Kinetic characterization of enzyme inhibitors using electrospray-ionization mass spectrometry coupled with multiple reaction monitoring

摘要

Electrospray ionization mass spectrometry coupled to multiple reaction monitoring (ESI-MS/MRM) has been applied for the first time to analyze enzyme inhibitor kinetics. Specifically, a known competitive inhibitor, guanosine 5'-monophosphate (GMP), and a synthetic, transition-state analogue inhibitor, guanosine 5'-[1D-(1,3,4/2)5-methyl-5-cyclohexene-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 K-i of 211.8 +/- 24.7 muM. The conventional analysis using GDP-[U-C-14]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 K-i 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 suicide-type inhibitor toward Fuc-TV. 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.