TitleActivity-Based Probes for Isoenzyme- and Site-Specific Functional Characterization of Glutathione S-Transferases.
Publication TypeJournal Article
Year of Publication2017
AuthorsStoddard, EG, Killinger, BJ, Nair, RN, Sadler, NC, Volk, RF, Purvine, SO, Shukla, AK, Smith, JN, Wright, AT
JournalJ Am Chem Soc
Date Published2017 11 15
KeywordsAnimals, Binding Sites, Catalytic Domain, Glutathione, Glutathione Transferase, Humans, Isoenzymes, Liver, Lung, Mice, Photoaffinity Labels, Protein Binding

Glutathione S-transferases (GSTs) comprise a diverse family of phase II drug metabolizing enzymes whose shared function is the conjugation of reduced glutathione (GSH) to endo- and xenobiotics. Although the conglomerate activity of these enzymes can be measured, the isoform-specific contribution to the metabolism of xenobiotics in complex biological samples has not been possible. We have developed two activity-based probes (ABPs) that characterize active GSTs in mammalian tissues. The GST active site is composed of a GSH binding "G site" and a substrate binding "H site". Therefore, we developed (1) a GSH-based photoaffinity probe (GSTABP-G) to target the "G site", and (2) an ABP designed to mimic a substrate molecule and have "H site" activity (GSTABP-H). The GSTABP-G features a photoreactive moiety for UV-induced covalent binding to GSTs and GSH-binding enzymes. The GSTABP-H is a derivative of a known mechanism-based GST inhibitor that binds within the active site and inhibits GST activity. Validation of probe targets and "G" and "H" site specificity was carried out using a series of competition experiments in the liver. Herein, we present robust tools for the characterization of enzyme- and active site-specific GST activity in mammalian model systems.

Alternate JournalJ. Am. Chem. Soc.
PubMed ID29068682
PubMed Central IDPMC6279235
Grant ListP41 GM103493 / GM / NIGMS NIH HHS / United States
P42 ES016465 / ES / NIEHS NIH HHS / United States
DE-AC06-76RL01830 / / Department of Energy / International