4. Core Concepts - Biological reactions are regulated by enzymes Flashcards
enzymes
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).
active site
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.
how is an enzyme substrate complex formed
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
what does temperature and pH affect in enzymes
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
how do enzymes affect activation energy
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.
graph for enzyme activation energy
lock and key hypothesis
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
diagram of lock and key
anabolism
two substrate molecuels combined
forms a single product molecule
catabolism
breaking down of complex substrate molecules into two or more product molecules
induced fit hypothesis
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
diagram of induced fir hypothesis
how do changes in pH affect amino acids
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
how does changes in charge on side groups affect ability of enzymes active site
change in charges on side groups
bonds may not be formed
enzyme may not be able to lower activation energy
enzyme is denatured
how do small changes in pH affect enyzmes
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
why do we use a buffer solution when investigating effect of pH on enzymes
buffer - maintians constant pH
diagram to show -
how substrate molecules form an enzyme substrate complex via bonding to amino acid side groups in the active site of enzymes
how do changes in temperature affect enzymes
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
diagram to show effect of increased temperature on enzymes
amino group
hydrogen bond
carboxyl group
investigating reactions involving enzymes
what can you measure
the disappearance of substrate
the appearance of product
During enzyme investigations you decide on the independent variable to change:
pH
temperature
concentration of substrate
concentration of enzyme.
during enzyme investigations what are the DVs you can measure
time
volume
mass
absorbance/transmission.
enzyme investigations
what can you do with DVs
You can then use these measurements to calculate the rate at which substrate disappears or product is made.
how to calculate rate from time
rate = 1 / time
If the dependent variable is a quantity: rate = quantity / time
how to caluclate rate from a graph:
rate between two times
initial rate
rate at a particular part
why is initial rate always highest
the concentration of substrate is the highest,
so there is the highest frequency of successful collisions
and the highest rate.
what happens to rate as reaction progresses
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.
Explain RofR at A
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.
Explain RofR at B
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.
Explain RofR at C
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.
Explain RofR at A
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.
Explain RofR at B
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.
Explain RofR at C
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.
Explain RofR at A
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.
Explain RofR at B
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.
Explain RofR at C
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.
Explain RofR at A
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.
Explain RofR at B
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.
Explain RofR at C
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.
Inhibition of enzymes
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.
Competitive inhibition
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.
Diagram of a competitive enzyme inhibitor
Eg competitive inhibitor
Non-Competitive Inhibition:
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.
Diagram for action of a non-competitive enzyme inhibitor
effect of substrate concentration on effect of enzyme inhibitors
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.
immobilising enzymes
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
uses of immobilised enzymes in industry
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.
uses of immobilised enzymes in medicine
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.
risk of hydrogen peroxide
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
improving enzymes investigations
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
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.
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
Explain what is meant by an immobilized enzyme
an immobilised enzyme is attached to an inert matrix so cannot move freely.
State two advantages of using immobilized enzymes in industry.
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
