Enzymes: properties, inhibition & immobilisation

Question 1

Define metabolism

Answer 1

The chemical reactions that take place in you body

Question 2

Define anabolic reactions

Answer 2

Building up molecules e.g. Protein synthesis

Question 3

What type of reactions break molecules down (e.g. Digestion)

Answer 3

Catabolic reactions

Question 4

Define enzymes

Answer 4

Globular proteins that are biological catalysts

Question 5

Name some properties of enzymes

Answer 5

• They sped up reactions without being changed or used up
• they have a high turnover number
• spherical globular shape with hydrophilic R groups making the enzymes soluble

Question 6

What would happen without enzymes?

Answer 6

Enzymes only catalyse reactions that are energetically favourable and would happen anyway but without enzymes reactions in cells would be too slow to be compatible with life

Question 7

Define active site

Answer 7

A small area with a specific 3D shape

Question 8

Explain what an extracellular site of enzyme action is

Answer 8

• some enzymes are secreted from cells by exocytosis and catalyse extracellular reactions
• e.g. analyse made in salivary glands moves down the salivary ducts to the mouths

Question 9

Explain what an intracellular (in solution) site of enzyme action is

Answer 9

• intracellular enzymes act in solution inside cells e.g. Enzymes that catalyse glucose breakdown in glycosis

Question 10

Explain what an intracellular (membrane bound) site of enzyme action is

Answer 10

• intracellular enzymes may be attached to membranes for example on the cristae of mitochondria and grana of chloroplasts

Question 11

How is an enzyme-substrate complex formed?

Answer 11

An enzyme acts on its substrate which it makes temporary bonds at the active site forming and enzyme-substrate complex

When the reaction is complete the products are released leaving the enzyme unchanged and the active site ready to receive another substrate molecule

Question 12

What are the two main models used to describe enzyme action?

Answer 12

Lock and key model & induced fit model

Question 13

Describe the lock and key theory

Answer 13

1) The unique shape of the active site means that an enzyme can only catalyse one type of reaction
2) other molecules with different shapes will not fit
3) enzyme specificity means that an enzyme is specific for its substrate
4) the enzyme is the lock and the substrate is the key with a complementary shape to fit in the enzyme

Question 14

Describe the induced fit theory

Answer 14

• The enzymes shape is altered when it binds to its substrate this suggests that it is flexible not rigid.
• an alternative model, the induced fit theory was proposed suggesting the enzyme shape alters slightly to accommodate the substrate
• e.g Lysozyme

Question 15

What is activation energy?

How do enzymes work with regard to activation energy?

Answer 15

The minimum energy required for molecules to react, breaking existing bonds in the reactants and making new ones

Enzymes work by lowering the activation energy (when a substrate entered the active site of an enzyme the shape of the molecule alters allowing reactions to occur at lower temperatures than in the absence of enzymes

Question 16

Describe and explain the course of enzyme controlled reaction

Answer 16

• when the enzyme and substrate are first mixed together there are many substrate molecules
• both enzyme and substrate molecules are in constant motion and collide
• substrate molecules bind to the active sites of the enzyme molecules. In a successful collision substrate is broken down and product is released
• more active sites are filled with substrate molecules
• the rate of reaction depends on number of free active sites (if all other conditions are optimal and and there is excess substrate
• the enzyme concentration is the limiting factor because it controls the rate of reaction
• as the reaction proceeds there is less substrate and more product. The substrate concentration is the limiting factor because it controls the rate of the reaction
• eventually all the substrate has been used and no more product can be formed so the line plateaus

Question 17

The effect on temperature on enzyme action

Answer 17

• increased temperature increases the kinetic energy of enzyme and substrate molecules
• they have more energy and so collide more often->increasing ROR
• At a certain temperature the reaction rare goes down because their increasing vibration breaks hydrogen bonds changing the tertiary structure
• this alters the shape of the active site and the substrate will not fit
• the enzyme is denatured (a permanent change )

Question 18

The effect of pH on the rate of enzyme action

Answer 18

• most enzymes have an optimum pH at which rate of reaction is highest
• small changes in pH around the optimum cause small irreversible changes in enzyme structure and reduce its activity
• extremes of pH denatures enzymes
  -the charges on the amino acid side
  chains of the enzymes active site are affected by hydrogen or hydroxyl ions

Question 19

What happens to the charges on the amino acid side chains at low pH?

Answer 19

Excess H+ ions are attracted to negative charges and neutralise them

Question 20

What happens to the charges on the amino acids at high pH?

Answer 20

Excess OH- ions neutralise the positive charges

Question 21

How does disruption of charges lead to denaturation?

Answer 21

Disruption of charges disrupts the ionic and hydrogen bonds maintaining the shape of the active site. The shape changes, denaturing the enzyme. No enzyme-substrate complexes form

Question 22

How does substrate concentration affect the rate of enzyme action?

Answer 22

• if the enzyme concentration is constant the rats of reaction increases as the substrate concentration increases
• at low substrate concentration the enzyme molecules have only a few substrate molecules to collide with so the sites are not working to their full capacity
• with more substrate the active sites are filled
• substrate concentration at this point is the limiting factor
• as more substrate is added the active sites beyond occupied
• rate of reaction at a maximum
• substrate concentration no longer a limiting factor

Question 23

How does enzyme concentration affect the rate of enzyme action?

Answer 23

-as the enzyme concentration increases there are more active sites available and therefore the rate of reaction increases

Question 24

define enzyme inhibition

Answer 24

-The decrease in rate of an enzyme controlled action by another molecule (an inhibitor)

Question 25

how do competitive inhibitors work?

Answer 25

-they have complementary molecular shape to the active site (and similar to that of the substrate) so they compete for the active site

Question 26

what enzyme breaks down succinic acid?

Answer 26

succinic dehydrogenase

Question 27

what does succinic dehydrogenase break down succinic acid into?

Answer 27

fumaric acid + 2H

Question 28

How do non-competitive inhibitors work?

Answer 28

• they bind to the enzymes allosteric site (a site other than the active site) so they do not compete with the substrate for the active site
• they affect bonds within the enzyme molecule and alter its overall shape, including that of the active site
• the substrate cannot bind with the active site and no enzyme-substrate complexes form

Question 29

briefly describe and explain the shape of a non-competitive inhibitor graph

Answer 29

• as the inhibitor concentration increases, more enzyme molecules are denatured and so the rate of reaction and the final mass of product decrease

Question 30

Give some examples of non-competitive inhibitors

Answer 30

• Heavy metal ions e.g. lead (Pb2+) and arsenic (As 3+)

Question 31

do all non-competitive inhibitors bind irreversibly?

Answer 31

no, some bind reversibly and some irreversibly

Question 32

Describe (enzyme) immobilisation

Answer 32

When enzymes are fixed, bound or trapped on an inert support or matrix (e.g alginate beads or cellulose microfibrils)

Question 33

how do glass columns work?

Answer 33

1) Substrate is added to the top of the column and
2) As it flows down its molecules bind to the enzyme molecules active sites
3) Both on the bead surface and inside the bead as the substrate molecules diffuse in

Question 34

Advantages of immobilised enzymes

Answer 34

• The enzymes are easily recovered so can be used over and over again–> reducing costs
• The product is not contaminated by the enzyme
• The polymer matrix creates a microenvironment for the enzymes so it is more stable at extremes of temperatures and pH
• Can be used in continuous processes–> the product of one reaction can be used as the substrate of another
• Several enzymes with different optima can be used at the same time

Question 35

Name some methods of immobilising enzymes

Answer 35

• ABSORBTION onto glass beads/clay particles/collagen
• ENTRAPMENT in cellulose mesh/alginate beads
• CHEMICALLY BONDED using glutaraldehyde

Question 36

Disadvantages of immobilising enzymes

Answer 36

• in the absorption process enzymes may become detached
• presence of alginate gel alters the shape the shape of the active site->reducing enzyme activity
• chemically bonding the enzyme is complex and expensive
• contamination is costly because the whole system has to be shut down and the vessel desterilised
• if the enzyme is held within a substance the substrate must first diffuse into the gel which takes time

Question 37

what is a biosensor?

Answer 37

An instrument that can detect a specific molecule in a mixture of molecules (e.g. glucose in blood)

Question 38

How do biosensors measure blood glucose concentration?

Answer 38

The electrode probe which has a specific immobilised enzyme – (glucose oxidase) on a membrane attached, is placed in the blood.

If glucose is present it diffuses through the membrane to the enzyme and forms an enzyme substrate complex – the product gluconic acid is formed

The reaction produces a small electrical current.
This is picked up by the electrode (transducer)
The current is then read by the monitor and the blood glucose concentration is displayed.
Normal levels are between 3.89-5.83 mmol dm-3

Question 39

Advantages of biosensors

Answer 39

Ability to detect very small concentrations of the target substance
They are very accurate
Easy to use
Quick to respond
Versatile and cost effective
They can be linked to data loggers and computers for on-line monitoring
They can be used for continuous monitoring because the immobilised are retained in the matrix/inside the alginate beads
The biological element can be regenerated and reused.

Question 40

Disadvantages of biosensors

Answer 40

Require extensive and expensive research and development from skilled workforce
Require accurate calibration