Objective

Grading

Background Information

Many details of how catalysis occurs have been obtained from the study of enzymatic reactions in biological systems, where specific protein molecules called enzymes function as homogeneous catalysts. They produce an increase in the rate of reaction by providing an alternate lower-energy pathway for the formation of products. This phenomenon of enzymatic catalysis makes biological reactions necessary for the maintenance of life possible. As biological catalysts, enzymes retain the characteristics of chemical catalysts: they increase the reaction rate, remain unchanged after the reaction, have no effect on the equilibrium constant (Keq)(Keq) size 12{ \( K rSub { size 8{ ital "eq"} } \) } {} or on the ultimate equilibrium conditions for a reaction, and are highly efficient. Enzymes help orient the reaction participants to be more likely to react, to discriminate between one possible reactant molecule and another with uncanny specificity, and sometimes to provide a coupling mechanism that ensures one reaction always is accompanied by another reaction in a specific sequence.

A molecule acted upon by an enzyme is referred to as the substrate of that enzyme. The presence or absence of a single atom, or a single charge, may decide whether a molecule is the optimum substrate or is rejected by the enzyme. The ability of the enzyme to select from among many possible molecules with which it could react is called enzyme specificity.

Although some molecules sufficiently resemble the optimum substrate of an enzyme to bind to the active site, they cannot undergo chemical reaction: they simply sit there, blocking the site rather like a bump on a log, preventing the enzyme from functioning with the true substrate. Such molecular impersonators are termed competitive inhibitors. This competitive inhibition can be reversible, since the impersonators can be flushed off the enzyme with a sufficient excess of true substrate. DFP (diisopropyl fluorophosphate, an organophosphate) is a potent and lethal nerve gas, i.e. an irreversible inhibitor as it irreversibly inhibits the enzyme acetylcholinesterase, which is essential for the conduction of nerve impulses.

Structure of DFP

Many organophosphorus compounds used as insecticides are deadly nerve toxins for exactly the same reason.

The ability of an enzyme to catalyze a specific reaction is termed its activity – a measure of the rate at which the reaction proceeds. Enzyme activity depends on several variables such as pH, temperature, concentration, and specificity of substrate, cofactors, and inhibitors. Vitamins and minerals, two important factors of human nutrition, play an essential role in the proper function of certain enzymes. Approximately one-third of known enzymes require a metallic ion for their activity. The term cofactor is used to group coenzymes and minerals within a general category.

An oxidase is an enzyme that catalyzes the transfer of hydrogen from some compound to molecular oxygen in order to form water. Polyphenoloxidase is a copper-containing enzyme that catalyzes the removal of hydrogen, as in the oxidation of dihydroxyphenols to the corresponding quinones. This type of oxidation is accompanied by a color change. Polyphenoloxidases, also known as o-diphenoloxidases, are distributed widely in the plant kingdom (e.g. champignon mushrooms, potatoes, bananas). They are responsible for the darkening of freshly cut surfaces of plants or fruits. For insects, o-diphenoloxidase is important both for melanin formation and for browning and hardening (sclerotization) of the cuticle. In this experiment, the observed color change is used as a measure of extent of the reaction.

Part III. The Specificity of Enzyme Activity

In this part of the experiment, you will evaluate the specificity of the substrate structure required for enzyme activity. The substrate structure varies to demonstrate the need for:

1. an aromatic or aliphatic ring
2. one or two hydroxy groups
3. proximity of hydroxy groups on the ring

The substrates include the following:

1. deionized water
2. cyclohexanol

Figure 1

1. 1,4 cyclohexanediol

Figure 2

1. phenol

Figure 3

1. catechol (o-hydroxyphenol)

Figure 4

1. resorcinol (m-hydroxyphenol)

Figure 5

1. hydroquinone (p-hydroxyphenol)

Figure 6

Caution: Some of the solutions used in the next step are toxic and can be absorbed through the skin. Avoid skin contact.

1. Prepare seven labeled 6-inch (15-cm) test tubes with labels and contents as follows:

Tube 1: 1.0 mL distilled waterTube 2: 1.0 mL 0.01 M cyclohexanolTube 3: 1.0 mL 0.01 M 1,4-cyclohexanediolTube 4: 1.0 mL 0.01 M phenolTube 5: 1.0 mL 0.01 M catecholTube 6: 1.0 mL 0.01 M resorcinolTube 7: 1.0 mL 0.01 M hydroquinone

To each tube add an additional 3.0 mL of distilled water. (These are the substrate tubes.)

1. Place all the labeled substrate test tubes into a 37 °C°C size 12{°C} {} water bath.
2. Transfer 3.0 mL of vegetable extract into each of another set of seven test tubes, and then place them into the constant temperature water bath. (These are the enzyme tubes.)
3. Allow the two sets of test tubes to remain in the constant temperature bath for five minutes.
4. Quickly add the contents of the enzyme tubes to the substrate tubes.
5. Remove all the test tubes from the water bath and compare the intensity of any developed color to Tube 1, which contains distilled water.
6. Arrange the test tubes in progressive intensity of color and record the sequence. Tube 1 (blank) is assigned a value of 0; the other tubes are then ranked 1, 2, 3, and so on.
7. What can you conclude about the development of a color?
8. On your report form, list the substrates that do not react with the enzyme.