• 3 Cell metabolism
• (a) Metabolic pathways. Anabolic (energy requiring) and catabolic (energy releasing) pathways — can have reversible and irreversible steps and alternative routes.

• 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

• https://www.youtube.com/watch?v=iIW5S
PY-vwI

• Anabolic
- biosynthesis
- require energy
• Catabolic
- Breakdown
- release energy

Carbon Dioxide
+
Water
Catabolic – Aerobic
Respiration
Glucose
+
Oxygen
Energy
Energy
ENERGY TRANSFER
ATP
ADP
+
Pi
Respiration
Synthesis
Catabolic
Energy
Energy e.g. Amino Acids
Anabolic – e.g.
Protein synthesis
Protein molecule
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
Starch in plants
Enzyme B
Intermediate 2
Enzyme C
Intermediate 3
Many enzyme controlled steps pyruvate

• 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
Starch in plants
Enzyme B
Intermediate 2
Enzyme C
Intermediate 3
Many enzyme controlled steps pyruvate

• 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 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 can be regulated at the
of the production of the enzyme itself

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.

•
• 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.
lactose
B-galactosidase 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 operator gene.
operon
Operator gene structural gene
The operator gene controls the switching on and off of the structural gene.

• 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.

• 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

• 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.

• 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)

• 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.

• 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

•
• 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

• 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.

• Temperature
• pH
• substrate concentration
• enzyme concentration
• inhibitors

This is the maximum rate of the reaction (37 o C)
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 ( o C)

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.

Each specific enzyme can only work over a particular range of pH
Each enzyme has its own optimum pH where the rate of reaction is maximum
B A 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

• Watch the bioluminescence film
• Carry out bioluminescence practical to show the effect of temperature on enzymes

• Do the phosphatase experiment to show effect of pH on enzymes

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 conc increases rate of reaction, to a point, as more active sites become occupied
• Beyond that point, the conc of enzyme becomes limiting

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.

• More substrate must be added to increase reaction rate

• Increasing enzyme conc increases rate of reaction, until enzyme conc is large
• Substrate conc is now the limiting factor

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.

• 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

• 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).

• http://www.educationscotland.gov.uk/highe rsciences/humanbiology/animations/enzy meaction.asp

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.
ONPG
β-galactosidase 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.

Cuvette number 20% galactose in buffer (CM3)
3
4
5
1
2
2
2
2
2
2
ONPG stock solution (CM3)
0.05
0.25
0.5
0.75
1.0
Buffer (CM3)
0.95
0.75
0.5
0.25
0
Absorbance
(units)
0.08
0.19
0.25
0.36
0.43
Each cuvette also contained β-galactosidase enzyme.
1. What was the purpose of including cuvette 1 in the experiment?
2. What was the effect of adding galactose to the rate of reaction?
3. What was the effect of increasing ONPG concentration on the rate of reaction?
4. Can you explain what might be 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.

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
2
3
1.0
1.0
1.0
0.05
0.5
1.0
1.95
1.5
1.0
0.13
0.12
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?

• 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=19162597-6771-
3752-7de6-0eee0d4f7250

• 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 .

• 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
Phenylphthalein phosphate
Phosphatase in beansprouts
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
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

• 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.

Aim: to investigate the effect of phosphate concentration on the inhibition of the enzyme phosphatase
Theory:

Phenylphthalein phosphate
Phosphatase in beansprouts
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 cm 3 from beaker 1
(containing plain buffer) to tube 1; then using the same syringe add 5 cm 3 from beaker 2 to tube 2 and continue this same procedure step wise to beaker 5.
Add 1 cm 3 of the substrate, phenolphthalein phosphate to each tube.
Add 1 cm 3 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 30 o C 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 cm 3 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

Now plot this data as a line graph

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

• 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!

a) speed up reactions and remain unchanged c) speed up reactions and are used up in the reaction b) slow down reactions and remain unchanged d) slow down reactions and are used up in the reaction

a) The place on a substrate where the enzyme binds.
c) The place on the product where the substrate binds.
b) The place on the substrate where the product binds.
d) The place on an enzyme where the substrate binds.

a)
b) temperature temperature c) d) temperature temperature

a) All types of substrate molecule.
c) One type of substrate molecule.
b) All types of product molecule.
d) One type of product molecule.

a) only one pH c) a range of pHs b) all pHs d) acidic pHs

a) Starch catalase maltose b) Starch amylase maltose c) Starch amylase glucose d) Starch catalase glucose

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.

b) Dead.
a) Its active site has changed shape.
c) Working at its fastest rate.
d) Attached to the substrate.

• 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.