Analysis of Enzyme Activity by Isothermal Titration Calorimetry

Abstract

The total time course of the exothermic heat flow of ATP hydrolysis catalyzed by the stable helicase domain derived from tomato mosaic virus replication protein (ToMV-Hel) was monitored by isothermal titration calorimetry (ITC) in 150 mM NaCl, 10 mM MgCl2, 0.5 mM tris(2-carboxyethyl)phosphine plus 100 mM phosphate buffer pH 7.5 at 15 °C. Analysis of the time course of the enzymatic reaction established a method for evaluating competitive inhibition of the product from the initial substrate/product concentration dependence of the apparent Michaelis constant and found that helicase activity is competitively inhibited by the hydrolysis product, ADP. Comparing the true Michaelis constant with the inhibition constant, the enzyme was estimated to have a 17-fold higher affinity for ATP than for ADP under these conditions. We will also present an example of using this method to screen for inhibitors of the ToMV-Hel using the ITC method of evaluating enzyme activity.