1: Investigate a factor affecting the initial rate of an enzyme-controlled reaction.

Question 1

What is a catalyst?

Answer 1

A chemical which speeds up reaction rates, remains unchanged at the end and so is re-usable.

Often, only a small number of catalysts are needed to turn over many reactions.

Question 2

How can the rate of an enzyme controlled reaction be measured?

Answer 2

The rate of an enzyme-controlled reaction can be calculated by measuring:

• Increase inn produce
• Decrease inn substrate

Proteases:
the time taken for a cloudy precipitate of a protein to disappear into solution.

Rate of reaction= rate of change of substrate/ product over time.

Question 3

Advantages of using BIOLOGICAL catalysts rather than CHEMICAL catalysts.

Answer 3

-more specific so less by-products.
-use physical forces rather than extreme heat or pH to speed a reaction.
… therefore they can be used in the body.

Question 4

What is the active site?

Answer 4

A pocket/cleft on the enzyme made up of specific amino acids, complementary to a specific substrate.

Question 5

What is the lock and key hypothesis?

Answer 5

1. The tertiary structure of the active site is complementary to the specific substrate.
2. Enzyme holds substrate in place as an Enzyme-Substrate Complex (ESC).
3. Substrate is converted into product via interactions and intermediates, forms an Enzyme-Product-Complex (EPC).
4. Product leaves active site.
5. Can also go in reverse.

Question 6

Factors affecting enzyme activity…

Answer 6

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

Question 7

What is the role of a protease enzyme?

Answer 7

Breaking down the peptide bond between amino acids, through HYDROLYSIS.

Question 8

Background:

Answer 8

Milk protein (Caesin) is broken down by protease enzymes such as trypsin. The white opaque colour of the milk is replaced by a clear solution. Therefore, a COLORIMETER can be used to measure the absorbance of the solution over time.

Question 9

Dependent variable:

Answer 9

The rate of reaction in absorbance units.

Question 10

Independent variable:

Answer 10

The concentration of Trypsin (an enzyme)

or another factor that affects the rate of an enzyme controlled reaction

Question 11

Control variables:

Answer 11

Temperature-control through use of a water bath/ thermometer.
Concentration of substrate
Volume of solution
pH: would effect the shape of the active sight. Enzyme would have the highest ROR at its optimum pH. A BUFFER might be used to maintain pH at a suitable level.

Question 12

Before beginning the experiment what must be done with the colorimeter>

Answer 12

Calibrate the colorimeter with a reference solution, which should be the colour of the trypsin-milk mixture when the casein has broken down.

Question 13

Outline the method:

Answer 13

1. Create five solutions, each of 10cm cubed volume.
   Dilute 1% trypsin stock with distilled water, using pipette, to produce test solutions of:
   0.2%
   0.4%
   0.6%
   0.8%
   (Multiply conc. by total volume to give volume of trypsin required)
2. Place 2cm^3 of trypsin and 2cm^3 of distilled water into a cuvette.
   Use this as a reference to set the colorimeter absorbance to zero.
3. Measure 2cm^3 of milk into a second cuvette.
4. Add 2cm^3 of trypsin solution to the milk in the cuvette. Mix and place the solution into the colorimeter quickly and start the stop clock.
5. Measure absorbance immediately and then at 15 second intervals for five minutes, or until there is little change in absorbance.
6. Rinse the cuvette with distilled water and repeat for each concentration.

Question 14

Risks:

Answer 14

Trypsin solution at conc. of 1% and above is an irritant.

wear eye protection, inform teacher if any trypsin gets in eyes. irrigate for at least 10 m minutes and see a doctor

Question 15

Analysis of results:

Answer 15

• record results in a suitable table.
• Plot a graph of absorbance against time.
• Use the graph to determine the initial rate of reaction for each conc. Do this by drawing a tangent of the initial part of each curve and calculating the gradient of each line.
• Draw a second graph to show the initial rate of reaction against the concentration of the enzyme.

Question 16

Explain the expected results.

Answer 16

The initial rate of reaction should be higher at higher enzyme concentrations. This is because there are more available active sites and therefore more successful collisions will occur between active sites and substrates, reducing the concentration of substrate and so decreasing the absorbance.

As the experiment involves a fixed volume of substrate (protein in the milk) the substrate will quickly become the LIMITING FACTOR (the factor that limits the Rate of Reaction) and the rate of reaction slows. The rate of reaction starts at a high level, before decreasing as the reaction progresses and the substrate is converted to product. This is why it is important to compare the INITIAL rate of reaction, because this is the point where the enzyme concentration is affecting the ROR and where controlled variables such as the substrate concentration are the same for all levels of the independent variable.