Download Higher Human Biology unit 1 section 5 ENZYMES

• 3 Cell metabolism
• (a) Metabolic pathways. Anabolic (energy
requiring) and catabolic (energy releasing)
pathways — can have reversible and
irreversible steps and alternative routes.
Cell Metabolism
• Is the collective term
for all the biochemical
reactions that occur in
a living cell
• Many of these are
steps in a complex
network of connected
and integrated
pathways that are
catalysed by enzymes
Anabolism and catabolism
• https://www.youtube.com/watch?v=iIW5S
PY-vwI
Two types of metabolic pathways
• Anabolic
- biosynthesis
- require energy
• Catabolic
- Breakdown
- release energy
e.g. Amino Acids
Carbon Dioxide
ATP
+
Water
ENERGY TRANSFER
Catabolic – Aerobic
Respiration
Glucose
Energy
Energy
Anabolic – e.g.
Protein synthesis
Energy
+
Oxygen
Respiration
Synthesis
Catabolic
Energy
ADP
Protein molecule
+
Pi
Protein
Anabolic
• Metabolic processes can
have reversible and
irreversible steps An
example of an
irreversible step- the
diffusion of glucose into
the cell which is
converted by an enzyme
into intermediate 1. This
keeps the concentration
of glucose in the cell low
allowing for further
diffusion of glucose into
the cell
glucose
Enzyme A
Glycogen
in animals
Intermediate 1
Enzyme B
Intermediate 2
Enzyme C
Intermediate 3
Many enzyme
controlled steps
pyruvate
Starch
in
plants
• Alternative routes
bypass steps in the
pathway
• Eg steps controlled by
enzyme A,B and C can be
bypassed when glucose
is converted into
sorbitol which then
returns to glycolysis
later in the pathway.
glucose
Enzyme A
Glycogen
in animals
Intermediate 1
Enzyme B
Intermediate 2
Enzyme C
Intermediate 3
Many enzyme
controlled steps
pyruvate
Starch
in
plants
Cell metabolism
• Metabolism describes all biochemical
reactions which occur within a cell.
• Metabolic pathways involve synthesis (building
up of molecules) reactions, termed anabolism,
and breakdown reactions, termed catabolism.
• Synthetic pathways require the input of
energy whereas break down pathways usually
release energy.
• Some pathways can be reversible, others
irreversible.
• Pathways may also have more than one route.
• (i) Control of metabolic pathways — presence or
absence of particular enzymes and the
regulation of the rate of reaction of key enzymes
within the pathway.
• Induced fit and the role of the active site of
enzymes including shape and substrate affinity.
Activation energy. The effects of substrate and
end product concentration on the direction and
rate of enzyme reactions. Enzymes often act in
groups or as multi-enzyme complexes.
• Enzyme induction experiments such as
ONPG and lactose metabolism in E. coli
and PGlo experiments.
Enzymes
• Enzymes are
biological catalysts
which are essential
to the maintenance
of life.
• They form an
enzyme-substrate
complex that
accelerates the rate
of reaction.
Enzyme action
• Enzyme action can be regulated at
the level of gene expression
• This means regulation of the
production of the enzyme itself
Control by gene expression
Some proteins are only required at certain
times. In order to prevent resources
being wasted, genes can be switched on
and off.
Jacob Monad Hypothesis- Switching genes on and off
Effect of B-galactosidase on lactose
Lactose is the
sugar found in
milk.
It is made from a
molecule of glucose
joined to a molecule
of galactose.
Control of metabolic pathways
• Metabolic pathways can be controlled by
switching on or off the gene for the
first enzyme in the pathway.
• If the gene to produce the first enzyme
is switched off, the enzyme is not
produced and the rest of the pathway
stops.
•
The enzyme B-galactosidase
can be used to breakdown
lactose into its component
molecules.
B-galactosidase
lactose
glucose
galactose
E.Coli has a gene
which codes for
the production of
B-galactosidase.
BUT!! It only produces the
enzyme when lactose is
present.
This is called enzyme induction.
Operon =
1 or more structural genes
with a neighbouring
operon
operator gene.
Operator gene
structural gene
The operator gene controls
the switching on and off of
the structural gene.
Switching genes
on and off.
• Some metabolic pathways (e.g. glycolysis
reactions in respiration) operate continuously.
• So the genes which code for these enzymes
are always expressed and ‘switched on’.
• However, other enzymes are only produced
when required by the cell, thereby saving
resources and energy.
The production of the enzyme B –
galactosidase by E. coli bacteria.
• In the absence of lactose (a sugar) the
lactose digesting enzyme ‘B – galactosidase’ is
not produced by the bacteria.
• And here is an animation:http://www.youtube.com/watch?v=oBwt
xdI1zvk
Intra- and extracellular signal
molecules.
• The molecules that effect a cell’s metabolism and
originate from outwith the cell (e.g. lactose) are
called extracellular signal molecules.
Hormones such as
Adrenaline are also
examples of extracellular
signal molecules.

Intra- and extra-cellular signal
molecules.
• Molecules that effect
a cell’s metabolism
and originate from
inside the cell itself
are called
intracellular signal
molecules.
•
Regulation of enzyme pathways can be
controlled by signal molecules which
may be:
a) Intra cellular (found within the cell)
b) Extracellular (found outside the cell)
Enzyme activation energy
Activation energy
• The energy required to break chemical
bonds in the reacting chemicals and to
start the reaction is called the
activation energy.
• Enzymes lower the activation energy
required.
Enzyme Properties
• Enzymes are globular
proteins
• They possess a small
region called the active
site where the reaction
occurs
• Enzymes are specific in
the reaction that they
catalyse
• Enzymes are only required
in small amounts and
remain unchanged at the
end of the reaction
Induced fit animation
• www.chem.ucsb.edu/~molvisual/ABLE/induced_fit/index.
html
•
http://courses.scholar.hw.ac.uk/vle/scholar/session.contr
oller?action=viewContent&back=topic&contentGUID=64
912796-af38-f1ae-e3f7-245e67abcfbc
• Short film:
• http://www.youtube.com/watch?v=ISw0hXK5dLM
Induced Fit
• Made of protein, enzymes possess a region
called the active site where the reaction
occurs. It has a specific shape that is
complementary to the shape of its substrate.
• The enzyme’s active site changes shape very
slightly as the substrate enters it, making the
fit even more precise. This is known as
induced fit.
• Enzymes are not directly involved in the
reaction, therefore they remain unchanged at
the end.
FACTORS AFFECTING ENZYME
ACTIVITY
•
•
•
•
•
Temperature
pH
substrate concentration
enzyme concentration
inhibitors
Temperature and Enzyme Activity
This is the maximum rate of the
reaction (37oC)
This is the optimum temperature.
Rate of
Reaction
As the temperature
increases, the
reaction rate
increases
As the temperature increases
beyond the optimum,
the active site is altered.
Substrate can no longer bind
to the enzyme. The enzyme
has been DENATURED
Temperature (oC)
Factors affecting enzyme activity
1. Temperature
As temperature increases up to the
enzyme’s optimum, rate of reaction
increases. Above the optimum, rate of
reaction dramatically decreases as the
enzyme becomes denatured. This
means that the shape of its active site
is permanently damaged, meaning that
the substrate can no longer fit it.
Enzymes and pH
Each specific enzyme can only work
over a particular range of pH
B
A
Each enzyme has its own optimum pH
where the rate of reaction is maximum
C
Enzyme A = amylase
optimum pH = 7
Enzyme B = pepsin
optimum pH = 2.5
Enzyme C = lipase
optimum pH = 9.0
Extremes of pH denature the enzyme
Bioluminescence
• Watch the bioluminescence film
• Carry out bioluminescence practical to
show the effect of temperature on
enzymes
Phosphatase
• Do the phosphatase experiment to show
effect of pH on enzymes
Factors affecting enzyme activity
2. pH
As pH increases up to the enzyme’s
optimum, rate of reaction increases.
Above the optimum, rate of reaction
dramatically decreases as the enzyme
becomes denatured. This means that
the shape of its active site is
permanently damaged, meaning that
the substrate can no longer fit it.
INCREASING SUBSTRATE
CONCENTRATION
INCREASING SUBSTRATE
CONCENTRATION
• Increasing
substrate conc
increases rate
of reaction, to a
point, as more
active sites
become
occupied
• Beyond that
point, the conc
of enzyme
becomes
limiting
Factors affecting enzyme activity
3. Substrate concentration (SC)
Increasing SC increases rate of reaction as
there as more active sites become occupied
by substrates. This is only until the point
where all active sites are filled and so rate
of reaction levels off. As there are no more
enzymes to react with more substrates,
enzyme concentration becomes the limiting
factor.
INCREASING ENZYME
CONCENTRATION
• More substrate must
be added to increase
reaction rate
INCREASING ENZYME
CONCENTRATION
• Increasing
enzyme conc
increases rate of
reaction, until
enzyme conc is
large
• Substrate conc is
now the limiting
factor
Factors affecting enzyme activity
4. Enzyme concentration (EC)
Increasing EC increases rate of reaction as
there as more active sites to join with
substrates. This is only until the point where
all substrates are used up and so rate of
reaction levels off. As there are no more
substrates to react with enzymes,
substrate concentration becomes the
limiting factor.
Metabolic Pathways and Enzymes
• A metabolic pathway usually involves
a group of enzymes
• Some enzymes are associated with
other enzymes involved in a
particular pathway to form
multienzyme complexes
• In reality, DNA polymerase isn’t just a
single enzyme. Rather, it is a
massive multi-enzyme complex
possessed of multiple catalytic
activities
• DNA polymerase and RNA
polymerase form part of multi
enzyme complexes
Multi enzyme complexes
• Metabolic pathways often involve a
group of enzymes not just one. These
are called multi enzyme complexes.
• Activation energy experiments, comparing
heat, manganese dioxide and catalase
action on hydrogen peroxide.
• Experiments on reaction rate with
increasing substrate concentration.
• DNA and RNA polymerases are part of
multi-enzyme complexes.
• Control of metabolic pathways through
competitive (binds to active site), noncompetitive (changes shape of active site)
and feedback inhibition (end product binds
to an enzyme that catalyses a reaction
early in the pathway).
Inhibitors
Enzyme regulation animation
• http://www.educationscotland.gov.uk/highe
rsciences/humanbiology/animations/enzy
meaction.asp
The experiment
This experiment uses the enzyme β-galactosidase. Its normal
substrate is lactose but a synthetic substrate, ONPG, is used
instead. When the enzyme is active, it breaks down the ONPG to
a yellow substance. Thus, the rate of reaction is proportional to
the intensity of the yellow colour formed.
β-galactosidase
ONPG
yellow substance + galactose
(ONP)
The intensity of the yellow colour can be measured using a
colorimeter. The higher the absorbance recorded the stronger
the colour.
Part 1: Addition of galactose
Cuvette number
20% galactose in
buffer (CM3)
ONPG stock
solution (CM3)
Buffer (CM3)
1
2
0.05
0.95
2
2
0.25
0.75
3
2
0.5
0.5
4
2
0.75
0.25
5
2
1.0
0
Absorbance
(units)
0.08
0.19
0.25
0.36
0.43
Each cuvette also contained β-galactosidase enzyme.
1.
2.
3.
4.
What was the purpose of including cuvette 1 in the experiment?
What was the effect of adding galactose to the rate of reaction?
What was the effect of increasing ONPG concentration on the rate of reaction?
Can you explain what might be happening?
So what is happening?
• This is competitive inhibition.
• The inhibitor molecule resembles the
shape of the substrate, allowing it to bind
to the active site.
• The inhibitor competes with the substrate
for the active site.
• This can be overcome by increasing the
substrate concentration.
Inhibitors
A second type of inhibition
This experiment uses iodine solution as an inhibitor. Again each cuvette also contains βgalactosidase enzyme.
Cuvette number
Iodine solution
(CM3)
ONPG stock
solution (CM3)
Buffer (CM3)
Absorbance
(units)
1
1.0
0.05
1.95
0.13
2
1.0
0.5
1.5
0.12
3
1.0
1.0
1.0
0.13
1. What is the effect of the addition of iodine to the rate of reaction? (Compare it to
cuvette 1 in the previous experiment.)
2. What is the effect of increasing the ONPG concentration on the rate of reaction?
3. Can you provide a theory as to what is happening this time?
So what is happening this time?
• This is called non-competitive inhibition.
• The inhibitor binds to the enzyme at a site
distinct from the active site.
• The binding of the inhibitor molecule causes
the active site to change shape.
• This prevents the substrate from binding.
• The opposite is also possible – the binding of
a molecule changes the shape of the active
site, allowing the substrate to bind.
• http://courses.scholar.hw.ac.uk/vle/scholar/
session.controller?action=viewContent&ba
ck=topic&contentGUID=19162597-67713752-7de6-0eee0d4f7250
Inhibitors
• Competitive inhibitors bind to the active
site and prevent the substrate from
binding.
• Non competitive inhibitors bind to a
point on the enzyme other than the
active site. They alter the shape of the
active site so that the substrate can no
longer fit in.
End-product Inhibition
• When end product D increases in concentration, it can bind to
the first enzyme in the pathway and reduce the efficiency of
conversion of A to B.
End point inhibition animation
• http://highered.mcgrawhill.com/olcweb/cgi/pluginpop.cgi?it=swf::535::535::/sites
/dl/free/0072437316/120070/bio10.swf::Feedback%20In
hibition%20of%20Biochemical%20Pathways
This lab is designed to show end product inhibition in the
following reaction
Phosphatase
Phenylphthalein in beansprouts
phosphate
Phenylphthalein +
Phosphate
The end product phosphate inhibits the enzyme phosphatase
How can we demonstrate this in the lab?
Extract phosphatase from
beansprouts and mix with
increasing
concentrations of
phosphate ( the inhibitor)
Mix the
Phosphatase/phosphate
mixture with the substrate
phenylphthalein phosphate
Allow each mixture to react for a given time and then stop
the reaction with sodium carbonate
Sodium carbonate in addition to stopping the reaction turns
the product phenylphthalein pink
As the concentration of phosphate increases inhibition of the
reaction should increase
Increased inhibition means that less phenylphthalein will be
produced
Increased inhibition means that as the phosphate concentrations
increase the final pink colour will get fainter
We can show the decrease in the pink colour by shining a
light beam through the test tube and measuring % transmission
of light.
If reaction is not inhibited pink colour will be intense and
% transmission low.
Light beam
Low % transmission
As the phosphate concentration increases inhibition increases
so the resulting pink colour lessens more light will pass through
the solution and the % transmission readings will rise
End product inhibition
• In order to control metabolic pathways
the end product of the pathway can
sometimes inhibit the activity of the
first enzyme in the pathway.
• This is called end product inhibition.
• It avoids the excessive production and
build up of the intermediate chemicals
in a pathway.
Enzyme end point inhibition experiment
Aim: to investigate the effect of phosphate
concentration on the inhibition of the enzyme
phosphatase
Theory:
Phosphatase
Phenylphthalein in beansprouts
phosphate
Phenylphthalein +
Phosphate
The end product phosphate inhibits the enzyme phosphatase
Carrying out the lab
Step 1 - Making up your sodium phosphate solutions
100ml of the following concentrations of sodium phosphate
need to be made up - 1M, 0.1M, 0.01M and 0M
Sample calculation
• The molecular weight of sodium phosphate is 138
• A 1 molar solution is produced when 138g are dissolved in 1 litre
of water.
• A 0.1 molar solution is produced when 13.8g are dissolved in 100ml
of water
• A 0.01 molar solution is produced when 1.38g are dissolved in 100ml
of water
Work out what weights of sodium phosphate need to be added
to 100 ml in order to produce each molarity required. Show your
results in a table
Tips - This is a quantitative experiment and requires great accuracy
• Weigh out sodium phosphate to the nearest 0.01g
• Rinse the boat with an eye wash bottle after adding beaker with
80ml of distilled water
• Dissolve thoroughly and add 80ml to 100ml volumetric flask
• Rinse beaker with distilled water into flask until 100ml line is
reached
Step 2 - Extracting phosphatase from beansprouts
Put about 20g of beansprouts in a mortar and grind to a
paste using the pestle or liquidise with a food processor
Filter the liquid through muslin into a clean centrifuge
tube.
Centrifuge at high speed for about five minutes.
Pour the liquid (the supernatant)
into a clean test tube being
careful not to disturb the pellet.
This liquid will be used as the
enzyme solution.
Step 3 - Starting the reaction
Collect 5 boiling tubes in a rack and label them 1 - 5.
Using a syringe add 5 cm3 from beaker 1
(containing plain buffer) to tube 1; then using the same
syringe add 5 cm3 from beaker 2 to tube 2 and continue
this same procedure step wise to beaker 5.
Add 1 cm3 of the substrate,
phenolphthalein phosphate to
each tube.
Add 1 cm3 of enzyme solution to
each tube and mix well.
To avoid serious cross contamination
with the stirring rod think about the
order you stir the test tubes.
Step 4 - Incubating
Incubate all tubes at 30oC for 20 minutes.
Do not incubate for longer.
The phosphate may be a competitive inhibitor.
This means that given sufficient time the enzyme will
break down all the substrate in all the tubes.
Tip - To get everyone’s tubes into the waterbath put your
boiling tubes in beakers and then into the bath.
Remember to fill the beakers with water from the bath
to ensure that they are incubating at the correct
temperature
Step 5 - Stopping the reaction and fixing the colour
Add 5 cm3 of 10% sodium carbonate solution to
each tube and mix as before.
Step 6 - Measuring % transmission
Using water as a blank, measure the intensity of the
pink colour using a colorimeter with a 550nm filter ie a
blue filter.
Analysing your results
1. Note your groups results in an appropriately formatted
table.
2. Plot a graph of your results and draw a best fit line
through the points.
3. Note all groups results in an appropriately formatted
table and calculate ‘average’ % transmission for each
molarity of sodium phosphate
4. Plot a graph of sodium phosphate molarity vs ‘average
% transmission’ and add error bars
Results
Now plot this data as a line graph
Conclusion
As you increase the concentration of
phosphate inhibitor, the percentage
transmission of light also increases. This is
because more light is able to travel through
the solution as less of the product has been
produced.
• Do catechol oxidase experiment
• Now do the T-F enzymes card sort
Poisons…. How do they work?
• Choose one historical event
• Use the websites provided to research
• Present your findings to the class
• Use the case study of poisons sheets to
help you!
Q1. Enzymes…
a) speed up reactions and
remain unchanged
b) slow down reactions and
remain unchanged
c) speed up reactions and are
used up in the reaction
d) slow down reactions and are
used up in the reaction
Q3. What is an active site?
a) The place on a substrate
where the enzyme binds.
b) The place on the substrate
where the product binds.
c) The place on the product
where the substrate binds.
d) The place on an enzyme
where the substrate binds.
Q4. Which graph shows the effect of
temperature on enzyme activity?
a)
activity
activity
b)
temperature
temperature
d)
activity
activity
c)
temperature
temperature
Q5. What will bind to an active site?
a) All types of substrate
molecule.
b) All types of product molecule.
c) One type of substrate
molecule.
d) One type of product molecule.
Q6. Enzymes will work at …
a) only one pH
b) all pHs
c) a range of pHs
d) acidic pHs
Q7. Which of the following is correct?
a) Starch
catalase
c) Starch
amylase
maltose
b) Starch
amylase
maltose
glucose
d) Starch
catalase
glucose
Q9. Which of the following is correct?
a) Amylase is a synthesis enzyme.
Catalase is a breakdown enzyme.
b) Phosphorylase is a synthesis enzyme.
Catalase is a breakdown enzyme.
c) Catalase is a synthesis enzyme.
Amylase is a breakdown enzyme.
d) Amylase is a synthesis enzyme.
Phosphorylase is a breakdown enzyme.
Q10. Which term best describes a
denatured enzyme?
a) Its active site has changed
shape.
b) Dead.
c) Working at its fastest rate.
d) Attached to the substrate.
Revision
• I am… you are……. cards
• Investigate the inhibition of beta
galactosidase by galactose and its
reversal by increasing ONPG
concentration.
• Experiments on product inhibition with
phosphatase.