• 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=iIW5SPY-vwI

Two types of metabolic pathways

• Anabolic - biosynthesis- require energy

• Catabolic- Breakdown- release energy

Respiration Protein Synthesis

Catabolic Anabolic

Glucose

+

Oxygen

Carbon Dioxide

+

Water

Energy

Energy

ATP

ADP

+

Pi

e.g. Amino Acids

Protein molecule

Energy

Energy

Catabolic – Aerobic Respiration

ENERGY TRANSFER Anabolic – e.g. Protein synthesis

• 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 B

Enzyme A

Intermediate 1

Intermediate 2

Enzyme C

Intermediate 3

Many enzyme controlled steps

pyruvate

Glycogen in animals

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 B

Enzyme A

Intermediate 1

Intermediate 2

Enzyme C

Intermediate 3

Many enzyme controlled steps

pyruvate

Glycogen in animals

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 can be regulated at the level of gene expression

• This means regulation of the production of the enzyme itself

Enzyme action

Control by gene expression

Some proteins are only required at Some proteins are only required at certain times. In order to prevent certain times. In order to prevent resources being wasted, genes can be resources being wasted, genes can be switched on and off.switched on and off.

Lactose is the Lactose is the sugar found in sugar found in milk.milk.

Effect of B-galactosidase on lactoseEffect of B-galactosidase on lactose

It is made from a It is made from a molecule of molecule of glucose joined to a glucose joined to a molecule of molecule of galactose.galactose.

Jacob Monad Hypothesis- Switching genes on and off

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-The enzyme B-galactosidase can be galactosidase can be used to breakdown used to breakdown lactose into its lactose into its component molecules.component molecules.

lactoselactose

B-B-galactosidasegalactosidase

galactosgalactosee

glucoseglucose

E.ColiE.Coli has a has a gene which gene which codes for the codes for the production of B-production of B-galactosidase.galactosidase.

BUT!! It only produces the BUT!! It only produces the enzyme when lactose is enzyme when lactose is present.present.

This is called This is called enzyme induction.enzyme induction.

Operon = Operon =

1 or more structural 1 or more structural genes with a genes with a neighbouring operator neighbouring operator gene.gene.

The operator gene controls The operator gene controls the switching on and off of the switching on and off of the structural gene. the structural gene.

operooperonn

OperatorOperator genegene

structuralstructural genegene

Switching geneson 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=oBwtxdI1zvk

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.controller?action=viewContent&back=topic&contentGUID=64912796-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

Rate ofReaction

Temperature (oC)

As the temperature increases, the reaction rate increases

This is the maximum rate of the reaction (37oC)

This is the optimum temperature.

As the temperature increasesbeyond the optimum,

the active site is altered. Substrate can no longer bind to the enzyme. The enzyme

has been DENATURED

Factors affecting enzyme activity

1. TemperatureAs 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 pHwhere the rate of reaction is maximum

AB C

Enzyme A = amylaseoptimum pH = 7

Enzyme B = pepsinoptimum pH = 2.5

Enzyme C = lipaseoptimum pH = 9.0

Extremes of pH denature the enzyme

Enzymes and pH

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. pHAs 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), non-competitive (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/highersciences/humanbiology/animations/enzymeaction.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)Absorbance

(units)

1 2 0.05 0.950.08

2 2 0.25 0.750.19

3 2 0.5 0.50.25

4 2 0.75 0.250.36

5 2 1.0 00.43

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?

Each cuvette also contained β-galactosidase enzyme.

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

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

This experiment uses iodine solution as an inhibitor. Again each cuvette also contains β-galactosidase enzyme.

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&back=topic&contentGUID=19162597-6771-3752-7de6-0eee0d4f7250

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.mcgraw-hill.com/olcweb/cgi/pluginpop.cgi?it=swf::535::535::/sites/dl/free/0072437316/120070/bio10.swf::Feedback%20Inhibition%20of%20Biochemical%20Pathways

This lab is designed to show end product inhibition in thefollowing reaction

Phenylphthalein phosphate

Phosphatasein 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 increasingconcentrations of phosphate ( the inhibitor)

Mix the Phosphatase/phosphate mixture with the substratephenylphthalein phosphate

Allow each mixture to react for a given time and then stopthe reaction with sodium carbonate

Sodium carbonate in addition to stopping the reaction turnsthe product phenylphthalein pinkSodium carbonate in addition to stopping the reaction turnsthe product phenylphthalein pink

As the concentration of phosphate increases inhibition of thereaction should increase

Increased inhibition means that less phenylphthalein will beproduced

Increased inhibition means that as the phosphate concentrationsincrease the final pink colour will get fainter

We can show the decrease in the pink colour by shining alight beam through the test tube and measuring % transmissionof light.

As the phosphate concentration increases inhibition increasesso the resulting pink colour lessens more light will pass throughthe solution and the % transmission readings will rise

Light beam Low % transmission

If reaction is not inhibited pink colour will be intense and % transmission low.

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:

Phenylphthalein phosphate

Phosphatasein beansprouts Phenylphthalein Phosphate+

The end product phosphate inhibits the enzyme phosphatase

Carrying out the lab

Step 1 - Making up your sodium phosphate solutions100ml of the following concentrations of sodium phosphateneed 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 addedto 100 ml in order to produce each molarity required. Show yourresults 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 apaste 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 beingcareful not to disturb the pellet.

This liquid will be used as the enzyme solution.

Step 3 - Starting the reaction

Add 1 cm3 of enzyme solution to each tube and mix well. To avoid serious cross contaminationwith the stirring rod think about the

order you stir the test tubes.

Add 1 cm3 of the substrate, phenolphthalein phosphate to each tube.

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.

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 willbreak 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 ablue filter.

Analysing your results

1. Note your groups results in an appropriately formatted table.

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

2. Plot a graph of your results and draw a best fit line through the points.

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

d) slow down reactions and are used up in the reaction

c) speed up reactions and are used up in the reaction

b) slow down reactions and remain unchanged

Q3. What is an active site?

a) The place on a substrate where the enzyme binds.

d) The place on an enzyme where the substrate binds.

c) The place on the product where the substrate binds.

b) The place on the substrate where the product binds.

Q4. Which graph shows the effect of temperature on enzyme activity?

a)

d)c)

b)

temperature

temperature

temperature

temperature

activ

ity

activ

ityac

tivity

activ

ity

Q5. What will bind to an active site?

a) All types of substrate molecule.

d) One type of product molecule.c) One type of substrate molecule.

b) All types of product molecule.

Q6. Enzymes will work at …

a) only one pH

d) acidic pHsc) a range of pHs

b) all pHs

Q7. Which of the following is correct?

a) Starch catalase maltose

d) Starch catalase glucosec) Starch amylase glucose

b) Starch amylase maltose

Q9. Which of the following is correct?

a) Amylase is a synthesis enzyme.

Catalase is a breakdown enzyme.

d) Amylase is a synthesis enzyme.

Phosphorylase is a breakdown enzyme.

c) Catalase is a synthesis enzyme.

Amylase is a breakdown enzyme.

b) Phosphorylase is a synthesis enzyme.

Catalase is a breakdown enzyme.

Q10. Which term best describes a denatured enzyme?

a) Its active site has changed shape.

d) Attached to the substrate.c) Working at its fastest rate.

b) Dead.

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.