A New Approach for Dynamics of Enzyme-Catalyzed Glutathione Conjugation by Electrospray Quadrupole/ Time-of-Flight Mass Spectrometry

• Published in: Analytical biochemistry Vol. 298; no. 1; pp. 83 - 92
• Main Authors: Ishigai, Masaki, Langridge, James I., Bordoli, Robert S.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01-11-2001
• Subjects: Dinitrochlorobenzene - chemistry; Dinitrochlorobenzene - metabolism; electrospray; enzyme; Enzyme Inhibitors - metabolism; Glutathione - analogs & derivatives; Glutathione - analysis; Glutathione - metabolism; glutathione conjugation; glutathione S-transferase; Glutathione Transferase - metabolism; Humans; Isoenzymes; Kinetics; Mass Spectrometry - methods; mechanism; Nitrobenzoates - chemistry; Nitrobenzoates - metabolism; noncovalent associations; Protein Binding - physiology; tandem MS; enzyme; glutathione conjugation; electrospray; glutathione S-transferase; noncovalent associations; mechanism; tandem MS
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1006/abio.2001.5339

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.