15 August 2014

#20.Factors affecting the rate of enzyme-catalysed reactions

These factors are:
- Temperature
- pH
- Enzyme concentration
- Substrate concentration 
- Inhibitor concentration




When an enzyme solution is added to a solution of its substrate, the molecules collide
With time, the quantity of substrate ↓(changed into product) --> frequency of collisions ↓--> rate of reaction gradually ↓.  The reaction rate is fastest at the start of the reaction (substrate concentration is greatest). --> When comparing reaction rates of an enzyme in different circumstances, we should
measure the initial rate of reaction.

1. Temperature
  • As to ↑, kinetic energy of reacting molecules ↑--> ↑ successful collision --> ↑ rate of reaction. 
  • At optimal to enzyme's activity is maximal --> rate is maximal.
  • Above this temperature, H bonds holding enzyme molecule in shape begin to break --> change tertiary structure of the enzyme (denaturation) --> active site is deformed ---> binding of substrate with enzyme --> ↓ rate of reaction. 


Enzymes in the human body generally have an optimum tof about 37oC, organisms that have evolved to live in much higher or lower temperatures may have much higher or lower optimum to.

2. pH
  • Most enzyme molecules only maintain their correct tertiary structure (exhibit maximum activity) within a very narrow pH range. 
  • Optimum pH - is the pH at which an enzyme has maximum activity. Biological buffers help maintain the optimum pH for an enzyme.
  • Changes in pH can make and break intra- and intermolecular bonds, changing the shape of the enzyme and, therefore, its effectiveness.
  • Most enzymes have an optimum pH that falls within the physiological range of 7.0-7.5.
  • Notable exceptions are the digestive enzymes pepsin and trysin:
    pepsin (active in the stomach) - optimum pH of 1.5
    trypsin (active in the small intestine) - optimum pH of 8.0.

3. Enzyme concentration

When there are more substrate than enzyme
  • limiting factor is Enzyme concentration
  • ↑ concentration of enzyme --> ↑ collisions between enzyme and substrate --> rate of the reaction (directly proportional to the enzyme concentration )
Increasing the enzyme concentration beyond a certain point does not change the rate of reaction BECAUSE when there are less substrate than enzyme: 
  • limiting factor is Substrate concentration
  • ↑ concentration of enzyme does NOT ↑ rate of reaction.

 Limiting factor: 
  • Factor that directly affects the rate of reaction at which a process occurs if its quantity is changed. 
  • Its value has to be ↑ in order to ↑ the rate.
4. Substrate concentration 

When there are more enzyme than substrate
  • limiting factor is Substrate concentration 
  • ↑ concentration of substrate  --> ↑ collisions between enzyme and substrate --> rate of the reaction 
Increasing the substrate concentration beyond a certain point does not change the rate of reaction BECAUSE when there are less enzyme than substrate: 
  • limiting factor is Enzyme concentration
  • ↑ concentration of substrate  does NOT ↑ rate of reaction.



5. Inhibitor concentration 

Inhibitor = a substance that slows down the rate at which an enzyme works.

Competitive inhibitors
  • Have similar shape to the enzyme's normal substrate
  • Can fit into the enzyme's active site, preventing the substrate from binding. 
  • The greater the proportion inhibitor : substrate, the more inhibitor molecules (not substrate molecules) will bump into an active site. 
  • Relative concentrations of the inhibitor and the substrate will affect the degree to which a competitive inhibitor slows down a reaction.
Non-competitive inhibitors
  • Have different shape than the substrate.
  • Do not bind to the active site
  • Bind to a different part of the enzyme --> changes the enzyme's shape (including the active site) --> substrate can not bind with enzyme. 
  • Relative concentrations of the inhibitor and the substrate does not affect the degree to which a non-competitive inhibitor slows down a reaction (if you add more substrate, it still won't be able to bind).


Investigating factors affecting the rate of enzyme-catalysed reactions


1. Temperature 

The effect of to on enzyme activity

You can use almost any enzyme reaction for this, such as the action of catalase on H2O2 in the #17 post. You could use the same method of collecting the gas that is described there, but here is another possible method:
  • Set up several conical flasks containing H2O(the same volume and concentration). Stand  each one in a water bath at a particular to. Use at least 5 to over a good range (0-90o C). Better to set up 3 sets of tubes at each t--> mean result for each to.
  • Take a set of test tubes and add the same volume of catalase solution to each one. Stand these in the same set of water baths.
  • Leave all the flasks and tubes to come to the correct to. Check  with a thermometer.
  • Take the first flask with H2O2, dry its base and sides and stand it on a sensitive top-pan balance. Pour in the catalase solution (same to) and immediately take the balance reading. 
  • Record the new balance readings every 30 seconds for about 3 minutes. The readings will  as O2 is given off.
  • Repeat with the solutions kept at other temperatures.
  • Work out the initial rate of each reaction, either taken directly from your readings, or by drawing a graph of mass lost (mass of O2) against time for each to, and then working out the gradient of the graph over the first 30 seconds or 60 seconds of the reaction.
  • Use your results to plot a graph of initial rate of reaction (y-axis) against to. 

   

2. pH


 The effect of pH on enzyme activity
  • You can adapt the method described in the #17 post for investigating the effect of to on the rate of breakdown of H2O2 by catalase.
  • Vary pH by using different buffer solutions added to each enzyme solution.
  • Keep toenzyme concentration, substrate concentration and total volume of reactants the same for all the tubes. 
  • Record, process and display results.





    3. Enzyme concentration


    The effect of enzyme concentration on rate of reaction

    You could use the following method to investigate the effect of enzyme concentration on the rate at which the enzyme catalase converts its substrate H2O2 to H2O and O2 .
    • Prepare a catalase solution as described in #17 post
    • Prepare different dilutions of this solution:

             and so on. The final 'solution' prepared should be 10 cm3 of distilled water.
    • Place each solution into a tube fitted with a gas syringe. Use small tubes --> there is not too much gas in the tube above the liquid, but leave space to add an equal volume of H2O2 solution at the next step. Labell tube with a waterproof marker. Better to prepare 3 sets of these solutions.
    • Place each tube in a water bath at 30oC.
    • Take another set of tubes and add 10 cm3 of H2O2 solution to each one. The concentration of H2O2 must be the same in each tube. Stand these tubes in the same water bath.
    • Leave all the tubes for 5 minutes --> correct to. Add the contents of 1 of the H2O2 tubes to the first enzyme tube. Mix thoroughly. 
    • Measure the volume of gas collected in the gas syringe after 2 minutes. If you are using 3 sets, then repeat using the other 2 tubes containing the same concentration of enzyme.  
    • Do the same for each of the tubes of enzyme. Record the mean volume of gas produced in 2 minutes for each enzyme concentration and plot a line graph to display your results. 
    • Note: if you find that you get measurable volumes of gas sooner than 2 minutes after mixing the enzyme and substrate --> take readings earlier. The closer to the start of the reaction you make the measurements, the better.




    4. Substrate concentration 


    The effect of substrate concentration on enzyme activity

    You can do this in the same way as described for investigating the effect of enzyme concentration, but this time keep the concentration of catalase the same and vary the concentration of hydrogen peroxide.


    Related post:
    Enzimes and reactions
    Following the course of an enzyme-catalysed reaction

     

     Syllabus 2015

    (d) [PA] investigate and explain the effects of temperature, pH, enzyme concentration and substrate concentration on the rate of enzyme-catalysed reactions;

    (e) explain the effects of competitive and non-competitive inhibitors on the rate of enzyme activity;



    Syllabus 2016  - 2018

    Factors that affect enzyme action

    Investigating the effects of factors on enzyme activity gives opportunities for planning and carrying out experiments under  controlled conditions.

    a)   investigate and explain the effects of the following factors on the rate  of enzyme-catalysed reactions:
    •   temperature
    •   pH (using buffer solutions)
    •    enzyme concentration
    •    substrate concentration
    •   inhibitor concentration

    c)   explain the effects of reversible inhibitors,  both competitive and non-competitive, on the rate  of enzyme activity




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