BL 4820 Biochemical Techniques -- Lecture 2 - Characterizing an Acid Phosphatase
1. General Principles Underlying Enzyme Assays
First, to analyzed the properties of an enzyme, you need to establish a valid assay method and identify the conditions to use to get the maximum activity using a specific substrate. To illustrate these general principles of enzymology, I will use the enzyme hexokinase as an example.
Hexokinase catalyzes the conversion of glucose to 6-phospho-glucose using Mg-ATP as the phosphate donor:
Glucose + Mg-ATP = Glucose-6-phosphate + Mg-ADP
This is basically an irreversible reaction since a lot of free energy is released during the reaction. But of greater importance to our discussion is that it is a two substrate enzyme. To make things simpler we will add a lot of Mg-ATP so that it is in excess. This makes the concentration of Mg-ATP (ie. [Mg-ATP]) fixed and [glucose] an allowed variable.
To optimize the assay conditions, you need to determine:
- Amount of Enzyme to use
- Optimum pH for the enzyme activity
- Optimum [Glucose]
To optimize these conditions for determining enzyme activity, you need a valid assay method. To do this, you can either measure the decrease in glucose during the enzyme catalyzed reaction or measure appearance of glucose-6-P. The assay will be much more sensitive if appearance of product is measured since you start the assay with no product present.
First, it should be determined in a control assay that in the absence of hexokinase, glucose and MgATP, the substrates and reactants in the reaction, do not form a significant amount of products (glucose-6-P and MgADP). Next, if you have a method available for measuring the amount of glucose-6-P formed during the reaction, then use it as the basis of the assay. The evaluation method for amount of glucose-6-P must be quantitative and give results in amounts of µmol glucose-6-P formed during measured periods of reaction time. This will allow you to calculate the reaction rate in terms of µmol/min. This will be called the enzyme catalyzed rate or enzyme activity.