4. Core Concepts - Biological reactions are regulated by enzymes

Question 1

enzymes

Answer 1

globular proteins

tertiary structure (can have tertiary structure)

synthesised by living cells

can act inside the cell (intercellular enzymes) or can be secreted by cells (extracellular enzymes).

Question 2

active site

Answer 2

a 3D space in the molecule into which specific substrate molecule(s) can fit and bind.

The active site has a specific shape, which is determined by the sequence of amino acids in the polypeptide;

if the sequence of amino acids changes then the active site will change shape, substrate will not bind to the active site because they are no longer complementary.

Question 3

how do enzymes work

Answer 3

substrate and enzyme collide successfully

substrate binds to active site by interactions with R groups/polar atoms of the amino acids in the active site - forms an enzyme substrate complex

Question 4

what does temperature and pH affect in enzymes

Answer 4

temp and pH affects ability of R groups and substrate to form bonds

bonds in substrate are distorted, puts strain on the bonds that are going to be broken, increase chance of breaking

breaking the bonds - brings new atoms in substrates closer together and new bonds can form

Question 5

how do enzymes affect activation energy

Answer 5

When an enzyme-substrate reaction forms,

the activation energy needed for the reaction to take place is reduced

– the reaction takes place faster - the enzyme acts as a biological catalyst.

enzyme is unchanged during the reaction.

Question 6

graph for enzyme activation energy

Answer 6

Question 7

lock and key hypothesis

Answer 7

active site - lock
substrate - key
substrate - is complimentary to active site so can bind

active site - fixed shape, substrate has to collide to form enzyme substrate complex

next - chemical changes take place, substrate molecule digested or combined (forming new products)

enzyme - not affected by reaction, can be reused

Question 8

diagram of lock and key

Answer 8

Question 9

anabolism

Answer 9

two substrate molecuels combined

forms a single product molecule

Question 10

catabolism

Answer 10

breaking down of complex substrate molecules into two or more product molecules

Question 11

induced fit hypothesis

Answer 11

As substrate molecule enters active site

forces attraction between substrate and R groups/polar atoms of amino acids in the active site are formed

This causes - change in shape of active sit , streonger bonds formed with substrate

weakesn bonds in substrate, lowers activation energy is reaction

when products released from substrate, active site returns to original shape

eg with enzyme lysozyme
-enzyme not affected by reaction, can be reused

Question 12

diagram of induced fir hypothesis

Answer 12

Question 13

how do changes in pH affect amino acids

Answer 13

amino acids - contain basic and acidic groups

change of pH changes bonding

causes changes to secondary and tertiary structure of a protein

reduces ability of substrate to bind to side groups of animo acid lining active site

Question 14

how does changes in charge on side groups affect ability of enzymes active site

Answer 14

change in charges on side groups

bonds may not be formed

enzyme may not be able to lower activation energy

enzyme is denatured

Question 15

how do small changes in pH affect enyzmes

Answer 15

cause small reversible changes in enzymes structure -
inactivation

large changes are irreversible – new bonds form that permanently change the 3D shape of the polypeptide chain- denaturation

ionic an dhydrogen bonds are disrupted

Question 16

why do we use a buffer solution when investigating effect of pH on enzymes

Answer 16

buffer - maintians constant pH

Question 17

diagram to show -
how substrate molecules form an enzyme substrate complex via bonding to amino acid side groups in the active site of enzymes

Answer 17

Question 18

how do changes in temperature affect enzymes

Answer 18

temperature increases, particles gain KE, up to optimun temp

below optimum temp, as temp increase enzymes and substrates have more KE, more successful collisions, more enzymes substrate complexes form, as RofR increases

above optimum temp, as temp increase, more energy given to particles, bonds in enzymes vibrate and they break (weak hydrogen bonds are broken first)

then loss of secondary and tertiary structure, 3D shape of active site changes, is denatured

Question 19

diagram to show effect of increased temperature on enzymes

Answer 19

Question 20

amino group

Answer 20

Question 21

hydrogen bond

Answer 21

Question 22

carboxyl group

Answer 22

Question 23

investigating reactions involving enzymes

what can you measure

Answer 23

the disappearance of substrate
the appearance of product

Question 24

During enzyme investigations you decide on the independent variable to change:

Answer 24

pH
temperature
concentration of substrate
concentration of enzyme.

Question 25

during enzyme investigations what are the DVs you can measure

Answer 25

time
volume
mass
absorbance/transmission.

Question 26

enzyme investigations

what can you do with DVs

Answer 26

You can then use these measurements to calculate the rate at which substrate disappears or product is made.

Question 27

how to calculate rate from time

Answer 27

rate = 1 / time

If the dependent variable is a quantity: rate = quantity / time

Question 28

how to caluclate rate from a graph:
rate between two times
initial rate
rate at a particular part

Answer 28

Question 29

why is initial rate always highest

Answer 29

the concentration of substrate is the highest,

so there is the highest frequency of successful collisions

and the highest rate.

Question 30

what happens to rate as reaction progresses

Answer 30

substrate is converted to product

so concentration decreases.

There is a lower frequency of successful collisions

and the rate decreases.

When all the substrate has been converted to product, the rate will decrease to zero.

Question 31

Explain RofR at A

Answer 31

as temperature increases, substrate molecules gain kinetic energy

increased frequency of successful collisions between substrate and enzyme molecules

increased frequency of enzyme-substrate complex formation

rate of reaction therefore increases.

Question 32

Explain RofR at B

Answer 32

at the optimum temperature, the maximum number of enzyme-substrate complexes are forming at the same time

the rate reaches a maximum – called Vmax

at this point substrate will be used up/product will be produced fastest.

Question 33

Explain RofR at C

Answer 33

at temperatures above the optimum, increased kinetic energy causes bonds in the enzyme to vibrate so much that they break

active sites of enzymes change shape and increasing numbers of enzyme molecules become denatured

the frequency of enzyme-substrate complex formation decreases until all enzyme molecules are denatured

and the reaction stops.

Question 34

Explain RofR at A

Answer 34

at the optimum pH, the shape of the active site enables bonds to form successfully with the substrate

greatest frequency of enzyme-substrate complex formation

and highest rate of reaction.

Question 35

Explain RofR at B

Answer 35

in low pH there is a high concentration of H + ions (acid conditions)

more amino groups will have a positive charge so will affect hydrogen and ionic bonding in the protein

this will change the 3D shape of the active site
as the pH becomes more acidic fewer bonds can form between the active site and the substrate molecules

fewer enzyme-substrate complexes form and the rate decreases.

Question 36

Explain RofR at C

Answer 36

at pH values above the optimum, not enough H + ions are present

increasing number of carboxylic acid groups have a negative charge

hydrogen and ionic bonding are affected and the 3D shape of the active site changes making it less able to form bonds with the substrate

as the pH becomes more basic, fewer bonds can form between the active site and the substrate molecules

fewer enzyme-substrate complexes form and the rate decreases.

Question 37

Explain RofR at A

Answer 37

at low substrate concentration, the number of substrate molecules is low and not all the active sites on the enzyme molecules are occupied

as the concentration of substrate increases, there is a greater frequency of enzyme-substrate complex formation and an increase in the rate of reaction

at low substrate concentrations the number of molecules of substrate acts as a limiting factor.

Question 38

Explain RofR at B

Answer 38

at higher substrate concentrations, more of the active sites become occupied at the same time

the frequency of enzyme-substrate complex formation increases at a slower rate

there is a smaller increase in the rate of reaction.

Question 39

Explain RofR at C

Answer 39

because there are a fixed number of active sites, eventually you reach a maximum rate of reaction – Vmax

all the active sites on the enzyme molecules are occupied – the active sites are said to be saturated

adding more substrate cannot cause an increase in the rate of reaction as no more active sites are available

at high substrate concentrations the number of molecules of enzyme acts as a limiting factor

only by adding more enzyme can the Vmax be increased.

Question 40

Explain RofR at A

Answer 40

at low enzyme concentrations, the number of enzyme molecules is low and all the active sites on the enzyme molecules are occupied

as the concentration of enzyme increases, more active sites become available

there is a greater frequency of enzyme-substrate complex formation and an increase in the rate of reaction

at low enzyme concentrations, the number of molecules of enzymes acts as a limiting factor.

Question 41

Explain RofR at B

Answer 41

at higher enzyme concentrations, more active sites become available
substrate concentration is limited

the frequency of enzyme-substrate complex formation increases at a slower rate

there is a smaller increase in the rate of reaction.

Question 42

Explain RofR at C

Answer 42

because there are a fixed number of substrate molecules eventually you reach a maximum rate of reaction – Vmax

adding more enzyme cannot cause an increase in the rate of reaction as no more substrate molecules are available

at high enzyme concentrations, the number of molecules of substrate acts as a limiting factor

only by adding more substrate can the Vmax be increased.

Question 43

Inhibition of enzymes

Answer 43

Inhibition of an enzyme occurs when enzyme action is slowed down or stopped by another substance.

This is needed in cells to control reactions by slowing down or stopping reactions which are no longer needed.

Question 44

Competitive inhibition

Answer 44

This occurs when a substance has a close structural resemblance (is a similar shape) to a substrate molecule

and can bind temporarily to the active site instead of the normal substrate.

This means that the active site is blocked for the substrate so the substrate cannot bind to the active site and there are fewer enzyme-substrate complexes

and the rate of reaction is decreased. This is reversible.

As substrate concentration is increased,

there are fewer inhibitor molecules in proportion to the number of substrate molecules.

Less competition occurs for the active site

and the maximum rate of the enzyme-controlled reaction can be achieved.

Question 45

Diagram of a competitive enzyme inhibitor

Answer 45

Question 46

Non-Competitive Inhibition:

Answer 46

This occurs when a substance has no structural resemblance to a substrate molecule but binds to the enzyme at a point other than the active site. This is called the allosteric site.

This changes the structure/3D shape of the active site. This means that the substrate cannot bind to the active site so fewer enzyme-substrate complexes are made and the rate of reaction is decreased.

Sometimes non-competitive inhibition is reversible, but the rate of reaction is not affected by substrate concentration, i.e. you cannot get back to the maximum rate of reaction by using higher concentrations of substrate.
Some non-competitive inhibitors are non-reversible.

Question 47

Diagram for action of a non-competitive enzyme inhibitor

Answer 47

Question 48

effect of substrate concentration on effect of enzyme inhibitors

Answer 48

Competitive inhibitor:

the Vmax of the enzyme is not affected
by adding increasing concentrations of substrate, Vmax is eventually reached as more active sites become occupied by the substrate rather than the competitive inhibitor.
Non-competitive inhibitor:

Vmax is reduced even at high substrate concentrations
fewer active sites are available as fixed concentration of enzymes.

Question 49

immobilising enzymes

Answer 49

in an inert (non-reactive) substance

eg alginate - a gel membrane

stabalises enzyme, reduces the ability of polypeptide chain to move

changes of temperature and pH have less effect of 3D shape

Question 50

uses of immobilised enzymes in industry

Answer 50

The enzyme can be recovered and reused:
* this reduces costs
* it also means that only small amounts of an enzyme are needed
* the product is also not contaminated by the enzyme
* several enzymes can be used at once each acting on a specific substrate.

Lower/higher temperatures can be used and still have higher yields than using the free enzyme.

An industrial example is the use of immobilised lactase, which is used to produce lactose-free milk:
* the enzyme is immobilised in alginate gel beads
* milk is passed over the beads and the enzymes digest the lactose into glucose and galactose
* the milk is not contaminated by the enzyme and the beads can be used many times.

Question 50

uses of immobilised enzymes in medicine

Answer 50

Because enzymes are specific to a particular substrate, they can be used as biosensors or analytical reagents.

The glucose oxidase electrode is one example of a biosensor that is important for diabetics, as it can detect glucose levels in the blood. The biosensor works as follows:

• the enzyme glucose oxidase is immobilised in a gel
• a small sample of blood is passed over the enzyme
• when glucose in the blood comes into contact with the enzyme, a reaction occurs, which releases energy (chemical)
• the energy released is converted into electrical impulses
• the more energy released, the higher the concentration of glucose in the blood
• a digital display of accurate concentration is available by referring to reference data stored in the processing unit.

Question 50

risk of hydrogen peroxide

Answer 50

Hazard Risk Control Measure
hydrogen peroxide is corrosive can irritate / damage skin and eyes when pouring hydrogen peroxide into test tube wear safety glasses / use a pipette to fill test tubes – don’t pour

Question 50

improving enzymes investigations

Answer 50

no instruction to wash forceps between dipping disc into potato paste / peroxide solution
enzyme / peroxide could be on forceps so reaction could start before timing
improve by washing and drying forceps each time after picking up a paper disc

length of time dipped into potato paste not specified
different amount of potato paste containing catalase absorbed onto different discs
could increase / decrease time
improve by keeping time paper disc held in potato disc the same each time, e.g. 5 seconds.
experiment carried out in the lab
temperature not controlled / could vary
which would change the kinetic energy of the molecules and affect rate
improve by using a thermostatically controlled water bath

Question 51

A student concluded that the CuSO4 was acting as a non-competitive inhibitor.
To what extent do your results agree with this conclusion? Explain your answer.

Answer 51

agree - non-competitive inhibitor
time taken with copper sulphate did not decrease at higher concentration of peroxide
if competitive you would expect time for disc to sink and rise at higher concentrations of peroxide

Question 52

Explain what is meant by an immobilized enzyme

Answer 52

an immobilised enzyme is attached to an inert matrix so cannot move freely.

Question 53

State two advantages of using immobilized enzymes in industry.

Answer 53

Any 2 from:
● the enzyme can be recovered and reused
● can use smaller quantities of enzyme
● the product is also not contaminated by the enzyme
● several enzymes can be used at once each acting on a specific substrate
● lower / higher temperatures pH can be used and still have higher yields than using free enzyme