BC 367 Experiment 4
Kinetic Properties of Acid Phosphatase
Phosphatases are enzymes that remove phosphate groups from substrates. Phosphorylated
compounds are widely distributed in living systems. They serve as storage forms for energy (e.g.,
ATP and phosphocreatine), as components of informational macromolecules (i.e., nucleotides
Phosphorylation-dephosphorylation reactions of proteins, mediated by protein kinases and
dehydrogenase, etc.). It is therefore not surprising that phosphatases of many kinds can be
extracted from many tissues. In general, these enzymes catalyze the hydrolysis of phosphate
monoesters as follows:
During this experiment, you will investigate the properties of an acid phosphatase from
wheat germ. Acid phosphatases have optimal activity at pH values from 4 to 6, and are distinct
from alkaline phosphatases with optimal activity in the pH range of 8-9. The function of the
wheat germ phosphatase has not been definitively established, but it has been proposed that it is
involved in the mobilization of the phosphate reserves of the seed.
Most of the phosphate within
a seed is esterified as inositol hexaphosphate (phytic acid).
Since the free phosphate
concentration in seeds is low enough to limit metabolic reactions, enzymes must be present to
catalyze the hydrolysis of the phosphate reserve substances.
During the first week, you will perform a kinetic study on wheat germ acid phosphatase to
determine its K
with p-nitrophenylphosphate as the substrate.
You will also determine
the nature of the inhibition of acid phosphatase by inorganic phosphate. For the second week,
you will examine the X-ray structure of acid phosphatase in the Schupf lab and meet with your
instructor to discuss your plans for the following week. During the third week, you will carry out
a set of your own kinetic experiments to investigate the effect of reaction conditions or a
potential inhibitor of your choice on the kinetic properties of wheat germ acid phosphatase.
Kinetics concerns the study of reaction rates and the conditions that affect reaction rates.
One of the primary concerns of kinetics deals with the study of the changes in the rate of a
reaction as a function of the reactant (substrate) concentrations. Such measurements can lead to
the calculation of rate constants and, where appropriate, of equilibrium constants.
be of great value for predicting or eliminating possible reaction mechanisms under consideration,
since an observed reaction rate must satisfy the proposed mechanism.