Title | Activity-Based Probes for Isoenzyme- and Site-Specific Functional Characterization of Glutathione S-Transferases. |
Publication Type | Journal Article |
Year of Publication | 2017 |
Authors | Stoddard, EG, Killinger, BJ, Nair, RN, Sadler, NC, Volk, RF, Purvine, SO, Shukla, AK, Smith, JN, Wright, AT |
Journal | J Am Chem Soc |
Volume | 139 |
Issue | 45 |
Pagination | 16032-16035 |
Date Published | 2017 11 15 |
ISSN | 1520-5126 |
Keywords | Animals, Binding Sites, Catalytic Domain, Glutathione, Glutathione Transferase, Humans, Isoenzymes, Liver, Lung, Mice, Photoaffinity Labels, Protein Binding |
Abstract | 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.
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DOI | 10.1021/jacs.7b07378 |
Alternate Journal | J. Am. Chem. Soc. |
PubMed ID | 29068682 |
PubMed Central ID | PMC6279235 |
Grant List | P41 GM103493 / GM / NIGMS NIH HHS / United States P42 ES016465 / ES / NIEHS NIH HHS / United States DE-AC06-76RL01830 / / Department of Energy / International |