DNA (see is p. Made Two Polynucleotide
of might Chains in a Double-Helix Structure

| | | | | | |
concentration of hydrogen 1 2 ). You 10
3) The andactive site changes shape andperoxide .
the enzyme delivery tube
For mooxygen produced . to denature, so the reaction stopped
To keep the pH constant, add equal volumes st human enzy mes have to work out the sooner. Not as much product was
and substrate no longer fit together. it’s around 37 °C isbu
per minute 0

| | | | | | | | | | |
of a suitable buffer solution to each tube. boiling tube t some Two DNA
1 ) initial rate of polynucleotide
reaction strands 0 j oin 1 0 together
20 30 by 40 50 60 made because not all the substrate
enzy memeasured Two j oined polynucleotide strands

| | | | | | | |
4) At this point, the enzyme is denatured s, like those used in hydrogen
(see below).bonding between the bases.
(A buffer solution is able to resist changes in pH biolog ical washing po Time (s) was converted to product before the
— it no longer functions as a catalyst. wders, 3 hydrogen bonds
when small amounts of acid or alkali are added.) bung can work well at 60 2 )| | | Each
| | | | | |base
| | | | | can only j oin with one particular partner — this is called
| | | | | | | | | | | | enzyme was denatured, so there is
trough of°C. The graph in yourbase
complementary
| | | | | | | | |

exampairing
could be (or specific base | | pairing).

| | |
|
still substrate left. C G
| | | | | | | | | | | |

| |
2 ) Set up the rest of the apparatus as shown
| | | | | | |
| | | | | | | |
based on any
|
| | | | | | | | | |
water var

| | | | | | | | | | |
iab le

| | | | | | |
3) Adenine always — e.g. pH , tem peratu
pairs with thymine (A trat
- T) and cytosine At 25always
˚C thepairs
pH Also in the diagram.
Affects Enzyme Activity hydrogen peroxide solution
or substrate concentration
with guanine (C - G ). . This
re, enz
You’llmeans
yme concen
that there
ion
are always equal
rate of reaction is remaining
amounts
constant and
2 hydrogen
3) Put each boiling tube in a water bath set to a and catalase enzyme
knowledge enzymes to hav e to use you r the volume of product is continuing to increase
bonds because
of adenineofand thymine expin lainawhaDNA t’s goi ng on. and equal
molecule not amounts
all of the substrate has been used up. A T

| |
different temperature (e.g. 1 0 °C, All 2enzymes
0 °C, 30have
°C and
an 40 °C) alongpH with
valueanother
. Most tube enzymescatalase
containing | | | | |
optimum human
| | | | | | | | | |
| | | | | | | | | | |

of cytosine and guanine.

| |
| | | | | | | | | | | |
| | | | | | | | | The two
(wait 5 minutes before movingwork onto best
the next step so the enzyme gets up to temperature).
at pH 7 (neutral), but there are exceptions. Pepsin, strands are antiparallel
4) Use a pipette to add the same for
volume and concentration
example, of catalase
works best at acidic pH 2 ,towhich
each is useful because 4) Two hydrogen bonds form between A and T, (they run in opposite directions)
and three hydrogen bonds form between C and G .
boiling tube. Then quickly attach the bung
it’s found and
in the delivery Above
stomach. tube. and below | the| | | | | optimum
| | | | | | | | | |pH,
| | | | | | | | | | | | |
| You Can Use a Tangent to Calculate the Initial Rate of Reaction
the H +
and OH –
ions found in acids and alkalis A negacantive mess contr up ol the
reaction, i.e. 5) Two antiparallel (running in opposite directions) polynucleotide strands
| | | | | | | | | | |
5) Record how much oxygen is produced in the first minute (60 s)

| | | | | | | | | | |
ionic
of the reaction. Use a stopwatch bonds and
to measure thehydrogen
time. bonds that hold thea enzyme’sboiling tubetertiary not containing twistrate
The initial to form the DNA
of reaction double-helix
is the .
rate of reaction right at the start of the reaction, close to time equals zero (t = 0)
DNA Double-Helix
structure in place. This makes the active site catalase, shou
change shape, ld also so be carried on6)
the DNA
graph.wasTo first
work out the initial rate of reaction carry out the following steps:
6) Repeat the experiment at each temperature three times, and use the observed in the 1 800s, but lots of scientists at the time
out at each temperature.
the enzyme is denatured . doubted
results to find an average volume of oxygen produced . Volumethat it could carry an genetic
by the 1 ) code
Drawbecause it has
a tangent a relatively
to the curve at t = 0, using a ruler. Do this by
|
| | | | | | | | | |
of product released
| | |
| | | | | | | | | | | | | | |

simple chemical composition.

enzyme-controlled reaction at 37 °C Some argued that genetic information
positioning the ruler so it’s an equal distance from the Antiparallel
curve
7) Calculate the average rate of reaction at each temperature by dividing
50 must be carried by proteins — which are at much moreofchemically
where it’s varied . it. Here you’ll have

Volume of product released (cm )

polynucleotide strands
both sides touching to

3
the volume of oxygen produced by the time taken (i.e. 60 s). The units will be cm 3 s-1 .
Enzyme Concentration Affects the Rate of Reaction 7) By 1 953, experiments had shown that DNA was where
estimate the carrier of the would
the curve geneticcontinue if it carried
Hydrogenon below
bonds between
40 code. This was also the year in which the double-helix structure, which bases, keeping the strands
zero. Then draw a line along the ruler. (For more on drawing
Bases coiled together
How Fast helps DNA to carry out its function, wastangents
determined by2Watson
see p. 2 8.) and Crick.
12)
) YouThe moreCan Measure
enzyme molecules there areSubstrate
the in a solution,is Broken Down 30
the more likely a substrate molecule is to collide with one and form 2) Then calculate the gradient of the tangent — this is the initial
an enzyme-substrate complex. So increasing the concentration The enzyme of amylase catalyses the breakdown RNA
20 is a Relatively Short Polynucleotide Chain Gradient = change in y axis ÷ change in x axis
rate of reaction.
the enzyme
mixture sampled increases the rate of reaction. of starch to maltose. The diagram shows how In this graph it’s: 40 cm 3 ÷ 8 s = 5 cm 3 s-1
RNA1 0 is made from a single polynucleotide chain (not a double one). PRA
QUE CTICE
2 ) But,each minute
if the amount of substrate is limited , there comes athe point when can be set up. You’ll need the
experiment ||
|| | | | | | | | | | | | | | | | |
It’s much shorter than most DNA polynucleotides. | | | | | | | STIO
NS
If you’re comparing the

Percentage of Bases in
there’s more than enough enzyme molecules to deal with apparatus
all the shown in the diagram as well as a

| | | | | | | | | | | | | | | |

| |
dropping pipette 0 50

DNA Sample (%)

| | | | | | | | | | | | |
test tube
available substrate, so adding more enzyme has no further stopwatch
effect.. A drop of iodine in potassium iodide 0 1 0 20 30 40 50 60 initia l rate of react ion for
drop of iodine 40 40
is put into each well on a spotting tile. A known two different reactions, you
Warm-Up Questions Time (s)

Volume of oxygen
starch solution in potassium iodide can work out the ratio P R ACT 30 37 oC
concentration of amylase and starch are then QU of

released (cm3 )
IC
and amylase the rates to give you EaSTIONE 30
mixed together in a test tube. A dropping pipette Q1 Name the bases in RNA. S 20
Substrate enzyme Concentration Affects the Rate of Reaction Up to a Point quick and easy comparison. 65 oC
spotting tile is used to put a drop of this mixture into one of the 20

| |
10
Warm-Up Question
|
| | | | | | | | | | | | | | | | | | | | | | | | |

wells containing the iodine solution on the spotting Exam Questions

tile at regular intervals and the resulting colour is observed. The iodine solution thedark
goes blue-black when

Guanine
Cytosine
10

Thymine
1 ) The higher the substrate concentration, the faster reaction — more

Adenine
steady increase Q1 You
Q1 The are
bar testing the effects
chart shows of pH onofthe
the percentage the action
bases inofaan enzyme.
DNA sample
starch is present but remains its normal
as more substrate browny-orange colour when there’s no starch around.
substrate molecules means a collision between substrate and enzyme is You can What other variables must you keep constant?
that are adenine and cytosine. On the chart, sketch bars to show the
see howarefast amylase is working by
molecules recording
more howso
likely and long it takes
more activefor thewill
sites iodine solution
be used. This noonly
to is longer
trueturn
up until a 0
percentages of thymine and guanine in the sample. [2 marks]
Rate of Reaction

available
blue-black when starch/amylase mixture ‘saturation’ point Repeat
is added. though.the experiment
After using
that, there different
are so concentrations
many substrate of
molecules Exam Question 0 10 20 30 40 50 60
Time (s)
amylase. all Makeactive sure that you alsothat
sites used repeat
the the experiment
enzymes three times
have about as muchat each amylase
as they can copeconcentration.
with Q2 a) Describe how nucleotides are j oined together in DNA.o [3 marks]
— increase in substrate Q1 A student carries out an enzyme-controlled reaction at 37 C and 65 oC. Her results are shown in the graph above.
concentration has no (all the active sites are full), and adding more makes no difference. b) Describe
Draw how
a tangent two single
to find polynucleotide
the initial strands
rate of reaction areoC.
at 65 j oined
Showto your
makeworking.
a double helix. [3 mark]
[1 marks]
The experiments above show you how you can investigate the effects of temperature and enzyme concentration on
further effect
the rate of enzyme-controlled reactions. 2 ) Substrate
You can concentration
also alter thesedecreases with time
experiments during a reaction
to investigate the effect(unless more
of a different
substrate is added to the reaction mixture), so
variable, such as pH (by adding a buffer solution with a different pH to each test tube) or substrate concentration if no other variables are Give
My rate
meof
a D,
reaction
give me
depends
an N, give
on what
me an
time
A! What
of day do
it is...
you get? — confused...
(you could serial dilutions to make
useConcentration changed,
substratethe rate of reaction
solutions will decrease
with different over timeThe
concentrations). too.keyThis makes the
to experiments
Substrate You
In your
needexam,
to learn
you the
could
structure
get asked
of DNA
about
—methods
the polynucleotide
used to measure
strands,
thethe
rate
hydrogen
of an enzyme-controlled
bonds, and don’treaction
forget or to
initial rate of reaction (the
like this is to remember to only change one variable — everything else should stay the same. reaction rate at the start ) the highest rate of reaction.
complementary
calculate the ratebase
frompairing.
a graph.Make
It’s worth
sure you
your
know
timethe
to memorise
differencesthe
between
examples
RNA and
and
learn
DNAthetoo
maths
— interesting
on these pages.
stuff.

Topic 1 A — Biological Molecules Topic Topic

1 B — 1More
A — Biological Molecules

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