Factors affecting enzyme reactions Flashcards
What the four factors that affect enzyme activity?
Enzyme concentration, substrate concentration, temperature, pH
How is a control set up in a practical measuring enzyme activity?
Replace the enzyme solution with distilled water or boiled enzyme solution.
How can a colorimeter determine the rate of reaction of trypsin and milk?
As trypsin digests the proteins in milk, the milk becomes more translucent and more light passes through. The decrease in absorbance can be measured by the colorimeter.
Outline the practical procedure used to measure the effect of enzyme concentration on enzyme activity, using trypsin and milk.
- Dilute stock solution of trypsin with distilled water to produce 4
concentrations. - Zero the colorimeter using 2 cm3 of milk suspension and 2 cm3
of distilled water. - To another cuvette, add 2 cm3 of milk suspension and 2 cm3 of the
stock trypsin solution. Mix, place in the colorimeter and measure
absorbance at 15 second intervals for 5 minutes. - Rinse the cuvette with distilled water.
- Repeat step 3 at all trypsin concentrations.
How is the rate of reaction calculated from time?
1/time
What is the effect of enzyme concentration on enzyme activity?
As the enzyme concentration increases, successful collisions to form ES complexes increase, so rate of reaction increases to an optimum.
Beyond the optimum, the rate plateaus as substance concentration becomes limiting.
How is the effect of substrate concentration on enzyme activity investigated?
Prepare a simple dilution of milk/substrate concentrations.
Add each solution to 1cm3 of trypsin of a fixed concentration. Record the absorbance immediately and every 15 seconds for 5 minutes.
What is the effect of substrate concentration on enzyme activity?
Enzyme activity increases initially as substrate concentration increases. Higher substrate concentration results in more successful collisions to form ES complexes.
Beyond a certain substrate concentration, enzyme activity plateaus as all the active sites are saturated and enzyme concentration is limiting.
How is the effect of pH on enzyme activity investigated?
Add fixed volumes of buffer solutions with a range of pH values to 1cm3 of trypsin and 2cm3 of milk, both of a fixed concentration.
Record the absorbance immediately and every 15 seconds for 5 minutes.
What is the effect of pH on enzyme activity?
Enzyme activity is highest at optimum pH.
Above or below, activity decreases as unsuitable pH disrupts the tertiary structure and changes the shape of the active site, causing partial denaturation.
Complete denaturation may happen at extreme pH values.
How is the effect of temperature on trypsin activity measured?
Prepare water baths with a range of temperatures. Place 2cm3 of trypsin solution and 2cm3 of milk in each water bath.
Leave for 5 minutes to allow the solutions to reach the temperature of the water bath.
Mix together. Record the absorbance immediately and every 15 seconds for 5 minutes.
What is the effect of temperature on enzyme activity?
Increasing t° increases enzyme activity to an optimum. Enzyme and substrates gain kinetic energy and move faster, so there are more successful collisions.
Beyond optimum temperature, enzyme activity decreases as the high temperature disrupts the tertiary structure of enzymes and denatures them.
State a hazard and safety precaution involved in this practical.
Students may have allergic reactions to enzymes, so avoid contact with skin and eyes. Wear eye protection.
