Kinetic Characterization of Enzyme Inhibitors Using Electrospray-Ionization Mass Spectrometry Coupled with Multiple Reaction Monitoring

Electrospray ionization mass spectrometry coupled to multiple reaction monitoring (ESIMS/MRM) has been applied for the first time to analyze enzyme inhibitor kinetics. Specifically, a known competitive inhibitor, guanosine 5ʹ-monophosphate (GMP), and a synthetic, transition-state analogue inhibitor, guanosine 5ʹ-[1D-(1,3,4/2)-5-methyl-5cyclohexene-1,2,3,4-tetrol 1-diphosphate] (1) have been characterized against recombinant fucosyltransferase (Fuc-T) V using ESI-MS/MRM. Dixon analysis with GMP yielded a signature plot for competitive inhibition. Nonlinear regression analysis gave a Ki of 211.8 ± 24.7 muM. The conventional analysis using GDP-[U-14C]Fuc yielded a similar Ki value of 235.6 ± 59.4 muM, confirming the validity of the MS-based method. The synthetic inhibitor 1 showed potent competitive inhibition with a Ki of 25.6 ± 2.8 muM. Although 1 possesses a chemically reactive allyl phosphate group, ESI-MS/MRM showed that there was no reduction in the concentration of 1 and no production of a predicted metabolite GDP during the assay. MS/MS also confirmed the absence of a possible pseudo-trisaccharide product. The results clearly show that 1 is neither a slow-reacting donor nor does it act as a suicidetype inhibitor toward Fuc-T V. ESI-MS/MRM is therefore a powerful tool for the kinetic characterization of enzyme inhibitors, providing complete disclosure of the mechanism of action of 1 as an inhibitor.