A new approach for dynamics of enzyme-catalyzed glutathione conjugation by electrospray quadrupole/time-of-flight mass spectrometry.
The dynamics of enzyme-catalyzed glutathione conjugation was studied by electrospray quadrupole/time-of-flight (Q-TOF) mass spectrometry with a nanospray interface. After incubation of human glutathione S-transferase A1-1 (GT) with glutathione (GSH) and an electrophilic substrate, electrospray indicated the presence of enzyme/product adducts such as [2GT + product], [2GT + GSH' + product], and [2GT + 2 products] as well as [2GT] and [2GT + GSH']. The relative abundance of GT/product adduct ions increased with incubation time. The wide m/z range of detection (m/z 300-5000) allowed the observation of product, suggested to be released from enzyme/product adducts, in the same mass spectrum. The noncovalent complexes of GT/product were completely replaced by GT/inhibitor complexes following the addition of GT inhibitor to the incubation mixture. Furthermore, a collision-activated decomposition analysis of these ion species provided us with useful information to interpret or identify ion species. The results suggest that electrospray Q-TOF mass spectrometry is a powerful approach for studying the dynamics of the enzyme reaction as well as the structure of enzyme complexes at high sensitivity.
Ishigai
M
M
Drug Metabolism & Pharmacokinetics Research Lab, Chugai Pharmaceutical Company, Ltd., Shizuoka, 412-8513, Japan. ishigaimsk@chugai-pharm.co.jp
Langridge
J I
JI
Bordoli
R S
RS
D004137:Dinitrochlorobenzene / Q000737:chemistry* / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000378:metabolism; D005978:Glutathione / Q000031:analogs & derivatives / Q000032:analysis* / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes; D007700:Kinetics; D013058:Mass Spectrometry / Q000379:methods*; D009579:Nitrobenzoates / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding / Q000502:physiology
D004137:Dinitrochlorobenzene; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D009579:Nitrobenzoates; D005982:Glutathione Transferase; C119995:glutathione S-transferase alpha; D005978:Glutathione; C043240:4-nitrobenzoyl chloride