ITC
Functional Application Areas
Enzyme Kinetics
Enzyme catalytic reactions are central to all biological pathways. A major portion of biochemical research is devoted to characterizing enzyme function, activity and structure, and how enzymes are inhibited and activated. Enzyme characterization is also key to drug discovery since many drug targets are enzymes. These include kinases and proteases.
Isothermal Titration Calorimetry (ITC) is well-established in the study of affinity of molecular interactions, and is now becoming a mainstream tool in the study of enzyme kinetics. The strength of the technique lies in the universal nature of ITC. Traditional enzyme assays utilize a probe to monitor either substrate depletion or product formation. These probes are system-dependent and must be optimized for each reaction under specific conditions. Also, the substrate may need to be modified which could interfere with the catalysis reaction. For optical methods, the experimental conditions can affect the detection system, preventing accurate measurements. This means that with traditional assay methods, many enzymes do not have practical assays.
ITC uses heat as a probe, and since every reaction generates or absorbs heat, there is no need for lengthy method development each time a new enzyme is assayed. ITC directly measures the heat change as catalysis proceeds, which is proportional to the rate of reaction. Todd and Gomez (2001) showed that Km and kcat from ITC experiments agreed favorably with traditional enzyme kinetics methods, and can be used with every class of enzyme, including those with no other direct assay methods.
The use of ITC to monitor the rate of enzymatic reactions is a non-destructive, sensitive, and direct assay. Multiple injections of substrate can be done in a single experiment, so Km and kcat can be determined in a single ITC experiment. With ITC, it is straightforward to vary experimental conditions such as pH and ionic strength, and one can get a complete analysis of catalysis and kinetics. ITC also provides valuable insights on the thermodynamics of enzymatic reactions.
References
Calorimetric Determination of Thermodynamic Parameters of Reaction Reveals Different Enthalpic Compensations of the Yeast Hexokinase Isozymes.
Bianconi M. L.
J Biol Chem 278, 18709-18713 (2003)
Calorimetry of Enzyme-catalyzed Reactions
Bianconi M. L.
Biophys Chem 126, 59-64 (2007)
Beta-d-Glucosidase Reaction Kinetics from Isothermal Titration Microcalorimetry
Jeoh T., Baker J. O., Ali M. K., Himmel M. E., and Adney W. S.
Anal Biochem 347, 244-253 (2005)
Enzyme Kinetics Determined Using Calorimetry: A General Assay for Enzyme Activity?
Todd, M.J., Gomez, J.
Anal. Biochem. 296, 179-187 (2001)
Reference List
ITC Enzyme Activity Studies Reference List
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