Biology 05LA – Wiinter Quarter 2008
Lab 5 – page 1
Enzymes are organic catalysts that participate in the chemical conversion of one organic
molecule to another. The molecule that is acted upon by an enzyme is known as its substrate and the
molecule that is formed by the reaction is referred to as its product.
Enzymes regulate the rate of a
reaction without being chemically changed in the course of the reaction.
Thus, one molecule of an
enzyme could theoretically catalyze an infinite number conversions of substrate to product.
When enzymes were first discovered, they were given a wide variety of names.
the enzyme that initiates the digestion of starch in the mouth, salivary amylase, was formally known as
The enzyme from the intestinal mucosa that splits lactose to glucose and galactose, beta-
galactosidase was formally referred to as lactase.
Currently, enzymes are named systematically by
what they do.
The ending "ase" identifies a substance as an enzyme.
This ending is preceded by a
stem which indicates a specific substrate or the general nature of the substrate or the type of action the
For example, succinic dehydrogenase removes two atoms of hydrogen from
succinic acid, while hydrolases insert a molecule of water across a bond and thereby hydrolyze a
Most enzymes are proteins and are sensitive to environmental conditions.
Enzymes, like most
proteins, can be irreversibly damaged or denatured by very high temperatures or pH extremes.
are fascinating exceptions to these general rules: enzymes whose critical component is not protein,
enzymes from organisms that thrive in super-heated ocean volcanic vents, and enzymes that operate
quite well at very acidic or very alkaline pH conditions normally viewed as deadly for all proteins and
All enzymes, even the unusual ones, have conditions of temperature, pH, concentration of
enzyme and concentration of substrates that promote their most efficient activity.
In today's exercises, we will examine bovine intestinal alkaline phosphatase.
normally operates in the intestines of cattle, away from the acidic stomachs (cattle have more than
one), breaking down a variety of phosphorylated compounds found in their food.
phosphatase can hydrolyze the artificial substrate p-nitrophenylphosphate, giving us a convenient way
to make quantitative measurements of its activity under various conditions.
The basis for this convenience is derived from the fact that p-nitrophenylphosphate (pNPP) is a
chromogenic substrate for alkaline phosphatase (and most other phosphatases).
This means that an
aqueous solution of pNPP, which is colorless, turns yellow when de-phosphorylated by alkaline
phosphatase to form p-nitrophenol.
This reaction is summarized as follows:
p-nitrophenylphosphate (colorless) + H
p-nitrophenol (yellow) + Pi (inorganic phosphate)
Further, a quantitative measure of the amount of yellow product (p-nitrophenol) generated in a