Experiment 1: Enzyme Function

Proteins are very specific three-dimensional molecules. Enzymes’ three-dimensional structure perfectly aligns specific molecules (substrates) in a way that encourages chemical bonding to occur between them, efficiently synthesizing specific molecules (products) necessary for cellular function. In solution, the substrates would eventually become products. The unique shape of enzymes allows them to play the role of matchmaker, bringing together substrates much faster than normal, generating specific products. The image to the right represents how the enzyme sucrase catalyzes the hydrolysis of sucrose into glucose and fructose.

Every unique combination of substrates requires their own specific enzyme. Amazingly, enzymes don’t react during the bonding of substrates. They are capable of maintaining their molecular structure and therefore are capable of repeatedly combining reactants to make the desired products without losing functionality.

Benzoquinone is an antimicrobial molecule produced by plants in order to prevent bacterial infections when their internal tissues are exposed. When the cells of exposed tissues come into contact with oxygen, an enzyme known as catecholase catalyzes benzoquinone from catechol and oxygen.

In this lab, you will measure the effect of the enzyme, catecholase, under varying conditions. Catecholase binds catechol and oxygen creating benzoquinone. Benzoquinone turns fruit brown. You will measure the effect of catecholase by determining the degree of brownness in a variety of experiments. 

The rate of enzyme efficiency can be affected by a number of factors. In this lab, you will experimentally alter the availability of reactants, pH, and temperature.

Enzyme efficiency relative to availability of reactants

You will examine the effect of the availability of substrates, by comparing the relative change in brownness by comparing two apples with different surface areas: one sliced and one grated.

Enzyme efficiency relative to pH

Enzymes are pH specific, meaning they work most efficiently with an optimal range of pH values. Above or below an enzyme’s optimal range can cause the enzyme to denature (altering their 3-D structure), and therefore lose their functionality. You will compare the relative change in brownness by comparing two apple slices immersed in two different pH solutions: neutral (H2O) and acidic (lemon juice).

Enzyme efficiency relative to temperature

Enzymes also have a specific optimal range of temperatures. Similar to pH, temperatures above or below an enzyme’s optimal range will cause the protein to denature, impeding their functionality.  

Materials

• ½ an apple
• Knife
• 3 - Petri dishes
• Grater
• Lemon juice
• Ice
• Plate warmer
• pH indicator
• 3 - 600ml beakers
• 3 - 100ml beakers
• Thermometer

Protocol

• In a large cup, warm 150ml of water to 40˚C. While that is warming, in a second cup create a 150ml ice bath. Fill a third large cup with 150ml of room temperature water.

• Cut your apple into six slices and assemble the following treatments:
  • Room temperature incubator. Place apple slice in dry, small cup
  • Hot incubator. Place apple slice in dry, small cup.
  • Cold incubator. Place apple slice in dry, small cup.
  • Lemon juice. Place apple slice on a plante. Flip apple every 5 minutes to moisten.
  • Water. Place apple slice on a plate. Flip apple every 5 minutes to moisten.
  • Grated apple. Place grated apple on a plate.
• Check your apples every 5 minutes and determine their level of brownness relative to the scale in The Biology Lab Primer.