Enzymes

Miss Rochford

Fifth Year Biology

In this topic:

• Sources of Energy

• What are enzymes?

• Enzyme functions and shape

• Role of enzymes in plants and animals

• Effects of pH & temp. on enzyme activity

• Bio-processing

Sources of Energy

Solar Energy

Cellular energy

• Primary source of energy for life on Earth

• Used for photosynthesis

• Energy stored in the bonds of biomolecules (carbohydrates or lipids)

• Used for respiration • Can pass along a food chain

Metabolism

Metabolism: the sum of all of the chemical reactions in an organism

• Enzymes are the driving force of metabolism

• Without enzymes, life would not exist

E

Enzyme: a biological catalyst. It speeds up chemical reactions without being used up or changed

Amylase

Starch molecule Maltose molecules

Enzymes

E

Enzyme: a biological catalyst. It speeds up chemical reactions without being used up or changed

Enzymes

• All enzymes are made of protein

• Most enzymes are globular proteins

• They are folded and have a 3D shape

Enzymes

• Enzymes only work on one type of reaction each

E Product: substance produced as a result of the action of an enzyme

E Substrate: substance enzyme acts on

Starch amylase Maltose

Substrate Enzyme Product

Metabolism Remember:

Anabolism USES energy to build larger

molecules from smaller molecules

Catabolism RELEASES energy by breaking large

molecules into smaller pieces

Catabolic Enzymes

Examples:

Catabolic enzymes: break large molecules into smaller pieces and release energy

Amylase

• Breaks starch into maltose

• Produced by saliva glands and the pancreas

Anabolic Enzymes

Examples:

Anabolic enzymes: use energy to build large molecules from smaller molecules

DNA Polymerase

• Forms and repairs DNA

• Found in almost all living things

Reversibility

• Enzyme reactions are reversible

– They can go in either direction

Active Site and Specificity

• Active sites are ‘specific’ – They will only accept one

substrate or set of substrates

Enzyme active site: where the substrate enters and is changed into a product(s)

Specificity: an enzymes ability to react with only one substrate

Theories of Enzyme Action

Lock and Key Model

Lock and Key Model

• 1894: Emil Fischer

• The theory:

– Enzymes have a rigid shape

– Substrate(s) fits into the active site of the enzyme

(like a key in a lock)

Induced Fit Model

Induced Fit Model

• 1958: Daniel Koshland Jr.

• Favoured by biologists

• The Theory:

– The active site is flexible

– The substrate ‘induces’ the active site to change into the correct shape

Enzyme Inhibitors

• Inhibitors attach to enzymes and destroy their shape

• This is known as denaturing

Nerve Gases

• Some nerve gases are inhibitors that attach to enzymes involved in our nerve transmissions (e.g. Sarin gas)

Cyanide

• Cyanide denatures an enzyme involved in respiration

Beneficial Enzyme Inhibitors

Inhibit nerve transmission enzymes in insects to kill them

Insecticide

Inhibit nerve transmission enzymes to prevent us from feeling pain

Painkillers

Affect bacterial enzyme action causing them to die Antibiotics

Leaving Cert Syllabus

You need to know two environmental conditions that affect enzyme action:

1. pH

2. Temperature

Effect of pH on enzyme activity

pH scale: an indication of how acidic or basic a substance is. It runs from 0 to 14.

Effect of pH on enzyme activity

• All enzymes have a pH at which they work best (optimum pH)

• A slight pH change can have a big effect on an enzyme’s activity

• Most enzymes work best at pH 6 – 8

• Stomach enzymes work best at low pH

Activity 8: To investigate the effect of pH on the rate of catalase activity

(Pg. 65)

Step 1

• Finely chop the celery.

• Weigh 5 g of the chopped celery.

Step 2

Add into a graduated cylinder:

• 20 ml buffer pH 4

• 1 drop wash-up liquid

• 5 g celery

Step 3

Add 2 ml of hydrogen peroxide to a boiling

tube.

Step 4

Put the graduated cylinder and the boiling tube in a

water bath at 25 °C.

Step 5

• Pour the hydrogen peroxide into the graduated cylinder

• record the volume immediately.

Step 6

• Time for 2 minutes

• Record the final volume.

Step 7

Repeat the steps for

different pH buffer solutions

e.g. 6, 7, 9, 10

Table of results:

pH of buffer

Initial volume (ml)

Final volume (ml)

Volume of foam produced

(ml)

Expected Graph

Effect of temperature on enzyme activity

• Why do we keep food in a fridge?

• Lower temp = slower enzyme activity

• Higher temp = faster enzyme activity

• This is to do with the speed of molecular movement

Effect of temperature on enzyme activity

Low Temperature Slows enzyme activity

Higher Temp. Speeds up enzyme activity

Too high a temp. Stops enzyme activity by changing

its shape (denaturing)

Effect of temperature on enzyme activity

Effect of temperature on enzyme activity

Optimum temperature

Human enzymes 37 °C (body temp)

Plant enzymes 20 - 30 °C

Activity 9: To investigate the effect of temperature on the rate of catalase activity

(Pg. 71)

Step 1

• Finely chop the celery.

• Weigh 5 g of the chopped celery.

Step 2

Add into a graduated cylinder:

• 20 ml buffer pH 9

• 1 drop wash-up liquid

• 5 g celery

Step 3

Add 2 ml of hydrogen peroxide to a boiling

tube.

Step 4

• Put the graduated cylinder and the boiling tube in an ice bath.

• Leave until they reach 4 °C

Step 5

• Pour the hydrogen peroxide into the graduated cylinder

• record the volume immediately.

Step 6

• Time for 2 minutes

• Record the final volume.

Step 7

Repeat the steps for

water baths at different

temperatures

e.g. 20°C, 30°C, 45°C, 60°C,

Table of results

Temperature (°C)

Initial volume (ml)

Final volume (ml)

Volume of foam produced (ml)

Expected Graph

Denaturation

How enzymes are denatured:

• High temperatures

• pH values outside the enzyme’s optimum

• Some chemicals

• Radiation

Denatured enzyme: an enzyme that has lost its shape and no longer functions properly

Activity 11: To investigate the effect of heat denaturation on catalase activity

(Pg. 82)

Step 1

• 5 g chopped celery into two boiling tubes

• Water baths at 100°C and 20°C for 10 minutes.

• Remove and cool.

Step 2

Add 20 ml of buffer pH 9 to two graduated

cylinders.

Step 3

Add one drop of washing up liquid to each graduated

cylinder.

Step 4

• Add the 5 g of boiled celery to one cylinder and label ‘A’.

• Add the 5 g of un-boiled celery to the other cylinder and label ‘B’.

Step 5

• Add 2 ml of hydrogen peroxide to two new boiling tubes.

• Place both tubes in the water bath at 25 °C

Step 6

Stand cylinders and boiling tubes

into the water bath until the

desired temperature is

reached.

Step 7

Add the hydrogen peroxide from each boiling tube to the

corresponding graduated cylinder.

Expected result:

Note the presence or absence of foam in each graduated

cylinder.

Table of results

Boiled

enzyme

Un-Boiled

enzyme

Foam present

or absent

Immobilised Enzymes

Immobilised Enzymes

Bioprocessing: The use of enzyme-controlled reactions to produce a product

Bioreactor: a vessel or container in which living cells or their products are used to make a product

Bioprocessing

Traditional Bioprocessing

• Used microorganisms (yeast and bacteria) to produce foodstuffs

Cheese Yoghurt Bread Wine & Beers

Modern Bioprocessing

• Uses purified enzymes

• Produces a vast range of products:

Antibiotics

Vaccines

Food colouring & flavours

Vitamins Enzymes

Perfume

Immobilised Enzymes

• Using freely dissolved enzymes is very wasteful

• They get removed at the end

• Immobilising means they can be used again

Immobilised Enzymes

Immobilised enzymes: enzymes attached to or trapped in an inert insoluble material

Physical Methods

Immobilised Enzymes

Immobilised enzymes: enzymes attached to or trapped in an inert insoluble material

Chemical Methods

Immobilising Techniques

Adsorption • Enzymes physically attached to inactive

supports • E.g.: glass beads, ceramics, cellulose

particles, polymers

Enclosed in a gel

• Gel used: sodium alginate comes from algae Holds enzyme in place Permeable to entry of substrate & exit of products

Advantages of Immobilised Enzymes

• Can be reused

• Cheaper process

• Product doesn’t need to be separated from enzymes

• Increased stability of the enzyme

Use of Immobilised Enzymes

• Expensive sweetener • Use glucose isomerase to convert

cheaper glucose to fructose

Fructose production

• Expensive enzyme that alters penicillin in antibiotic research

• Cheaper to use when immobilised

Penicillin acylase

• Expensive enzyme • Converts lactose to glucose & galactose • Used in soft toffee and caramel

Lactase

Activity 10: To prepare an enzyme immobilisation and examine its application

(Pg. 76)

Step 1

In a beaker:

• 10 ml distilled water

• add 0.4 g sodium alginate

• stir.

Step 2

Separate beaker:

• add 2 g of yeast to 10 ml of distilled water

• Stir

Step 3

Separate large beaker:

• dissolve the calcium chloride in water

Step 4

Add:

• yeast suspension

• to the alginate solution

Step 5

Draw the mixture into a 20 ml

syringe.

Step 6

Height of 10 cm:

• Drop from the syringe into the calcium chloride

• Only one drop at a time.

• Leave to harden for 10 minutes.

Step 7

• Filter the hardened beads through a sieve

• Rinse with water.

Step 8

• Mix 2 g of yeast in 10 ml of distilled water

• Pour into one of the separating funnels

Step 9

Pour the beads into the second funnel.

Step 10

Separate Beaker

• Dissolve 1 g of sucrose in 100 ml of distilled water.

• Pour 50 ml into each separating funnel.

Step 11

Immediately test the

products in the beakers with

glucose strips.

Step 12

Repeat glucose strip test every 2

minutes until glucose

appears in both beakers.

Step 13

• Empty each funnel into the beakers

• Compare the turbidity

Table of results Time

(minutes)

Free yeast –

presence of glucose

Immobilised yeast –

presence of glucose

0

2

4

6

8

10

Free yeast Immobilised yeast

Turbidity of solution

Energy Carriers

ATP

• Found in all organisms

• Most important energy carrier

• Directly supplies energy for metabolism

ATP = Adenosine triphosphate

ATP

• Made of:

Adenine base (also found in DNA)

Ribose: a 5-carbon sugar

3 phosphates

ATP and ADP

• This is an anabolic reaction

• Energy in ATP is stored in the 3rd phosphate bond

ADP = Adenosine diphosphate

Releasing energy from ATP

• When the 3rd phosphate bond is broken:

Energy is released

ADP is formed again

A phosphate is released

Where does this all happen?

Mitochondria Cells

ATP

ADP P

Energy

water

Oxidation and Reduction

Oxidation

Is

Loss of electrons

Reduction

Is

Gaining electrons

OIL RIG

NAD+ and NADP+

• Electrons and protons are really important parts of respiration and photosynthesis

• Protons are represented as H+

NAD+ and NADP+

• When electrons gain energy, H+ are released

• H+ are too unstable to exist on their own

– They get transferred by the energy carrier molecules

NAD+ NADP+

Respiration energy carrier

Photosynthesis energy carrier

NAD+ and NADP+

NAD+ Nicotinamide adenine dinucleotide

NADP+ Nicotinamide adenine dinucleotide

phosphate

NAD+ and NADP+

• Each energy carrier can gain an electron (e-) by reduction

NAD+

NADP+

e-

e-

NAD

NADP

NAD+ and NADP+

• They gain another electron (e-) by reduction

NAD

NADP

e-

e-

NAD-

NADP-

NAD+ and NADP+

• They then attract a Hydrogen Ion (H+) because of their negative charge

NAD-

NADP-

H+

H+

NADH

NADPH

In summary:

In photosynthesis:

In respiration:

To release energy for anabolic reactions:

In respiration:

In photosynthesis:

Chapter 9: Enzymes

DONE!!