0.7 The curious case of catalase

The curious case of catalase

Objective

• To prepare the enzyme o-diphenoloxidase from a vegetable or fruit.
• To study the effects of temperature.
• To study the specificity of enzyme activity.
• To observe the change in enzyme activity due to changes in pH.
• To study the effect of an inhibitor on your prepared enzyme.
• To put you off potatoes for life!

Grading

• Pre-Lab (10%)
• Lab Report Form (80%)
• TA Points (10%)

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 $(K_{\text{eq}})$ 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.