Activity-Based Probes for Isoenzyme- and Site-Specific Functional Characterization of Glutathione S-Transferases

J Am Chem Soc. 2017 Nov 15;139(45):16032-16035. doi: 10.1021/jacs.7b07378. Epub 2017 Nov 1.


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Binding Sites
  • Catalytic Domain
  • Glutathione / metabolism
  • Glutathione Transferase / antagonists & inhibitors
  • Glutathione Transferase / chemistry
  • Glutathione Transferase / metabolism*
  • Humans
  • Isoenzymes / antagonists & inhibitors
  • Isoenzymes / chemistry
  • Isoenzymes / metabolism
  • Liver / enzymology
  • Lung / enzymology
  • Mice
  • Photoaffinity Labels / chemistry
  • Photoaffinity Labels / metabolism*
  • Protein Binding


  • Isoenzymes
  • Photoaffinity Labels
  • Glutathione Transferase
  • Glutathione