Analysis of enzyme kinetics using electrospray ionization mass spectrometry and multiple reaction monitoring: fucosyltransferase V.
An accurate, rapid, and versatile method for the analysis of enzyme kinetics using electrospray ionization mass spectrometry (ESI-MS) has been developed and demonstrated using fucosyltransferase V. Reactions performed in primary or secondary amine-containing buffers were diluted in an ESI solvent and directly analyzed without purification of the reaction products. Decreased mass resolution was used to maximize instrument sensitivity, and multiple reaction monitoring (MRM), in the tandem mass spectrometric mode, was used to enhance selectivity of detection. The approach allowed simultaneous monitoring of multiple processes, including substrate consumption, product formation, and the intensity of an internal standard. MRM gave an apparent K(m) for GDP-L-fucose (GDP-Fuc) of 50.4 +/- 5.5 microM and a k(cat) of 1.46 +/- 0.044 s(-1). Under the same conditions, the conventional radioactivity-based assay using GDP-[U-(14)C]Fuc as substrate gave virtually identical results: K(m) = 54.3 +/- 4.6 microM and k(cat) = 1.49 +/- 0.039 s(-1). The close correlation of the data showed that ESI-MS coupled to MRM is a valid approach for the analysis of enzyme kinetics. Consequently, this method represents a valuable alternative to existing analytic methods because of the option of simultaneously monitoring multiple species, the high degree of specificity, and rapid analysis times and because it does not rely on the availability of radioactive or chromogenic substrates.
Norris
A J
AJ
Crump Institute for Molecular Imaging, Departments of Molecular and Medical Pharmacology, Chemistry and Biochemistry, University of California, Los Angeles, California 90095, USA.
Whitelegge
J P
JP
Faull
K F
KF
Toyokuni
T
T
D000606:Amino Sugars / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D002021:Buffers; D002240:Carbohydrate Sequence; D004579:Electron Transport; D005643:Fucose / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D005647:Fucosyltransferases / Q000737:chemistry* / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007477:Ions / Q000737:chemistry; D007700:Kinetics; D016256:Lewis X Antigen / Q000031:analogs & derivatives; D008969:Molecular Sequence Data; D009711:Nucleotides / Q000737:chemistry / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D014312:Trisaccharides / Q000737:chemistry / Q000378:metabolism
D000606:Amino Sugars; D002021:Buffers; C418802:GDP-6F-fucose; D007477:Ions; D016256:Lewis X Antigen; D009711:Nucleotides; D014312:Trisaccharides; C110530:galactosyl-(1,4)-fucopyranosyl-(1,3)-N-acetylglucosamine; D006153:Guanosine Diphosphate; C065276:2'-deoxyguanosine 3',5'-diphosphate; D005643:Fucose; C000458:N-acetyllactosamine; D005647:Fucosyltransferases; C418611:alpha3-fucosyltransferase V