Isothermal titration calorimetry (ITC) measures the heat absorbed or released from biochemical processes. Although the primary application of ITC has been in binding studies, the reaction enthalpy detected by ITC can be used as a probe to quantify enzyme activity. Kinases are an important class of enzymes that are involved in many types of cellular regulation, but kinetic studies of kinases by ITC have been limited. T4 polynucleotide kinase (T4 PNK) is proposed as a model system to optimize and validate the ITC analysis conditions for a kinase. T4 PNK typically phosphorylates an oligonucleotide, but a single nucleotide can serve as a substrate if a 3-phosphate group is present. We used ITC to study the T4 PNK-catalyzed conversion of 3-AMP to adenosine-3,5-bisphosphate. To initiate the reaction, a single injection of 3-AMP substrate was made into the ITC cell containing T4 PNK and ATP at pH 7.6 and 30oC. The shape of the resultant Differential Power versus Time curve is determined by the kinetic properties of the enzyme. Integration of the area under the curve yields reaction enthalpy. The apparent ∆H for the reaction was found to vary depending on the type of buffer used. This result was explained by differential protonation of reactants and products at pH 7.6, and indicated the release of approximately 0.6 protons per mole for the forward reaction. Extrapolation was used to estimate an intrinsic (i.e. buffer-independent) ∆H of -25 kJ/mol. The Michaelis-Menten constant, KM, was 1.3E-05 M, and the turnover number, kcat, was 8/sec. The reaction enthalpy and kinetic parameters for T4 PNK phosphorylation of 3-AMP were assessed successfully by single injection ITC analysis. This finding supports the potential to analyze more complex kinase systems using ITC.
Support or Funding Information
This work was supported by the U.S. Army Research Office grant W911NF-15-1-0216.
