How to obtain enzyme kinetic constants using Isothermal Titration Calorimetry
contributed by Malvern Panalytical |
The use of ITC for obtaining enzyme kinetic constants
Enzymes are proteins that function as biological catalysts and they play a crucial role in nearly all processes that take place in living organisms. Therefore studies of these molecules and the reactions they catalyze have been a core activity for biochemists for at least the past fifty years. Apart from the fundamental scientific interest, investigations into enzyme function are important in order to develop novel therapeutics against diseases such as cancer. A large group of drugs currently used in the clinic exert their primary biological effects by inhibiting a specific enzyme (1). Development of next generation inhibitors requires that we understand the way the enzyme binds and processes the natural substrate.
To understand any biochemical phenomena it is necessary to study the enzyme reactions which make up that system individually. The aim is to learn how and why the enzyme recognizes its substrate and the mechanism it employs in catalyzing the reaction to form the product. Modern techniques in molecular biology, sequencing, and proteomics have led to an upsurge in the number of enzymes that can be routinely expressed as recombinant proteins. Substrates or substrate analogues can also be produced using the same technology or chemically synthesized. Once the two components have been obtained it is then necessary to develop an assay technique that allows the enzyme-catalyzed reaction to be studied. When these data are combined with structural studies a picture of the enzyme mechanism can emerge that allows potential inhibitors to be developed based on a rational approach.
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