Enzymes Lab #6

Enzyme

A group of proteins that act as biological catalysts to increase the rate of a reaction and decrease the amount of activation energy necessary to start the reaction.

Catalyst

• A chemical agent that helps molecules reach their transition state without being consumed or chemically altered by the reaction.

• Works better than increasing the temperature, which can destroy structural components of the cell AND increase speeds of reaction limiting the cell’s ability to precisely regulate metabolic pathways.

Substrate

• The reactants that bind to the enzyme’s active site.

• Contributes to the high level of complexity of metabolism of typical cells involving thousands of different enzymes

• Fits perfectly into the enzyme like a lock and key (induced fit hypothesis)

Enzyme-substrate complex

The combination of the enzyme and substrate being bonded together.

Active Site

The area of the enzyme in which the substrate binds to.

Activation Energy

The amount of energy required to bind the substrate to the enzyme’s active site.

Hydrolysis

Reactions that split substrate molecules into smaller product molecules by the addition of water

Dehydration Synthesis

Piecing substrate molecules together to form a larger product upon the removal of of a water molecule.

Anabolic Reactions

Reactions that build complex molecules from simpler ones (endergonic)

Catabolic Reactions

Break down complex molecules into simpler ones and release energy (exergonic)

Peroxidase

• The enzyme used from this lab; extracted from rutabaga tissue.

• Large protein containing several hundred amino acids

• Iron ion is located in the active site, which allows hydrogen peroxide (substrate) to turn into water and oxygen.

Rutabaga

• Brassica napus

• Used to study the catabolism of the substrate hydrogen peroxide

Hydrogen Peroxide

(H2O2) - The substrate in this experiment; 2H2O2 is catalyzed/broken down into 2H2O and O2

Dye-coupled Reaction

• We use a dye-coupled reaction in order to look for evidence of oxygen chemical activities.

• Peroxidase is not affected by the dye, but the products of the Peroxidase reaction are

Guaiacol

The dye that is used in this experiment. Initially colourless, but turns orange in the presence of oxygen.

What happens when the guaiacol darkens?

The light gets absorbed by the oxidized dye. The rate at which the solution darkens is proportional to the enzyme activity (reaction rate)

Mortar

The bowl used to crush the rutabaga

Pestle

The hand-held device used to crush the rutabaga

Buffer

• pH 7 buffer used to grind the enzyme with the sand

• 5 buffer used to test change in concentration

• Used to maintain the optimal pH for the enzyme to function

Blank in this specific experiment

Contains substrate, buffer, and dye, but does not contain peroxidase

Centrifuge Tube

Used to separate the buffer containing the enzymes from the unwanted plant material (also used cheesecloth)

Independent Variable

• What you manipulate (concentration in this experiment)

• Graphed on the horizontal x-axis

Dependent Variable

• What you are influencing (reaction rate/time in this experiment) on the vertical y-axis