A strategy for the determination of enzyme kinetics using electrospray ionization with an ion trap mass spectrometer

A simple and rapid means of enzyme kinetic analysis was achieved using elec trospray ionization mass spectrometry and a one-point normalization factor. The model system used, glutathione S-transferase from porcine liver, is a two-substrate enzyme catalyzing the conjugation of glutathione with a varie ty of compounds containing an electrophilic center. An internal standard th at is structurally similar to the product was added to the reaction quench solution, and a single-point normalization factor was used to determine the product concentration without the need of a calibration curve. Kinetic par ameters, such as K-m, V-max and K-i (for thyroxine), obtained by electrospr ay mass spectrometry agreed with those obtained from traditional UV-vis spe ctroscopy, and competitive vs noncompetitive inhibition reactions could be delineated via mass spectrometry. These results suggest that our method can be applied to enzymatic processes in which spectrophotometric or spectrofl uorometric assays are not feasible or when the relevant substrates do not i ncorporate chromophores or fluorophores. Ibis new method is competitive wit h traditional UV assays in that it is facile and it involves very little an alysis time.