Lab 7: Enzymes and Amylase Activity

Question 1

Define enzymes.

Answer 1

Enzymes are biological catalysts that speed up chemical reactions.

Question 2

What type of macromolecules are enzymes?

Answer 2

Enzymes are typically proteins made of amino acids.

Question 3

What does it mean to be a catalyst?

Answer 3

A catalyst is a substance that speeds up the rate of a chemical reaction without
being used up by the reaction.

Question 4

Name two advantages of having enzyme-catalyzed chemical reactions in living cells?

Answer 4

Speed: Enzymes accelerate chemical reactions, allowing cells to carry out essential processes rapidly.

Specificity: Enzymes are highly specific, meaning they catalyze specific reactions with precise substrates. This specificity ensures that only the intended reactions occur, preventing unwanted side reactions and maintaining cellular homeostasis.

Question 5

What is a substrate?

Answer 5

Reactants in an enzymatic reaction that only bind to specific enzymes.

Question 6

Where on enzymes do substrates typically bind to during a chemical reaction?

Answer 6

The active site.

Question 7

When an enzymatic reaction is in process, will the following increase, decrease, or remain the same?
1. Substrate
2. Product
3. Enzyme

Answer 7

1. decrease
2. increase
3. stays the same

Question 8

How can enzymatic activity be measured?

Answer 8

Spectrophotometry: measures changes in absorbance of light caused by the enzymatic reaction. i.e. absorbance monitored at specific wavelengths.

Colorimetric assays: use color changes to quantify enzyme activity by looking at the conversion of a colorless substrate to a colored product by the enzyme, which can be detected using a spectrophotometer.

Question 9

What are cofactors?

Answer 9

Non-protein chemical compounds or ions that are required for the proper functioning of enzymes.

Question 10

What type of bond attaches substrate to enzymes?

Answer 10

weak, non-covalent bonds

Question 11

What is an induced fit?

Answer 11

When a substrate binds to the active site of an enzyme, the enzyme undergoes a conformational change or a structural rearrangement to accommodate the substrate more effectively.

Question 12

What is activation energy?

Answer 12

The initial amount of energy required or a reaction to take place.

Question 13

Enzymes (slow down/speed up) the rate of a reaction by (lowering/raising) the activation energy.

Answer 13

1. speed up
2. lowering

Question 14

What is the activation energy barrier?

Answer 14

The minimum amount of energy that must be overcome for a chemical reaction to occur.

Question 15

Name three factors that effects enzymatic activity.

Answer 15

1. Temperature
2. pH
3. Substrate/Enzyme concentration

Question 16

What does hydrolysis mean?

Answer 16

A chemical reaction where a water molecule is used to BREAK DOWN a larger molecule into smaller fragments.

Question 17

Starch is a polymer of…

Answer 17

Glucose

Question 18

Maltose is a (monosaccharide/reducing disaccharide) made of #__ ______ molecules.

Answer 18

Reducing disaccharide made of 2 glucose molecules.

Question 19

What is Alpha amylase?

Answer 19

An enzyme that catalyzes the hydrolysis of α-1,4 glycosidic
linkages of starch into maltose.

Question 20

How is energy extracted from starch?

Answer 20

Amylase catalyzes this by hydrolyzing starch down into smaller sugars.

Starch + water – Amylase –> Maltose

Question 21

What are the three sources of amylase?

Answer 21

1. Bacteria
2. Fungal
3. Human

Question 22

How can concentration of maltose be measured?

Answer 22

Using colorimetric assay, which means combining
maltose with a certain reagent (dinitrosalicylic acid, DNS) that causes a color change.

Question 23

What are reducing substances?

Answer 23

Molecules that have the ability to donate electrons, thereby reducing another molecule by transferring electrons to it.

Question 24

What happens between maltose and DNS, and why?

Answer 24

Maltose participates in an oxidation-reduction reaction with DNS due to its carbonyl group. DNS is reduced to 3-amino, 5-nitrosalicylic acid and maltose is oxidized to maltonic acid. This causes a color change from yellow to orange.

Question 25

Why is the color change after combining maltose and DNS significant for measuring maltose concentration?

Answer 25

The change in color results in a change in absorption of light, and absorbance is measured in a
spectrophotometer at a wavelength of 540 nm. The intensity of color from the reaction, and the
absorbance of light, is proportional to the concentration of maltose and is used to estimate the
concentration of maltose in any given solution.

Question 26

Name two reasons why we boil our solution after adding DNS?

Answer 26

1. Reaction activation: To help activate the chemical reaction between DNS and the reducing sugars present in the solution.
2. Denaturization: Boiling also denatures or inactivates the enzyme (amylase) present in the solution. This step is important to stop the enzymatic reaction and to prevent further breakdown of starch into reducing sugars.

Question 27

How is the standard maltose curve calculated?

Answer 27

By preparing a series of maltose solutions with known concentrations and measuring their absorbance at a specific wavelength, a standard curve relating absorbance to maltose concentration can be generated.

Question 28

What is the standard maltose curve used for in this experiment?

Answer 28

This standard curve serves as a reference for quantifying the amount of maltose present in the reaction samples based on their absorbance values. The absorbance of the reaction samples containing maltose is measured, and the absorbance values are then compared to those on the standard curve to determine the corresponding maltose concentrations.

Question 29

How can we determine an enzymes optimal temperature with this experiment?

Answer 29

By performing the experiment at different temperatures and analyzing the results, you can determine the temperature range at which the enzyme functions most efficiently. This optimal temperature reflects the temperature at which the enzyme’s catalytic activity is highest, and deviations from this temperature may lead to decreased enzyme activity due to denaturation or other factors.

Question 30

What is the formula used to calculate the final concentration (mg/ml) of maltose in each tube?

Answer 30

C1V1 = C2V2

C1 is the initial concentration of the solution you’re diluting (the stock solution).

V1 is the volume of the initial solution you’re diluting.

C2 is the final concentration you want to achieve after dilution.

V2 is the final volume of the diluted solution (the total volume after dilution).

Question 31

Describe the procedure of the amylase activity lab.

Answer 31

The procedure involves preparing a series of test tubes containing starch solution and amylase enzyme. After incubating the tubes at various temperatures, the reaction is stopped by boiling the solution. Then, DNS reagent is added to detect the presence of maltose, followed by spectrophotometric analysis to measure the absorbance.

Question 32

What is the purpose of DNS reagent in the amylase activity lab?

Answer 32

The purpose of DNS reagent is to detect the presence of reducing sugars, such as maltose, formed during the hydrolysis of starch by amylase. It reacts with reducing sugars to produce a colored product that can be quantitatively measured.

Question 33

What formula is used in the amylase activity to get from maltose produced (mg/ml) to amylase activity (mg/ml/min)?

Answer 33