core practical 4: investigate teh effect of temperature,pH, enzyme concentration and substrate concentration on the initial rate of enzyme catalysed reactons

Name the 4 factors that affect the initial rate of enzyme-catalysed reactions

1. Temperature
2. pH
3. Enzyme concentration
4. Substrate concentration

enzymes at low temp

• Lower temperatures either prevent reactions from proceeding or slow them down:

  • Molecules move relatively slow

  • Lower frequency of successful collisions between substrate molecules and active site of enzyme

  • Less frequent enzyme-substrate complex formation

  • Substrate and enzyme collide with less energy, making it less likely for bonds to be formed or broken (stopping the reaction from occurring)

enzymes at hgiehr temp

• Molecules move more quickly

• Higher frequency successful collisions between substrate molecules and active site of enzyme

• More frequent enzyme-substrate complex formation

• Substrate and enzyme collide with more energy, making it more likely for bonds to be formed or broken (allowing the reaction to occur)

what does it man when an enzyme denatures

• Bonds (eg. hydrogen bonds and ionic bonds) holding the enzyme molecule in its precise shape start to break

• This causes the tertiary structure of the protein (ie. the enzyme) to change

• This permanently damages the active site, preventing the substrate from binding

• Denaturation has occurred if the substrate can no longer bind

enzymes at extremes of pH

• Hydrogen and ionic bonds hold the tertiary structure of the protein (ie. the enzyme) together

• Below and above the optimum pH of an enzyme, solutions with an excess of H⁺ ions (acidic solutions) and OH^- ions (alkaline solutions) can cause these bonds to break

• This alters the shape of the active site, which means enzyme-substrate complexes form less easily

• Eventually, enzyme-substrate complexes can no longer form at all

• At this point, complete denaturation of the enzyme has occurred

effect on enzyme concentrationon rate of reactio

• Enzyme concentration affects the rate of reaction

• The higher the enzyme concentration in a reaction mixture, the greater the number of active sites available and the greater the likelihood of enzyme-substrate complex formation

• As long as there is sufficient substrate available, the initial rate of reaction increases linearly with enzyme concentration

• If the amount of substrate is limited, at a certain point any further increase in enzyme concentration will not increase the reaction rate as the amount of substrate becomes a limiting factor

effect of susbtrate concentration on rate of reaction

• The higher the substrate concentration the faster the rate of reaction

• More substrate molecules means more collision between enzyme and substrate so the more likely an active site will be used by a substrate

• The is only the case up until a certain concentration of substrate, at which point a saturation point is said to have been reached

  • At this point all active sites are occupied and increasing the substrate concentration will not affect the rate of the reaction 

• Substrate concentration will decrease over time (if no new substrate is added) 

• The rate of reaction will therefore decrease over time

• This means the initial rate of reaction will be fastest throughout the reaction 

describe how a suitable pH could be decided on experimentally

• carry out the experiment described at constant temp/substrate/enzyme concentration

• at a range of pHs controlled by buffer

• choose the pH which gives a time which is not too short/too long for practicality

Name the enzyme used in investigating the effects of different factors on rate of enzyme-catalysed reaction

trypsin which breaks down milk protein (casin). The opaque white colour of milk is replaced by a clear solution.

How can colorimeter can be used to measure the absorbance of the solution?

More light passes through the transparent and lighter solutions, so a colorimeter can be used to measure the absorbance of the solution which in turn indicates the rate of reaction of the experiment.

what is buffer slution

• Buffer solutions each have a specific pH

• Buffer solutions maintain this specific pH, even if the reaction taking place would otherwise cause the pH of the reaction mixture to change

• A measured volume of the buffer solution is added to the reaction mixture

• This same volume (of each buffer solution being used) should be added for each pH value that is being investigated

The progress of enzyme-catalysed reactions can be investigated by:

• Measuring the rate of formation of a product 

• Measuring the rate of disappearance of a substrate 

Effect of enzyme concentration on the rate of reaction method

1. Add a set volume of hydrogen peroxide solution to a boiling tube

2. Add a set volume of buffer solution to the same boiling tube

3. Invert a full measuring cylinder into a trough of water

4. Place the end of the delivery tube into the open end of the measuring cylinder and attach the other end to a bung

5. Add a set volume of one concentration of catalase to the boiling tube and quickly place the bung into the boiling tube

6. Record the volume of oxygen collected in the measuring cylinder by the water displaced every 10 seconds for 60 seconds

7. Repeat the experiment twice more and calculate the average volume of oxygen produced at each 10 second interval

8. Repeat the whole experiment for the different concentrations of catalase

9. Plot the average volume of gas produced against time for each concentration

10. Compare the initial rate of reaction of each of the concentrations

Explain how optimum temperature can be determined

• find the rate of production of glucose / find the {concentration / eq} of glucose after set time at a (suitable) range of temperatures

• The colorimeter will measure how the absorbance of the starch solution change over a period of time once amylase is added to it

• idea that optimum temperature is the temperature at which the reaction is quickest
  e.g add a known volume of a known concentration of enzyme to the cellulose / add a known mass of cellulose (to a known volume of water)

• idea of equilibration to the appropriate temperature / idea of sugar test / use of Benedict's / keeping pH constant

• This can be repeated for a range of different starch concentrations/temps/pH and a graph of absorbance against time can be plotted

• repeat at narrow range using more concentrations/temp around where optimum thought to be from graph

results of effect of concentration experiment

• As the concentration of catalase increases the volume of oxygen produced would increase

• This is because there would be more available active sites for hydrogen peroxide to use

• The volume of oxygen would plateau out after the initial rate of reaction due to the substrate decreasing, having been converted into the product (oxygen)

Ideally, you would repeat the procedure for each factor several times. Explain why it is important to measure the initial rate of reaction rather than an average rate over a longer period of time.

• substrate not limiting

• As the reaction continues, substrate is used up and product accumulates, which can slow the reaction or inhibit the enzyme. Measuring the average rate over a longer period would therefore not accurately reflect the enzyme's true activity. The initial rate provides a more reliable and consistent comparison between different conditions, as it is measured before any significant changes in substrate or enzyme activity occur.

  
  

describe how to keep sucrose concentration same for all solutions

• dissolve a stated mass of sucrose in stated volume of water/ use a stock solution of sucrose

• use the same volume of this solution for each pH

how initial rate of reaction determined for rate at which sucrose used up

the rate slows down as {sucrose is used up / sucrose becomes limiting} (1)

 so (rates / results} cannot be validly compared (1)

how to measure initial rate of reaction for sucrose beign used up

 measure the mass of {sucrose broken down / products formed} at intervals over time (1)

 plot a graph of mass (y) against time (x) (1)

 find the gradient of the graph at its start (1)

desribe procedure to compare percentage germination of pollen collected

• credit suitable methods used (to observe germination of pollen grains) (1) eg agar plates, filter paper

• sucrose solution added (1)

• suitable time quoted before germination rate assessed (1) > or = 15 minutes < or = 5 days

• control of one relevant abiotic variable (1)

• control of one relevant biotic variable (1)

• view through microscope / count germinated & non-germinated / count germinated & total (and work out percent)

(1) • reference to relevant health and safety issue (1) eg pollen allergy and bee sting