Enzymes:

• Amylase - produced by the salivary glands of mammals.
• Pepsin - located in the stomachs of mammals.
• Taq Polymerase - naturally occurring enzyme found in the bacterium Thermus aquaticus.

Substrates:

• Nucleotide - monomer of all nucleic acids (DNA and RNA) which are made of many nucleotides linked together; when many nucleotides are linked together, either DNA or RNA is formed.
• Protein - made up of many amino acids that are linked together by peptide bonds; when a protein breaks down, amino acids are released.
• Lipids - in this case, they are fats that are made up of glycerol and fatty acids; in this case, when a lipid is broken down, fatty acids will be released.
• Starch - polysaccharide (very large carbohydrate) that is made up of many monosaccharides that are linked together; when starch is broken down, monosaccharides are released.

Instructions (if absent on day of lab):

1. For trial 1, set the temperature to 50o C but do not change the pH that is set to 7.
2. Ensure that all four substrates are set to 500 mM (they should be set there by default).
3. Select "Add Amylase" and only “Add Amylase” (do not select the other three enzymes), click "run", and watch the four graphs to the right; the graph that shows a line higher than 0 mM is displaying evidence that the substrate is acted on.

• If the Disaccharides Produced graph shows an upward sloping line, it acts on carbohydrates.
• If the Amino Acids Produced graph shows an upward sloping line, it acts on proteins.
• If the DNA Produced graph shows an upward sloping line, it acts on nucleotides.
• If the Fatty Acids Produced graph shows a flattish line greater than 0 mM, it acts on lipids.

1. Record in the data table, for “Trial 1”, which substrate amylase acted on.
2. Repeat steps 2-4 with “Add Pepsin” at a pH of 2 and a temperature of 50o C.
3. Repeat steps 2-4 with “Add Taq Polymerase” at a temperature of 70o C and a pH of 7.

1. Select “Add Amylase” and set the substrates to these settings:

1. Ensure that the pH is set to 7 and you will set the temperature to 0o C.
2. Click, “Run” and record the disaccharides produced in the data table for trial 2 under Amylase; click “Reset” after you have recorded the data from the graph onto your data table.
3. Increase the temperature to 10o C, click “Run”, and record your data.
4. Continue increasing the temperature by factors of 10o C until the disaccharides produced is 0 mM or until you reach the highest temperature setting (100o C).
5. Once you have recorded all of the data (Trial 2 data table for temperature) for this section, you may click “Reset”.

1. Select “Add Pepsin” and set the substrates to these settings:

1. Ensure that the pH is set to 2 and you will set the temperature to 0o C.
2. Click, “Run” and record the amino acids produced in the data table for trial 2 under Pepsin; click “Reset” after you have recorded the data from the graph onto your data table.
3. Increase the temperature to 10o C, click “Run”, and record your data.
4. Continue increasing the temperature by factors of 10o C until the amino acids produced is 0 mM or until you reach the highest temperature setting (100o C).
5. Once you have recorded all of the data (Trial 2 data table for temperature) for this section, you may click “Reset”.

1. Select “Add Taq Polymerase” and set the substrates to these settings:

1. Ensure that the pH is set to 7 and you will set the temperature to 40o C.
2. Click, “Run” and record the amino acids produced in the data table for trial 2 under Taq Polymerase; click “Reset” after you have recorded the data from the graph onto your data table.
3. Increase the temperature to 10o C, click “Run”, and record your data.
4. Continue increasing the temperature by factors of 10o C until the DNA produced is 0 mM or until you reach the highest temperature setting (100o C).
5. Once you have recorded all of the data (Trial 2 data table for temperature) for this section, you may click “Reset”.

1. Select “Add Amylase” and set the substrates to these settings:

1. Ensure that the temperature to 25o C and that the pH is set to 0.
2. Click, “Run” and record the disaccharides produced in the data table for trial 2 under Amylase; click “Reset” after you have recorded the data from the graph onto your data table.
3. Increase the pH to 0, click “Run”, and record your data.
4. Continue increasing the pH by factors of 1 until the disaccharides produced reads 0 mM or until you hit the highest pH setting (14).
5. Once you have recorded all of the data (Trial 2 Data Table for pH) for this section, you may click “Reset”.

1. Select “Add Pepsin” and set the substrates to these settings:

1. Ensure that the temperature is set to 25o C and that the pH is set to 0.
2. Click, “Run” and record the amino acids produced in the data table for trial 2 under Amylase; click “Reset” after you have recorded the data from the graph onto your data table.
3. Increase the pH to 5, click “Run”, and record your data.
4. Continue increasing the pH by factors of 1 until the amino acids produced reads 0 mM or until you hit the highest pH setting (14).
5. Once you have recorded all of the data (Trial 2 Data Table for pH) for this section, you may click “Reset”.

1. Select “Add Taq Polymerase” and set the substrates to these settings:

1. Ensure that the temperature to 80o C and that the pH is set to 0.
2. Click, “Run” and record the amino acids produced in the data table for trial 2 under Amylase; click “Reset” after you have recorded the data from the graph onto your data table.
3. Increase the pH to 5, click “Run”, and record your data.
4. Continue increasing the pH by factors of 1 until the DNA produced reads 0 mM or until you hit the highest pH setting (14).
5. Once you have recorded all of the data (Trial 2 Data Table for pH) for this section, you may click “Reset”.

Read This After the Lab

Temperature Effects on Enzymes

1. Optimal Temperature: Enzymes have an optimal temperature range where they function most efficiently. Increased kinetic energy within this range enhances enzyme-substrate interactions.
2. Inhibition: Low Temperatures - At temperatures below the optimal range, enzyme activity decreases as molecular motion slows, resulting in fewer collisions with substrates. High Temperatures - Excessive heat can lead to dysfunction without immediate denaturation, reducing reaction rates.
3. Denaturation: At high temperatures, enzymes can denature, meaning their three-dimensional structure is disrupted. This process breaks hydrogen bonds and alters the active site, rendering the enzyme inactive.

pH Effects

1. Optimal pH: Each enzyme has an optimal pH range that maintains its shape and charge properties necessary for binding substrates.
2. Inhibition: Extreme pH Values: Deviations from the optimal pH can inhibit enzyme activity by affecting the ionization of amino acids at the active site, disrupting substrate binding.
3. Denaturation: Extreme pH levels can also lead to denaturation, altering the enzyme's structure and preventing it from functioning properly.

Consequences of Inhibition and Denaturation

• Inhibition decreases the rate of reaction, while denaturation typically results in a complete loss of enzymatic function.
• Irreversibility: Denaturation is generally irreversible, meaning the enzyme cannot regain its functional shape once it has been denatured.

Enzymes:

• Amylase - produced by the salivary glands of mammals.
• Pepsin - located in the stomachs of mammals.
• Taq Polymerase - naturally occurring enzyme found in the bacterium Thermus aquaticus.

Substrates:

• Nucleotide - monomer of all nucleic acids (DNA and RNA) which are made of many nucleotides linked together; when many nucleotides are linked together, either DNA or RNA is formed.
• Protein - made up of many amino acids that are linked together by peptide bonds; when a protein breaks down, amino acids are released.
• Lipids - in this case, they are fats that are made up of glycerol and fatty acids; in this case, when a lipid is broken down, fatty acids will be released.
• Starch - polysaccharide (very large carbohydrate) that is made up of many monosaccharides that are linked together; when starch is broken down, monosaccharides are released.