A method to determine ¹⁸O kinetic isotope effects in the hydrolysis of nucleotide triphosphates

A method to determine ¹⁸O kinetic isotope effects (KIEs) in the hydrolysis of GTP that is generally applicable to reactions involving other nucleotide triphosphates is described. Internal competition, where the substrate of the reaction is a mixture of ¹⁸O-labeled and unlabeled nucleotides, is employed, and the change in relative abundance of the two species in the course of the reaction is used to calculate KIE. The nucleotide labeled with ¹⁸O at sites of mechanistic interest also contains ¹³C at all carbon positions, whereas the ¹⁶O-labeled nucleotide is depleted of ¹³C. The relative abundance of the labeled and unlabeled substrates or products is reflected in the carbon isotope ratio (¹³C/¹²C) in GTP or GDP, which is determined by the use of a liquid chromatography-coupled isotope ratio mass spectrometer (LC-coupled IRMS). The LC is coupled to the IRMS by an Isolink interface. Carbon isotope ratios can be determined with accuracy and precision greater than 0.04% and are consistent over an order of magnitude in sample amount. KIE values for Ras/NF1³³³-catalyzed hydrolysis of [β¹⁸O₃,¹³C]GTP were determined by change in the isotope ratio of GTP or GDP or the ratio of the isotope ratio of GDP to that of GTP. KIE values computed in the three ways agree within 0.1%, although the method using the ratio of isotope ratios of GDP and GTP gives superior precision (<0.1%). A single KIE measurement can be conducted in 25 min with less than 5 μg nucleotide reaction product.