title: Kinetic characterization of enzyme inhibitors using electrospray-ionization mass spectrometry coupled with multiple reaction monitoring.
abstract: 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 Ki of 211.8+/-24.7 microM. The conventional analysis using GDP-[U-14C]-Fuc yielded a similar Ki value of 235.6+/-59.4 microM, confirming the validity of the MS-based method. The synthetic inhibitor 1 showed potent competitive inhibition with a Ki of 25.6+/-2.8 microM. 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-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.
authors.affiliation: Crump Institute for Molecular Imaging, Department of Molecular and Medical Pharmacology, UCLA School of Medicine, Los Angeles, California 90095-1770, USA.
authors.forename: A J
authors.initials: AJ
authors.lastname: Norris
authors.forename: J P
authors.initials: JP
authors.lastname: Whitelegge
authors.forename: K F
authors.initials: KF
authors.lastname: Faull
authors.forename: T
authors.initials: T
authors.lastname: Toyokuni
mesh_terms: D004791:Enzyme Inhibitors / Q000737:chemistry*; D005647:Fucosyltransferases / Q000037:antagonists & inhibitors / Q000737:chemistry; D006151:Guanosine / Q000031:analogs & derivatives / Q000737:chemistry*; D006157:Guanosine Monophosphate / Q000737:chemistry; D007202:Indicators and Reagents; D007700:Kinetics; D012044:Regression Analysis; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D004791:Enzyme Inhibitors; D007202:Indicators and Reagents; C446703:guanosine 5'-(1D-(1,3,4-2)-5-methyl-5-cyclohexene-1,2,3,4-tetrol 1-diphosphate); D006151:Guanosine; D006157:Guanosine Monophosphate; D005647:Fucosyltransferases