A new approach for dynamics of enzyme-catalyzed glutathione conjugation byelectrospray quadrupole/time-of-flight mass spectrometry

The dynamics of enzyme-catalyzed glutathione conjugation was studied by ele ctrospray quadrupole/ time-of-flight (Q-TOF) mass spectrometry with a nanos pray interface. After incubation of human glutathione S-transferase A1-1 (G T) with glutathione (GSH) and an electrophilic substrate, electrospray indi cated 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 incubatio n time. The wide m/z range of detection (m/z 300-5000) allowed the observat ion of product, suggested to be released from enzyme/product adducts, in th e same mass spectrum. The noncovalent complexes of GT/product were complete ly replaced by GT/inhibitor complexes following the addition of GT inhibito r to the incubation mixture. Furthermore, a collision-activated decompositi on analysis of these ion species provided us with useful information to int erpret 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 sensit ivity. (C) 2001 Academic Press.