Enzyme Kinetics Experiment Paper

ENZYME KINETICS

Enzymes, considered the catalysts of biological systems, are remarkable molecular devices that determine the pattern of chemical transformations. Enzymes accelerate reactions and most reactions in biological systems do not occur in the absence of enzymes.

 

Enzyme Kinetics is that branch of enzymology that deals with factors affecting the rates of enzyme-catalyzed reactions. The most important factors include enzyme concentration, ligand concentrations [substrates, products, inhibitors and activators], pH ionic strength and temperature. When all these factors are studied properly, a great deal can be learned about the nature of reactions that involve an enzyme.

 

Experiment Design

 

The experiment is designed to determine the student’s understanding of the principles on enzyme kinetics. These include effects of pH, temperature and substance concentrations on the enzyme. This will also give the student the opportunity to apply knowledge learned in enzyme kinetics.

 

Learning Outcomes

 

At the end of the plenary discussion of the results of the experiments on Enzymes, the student should be able to:

  1. Explain the general characteristics of enzymes
    1. enzymes as proteins
    2. mechanisms of action
    3. specific and relative specificity
    4. characteristics of active site
  2. Explain the effects of the different factors on the rate rate or velocity of an enzyme-catalyzed reaction
    1. nature of substrate
    2. pH
    3. temperature
    4. substrate concentration
  3. Explain the concepts of the following topics related to the experiment:
    1. Assay of alpha amylase
    2. Effects of amylase on the different substrates
    3. Effects of above factors on amylase

4.Demonstrate the effects of substrate concentration by plotting reaction velocity

    1. Michaelis-Menten equation and plot
    2. Lineweaver-Burke equation and plot
    3. Concept of Km or Michaelis-Menten constant
    4. Calculation of Km value

5. Demonstrate the effects of inhibitors on enzyme kinetics 6. Discuss the biomedical importance of enzymes.

 

OBJECTIVE

 

This experiment aims to find out the effect of several factors on the activity of the enzyme alpha amylase. The factors to be considered include:

  1. Nature of the substrate
  2. pH
  3. Temperature
  4. Substrate concentration
  5. Inhibitors

 

PRINCIPLE OF THE ASSAY

This experiment shall make use of alpha amylase as the enzyme. This enzyme is a type of hydrolase that acts on the α(1,4) glycosidic bonds present in starch or similar polymers, yielding glucose.

 

There are several ways to measure enzyme activity. Direct methods include measuring enzyme-substrate binding and catalysis. However, this is not possible in a routine laboratory setting. Hence, for this experiment, we are going to measure enzyme activity indirectly by measuring the amount of product that is formed (in this case, glucose).

 

The enzymatic reaction is allowed to take place in vitro under various experimental conditions, producing glucose. The glucose that is formed is acted upon by glucose oxidase to produce gluconic acid and hydrogen peroxide. Hydrogen peroxide, in turn reacts with a chromogenic oxygen acceptor phenolaminophenazone in the presence of peroxidase to form quinoneimine, which has a peak absorbance of 540nm. The absorbance is then used to compute for the concentration of glucose, which is used to derive the enzyme activity.

 

 

SPECIMEN

Commercially prepared alpha amylase reagent (recombinant).

 

EXPERIMENT 3A: Enzyme Specificity

 

PROCEDURE SUMMARY

  1. A set of 5 test tubes containing 0.5ml of 1% solution of substrates is prepared and labeled from 1 to 5 (Set A).

 

TEST TUBE NO.

SUBSTRATE

1

Starch

2

Glycogen

3

Inulin

4

Dextrin

5

Cellulose

  1. To each of the test tube, 1ml of 0.05 M phosphate buffer at pH 6.7 and 0.5ml of normal saline (0.9% NaCl).
  2. To each of these tubes, 0.5ml of alpha amylase preparation shall be added.

TEST TUBE NO.

SUBSTRATE

Sample

1

Starch

0.5ml amylase

2

Glycogen

0.5ml amylase

3

Inulin

0.5ml amylase

4

Dextrin

0.5ml amylase

5

Cellulose

0.5ml amylase

 

  1. Incubate at 37oC for 15 minutes.
  2. Stop the reaction by placing the test tubes to a boiling water bath.
  3. Cool the solution in ice water bath.
  4. Prepare another set of 5 tubes (set B) and add 1ml of glucose oxidase colorimetric reagent for each tube.
  5. Obtain 50μl of sample from each of the set A tubes then transfer to the corresponding set B tubes containing the glucose reagent.
  6. Measure absorbance of the set B tubes at 540nm using the spectrophotometer.
  7. In this experiment, the values for the enzyme activity shall be computed for you (shall be shown during the virtual experiment session).
  8. Record your results on the table below.

 

Test Tube #

Substrate

Sample

Results

1

Starch

0.5ml amylase

 

2

Glycogen

0.5ml amylase

 

3

Inulin

0.5ml amylase

 

4

Dextrin

0.5ml amylase

 

5

Cellulose

0.5ml amylase

 

 

Research Questions

  1. Based on the results and your knowledge about the chemistry of the substrates used, how does the nature of the substrates affect the activity of the enzyme?

 

 

 

 

 

 

 

 

 

 

 

 

 

  1. Explain why most enzymes are very specific with substrates.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

  1. What do you think would be the biochemical implications of your findings?

 

 

 

 

 

 

 

 

 

 

 

 

EXPERIMENT 3B: Effects of Temperature on Enzyme Activity

 

PROCEDURE Summary

  1. For the next phase of our experiment, we will be testing the effects of temperature on enzyme activity.
  2. A set of 8 tubes shall be prepared, containing the following:
    1. 0.5ml 1% starch solution
    2. 1.0ml of phosphate buffer 6.7 pH
    3. 0.5ml of 0.9% NaCl
  3. Label the tubes with numbers 1A to 8A (Set A tubes).
  4. The tubes shall then be placed in different water baths with the following temperature:

 

Tube #

o

Temp ( C)

1

2

2

8

3

16

4

32

5

40

6

50

7

60

8

80

 

  1. After 10 minutes, 0.5ml of alpha amylase preparation shall be added to the tubes.

Tube #

o

Temp ( C)

Enzyme

1

2

0.5ml amylase

2

8

0.5ml amylase

3

16

0.5ml amylase

4

32

0.5ml amylase

5

40

0.5ml amylase

6

50

0.5ml amylase

7

60

0.5ml amylase

8

80

0.5ml amylase

 

  1. The reaction shall be allowed to run for 15 minutes.
  2. After the 15-minute incubation, all test tubes shall be transferred to the boiling water bath for 5 minutes.
  3. Prepare a second set of 8 tubes and label from 1 to 8 (Set B).
  4. One ml of the glucose oxidase colorimetric reagent shall be added to these tubes.
  5. Transfer 50μl from each of the set A tubes and transfer them to the corresponding set B tubes.
  6. Measure absorbance of the set B tubes at 540nm using the spectrophotometer.

 

 

 

 

  1. Record your results on the table below.

Tube #

o

Temp ( C)

Enzyme

Activity

1

2

0.5ml amylase

 

2

8

0.5ml amylase

 

3

16

0.5ml amylase

 

4

32

0.5ml amylase

 

5

40

0.5ml amylase

 

6

50

0.5ml amylase

 

7

60

0.5ml amylase

 

8

80

0.5ml amylase

 

  1. Using a mathematics (graphing) paper, prepare a line graph using the data from the previous slide. The graph should use the following data as coordinates of the points in the line graph:
    1. Temperature (x axis)
    2. Enzyme activity (y axis)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Research Questions

  1. Describe the shape of the curve produced by plotting the data obtained from this phase of the experiment.

 

 

 

 

 

 

 

 

 

 

 

 

 

  1. How does temperature affects the activity of enzymes?

 

 

 

 

 

 

 

 

 

 

 

 

 

  1. What could be the possible molecular explanation for these findings?

 

 

 

 

 

 

 

 

 

 

 

 

 

  1. What do you think would be the biomedical implications of these findings for enzymes in general?

 

 

 

 

 

 

 

 

 

 

 

 

 

 

EXPERIMENT 3D: Effects of pH on Enzyme Activity

PROCEDURE SUMMARY

  1. For the next phase of our experiment, we will be investigating the effects of pH on enzyme activity.
  2. A set of 13 tubes shall be prepared, containing the following:
    1. 0.5ml 1% starch solution
    2. 0.5ml of 0.9% NaCl
  3. Label the tubes with numbers 1A to 13A (Set A tubes).
  4. To each of these tubes, 1ml of buffer ranging from 3 to 9 shall be added as shown in the table below.

 

Tube #

pH

Enzyme

1

3.0

0.5ml amylase

2

3.5

0.5ml amylase

3

4.0

0.5ml amylase

4

4.5

0.5ml amylase

5

5.0

0.5ml amylase

6

5.5

0.5ml amylase

7

6.0

0.5ml amylase

8

6.5

0.5ml amylase

9

7.0

0.5ml amylase

10

7.5

0.5ml amylase

11

8.0

0.5ml amylase

12

8.5

0.5ml amylase

13

9.0

0.5ml amylase

 

  1. To each of these tubes, 0.5ml of alpha amylase preparation shall be added.
  2. Incubate the tubes at a 37oC water bath for 15 minutes.
  3. Stop the reaction by placing all the tubes in the boiling water bath.
  4. Cool the test tubes to room temperature.
  5. Prepare a second set of 13 tubes and label from 1 to 8 (Set B).
  6. One ml of the glucose oxidase colorimetric reagent shall be added to these tubes.
  7. Transfer 50μl from each of the set A tubes and transfer them to the corresponding set B tubes.
  8. Measure absorbance of the set B tubes at 540nm using the spectrophotometer.
  9. Record your results on the table provided below.

Tube #

pH

Enzyme

Activity

1

3.0

0.5ml amylase

 

2

3.5

0.5ml amylase

 

3

4.0

0.5ml amylase

 

4

4.5

0.5ml amylase

 

5

5.0

0.5ml amylase

 

6

5.5

0.5ml amylase

 

7

6.0

0.5ml amylase

 

8

6.5

0.5ml amylase

 

9

7.0

0.5ml amylase

 

10

7.5

0.5ml amylase

 

11

8.0

0.5ml amylase

 

12

8.5

0.5ml amylase

 

13

9.0

0.5ml amylase

 

 

 

 

 

 

  1. Using a mathematics (graphing) paper, prepare a line graph using the data from the previous slide. The graph should use the following data as coordinates of the points in the line graph:
    1. pH (x axis)
    2. Enzyme activity (y axis)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Research Questions

  1. Describe the shape of the curve produced by plotting the data obtained from this phase of the experiment.

 

 

 

 

 

 

 

 

 

 

 

  1. How does pH affects the activity of enzymes?

 

 

 

 

 

 

 

 

 

 

 

  1. What could be the possible molecular explanation for these findings?

 

 

 

 

 

 

 

 

 

 

 

  1. What do you think would be the biomedical implications of these findings for enzymes in general?

 

 

 

 

 

 

 

 

 

 

 

 

 

EXPERIMENT 3E: Effects of Substrate Concentration on Enzyme Activity SCENARIO:

    1. As part of your requirement for research, you were tasked to determine the kinetic parameters of a recently isolated enzyme.
    2. Your task is to determine the Km and Vmax of the novel enzyme given the following data that you obtained after allowing the reaction to run at different substrate concentrations.
    3. The results of the experiment are provided in the table below.

Substrate (μM)

-1

Enzyme Velocity (nmol•min )

0.20

1.43

0.26

1.67

0.33

2.08

1.00

3.33

    1. Prepare the Michaelis-Menten plot for this series of enzyme reactions.

 

 

 

 

 

 

 

 

 

 

    1. Prepare the Lineweaver-Burke Plot for this series of reaction by computing for the inverse of the substrate concentration and reaction velocity. Plot the values using a mathematics (graphing) paper).

Substrate (μM)

-1

Enzyme Velocity (nmol•min )

S-1

Vo

0.20

1.43

 

 

0.26

1.67

 

 

0.33

2.08

 

 

1.00

3.33

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

    1. Determine by inspection of the graph the values of Km and Vmax.

Km:______

Vmax:______

 

Research Questions

  1. What is the effect of substrate concentration with the enzyme activity?

 

 

 

 

 

 

 

  1. Explain the biomedical implications of your findings regarding the relationship between substrate concentration and enzyme velocity.

 

 

 

 

 

 

EXPERIMENT 3F: Effects of Inhibitors on Enzyme Activity SCENARIO:

  1. A novel cholinesterase inhibitor is currently being investigated as a potential pharmacologic agent for the treatment of Alzheimer disease.
  2. You have been tasked to perform preclinical study regarding the drug as a prerequisite prior to clinical trials.
  3. One of your task is to perform an enzyme inhibition study to determine the type of inhibition this drug exerts on acetylcholinesterase.
  4. After running the test, you were able to obtain these results.

 

Substrate (mmol/L)

V (no inhibitor) o

V (25nM inhibitor) o

V (50nM inhibitor) o

0.4

0.2198

0.1634

0.1121

0.6711

0.2896

0.2237

0.1621

1

0.3279

0.2809

0.2083

2

0.4

0.3497

0.2941

 

  1. Given the above data, your tasks shall be:
    1. Prepare a Michaelis-Menten and Lineweaver-Burk plot of the results obtained.
    2. Using the Lineweaver-Burk plot, determine graphically, the Km and Vmax of the uninhibited reaction series and the inhibited reaction series (containing 25nM and 50nM inhibitors).
    3. Visually inspect the line formed in the Lineweaver-Burk plot and determine the type of inhibition involved in this case.
    4. Show your solutions.

 

Research Questions

  1. Enumerate and discuss the different types of enzyme inhibition.
    1. Explain the changes in the enzyme kinetic parameters (Km and Vmax) for each type of inhibition.

 

 

 

 

 

 

 

 

 

 

 

 

 

  1. Explain the biomedical significance of enzyme inhibition.
    1. In what situation would you want to inhibit an enzyme activity and in what situation is enzyme inhibition detrimental to the homeostasis of an organism?

 

 

 

 

 

 

 

 

 

 

 

 

 

  1. Discuss the uses of enzymes and give examples for each:
    1. Targets of pharmacologic agents.
    2. Diagnostic markers.
    3. Research tools.
    4. Therapeutic agents.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

References (cite at least 3 reputable references)

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